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n-Hexane Layer. Filtrate. Add anhydrous Na2SO4. (suitable amount), let stand, filter. Remove solvent by spraying nitrogen ... once the ethyl ether liquid inside has turned yellow. 4. Leave test ...... GC and HPLC incorporated methods are almost always used for ...... Add pure water to soy sauce until diluted by 10 fold. 2. Filter ...

C180-E059B

Index C H O

1. Food Product Components

1. 1 Analysis of Fatty Acids (1) – GC/MS-----------------------------------------------------------1 Analysis of Fatty Acids (2) – GC/MS-----------------------------------------------------------2 Analysis of Fatty Acids (3) / Derivatization - Fat Extraction Method------------------------------------------------------3 Analysis of Fatty Acids (4) / Derivatization - Preparation of Methyl Fatty Acids----------------------------------------4 Analysis of Fatty Acids (5) / Derivatization - Alkali Hydrolysis of Fat------------------------------------------------------5 Analysis of Fatty Acids (6) / Derivatization (1) - Preparation of Methyl Ester Derivative----------------------------------------------------6 Analysis of Fatty Acids (6) / Derivatization (2) - Methyl Ester Derivative---------------------------------------------------------------------7 1. 2 Fatty Acids (Fish Oil) - GC-----------------------------------------------------------------------8 1. 3 Triglycerides - GC--------------------------------------------------------------------------------9 1. 4 Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (1) - IR---------------10 Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (2) - IR---------------11 1. 5 Application of the ELSD-LT Low Temperature Evaporative Light Scattering Detector – LC-----------12 1. 6 Analysis of Decenoic Acid in Royal Jelly - LC-----------------------------------------------13 1. 7 Analysis of Fatty Acids - LC-------------------------------------------------------------------14 1. 8 Analysis of Organic Acid in Beer - LC--------------------------------------------------------15 1. 9 Analysis of Amino Acid in Cooking Vinegar Using Precolumn Derivatization (1) - LC-----------------------------------------------------------------------16 Analysis of Amino Acid in Cooking Vinegar Using Precolumn Derivatization (2) - LC-----------------------------------------------------------------------17 1. 10 Analysis of Amino Acids in Fermented Foods – LC-----------------------------------------18 1. 11 Simultaneous Analysis of D- and L-Amino Acids (1) - LC--------------------------------19 Simultaneous Analysis of D- and L-Amino Acids (2) - LC---------------------------------20 1. 12 Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (1) – LC-----------------------------21 Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (2) – LC-----------------------------22 1. 13 Analysis of Theanine in Green Tea – LC-----------------------------------------------------23 1. 14 Obligation to Display Nutritive Components in Processed Foods - UV-------------------24 1. 15 Analysis of Trace Amounts of Vitamins B1 and B2 in Food Products Using Fluorescence Photometry (1) - RF-------------------------------------------------25 Analysis of Trace Amounts of Vitamins B1 and B2 in Food Products Using Fluorescence Photometry (2) - RF-------------------------------------------------26 1. 16 Analysis of Water Soluble Vitamins Using Semi-micro LC System - LC----------------27 1. 17 Analysis of Vitamin B Group - LC------------------------------------------------------------28 1. 18 Analysis of Tocopherol in Milk - LC----------------------------------------------------------29 1. 19 Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (1) – LC-----------30 Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (2) – LC-----------31 1. 20 Analysis (Measurement of K Value) of Nucleotide in Tuna Meat - LC--------------------32 1. 21 Analysis of Oligosaccharide in Beer - LC----------------------------------------------------33 1. 22 Analysis of Oligosaccharides in Beer Using the ELSD-LT Low Temperature Evaporative Light Scattering Detector – LC----------34 1. 23 Analysis of Saccharides in Fermented Foods – LC-----------------------------------------35 1. 24 Analysis of Nonreducing Sugar Using Postcolumn Derivatization with Fluorescence Detection - LC----------------------------------------------------------------36 1. 25 Analysis of Sugar in Yogurt - LC--------------------------------------------------------------37 1. 26 Analysis of Alliin in Garlic - LC----------------------------------------------------------------38 1. 27 Analysis of Catechins in Green Tea - LC-----------------------------------------------------39 1. 28 Analysis of Chlorogenic Acid in Coffee - LC-------------------------------------------------40 1. 29 Analysis of Lycopene and β-Carotene in Tomato - LC-------------------------------------41 1. 30 Melting of Fats and Oils - TA------------------------------------------------------------------42 1. 31 Gelatinization of Starch - TA-------------------------------------------------------------------43

2. Food Additives 2. 2. 2. 2. 2.

1 2 3 4 5

2. 6 2. 7 2. 8 2. 9 2. 10 2. 11 2. 12 2. 13 2. 14 2. 15

Propionic Acid in Cookies and Bread - GC---------------------------------------------------44 Saccharine and Sodium Saccharine - GC----------------------------------------------------45 Ethylene Glycols in Wine - GC-----------------------------------------------------------------46 Sorbic Acid, Dehydroacetic Acid and Benzoic Acid - GC-----------------------------------47 Analysis of Preservatives in Food Products with Absorption Photometry (1) - UV-------------------------------------------------------------------------48 Analysis of Preservatives in Food Products with Absorption Photometry (2) - UV-------------------------------------------------------------------------49 Color Control of Food Products (1) - UV-----------------------------------------------------50 Color Control of Food Products (2) - UV-----------------------------------------------------51 Analysis of Sweetener in Soft Drink - LC----------------------------------------------------52 Analysis of Fungicide in Oranges - LC-------------------------------------------------------53 Analysis of Phenol Antioxidant in Foods - LC-----------------------------------------------54 Analysis of L-Ascorbic Acid 2-Glucoside - LC---------------------------------------------55 Analysis of EDTA in Mayonnaise - LC-------------------------------------------------------56 Analysis of Benzoyl Peroxide in Food Product - LC----------------------------------------57 Analysis of p-Hydroxybenzoates in Soy Sauce - LC----------------------------------------58 Analysis of Potassium Bromate in Bread - LC----------------------------------------------59 Simultaneous Analysis of Water-soluble Tar Pigments - LC------------------------------60

3. Residual Pesticides 3. 1 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) – GC------------61 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) – GC------------62 3. 2 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) – GC------------63 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) – GC------------64 3. 3 Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (1) – GC----------65 Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (2) – GC-------------66 3. 4 Simultaneous Analysis of Pesticides (1) - GC/MS------------------------------------------67 Simultaneous Analysis of Pesticides (2) - GC/MS------------------------------------------68 3. 5 Analysis of Pesticides Using NCI (1) - GC/MS----------------------------------------------69 Analysis of Pesticides Using NCI (2) - GC/MS----------------------------------------------70 3. 6 Analysis of Pesticide Residue in Foods Using GC/MS (1) – GC/MS----------------------71

Analysis of Pesticide Residue in Foods Using GC/MS (2) – GC/MS----------------------72 3. 7 Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (1) – GC/MS-----------73 Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (2) – GC/MS-----------74 3. 8 Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (1) – GC/MS----------75 Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (2) – GC/MS----------76 3. 9 Analysis of Pesticides with Specific Threshold Levels in Foods (1) – LC----------------77 Analysis of Pesticides with Specific Threshold Levels in Foods (2) – LC----------------78 3. 10 GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (1) – LC------------79 GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (2) – LC------------80 3. 11 Analysis of Carbamate Pesticides – LC------------------------------------------------------81 3. 12 Analysis of Imazalil in Oranges - LC----------------------------------------------------------82 3. 13 Analysis of Pesticide Residue in Agricultural Products Using LC/MS (1) – LC/MS--------------83 Analysis of Pesticide Residue in Agricultural Products Using LC/MS (2) – LC/MS--------------84 3. 14 Analysis of N-methylcarbamate Pesticides Using LC/MS (1) – LC/MS------------------85 Analysis of N-methylcarbamate Pesticides Using LC/MS (2) – LC/MS------------------86 3. 15 Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (1) – LC/MS--------87 Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (2) – LC/MS--------88 3. 16 Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (1) – LC/MS----------------------89 Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (2) – LC/MS----------------------90

4. Aromas and Odors 4. 1 4. 2 4. 3 4. 4 4. 5 4. 6 4. 7 4. 8 4. 9 4. 10 4. 11 4. 12 4. 13 4. 14

Aromatic Components of Alcohols - GC-----------------------------------------------------91 Aromatic Components of Tea - GC-----------------------------------------------------------92 Essential Oil (Headspace Analysis) - GC-----------------------------------------------------93 Essential Oil (Direct Analysis) - GC-----------------------------------------------------------94 Diketones - GC----------------------------------------------------------------------------------95 Fruit Fragrances - GC---------------------------------------------------------------------------96 Vegetable Fragrances - GC--------------------------------------------------------------------97 Flavor of Rice – GC-----------------------------------------------------------------------------98 Flavoring Agent for Food Product - GC------------------------------------------------------99 Analysis of Fishy Smell in Water (1) - GC/MS---------------------------------------------100 Analysis of Fishy Smell in Water (2) - GC/MS---------------------------------------------101 Analysis of Alcohols (1) - GC/MS-----------------------------------------------------------102 Analysis of Alcohols (2) - GC/MS-----------------------------------------------------------103 Analysis of Strawberry Fragrances - GCMS------------------------------------------------104 Analysis of Beverage Odors (1) - GC/MS---------------------------------------------------105 Analysis of Beverage Odors (2) - GC/MS---------------------------------------------------106 Analysis of Fragrant Material (1) - GC/MS-------------------------------------------------107 Analysis of Fragrant Material (2) - GC/MS-------------------------------------------------108 Analysis of Fragrant Material (3) - GC/MS-------------------------------------------------109

Na Mg

Ca

5. Inorganic Metals

5. 1 Analysis of Inorganic Ions in Milk (1) - LC-------------------------------------------------110 Analysis of Inorganic Ions in Milk (2) - LC-------------------------------------------------111 5. 2 Analysis of Pb in Milk Using Atomic Absorption Spectrophotometry - AA--------------112 5. 3 Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (1) - AA--------------113 Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (2) - AA--------------114 5. 4 Analysis of Cadmium in Rice (1) – AA------------------------------------------------------115 Analysis of Cadmium in Rice (2) – AA------------------------------------------------------116 5. 5 Analysis of Inorganic Components in Powdered Milk (1) - ICP-AES-------------------117 Analysis of Inorganic Components in Powdered Milk (2) - ICP-AES-------------------118 5. 6 Analysis of Canned Beverage (Green Tea) - ICP-AES ------------------------------------119 5. 7 Analysis of Inorganic Components in Processed Food Products - ICP-AES-------------120 5. 8 Analysis of Cooking Oil – ICP-AES----------------------------------------------------------121 5. 9 Analysis of Pastry – ICP-AES----------------------------------------------------------------122 5. 10 Analysis of Nutrition Function Food Products (1) – ICP-AES---------------------------123 Analysis of Nutrition Function Food Products (2) – ICP-AES---------------------------124 5. 11 Analysis of Plants – ICP-AES----------------------------------------------------------------125 5. 12 Analysis of Powdered Milk Using ICPM-8500 – ICP/MS---------------------------------126 5. 13 Analysis of Plants Using ICPM-8500 – ICP/MS-------------------------------------------127

6. Others 6. 1 Analysis of Organotin Compounds Using Capillary GC-FPD – GC-----------------------128 6. 2 Analysis of Organotin in Fish (1) – GC/MS-------------------------------------------------129 Analysis of Organotin in Fish (2) – GC/MS-------------------------------------------------130 6. 3 Analysis of Shellfish Toxins (1) - LC--------------------------------------------------------131 Analysis of Shellfish Toxins (2) - LC--------------------------------------------------------132 6. 4 Analysis of Oxytetracycline - LC-------------------------------------------------------------133 6. 5 Analysis of Closantel - LC--------------------------------------------------------------------134 6. 6 Simultaneous Analysis of Synthetic Antibacterial Agent (1) - LC------------------------135 Simultaneous Analysis of Synthetic Antibacterial Agent (2) - LC------------------------136 6. 7 Analysis of Enrofloxacin in Broiled Eel (1) - LC--------------------------------------------137 Analysis of Enrofloxacin in Broiled Eel (2) - LC/MS---------------------------------------138 6. 8 Analysis of Malachite Green in Farmed Fish – LC-----------------------------------------139 6. 9 Analysis of New Type Quinolone Antibacterial Agents in Poultry - LC/MS----------140 6. 10 Analysis of Aminoglycoside Antibiotics(1)-LC/MS----------------------------------------141 Analysis of Aminoglycoside Antibiotics(2)-LC/MS----------------------------------------142 6. 11 Analysis of Fumonisin in Sweet Corn (1) - LC---------------------------------------------143 Analysis of Fumonisin in Sweet Corn (2) - LC---------------------------------------------144 6. 12 Analysis of Aflatoxins (1) – LC--------------------------------------------------------------145 Analysis of Aflatoxins (2) – LC--------------------------------------------------------------146 Analysis of Aflatoxins (3) – LC--------------------------------------------------------------147 6. 13 Analysis of Aflatoxins Using LC/MS (1) – LC/MS-----------------------------------------148 Analysis of Aflatoxins Using LC/MS (2) – LC/MS-----------------------------------------149 6. 14 Analysis of Patulin Using LC/MS-LC/MS---------------------------------------------------150 6. 15 Analysis of Diarrhetic Shellfish Poison (DSP) by LC/MS (1) – LC/MS------------------151 Analysis of Diarrhetic Shellfish Poison (DSP) by LC/MS (2) – LC/MS------------------152

C H O

1. Food Product Components

1.1 Analysis of Fatty Acids (1) - GC/MS ■ Explanation Fatty Acids exist in a great many food products. And derivatization process is used to measures them. The aims of derivatization process are as follows. 1) Weaken the polarity of compounds. 2) Lower the boiling point. 3) Increase molecular ion peak and ion intensity in high mass region. In the case of fatty acids, derivatization process is used to achieve item 1). The methyl esterization or trimethylsilylation can be used but generally methyl esterization employing diazomethane is used for the derivatization.

cases, the Ci mass spectrum is measured. With the Ci mass spectrum, the ion denoting the molecular weight appears as an ion (M+1) with added proton in the molecular weight for detection of molecular weight + 1 ion. Measuring the Ei and Ci mass spectra enables qualitative analysis of compounds in fatty acid methyl ester measuring. Also, the columns used in this measuring include the slightly polar column DB-1 and polar column DB-WAX. The polarity column produces peaks in the saturated and unsaturated order while the slightly polar column produces peaks in the reverse order. ■ Analytical Conditions : GCMS-QP5000 Instrument : DB-WAX Column (30m×0.25mmI.D. df=0.25µm) Column Temp. : 60˚C-10˚C/min-250˚C : 250˚C Inj. Temp. : 250˚C I/F Temp. : He(100kPa) Carrier Gas Reagent Gas : Isobutane

Normally, the molecular ion peak that displays the molecular weight is detected for the Ei mass spectrum's saturated fatty acid methyl ester and, as determination of molecular weight is easy, a carbon count is possible. However, the molecular ion peak often does not appear when the level of unsaturation increases, which means that not only molecular weight but also the carbon count and unsaturated level cannot be determined. In such

74

43

x

5.0

C18:0 87 55

97 111 129

50

255

143 157

100

185 199

150

213 227

200

267

250

298(M)

300

350

400

400

450

Fig. 1.1.1 Ei mass spectrum of C18:0

299

(M+H)

C18:0

113 131

100

165

150

196

200

219

244

250

+

265

300

350

Fig. 1.1.2 Ci mass spectrum of C18:0

1

1.1 Analysis of Fatty Acids (2) - GC/MS

41

x

5.0

C20:5 79 67 55 93 119 145 159

50

100

181 199

252

200

250

150

300

350

400

400

450

Fig. 1.1.3 Ei mass spectrum of C20:5

317

(M+H)

C20:5

109

175

135 149

100

195

150

285

221 235

200

+

267

250

300

350

317

265

Fig. 1.1.4 Ci mass spectrum of C20:5

355

291

345 383 381 345

311

319

325

291

293

319

369

327

299

295 295 293 293

263

297

297

263

267

271 269

243

343

2779198

TIC 243.00 271.00 269.00 267.00✻10.00 265.00✻10.00 263.00✻10.00 299.00✻10.00 297.00 295.00✻10.00 293.00✻10.00 291.00✻10.00 327.00✻10.00 325.00✻10.00 319.00✻10.00 317.00 355.00✻10.00 331.00✻10.00 369.00 345.00✻10.00 383.00✻10.00 343.00✻10.00 381.00✻10.00

Fig. 1.1.5 Mass chromatogram of protonized molecules for fatty acid methyl ester

2

C

Food Product Components

H O

1.1 Analysis of Fatty Acids (3) / Derivatization - Fat Extraction Method ■ Pretreatment for Fatty Acid Analysis Fat must be extracted from the food product and hydrolysis and methylation performed for GC and GC/MS analysis of fatty acids in food products. Here,

1. Fat extraction

several representative pretreatment methods will be introduced from the numerous methods available.

2. Preparation of methylated fatty acid

GC, GC/MS analysis

Alternatively 1. Fat extraction

4. Methylation of fatty acid

3. Alkali hydrolysis of fat

GC, GC/MS analysis

1. Fat Extraction This shows an example of fat extracted from a sample. References Standard Methods of Analysis for Hygienic Chemists and Notes 1990 Appended supplement (1995) Pharmaceutical Society of Japan Edition, published by Kanehara & Co., Ltd (1995) Sample 5g

(Precisely measured) Add 16mL of H2O Homogenize Separate sample into separating funnel with 100mL of CHCR3 : MeOH (2:1) Shaking extraction for 5 min

Water Layer

Water Layer

CHCR3Layer Shaking extraction for Shaking extraction for 5 min 100mL of CHCR3 : MeOH (2:1) Two Times CHCR3Layer

Rinse with 100mL of 0.5% NaOH Dehydrate and filter with anhydrous Na2SO4 (suitable amount) Filtrate

Remove solvent by spraying nitrogen gas at 40˚C or less Fat Extract 3

Fig. 1.1.6 Fat extraction method

1.1 Analysis of Fatty Acids (4) / Derivatization - Preparation of Methyl Fatty Acids 2. Preparation of Methylated Fatty Acid This shows a transmethylation method for extracting fat using an alkali catalyst that does not require fat extraction of food oils, etc. This easy method just requires hydrolysis and fatty acid extraction so labor is reduced.

Note, however, that amide-bonded fatty acid and free fatty acid do not methylate.

Reference Standard Methods of Analysis for Hygiene Chemists and Notes 1990 Appended supplement (1995) Pharmaceutical Society of Japan Edition, published by Kanehara & Co., Ltd

20mg of Extracted Fat

(Precisely measured)

Dissolve in 1mL of benzene Add 2mL of 0.5N sodium methoxide (diluted with anhydrous MeOH) and shake, and leave for 10 min at room temperature Add 0.5N acetic acid water solution to neutralize Add 5mL of hexane and perform shaking extraction for 1 min Water Layer

Hexane Layer Re-extract with 5mL of hexane

Water Layer

Hexane Layer

Add small amounts of anhydrous Na2SO4 + NaHCO3 (2+1), leave for 30 min, and filter Filtrate

Remove solvent by spraying nitrogen gas at 40˚C or less Dissolve in 5mL of hexane GC, GC/MS

Fig. 1.1.7 Preparation of methylated fatty acid

4

C

Food Product Components

H O

1.1 Analysis of Fatty Acids (5) / Derivatization - Alkali Hydrolysis of Fat 3. Alkali Hydrolysis of Fat Extracted fat is triacylglycerol which emerges as glycerol and potassium salt's fatty acid (water soluble) using alkali. Fatty acid hardly separates when acidified, which

enables extraction with non-polar solvent. Here, an example of alkali hydrolysis is introduced.

Reference Organic Chemistry Testing Guidebook No. 5, Handling Biological materials, Toshio Goto, Tetsuo Shiba, Teruo Matsuura ed, Kagaku-Dojin Publishing Company, INC (1991)

5g of Extracted Fat (Precisely measured) Add 15mL of 20% (w/w) KOH (in 40% EtOH water solution) Reflux for approximately 1 hr in 85˚C heated bath After cooling, separate reaction liquid using separatory funnel, and add 6N-HCr to adjust to pH1. Perform shaking extraction with diethyl ether (3 extractions: 30mL, 20mL and 20mL)

Water Layer

Diethyl Ether Layer Combine diethyl ether with separatory funnel, clean 3 times with 20mL of saturated Na2CO3 water solution, and then rinse 3 times with 20mL of water Add anhydrous Na2SO4, stir, leave for 1 hr, and filter Filtrate Remove solvent using rotary evaporator or nitrogen gas spraying Fatty Acid Fig. 1.1.8 Alkali hydrolysis of fat

5

1.1 Analysis of Fatty Acids (6) / Derivatization (1) - Preparation of Methyl Ester Derivative 4. Methyl Ester Derivative Preparation Method High-class fatty acids are generally derived into methyl

ester. The currently used methods are introduced here.

(1) Methyl Esterization using BF3-CH3OH

20mg of Fatty Acid (Remove solvent if it is in solution) Boil approximately 3mL of BF3-CH3OH over a water bath for 2 min Shake 20mL of n-hexane + 20mL of distilled water

Water Layer

n-Hexane Layer Add anhydrous Na2SO4 (suitable amount), let stand, filter Filtrate Remove solvent by spraying nitrogen gas over 50˚C water bath GC,GC/MS

Fig. 1.1.9 Methyl esterization using boron trifluoride-methanol

(2) Methyl Esterization Using H2SO4-CH3OH

20mg of Fatty Acid Boil approximately 20mL of H2SO4-CH3OH over a water bath for 1 hr Shake 20mL of n-hexane + 20mL of distilled water

Water Layer

n-Hexane Layer • Repeatedly rinse with 10mL batches of distilled water to neutralize • Add anhydrous Na2SO4 (suitable amount), let stand, filter Filtrate Remove solvent GC,GC/MS

Fig. 1.1.10 Methyl esterization using sulphuric acid-methanol

6

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Food Product Components

H O

1.1 Analysis of Fatty Acids (6) / Derivatization (2) - Methyl Ester Derivative

Fig. 1.1.11 Methyl esterization using diazomethane

(3) Methyl Esterization Using CH2N2 A diazomethane generator is assembled as shown in the diagram. And ethyl ether (I), 50% potassium hydroxide water solution (II), 10mg of fatty acid + 2mL of ethyl ether (III) and acetic acid are sealed in tubes. 1. A suitable amount of nitrogen gas is passed through test tube I. 2. Some 0.5 to 1mL of N-methyl-N’-nitroso-ptoluenesulfonamide with 20% ethyl ether is injected into test tube II to create diazomethane. 3. Remove test tube III from diazomethane generator once the ethyl ether liquid inside has turned yellow. 4. Leave test tube III to stand for 10 min to enrich the ethyl ether, and inject into GC or GCMS. ● Notes and coutions - Handle diazomethane with care, as it is carcinogenic. - For the above reason, only adjust small amounts and be sure to use a ventilating hood. - Do not use ground glass stoppers because there is a danger of explosion. - Small amounts of ether solution (100mL or less) can be stored in a refrigerator for several days. ● Several relatively easy-to-handle diazomethane generators are available in market.

7

(4) Methyl Esterization Using Dimethylformamide Dialkylacetals (CH3) 2NCH(OR)2 Add 300µL of esterification reagent to some 5 to 50mg of fatty acid. Dissolve the sample and inject the resultant reaction liquid into the GC or GCMS. (Normally it is best to heat this at 60˚C for 10 to 15 min.) (5) Methyl Esterization Using Phenyltrimethyl Ammonium Hydroxide (PTAH) Dissolve the fatty acid in acetone, add PTAH/methanol solution (1 to 1.5M%), thoroughly stir sample and reaction reagent, leave to stand for 30 min, and induct into GC or GCMS. This methyl esterization using on-column injectionn is a method where the PTAH/methanol reagent and fatty acid are mixed in advance, injected into the GC and made to react in a GC injector. Compared to other methods treatment is quick and simple and there is no volatile loss because the reaction is in a GC injector. Furthermore, harmful, dangerous reagents are not required.

1.2 Fatty Acids (Fish Oil) - GC ■ Explanation Among high-class fatty acids, unsaturated fatty acids are currently in the limelight, for example, much attention is being given to the antithrombogenic effect of eicosapentaenoic acid, etc. From the outset, gas chromatographs have been used to separate and quantify high-class fatty acids. High-class fatty acids have absorptivety and high boiling points, which means that derivatization (usually methyl esterization) is performed for GC analysis. This example introduces capillary column analysis of fatty acid methyl ester in fish oil. Fig. 1.2.1 shows constant pressure analysis at 110kPa and Fig. 1.2.2 shows programmed pressure analysis from 110kPa to 380kPa. Programmed pressure analysis provides quicker analysis with improved sensitivity because separation hardly changes.

■ Pretreatment Methyl esterization of fatty acids in fish oil is performed in accordance with Fig. 1.1.11 followed by GC analysis.

■ Analytical Column Column Temp. Inj. Temp. Det. Temp. Carrier Gas

Conditions

: CBP20 (25m×0.22mm I.D. df=0.25µm) : 210˚C : 230˚C : 230˚C(FID) : He 100kPa (0.52mL/min at 210˚C) Injection Method : Split 1:100

380kPa(10min)

Pressure Pressure 110kPa

180kPa 1= C18

C16

10kPa/min 1min

5=

2=

C20

C18

3=

1=

C18

C18

C16

30kPa/min

110kPa

1=

C22

3= C18

6=

C22 1=

C20 5=

C20

+

C14

5=

Fig. 1.2.1 Analysis of fatty acid methyl ester in fish oil (constant pressure)

18.0

8.0

6.0

4.0

2.0

0.0

38.0

36.0

34.0

(min)

12.0

C20 32.0

30.0

28.0

26.0

24.0

22.0

20.0

18.0

C22

4=

5=

C22 16.0

14.0

1=

C24

16.0

+ 4=

C20 12.0

8.0

4= C18 3= C18

C18

6=

C22

14.0

1=

C24

2= C18

10.0

C18

6.0

4.0

2.0

0.0

C14

1=

C16

1=

C22

10.0

1=

C20

1=

C16

(min)

Fig. 1.2.2 Analysis of fatty acid methyl ester in fish oil (programmed pressure)

8

Food Product Components

C H O

1.3 Triglycerides - GC ■ Explanation Triglycerides are compounds with a high boiling points and strong absorptivity. Separation is poor in analysis of these compounds when a short column filled with highly heat resistant packing is used with the packed-column GC. In comparison to this kind of column a capillary column filled with fused silica offers minimal absorptivity at high separation and excellent heat resistance. However, even better heat resistance is required for high-boiling-point compounds like triglycerides. Stainless steel capillary columns or aluminum coated ones are extremely heat resistant and, as such, are suitable for analysis of triglycerides. Also, cold on-column injector suppress discrimination of samples.

■ Analytical Conditions : CBM65 (25m×0.22mm I.D. df=0.10µm) Column Column Temp. : 50˚C(1min)-20˚C/min-240˚C -6˚C/min-390˚C : 390˚C(FID) Det. Temp. : He(1.5mL/min) Carrier Gas Injection Method : Cold on-column

C50

C40

C40

C54

C30

C30

min

Fig. 1.3.1 Analysis of triglyceride in butter

9

Fig. 1.3.2 Analysis of triglyceride in palm oil

44

40

36

32

28

24

20

16

12

8

4

0

56

52

48

44

40

36

32

28

24

20

16

12

8

4

0

C50

min

1.4 Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (1) - IR ■ Explanation Food products are mixtures of various compounds that require liquid chromatography (LC) or gas chromatography (GC) separation procedures for component analysis. However, injections of a large amount of samples are difficult due to column load restrictions in chromatography, so at maximum the amount of component existing in one peak of a chromatogram will be only in the µg order. Nevertheless, if a FTIR is used, infrared measuring is possible and components can be quantified. Here, component analysis of food products using a preparative LC-FTIR method will be introduced. ■ Pretreatment Red wine that has been filtered through a membrane filter was injected into an LC. Fig. 1.4.1 shows a chromatogram detected by the UV detector. The separated substances in peaks A to C have been collected, but because there are numerous coexisting substances in the collected substances, the collected substance is reinjected into the LC using a mobile phase of water, and the chromatogram measured. The separated substances in the largest peak obtained from this operation is collected, the mobile phase vaporized from within the collected substance, this collected substance is mixed with KBr powder and measured using a diffuse reflection method. Fig. 1.4.2 shows the infrared spectrum of peak A. Absorption of coexisting substances is overlaid but tartaric acid can be clearly confirmed.

Fig. 1.4.3 shows the infrared spectrum of peak B. The carboxylic acid peak can be confirmed in the region of 1730cm-1 and, as glucose (a coexisting substance) is equal to the holding time, glucose absorption has mostly become infrared spectrum. Fig. 1.4.4 shows the infrared spectrum of peak C. In this case there is no interference from other components and the spectrum is only for succinic acid.

■ Analytical Conditions : LC-VP Series Instrument : Shim-pack SCR-102H Column (300mmL.×8.0mm I.D.) Mobile Phase : 5mM Trifluoroacetic Acid Aqueous Solution : 1mL/min Flow Rate Column Temp.: 50˚C : UV-VIS Detector 210nm Detector Instrument Resolution Accumulation Appodization Detector

: FTIR : 4cm-1 : 50 : Happ-Genzel : Pyroelectric Detector

10

C

Food Product Components

H O

1.4 Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (2) - IR

% 100.00

100.00

88.00

88.00

76.00

76.00

64.00

64.00

52.00

52.00

40.00

40.00

A C

-

-

-

-

5

10

15

20

B

Fig. 1.4.1 LC chromatogram of red wine

Fig. 1.4.2 Infrared spectrum of peak A (T: tartaric acid peak)

%

% 100.00

100.00

100.00

100.00

86.00

86.00

86.00

86.00

72.00

72.00

72.00

58.00

58.00

58.00

44.00

44.00

44.00

30.00

30.00

30.00

G 72.00

G

G

58.00

G G 44.00

G 30.00

Fig. 1.4.3 Infrared spectrum of peak B (G: glucose)

11

Fig. 1.4.4 Infrared spectrum of peak C (succinic acid)

1.5 Application of the ELSD-LT Low Temperature Evaporative Light Scattering Detector - LC - Analysis of Triglyceride in Cooking Oil ■ Explanation The ELSD-LT Low Temperature Evaporative Light Scattering Detector is an HPLC general-purpose detector that converts the target components into minute particles by evaporating the mobile phase and makes lightscattering measurements of the particles. The ELSD-LT can, in principle, detect nearly all nonvolatile compounds and so it is ideal for the analysis of compounds such as sugars, oils, and surfactants, which have a low absorbency and are difficult to detect with UV detectors. It can also be used for gradient elution, which is not possible using a differential refractive index detector (RID), allowing application in a wide variety of fields. Application of the ELSD-LT to the analysis of triglyceride in cooking oil is described here as an example.

■ Analytical Conditions : Shim-pack VP-ODS (250mmL.×4.6mm I.D.) Column Mobile Phase : A : Acetonitrile B : Acetone Linear Gradient B 50% → 70% (10 to 40min) : 1.0mL/min Flow Rate Temperature : 30˚C : ELSD-LT Detection Temperature : 35˚C Gain : 5 Nebulizer Gas : Air Gas Pressure : 350kPa Mobile phase

Gas Nebulization

Evaporation

Detection Light Photomultiplier

Fig. 1.5.1 Operating principle of ELSD-LT

■ Analysis of Triglyceride in Cooking Oil Cooking oil contains many types of triglyceride, which differ according to the acyl group. Quality control of cooking oils is also carried out by performing pattern analyses of these triglycerides. Fig. 1.5.2 and 1.5.3 show the results obtained by preparing 2.0g/L solutions of commercial safflower oil

and sesame oil (solvent: acetonitrile/acetone = 1/1, v/v) and injecting 20µL of these solutions. In general, elution is faster for triglycerides with smaller acyl carbon numbers or with a larger numbers of double bonds.

40

mV

mV

15

20

10

5

0

0 0

20

40 min

Fig. 1.5.2 Chromatogram of safflower oil

60

0

20

40

60

min

Fig. 1.5.3 Chromatogram of sesame oil

12

C

Food Product Components

H O

1.6 Analysis of Decenoic Acid in Royal Jelly - LC ■ Explanation Royal jelly is widely known as a food product and herbal medicine, and its peculiar component is 10-hydroxy-δdecenoic acid (10-HAD). The amount of this and the investigation method are vital points in composition standards for royal jelly. The following is an analysis example.

■ Pretreatment Distilled water is added to a specific amount of sample, dissolved through mixing, a specific amount of internal standard (benzoic acid) was added and the mixture filtered through a disposable 0.45µm filter. ■ Analytical Conditions Column : STR ODS-2(150mmL.×4.6mm Ι.D) Mobile Phase : 10mM Sodium Phosphate Buffer (pH2.6)/Methanol=55/45 (v/v) Flow Rate : 1.0mL/min Temperature : 40˚C Detection : UV-VIS Detector 210nm

Internal standard substance

10-HDA

Reference Study group text related to royal jelly composition standard testing method provided by Japan Royal Jelly Fair Trade Council

0

10

Fig. 1.6.1 Analysis of decenoic acid in raw royal jelly

13

min

1.7 Analysis of Fatty Acids - LC ■ Explanation Fatty acid can be detected using carboxyl group absorbent (210nm) in the same way as organic acid, etc. However, this kind of short wavelength is susceptible to impurities and some samples are difficult to analyze. Here, a prelabel agent is derived into a fluorescent substance and detected using a fluorescent detector. The compound labeling agent ADAM (9-Anthryldiazomethane) possessing the carboxyl group is a prelabel agent that targets the methylating agent (diazomethane) reaction. Here, direct analysis using UV absorption detection and prelabel derivatization detection using ADAM agent will be introduced.

Reference Shimadzu HPLC Food Analysis Applications Data Book (C190-E047) ■ Analytical Conditions : Shim-pack CLC-ODS (150mmL.×6.0mm I.D.) Column Mobile Phase : Acetonitrile/Water = 95/5 (v/v) : 1.0mL/min Flow Rate Temperature : 45˚C : Fluorescence Detector Detection Ex : 365nm Em : 415nm

■ Peaks

■ Peaks

1. Lauryl acid

1. Lauryl acid

2. Myristic acid

2. Myristic acid

3. Linoleic acid

3. Palmitic acid

4. Palmitic acid

4. Stearic acid

1

5. Oleic acid 6. Stearic acid 2 1 4 3

5

2

6

3 4

0

5

10

15 (min)

Fig. 1.7.1 Analysis of fatty acid using UV absorption detection

+ CHN 2 ADAM (9-Anthryldiazomethane)

0

10

20

30

40

50

60

(min)

Fig. 1.7.2 Analysis of high-class fatty acid using precolumn derivatization method with ADAM

HOCOR

-N 2 CHN 2 OCOR (Ex365nm Em412nm)

Fig. 1.7.3 Reaction equation for ADAM and fatty acid

14

C

Food Product Components

H O

1.8 Analysis of Organic Acid in Beer - LC ■ Explanation In the case of analysis of organic acid using absorptiometry, carboxyl group absorption at 200 to 210nm is used, but some samples are difficult to analyze because of poor selectivity and impurity interference at this wavelength. In such cases, a conductivity detector that detects ionized substances at selectively high sensitivity is used. References Hayashi, Shimadzu Review 49 (1), 59 (1992) Shimadzu LC Application Report No. 18 Shimadzu HPLC Food Analysis Applications Data Book (C190-E047)

■ Analytical Conditions : Shim-pack SCR-102H×2 Column (300mmL.×8.0mm I.D.) : 5mM p-Toluenesulfonic Acid Mobile Phase : 0.8mL/min Flow Rate : 45˚C Temperature Reaction Reagent : 5mM p-Toluenesulfonic Acid 20mM Bis-Tris 100µM EDTA : Reaction Reagent Flow Rate 0.8mL/min : 48˚C Cell Temperature : Conductivity Detector Detection

■ Pretreatment Beer is injected in without any pretreatment.

■ Peaks

1

1. 2. 3. 4. 5. 6. 7. 8. 9.

Phosphoric acid Citric acid Pyruvic acid Malic acid Succinic acid Lactic acid Formic acid Acetic acid Levulinic acid

8

2 4 5

9

3 6 7

0

5

10

15

20 (min)

Fig. 1.8.1 Analysis of beer Column oven Injector Pump

Conductivity detector Analysis column

Mixer

Mobile phase

Reaction agent

15 Fig. 1.8.2 Flowchart of organic acid analysis system

1.9 Analysis of Amino Acid in Cooking Vinegar Using Precolumn Derivatization (1) - LC ■ Explanation A separation method (precolumn derivatization method) exists using reversed phase chromatography with a derivatization reaction performed on the sample pretreatment stage. Here, the analysis example shows an OPA (o-phthalaldehyde) precolumn derivatization method. ■ Pretreatment See next page for details.

■ Analytical Conditions : Shim-pack CLC-ODS Column (150mmL.×6.0mm I.D.) with Guard Column Precolumn : Shim-pack GRD-ODS (250mmL.×4.0mm I.D.) Mobile Phase : (A)10mM Sodium Phosphate Buffer (pH 6.8) (B)A/Acetonitrile = 2/1 (C)80% Acetonitrile Water Solution Gradient Method : 1.0mL/min Flow Rate Temperature : 45˚C : Fluorescence Detector Detection Ex : 350nm Em : 460nm (Primary Amino Acid) Ex : 485nm Em : 530nm (Secondary Amino Acid)

LEU PHE ILE

LYS

ORN

HIS α––ABA

TAU

PEA GLN

SER

GLY

ASN

THR

MET

TYR

ALA

VAL

ASP

n ––V A L ( I . S . )

GLU

Ex350nm Em460nm

ARG

PRO

Ex485nm Em530nm

(min)

Fig. 1.9.1 Analysis of cooking vinegar using prelabel amino acid analysis method

16

C

Food Product Components

H O

1.9 Analysis of Amino Acid in Cooking Vinegar Using Precolumn Derivatization (2) - LC P re tre a tme n t S a mp le

1 0 ~ 20µL -M e rc a pt O PA re a ge nt

re a ge nt 1 ) 2)

200µL

200µL

M ix in g N B D -C l re a ge nt 3 )

200µL

M ix in g an d W a iting In jec tio n 1)

1 0µL

to H PLC 10 µL 10mL 20mg 3mL 10mL 100mg 10mL

-M e rc a p t p ro p io n ic a c id

1 0 0 mM B o ra te bu ffer (p H 9.0) 2 ) O PA A ce to n itrile 1 0 0 mM B o ra te bu ffer (p H 9.0) 3 ) N BD -Cl A ce to n itrile Fig. 1.9.2 Pretreatment conditions B.CONC B.CONC B.CONC

5 15 20

16.0 18.0 25.0

B.CONC B.CONC B.CONC

27 30 45

30.0 39.0 39.01 42.0

B.CONC B.CONC B.CONC B.CONC

50 65 70 75

48.0 48.01

B.CONC B.CONC

80 100

49.0 53.0 54.0

SV SV B.CONC

1 0 100

54.01 54.02

B.CONC STOP

0

0.01 4.0 8.0

17

Fig. 1.9.3 Gradient conditions

1.10 Analysis of Amino Acids in Fermented Foods - LC

Abbreviation

Amino acid

P-SER TAU

o-Phosphoserine Taurine

P-ET-AMINE

o-Phosphoethanolamine

ASP OH-PRO THR SER ASN GLU GLN SAR α-A-A-A PRO GLY ALA CTRULINE α-A-B-A VAL CYS

L-Aspartic Acid Hydroxy-L-proline L-Threonine L-Serine L-Asparagine Glutamic Acid L-Glutamine Sarcosine α-Aminoadipic Acid L-Proline Glycine L-Alanine L-Citrulline DL-α-Amino-n-butyric Acid L-Valine L-Cystine

0

60

80

Abbreviation

Fig. 1.10.1 Analysis of soy sauce

120

ARG

OH-LYS (ORNITIHNE) LYS NH4,E-AMINE

TRP

(HIS) 3-ME-HIS (1-ME-HIS) (CARNOSINE) (ANSERINE)

PHE TYR

γ-A-B-A

(CYS) MET

ILU (CYSTATHIONINE)

VAL (CTRULINE) ( α-A-B-A)

GLN

ASN

OH-PROH

(SAR) (α-A-A-A) PRO

THR

ARG

(P-SER) TAU (P-ET-AMINE)

Amino acid

L-Methionine MET L-Isoleucine ILU CYSTATHIO L-Cystathionine NINE L-Leucine LEU L-Tyrosine TYR L-Phenylalanine PHE β-Alanine β-ALA DL-β-Aminoisobutyric Acid β-A-I-B-A γ-Aminobutyric Acid γ-A-B-A L-Tryptophan TRP L-Histidine HIS L-3-Methylhistidine 3-ME-HIS L-1-Methylhistidine 1-ME-HIS L-Carnosine CARNOSINE L-Anserine ANSERINE Hydroxylysine OH-LYS L-Ornithine ORNITHINE L-Lysine LYS L-Arginine ARG

GLU

LEU

100

min

LYS NH4,E-AMINE

OH-LYS (ORNITHINE)

HIS (3-ME-HIS) (1-ME-HIS) (ANSERINE) (CARNOSINE)

γ-A-B-A

TRP

ILE (CYSTATHIONINE) LEU TYR PHE β-ALA (β-A-I-B-A)

(CYS) MET

GLY

40

(CTRULINE) (α-A-B-A) VAL

SER

GLU (SAR) (α-A-A-A) PRO

(GLN)

(ASN)

ASP

20

(OH-PRO) THR

P-SER (TAU) (P-ET-AMINE)

SER

ALA

ASP

■ Analysis of Soy Sauce and Sweet Sake The soy sauce was diluted by a factor of 200 in citric-acid (lithium) buffer solution (for sample dilution) and the sweet sake was diluted by a factor of 10. After filtration with a membrane filter, 10µL of each solution was injected.

Table 1.10.1

(β-ALA) ( β-A-I-B-A)

Shimadzu's Amino Acid Analysis System incorporates the Na type method, which enables protein hydrolysis and amino-acid analysis, and the Li type method, which enables the analysis of free amino acids. The analysis of amino acids in fermented foods using the lithium method is described here as an example.

■ Analytical Conditions : Shim-pack Amino-Li (100mmL. × 6.0mm I.D.) Column : AminoAcid Mobile-Phase Kit (Lithium type) Mobile Phase Gradient Elution method : 0.6mL/min Flow Rate : 39˚C Temperature Reaction Reagent : AminoAcid Reaction Reagent Kit Flow Rate of Reaction Reagent : 0.3mL/min ReactionTemperature : 39˚C : RF-10AXL Detection Ex : 350nm Em : 450nm

GLY ALA

■ Explanation The detection method is an important factor to improve sensitivity and selectivity in the analysis of amino acids using HPLC. For this reason, a wide variety of pre- and post-column derivatization methods have been developed. Of these methods, post-column fluorescent derivatization using o-phthalaldehyde as the reagent offers significant advantages in terms of detection sensitivity, selectivity, and operational ease and is used in a number of fields, including foods.

140 0

10

20

30

40

50

60

70 min

80

90

100

110

120

130

140

Fig. 1.10.2 Analysis of sweet sake

18

C

Food Product Components

H O

1.11 Simultaneous Analysis of D- and L-Amino Acids (1) - LC ■ Analytical Conditions

■ Explanation Measurement of optical purity in the food product field is vital. In the case of amino acid, optical separation of configured amino acid is necessary because, in particular, optical purity greatly affects synthetic peptide and its physiological activity in derivatives. Optical isomer separation methods in LC are broadly divided among the Chiral column solid phase method, Chiral mobile phase method and the Chiral derivatization method. This explanation introduces the Chiral derivatization method. OPA/N-acetylcysteine agent was used as the derivatization agent.

Column

: Develosil ODS-UG-5 (200mmL.×6.0mm I.D.) with Guard Column : Shim-pack GRD-ODS Precolumn (250mmL.×4.0mm I.D.) Mobile Phase : (A) 50mM Sodium Acetate (B) Methanol (A)→(B)Gradient Method : 1.2mL/min Flow Rate Temperature : 35˚C : Fluorescence Detector Detection Ex : 350nm Em : 450nm

References N. Nimura and T. Kinoshita, J. Chromatogr., 352, 169 (1986) Murakita, et al Clinical Chemistry, Supplement No. 2, pp71b, 21 (1992) Murakita, et al Summary of Symposium on Separation Science and Related Techniques, pp101 (1993) Shimadzu Application News No. L235(C190-E063)

■Peaks 1 . D-ASP 2 . L-ASP 3 . L-GLU 4 . D-GLU 5 . D-SER 6 . L-SER

3 2

7 . D-THR 8 . L-THR 9 . L-ARG 10. D-ARG 11. D-ALA 12. L-ALA

13. 14. 15. 16. 17. 18.

L-TYR D-TYR L-VAL D-MET L-MET D-VAL

each component c.a. 200pmol 12

5

1

22

11 13 14

7

4

21

23

6 8

10

15

24

18

20

9 15

0

20

17

40

Fig. 1.11.1 Analysis of D-, L-amino acid standard solution

19

19. 20. 21. 22. 23. 24.

19

60 min

D-PHE L-PHE L-ILE D-ILE D-LEU L-LEU

1.11 Simultaneous Analysis of D- and L-Amino Acids (2) - LC

CHO HS-CH2CHCOOH CHO

NHCOCH3

OPA

N-acetyl-L-cysteine R-CH-COOH NH2 D,L-amino acid

COOH C

CH3COHN

COOH

H

CH3COHN

CH2

S

C

H

CH2

S

COOH N

C

H

H

N

R

C

COOH

TIME

FUNCTION

VALUE

16 24 29 50 59 59.01 64 64.01 65

BCONC BCONC BCONC BCONC BCONC BCONC BCONC BCONC STOP

24 24 40 40 67 80 80 0

R

Fig. 1.11.2 Chiral derivatization reaction

Fig. 1.11.3 Gradient conditions

standard solution 300µL buffer solution *1

200µL

mix reagent A*2

100µL

reagent B*3

100µL

mix

mix and wait

inject *1 *2 *3

3min

20µL

0.1N sodium tetraborate 2% N-acetyl - L-cysteine (0.1N sodium tetraborate solution) 1.6% o-Phthalaldehyde (methanol solution)

Fig. 1.11.4 Derivatization conditions

20

C

Food Product Components

H O

1.12 Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (1) - LC ■ Analytical Conditions : Shim-pack VP-ODS (150mmL. × 4.6mm I.D.) Column : 20mM Sodium Phosphate Buffer Solution containing Mobile Phase 10mM Sodium 1-Hexane Sulfonate (pH 2.5) : 0.8mL/min Flow Rate : 45˚C Temperature Reaction Reagent : Amino Acid Reaction Reagent Kit, Solution B Flow Rate of Reaction Reagent : 0.4mL/min Reaction Temperature : 45˚C : SUS, 2m × 0.8mm I.D. Reaction Coil : Fluorescence Detector Detection Ex : 350nm Em : 450nm

■ Explanation 4-amino-n butyric acid (gamma-aminobutric acid, GABA) is a type of amino acid that is common in animals and plants. It stimulates blood circulation in animals and supports the metabolic function. A large amount of this substance is present in rice, particularly unpolished rice, and so it is an important consideration when considering the role of unpolished rice as a health food. GABA can be analyzed simply with HPLC by combining separation using reverse-phase ion-pair mode and detection using post-column derivatization. The batch analysis of other amino acids as well as GABA is also possible using Shimadzu's Amino Acid Analysis System.

■ Pretreatment

Simple analysis of GABA using the reverse-phase ionpair mode and batch analysis of 18 amino acids using the cation exchange mode are described here as examples.

5g of cooked rice ←25mL of 80% ethanol solution Mixing, homogenizing, and centrifugation Supernatant

Residue ←20mL of 80% ethanol solution Mixing, homogenizing, and centrifugation

■ Analysis by reverse-phase ion-pair

Chromatography with post-column derivatization

Supernatant

Fig.1.12.1 shows a chromatogram obtained by this method after GABA is extracted by 80% ethanol solution from polished and unpolished rice then replaced by solvent. the analyzed unpolished and polished rice (cooked with rice-cooker) contained GABA 2.7mg and 0.3.mg respectively in 100g.

Residue

Volume increased to 50mL with 80% ethanol solution→1mL sample collected Evaporation to dryness 1mL of mobile phase→ Injection into HPLC

15 ■Peak 1.GABA

10

mV

1

5

Unpolished rice

0 Polished rice 0

5

10 min

15

20

Fig. 1.12.1 Chromatograms of polished and unpolished rice

21

1.12 Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (2) - LC ■ Explanation Batch Analysis of GABA and Other Amino Acids Using Shimadzu's Amino Acid Analysis System As an example, Fig. 1.12.2 shows the results obtained when GABA and other amino acids in polished and unpolished rice were measured with Shimadzu's Amino Acid Analysis System using the sodium method. Using this system, it was possible to separate 18 amino acids obtained by protein hydrolysis and GABA. The quantitative results are shown in Table 1.12.1. The overlapping of peaks was observed, however, with some of the amino acids analyzed in this batch, such as threonine. To perform the batch separation and quantitative analysis of these acids, use the lithium method, which enables the separation of even more amino acids.

■ Analytical Conditions : LC-VP Amino Acid Analysis System Instrument : Shim-pack Amino-Na (150mmL. × 6.0mm I.D.) Column Ammonia Trap Column : Shim-pack ISC-30/S0504 (50mmL. × 4.0mm I.D.) : Amino Acid Mobile Phase Kit (Na type) Mobile Phase Gradient Elution Method : 0.4mL/min Flow Rate : 60˚C Temperature Reaction Reagent : Amino Acid Reaction Reagent Kit (Solutions A and B) Flow Rate of Reaction Reagent : 0.3mL/min Reaction Temperature : 60˚C : Fluorescence Detector Detection Ex : 350nm Em : 450nm

Table 1.12.1 Results of Quantitative Analysis (mg/100g) Polished rice

3.4 1.7 1.3 4.5 0.8 0.6 2.9 0.4 0.3 2.8 1.0

1.7 1.2 0.6 1.9 n.d. 0.2 0.8 0.1 n.d. 0.4 n.d.

■Peaks 1. Asp 7. Ala 2. Thr 8. Val 3.Ser 9. Phe 4. Glu 10. GABA 5. Pro 11. Arg 6. Gly

40

10

mV

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Phenylalanine 4-amino-n-butyric acid Arginine

Unpolished rice

60

1

20

23

7

4 6

5

8

9

11 Unpolished rice

0 Polished rice

n.d. : not detected 0

20

40 min

Fig. 1.12.2 Chromatograms of polished and unpolished rice

22

Food Product Components

C H O

1.13 Analysis of Theanine in Green Tea - LC ■ Explanation Theanine is the predominant amino acid in green tea, and is known as the component responsible for the good, sweet taste of green tea. Regarding the efficacy of theanine, various types of research have been conducted, and these researches have reported its efficacy to soothe the excited nerves induced by caffeine and other substances, and to lower the blood pressure. Theanine analysis can be conducted simultaneously with other amino acids by the post-column derivatization method with fluorescence detection using OPA (ophthalaldehyde) as the reaction reagent. Here we introduce the simultaneous analysis of theanine together with other amino acids in a commercially sold green tea beverage using the Shimadzu amino acid analysis system.

■ Analytical Conditions : Shim-pack Amino-Li Column (100 mmL. × 6.0 mm I.D.) : Amino Acid Mobile Phase Kit Mobile Phase (Li type) Gradient Elution method Mobile Phase Flow Rate : 0.6 mL/min Column Temperature : 39˚C Reaction Reagent : Amino Acid Reagent Kit Reaction Reagent : 0.3 mL/min Flow Rate Reaction Temperature : 39˚C : RF-10AXL Detection Ex : 350 nm Em : 450 nm Injection Volume : 20 µL

■ Pretreatment Sample 5 mL

NH2 CH

CH2

CH2

COOH

C

NH

CH2

Filter using disposable filter (0.45 µm)

CH3

O

2 mL Make the volume 10 mL with mobile phase A

Fig. 1.13.1 L-theanine structural formula

Inject 20 µL

■ Peaks 1.ASP 2.THR 3.SER 4.GLU 5.GLN

6 1

4

4

mV

3

6. 7. 8. 9. 10.

THEANINE ALA VAL ILU LEU

11.TYR 12.PHE 13.β-ALA 14.γ-A-B-A 15.TRP 16.ARG

2 3 7

1

16

14

5 2

1112 9 10 13

8

15

0 0

10

20

30

40

50

60

70

80

90

100

Fig. 1.13.2 Chromatogram of green tea

23

110

120

130

140

1.14 Obligation to Display Nutritive Components in Processed Foods - UV Abs. 0.5

0.5

Vitamine C 1.5mg/dL 0.250

0.250

■ Explanation Japanese government national health policy since 1986 dictates that processed food must display nutritive components. Within the regulations governing this policy, energy, proteins, lipid, saccharine and table salt can be displayed. The above policy also includes directives for nutritive component analysis method standards and analyzers. Here, analysis of vitamin C using a spectrophotometer for ultraviolet and visible region will be introduced.

1.0mg/dL

0.5mg/dL

0 650.0

550.0

500.0

■ Analytical Conditions Instrument : UV Spectrophotometer : Candy Sample : Metaphosphoric Acid Solution Solvent : 10mm Cell : 0∼0.5ABS Range : 2nm Slit

450.0 0

■ Pretreatment Reducing vitamin C is converted into oxidized vitamin C, and red osazone created through reaction of 2,4Dinitrophenylhydrazine. This osazone is dissolved in 85% sulfuric acid and measured using a spectrophotometer. Here, vitamin C in a nutritious candy was dissolved using metaphosphoric acid solution and measured.

Fig. 1.14.1 Absorption spectrum of vitamin C 0.270

K 5.7000

B -0.0009

R 2 ** 0.9997

ABS.

0 0

CONC.

1.500

Fig. 1.14.2 Calibration curve for vitamin C

STD. NO. 01 02 03 04

CONC. 0 0.5000 1.0000 1.5000

ABS. 0 0.0870 0.1780 0.2620

CONC.=K✽ABS.+B K 5.7000

NO. 01 02

B -0.0009

R✽✽2 0.9997

ABS. 0.1940 0.1640

C= C=

CONC. 1.1048 0.9338

Balance Food Candy

Fig. 1.14.3 Quantitative results for vitamin C

24

Food Product Components

C H O

1.15 Analysis of Trace Amounts of Vitamins B1 and B2 in Food Products Using Fluorescence Photometry (1) - RF ■ Explanation Vitamins are one valuable form of nutrition. They help to condition physiological function in minute amounts and have been much used in physiology and pharmacology from ancient times. Vitamin analysis differs for the characteristics (water soluble, fat soluble) and types. And the Japanese Pharmacopoeia and Standard Methods of Analysis for Hygienic Chemists state that on the whole analysis should be conducted using chromatography, absorptiometry and fluorescence photometry. The latter being often used where the vitamin is chemically processed to increase its unique fluorescence for measuring. Here, a measuring example using a fluorescence photometry will be introduced.

■ Pretreatment Vitamin B2 - or riboflavin as it is commonly known - is copiously contained in milk, eggs and grains and promotes growth in animals. A riboflavin deficiency leads to various inflammations such as oral ulcers and vision impairment. Water-solution riboflavin is lime green and shows a green fluorescence. And when it is in an alkali solution, and an ultraviolet is irradiated onto that solution, it becomes a lumiflavin with strong fluorescent properties uniquely inactive.

Fig. 1.15.1 shows the creation process for lumiflavin. And measurement of lumiflavin provides a good way for quantifying vitamin B2, which also has been adopted for the Standard Methods of Analysis for Hygienic Chemists. Here, vitamin B2 copiously found in Soya beans was pretreated in accordance with the Standard Methods of Analysis for Hygienic Chemists and measured. Photolysis was performed in an alkali solution on the vitamin B2 that had been hot-water extracted. And after oxidation, the liquid extracted with chloroform was measured. Vitamin B 2 itself is fluorescent and that excitation and fluorescent spectrum is shown in Fig. 1.15.2. Fig. 1.15.3 shows the spectrum after pretreatment. Fig. 1.15.4 shows the data for processed and measured Soya bean. A comparison with the standard product shows that 2 µg of vitamin B2 exist in 1g of Soya bean.

■ Analytical Conditions Instrument : RF Spectrofluorophotometer : Vitamin B2 in Soya bean Sample : Chloroform Solvent Excitation : 469nm : Ex : 10nm Em : 10nm Slit

CH2(CHOH)3CH2OH N

H3 C

N O

H3 C

N O

Riboflavin CH3

Photolysis

H3 C

N

H3C

N

N O NH O

Excitation spectrum

Relative fluorescent intensity

NH

Fluorescent spectrum

Lumiflavin

400

Fig. 1.15.1 Creation process of lumiflavin

25

450

500

550

600nm

Fig. 1.15.2 Excitation and fluorescent spectrum of vitamin B2 (riboflavin)

1.15 Analysis of Trace Amounts of Vitamins B1 and B2 in Food Products Using Fluorescence Photometry (2) - RF Excitation spectrum

Fluorescent spectrum

A

450

500

550

600nm

Relative fluorescent intensity

Relative fluorescent intensity

400

A: Sample solution + standard sample B: Sample solution only C: Sample blank

B

C

Fig. 1.15.3 Excitation and fluorescent spectra of lumiflavin created from photolysis 450

500

550

600nm

Fig. 1.15.4 Measurement of vitamin B2 in soya bean Soya bean

Homogenize

Hot-water extraction

Test solution + standard solution

Test solution + purified water

Test solution + purified water

Add 1N NaOH

Photolysis

Dark-location storage

Add acetic acid, 4% KMnO4

Chloroform extraction

Measuring solution A

Measuring solution B

Measuring solution C

Fig. 1.15.5 Pretreatment for vitamin B2 analysis

26

C

Food Product Components

H O

1.16 Analysis of Water Soluble Vitamins Using Semi-micro LC System - LC ■ Pretreatment A 0.45 µm membrane filter was used for filtration.

■ Explanation A column with an inner diameter of 4 to 6mm is usually used in HPLC analysis, but in recent years semi-micro scale columns are being employed in this area and will undoubtedly become the mainstream column for the following reasons. (1) Mass sensitivity (sensitivity based on mass) is increased. (2) The amounts of mobile phase and sample used are reduced. Fig. 1.16.1 shows a semi-micro LC analysis example of the vitamin B group and caffeine in a vitamin drink. Some 2µL of sample was injected.

■ Analytical Conditions : STR ODS-II (150mmL. × 2.0mmI.D.) Column Mobile Phase : 10mM Phosphate Buffer (pH 2.6) containing 5mM Sodium Hexanesulfonate Acid /Acetonitrile = 9/1 (v/v) Flow Rate : 0.2mL/min Temperature : 25˚C : UV-VIS Detector 240nm Detection

Reference Shimadzu Application News No. L239 (C190-E065)

4

1

5

2 3

0

1

2

3

4

5

6

7

8

9

10

11

12min

Fig. 1.16.1 Analysis of vitamin B group and caffeine in vitamin drink

27

1.17 Analysis of Vitamin B Group - LC ■ Explanation Quantification methods for vitamins have shifted from biological methods to chemical methods. GC and HPLC incorporated methods are almost always used for fat-soluble Vitamins whereas GC analysis of water-soluble vitamins is complicated to the point that it is impractical thus the HPLC analysis method is the most favored. Ion conversion and normal-phase partition chromatography are used for separation but, from the point of view of column durability and analysis stability, reversed phase chromatography has become the mainstream method. There are individual test methods for each vitamin, and chromatography simultaneous analysis capabilities for samples with comparatively few impurities and large amounts of target components are often found in medical products and drink materials. Here, the conditions for simultaneous analysis and the analysis example itself are shown for the vitamin B group.

Reference Shimadzu HPLC Application Report No. 14(C196-E035)

■ Analytical Conditions : Shim-pack CLC-ODS(150mmL. × 6.0mm I.D.) Column Mobile Phase : 100mM Sodium Phosphate Buffer (pH 2.1) containing 0.8mM Sodium Octanesulfonate /Acetonitrile = 9:1 (v/v) Temperature : 40˚C Flow Rate : 1.5mL/min : UV-VIS Detector 210nm or 270nm Detection

■Peaks 1. Nicotinic acid 2. Nicotinamide 4. Pyridoxine 5. Riboflavin phosphate 6. Thiamine 7. Caffeine 8. Folic acid 10. Riboflavin

■Peaks 1. Nicotinic acid 2. Nicotinamide 3. Pantothenic acid 4. Pyridoxine 5. Riboflavin phosphate 6. Thiamine 7. Caffeine 8. Folic acid 9. Biotin 10. Riboflavin

0

5

10

15(min)

Fig. 1.17.1 Analysis example (210nm) of vitamin B group

0

5

10

15(min)

Fig. 1.17.2 Analysis example (270nm) of vitamin B group

28

C

Food Product Components

H O

1.18 Analysis of Tocopherol in Milk - LC ■ Explanation LC vitamin analysis is broadly separated into watersoluble vitamin analysis and fat-soluble vitamin analysis. Use of HPLC enables simultaneous analysis of the components, which has made it a popular form of analysis from the outset. Here, analysis of fat-soluble vitamin tocopherol is introduced.

■ Pretreatment 1. Add chloroform to sample for extraction. 2. After vaporizing and dry hardening the chloroform layer, the sample is dissolved in a small amount of hexane and then concentrated. 3. The dissolved liquid sample is injected.

■ Analytical Conditions : Shim-pack CLC-NH2 Column (150mmL.×6.0mm I.D.) Mobile Phase : n-Hexane/Isopropyl Alcohol = 100/4 (v/v) Temperature : 40˚C : 1.5mL/min Flow Rate : UV-VIS Detector 297nm Detection

References Shimadzu HPLC Food Analysis Applications Data Book (C190-E047) Shimadzu HPLC Application Data book (C190-E001)

Peaks 1. α-tocopherol 2. β-tocopherol 3. γ-tocopherol 4. δ-tocopherol 3

4

1

2

0

5

10

15(min)

Fig. 1.18.1 Analysis of tocopherol types in milk

29

1.19 Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (1) - LC ■ Explanation In April 2002, the Food with Health Claims System was established in Japan. The aim of this system is to regulate the wide variety of so-called "health foods" by recognizing the health claims of foods that satisfy certain requirements. Foods with health claims are divided into two categories: food for specific health use and food with nutrient function claims. Foods with nutrient function claims are subject to specific standards. The content of 12 vitamins and 2 minerals must lie between the lower and upper limits for the recommended daily intake. The analysis of the water-soluble vitamins that are specified nutrients for food with nutrient function claims is described here as an example.

■ Analysis of Food with Nutrient Claims Fig. 1.19.1, 1.19.2, and 1.19.3 show analysis examples for commercial tablet-shaped sweets (sample A) and for multivitamin tablets (sample B). The vitamin B group includes vitamins that only dissolve in dilute alkalis and so the following pretreatment was carried out. 20mL of each sample solution was injected.

Fig.1.19.1 (dosage per day)

Pound in a mortar Fig.1.19.2

[Fig. 1.19.2] : Shim-pack VP-ODS (150mmL. × 4.6mm I.D.) Column Mobile Phase : A: 100mM Sodium Phosphate Buffer Solution (pH 2.1) B: Acetonitrile A/B = 8/1 (v/v) : 1.2mL/min Flow Rate Temperature : 40˚C : SPD-10AVVP 550nm Detection [Fig. 1.19.3] : Asahipak NH2P-50 4E (250mmL. × 4.6mm I.D.) Column Mobile Phase : A: 100mM Phosphate (Triethanolammonium) Buffer Solution (pH 2.2) B: Acetonitrile A/B = 1/4 (v/v) : 1.0mL/min Flow Rate Temperature : 40ºC : SPD-10AVVP 240nm Detection

■ Pretreatment Sample

■ Analytical Conditions [Fig. 1.19.1] : Shim-pack VP-ODS (150mmL. × 4.6mm I.D.) Column Mobile Phase : A: 100mM Sodium Phosphate Buffer Solution containing 0.8mM Sodium 1-octanesulfonate (pH 2.1) B: Acetonitrile A/B = 10/1 (v/v) : 1.2mL/min Flow Rate Temperature : 40˚C : UV (LC-2010) 210nm Detection

Fig.1.19.3

(10 times of dosage per day) (dosage per day)

Add 1mM NaOH 10mL

Mixing

Mixing Add each buffer

100mL

Filtration

Inj. 20µL

30

Food Product Components

C H O

1.19 Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (2) - LC

30

40

Sample A

Sample B

12 3

■ Peaks

1 2 3

1. Nicotinamide 2. Ca pantothenate 3. Vitamin B6 4. Vitamin B1 5. Folic acid 6. Vitamin B2

30

mAU

mAU

20

4

20 4

10

6

6

10

5

5 0

0 5

0

10

15

min

0

5

10

min

15

Fig. 1.19.1 Chromatograms of commercial foods with nutrient claims

0.2

■ Peak 0.15

1

1

■ Peak 1. Vitamin C

40

1. Vitamin B12

30

mAU

mAU

0.1 20

0.05 10

0 0

2

4

min

6

8

10

Fig. 1.19.2 Chromatogram of sample B

31

0

2

4

6

8

min

Fig. 1.19.3 Chromatogram of sample B

10

1.20 Analysis (Measurement of K Value) of Nucleotide in Tuna Meat - LC ■ Explanation Nucleic acid base and nucleotide are usually analyzed using reversed phase chromatography as they can be simultaneously analyzed. Here, a separation example using reversed phase chromatography for 8 adenine derivative components is shown. This form of analysis is applied to measuring of fish freshness indicated by the K value (freshness constant) because the 4 kinds of nucleotides, hypoxanthine and inosine can be individually quantified.

K=

Hyp+Ino Hyp+Ino+IMP+AMP+ADP+ATP

Reference Shimadzu HPLC Food Analysis Applications Data Book (C190-E047)

■Peaks 1.Hyp 2.IMP 3.Adenine 4.Ino 5.AMP 6.ADP 7.Adenosine 8.ATP

Fig. 1.20.1 Analysis of adenine derivative components

■ Pretreatment 1. Add 25mL of 1M perchloric acid to 10g of tuna meat and homogenize. 2. Centrifugally separated (3000 rpm for 5 min). 3. Skim off top layer, and add 1M potassium bicarbonate solution to adjust sample to pH 6.5. 4. Remove the created potassium perchlorate sediment, and filter top layer through membrane filter. 5. Inject 5µL of filtered solution. ■ Analytical Conditions Column : STR ODS-2 (150mmL×.4.6mm I.D.) Mobile Phase : A : 100mM Phosphate(Triethylammonium)Buffer(pH 6.8) B : Acetonitrile A / B = 100/1 (v/v) Temperature : 40˚C Flow Rate : 1.0mL/min Detection : UV-VIS Detector 260nm

■Peaks 1.Hyp 2.IMP 3.Adenine 4.Ino 5.AMP 6.ADP 7.Adenosine 8.ATP

Fig. 1.20.2 Analysis of tuna meat

32

C

Food Product Components

H O

1.21 Analysis of Oligosaccharide in Beer - LC ■ Explanation In the case of analysis of sugars using the partition method, the mobile phase is a mixture of water and acetonitrile used with an aminopropyl column. The elation position can be adjusted by changing the water to acetonitrile ratio. Fig. 1.21.1 shows an analysis example of monosaccharide and oligosaccharide standard solutions and Fig. 1.21.2 shows an analysis example of oligosaccharide in beer.

■ Analytical Conditions : Shim-pack CLC-NH2 (150mmL.×6.0mm I.D.) Column Mobile Phase : Acetonitrile/Water = 60/40 (v/v) Temperature : 25˚C : 1.0mL/min Flow Rate : Refractive Index Detector Detection

References Shimadzu HPLC Food Analysis Applications Data Book (C190-E047) Mikami, Egi, Shimadzu Review, Nos. 44 (3), 47 (1987) Shimadzu HPLC Application Report No. 11 (C196E036)

■ Peaks ■ Peaks

1. Fructose

1. Glucose

2. Glucose

2. Maltose

3. Maltose

3. Maltotriose

4. Maltotriose

4. Tetraose (L-DP-4)

5.L-DP-4 2 1

5. Pentaose (L-DP-5) 1

6. Hexaose (L-DP-6)

6.L-DP-5 7.B-DP-5

7. Heptaose (L-DP-7)

8.L-DP-6

L : Straight-chain structure

2 3

9.B-DP-6

B : Branching structure

10.B-DP-7

DP: Sugar number

11.B-DP-8 12.B-DP-9

4

13.B-DP-10

5 4

6 7

5 3 6

0 0

5

10

10

10

11

12 13

15

20

(min)

15(min)

Fig. 1.21.1 Analysis of sugar and oligosaccharide standard samples

33

5

78 9

Fig. 1.21.2 Analysis of oligosaccharides in beer

1.22 Analysis of Oligosaccharides in Beer Using the ELSD-LT Low Temperature Evaporative Light Scattering Detector - LC ■ Analytical Conditions : NH2P-50 (250mmL. × 4.6mm I.D.) Column Mobile Phase : (1)Acetonitrile/Water = 6/4 (v/v) Fig. 1.22.1 (2)A : Acetonitrile B : Water Linear Gradient B 30% → 60% Fig. 1.22.2 and 1.22.3 : 1.0mL/min Flow Rate Temperature : 40˚C : ELSD-LT Detection Temperature : 35˚C Gain : 7 Nebulizer Gas : N2 Gas Pressure : 350kPa

■ Explanation Combining the GE method with the ELSD-LT enables efficient separation when analyzing oligosaccharides. Fig. 1.22.1 shows the results of analyzing the oligosaccharides in beer using the isocratic elution method and the GE method. 10µL of beer was injected after filtering with a membrane filter. There are branched oligosaccharides (1 -> 6 glycosidic linkage), linear oligosaccharides (1 -> 4 glycosidic linkage), and other types of oligosaccharide. In general, the different types of oligosaccharide are mixed together when eluted. The elution times for monosaccharides as well as linear disaccharides, trisaccharides, and heptasaccharides are indicated in the chromatogram. It can be seen that the GE method enables the efficient separation and detection of oligosaccharides up to 20-mer. The results of analyzing commercial beers under the same GE conditions are shown in Fig. 1.22.2 and 1.22.3. 2500

■ Elution Times 1.Glucose 2.Maltose 3.Maltotriose 4.Maltoheptaose

2000

mV

1500

1

1000

2

4

3

Gradient elution method

500

0

-500

2

4

Isocratic elution method

4

12 3 0

6

8

10

12

14 min

16

18

20

22

24

26

28

1200

1200

1000

1000

800

800

600

600

mV

mV

Fig. 1.22.1 Analysis of oligosaccharides in beer

400

400

200

200

0

0

0

2

4

6

8

10

12

14 min

16

18

20

22

Fig. 1.22.2 Chromatogram of beer A

24

26

28

0

2

4

6

8

10

12

14 min

16

18

20

22

Fig. 1.22.3 Chromatogram of beer B

24

26

28

34

C

Food Product Components

H O

1.23 Analysis of Saccharides in Fermented Foods - LC ■ Explanation Foods created by the fermentation process are attracting a great deal of interest because of the many benefits they offer. It is often the case, however, that foods with many benefits contain a large number of constituents, and, especially when the target constituent is only present in a very small amount, analysis can be difficult. In order to minimize the influence of impurities when analyzing this kind of sample, a selective and highly sensitive detection method is required. In terms of selectivity and sensitivity, post-column fluorescent derivatization is suited to the analysis of saccharides in fermented foods. The batch analysis of saccharides in miso and sake lees with Shimadzu's Reducing Sugar Analysis System, which uses an original arginine reagent, is described here as an example.

■ Analysis of Miso and Sake Lees Miso and sake lees were pretreated in the way shown on the right and 10µL of each were injected.

■ Analytical Conditions : Shim-pack ISA-07/S2504 (250mmL. × 6.0mm I.D.) Column : Guard column ISA (50mmL. × 4.0mm I.D.) Guard Column : A: 0.1M Borate (Potassium) Buffer Solution Mobile phase (pH8) B: 0.4M Borate (Potassium) Buffer Solution (pH9) A → B/Linear Gradient Elution Method : 0.6mL/min Flow Rate : 65˚C Temperature Reaction Reagent : 1% L-Arginine, 3% Boric Acid Solution Flow Rate of Reaction Reagent : 0.5mL/min Reaction Temperature : 150˚C : RF-10AXL Detection Ex : 320nm Em : 430nm ■ Pretreatment 2mL of water added to 200mg of miso or sake lees

Mixing

Diluted by factor of 10 with water

Centrifugation (3,000rpm, 5min)

10µL injected

Supernatant Filtration

mV 10

5

mV10

5

■ Peaks 1. Ribose 2. Fructose 3. Galactose 4. iso-Maltose 5. Glucose

4

2

4

■ Peaks 1. Ribose 2. Mannose 3. iso-Maltose 4. Glucose 5

3

3 1

1

2 0

0

0

20

40

60

Fig. 1.23.1 Analysis of miso

35

80 min

0

20

40

60

Fig. 1.23.2 Analysis of sake lees

80 min

1.24 Analysis of Nonreducing Sugar Using Postcolumn Derivatization with Fluorescence Detection - LC ■ Explanation Nonreducing sugars such as sucrose, raffinose and stachyose can be analyzed at high sensitivity and high selectivity by adding taurocyamine (as a fluorescent reaction agent for postcolumn fluorescence detection) to reducing sugar. Fig. 1.24.1 shows an analysis example for mixed standard solutions of sucrose, raffinose and stachyose. Some 500pmol of each component was injected. Reference T. Kinoshita, et al, J. Liquid Chromatogr., No. 14 (10), 1929 (1991)

■ Analytical Conditions Column Mobile Phase Temperature Flow Rate Reaction Reagent

: Asahipak NH2P-50 (250mmL.×4.6mm I.D.) : Acetonitrile/Water = 65/35 (v/v) : 40˚C : 1.0mL/mi : 0.1M Potassium Tertraborate Solution containing 20mM Taurocyamine, 1mM Sodium Periodate (adjust to pH 10.5 using 10MKOH solution) Reaction Reagent Flow Rate : 1.0mL/min Reaction Temperature : 150˚C : Fluorescence Detector Detection Ex : 320mm Em : 450nm

■ Peaks 1. Sucrose 2. Raffinose 3. Stachyose

1. mobile phase 2. pump 3. injector 4. column 5. column oven 6. reagent 7. pump 8. reaction oven 9. cooling coil

■ Peaks 1. Sucrose 2. Raffinose 3. Stachyose

3 2

10. detector 11. coil 12. waste 1. mobile 2. pump

1

2

3. injecto

7

4. colum 5. colum

3

6. reage

6

7. pump

4 1

0

5

10

8. reactio 5

9. coolin

(min)

10. detect

10 9

11

11. coil 12. waste

8 12

Fig. 1.24.1 Analysis of nonreducing oligosaccharide

Fig. 1.24.2 Flowchart diagram of nonreducing sugar analysis system

36

Food Product Components

C H O

1.25 Analysis of Sugar in Yogurt - LC ■ Explanation The ligand conversion chromatography column SCR-101 series consists of the 101N, 101C and 101P types with ends made respectively of Na, Ca and Pb. And the retaining behavior of sugars differs with each one. In particular, in the case of sugar alcohol analysis, 101C or 101P is recommended. Also, glucose and galactose separation is possible with the 101C type. Fig. 1.25.1 shows an analysis example of a Japanese pickle liquid and Fig. 1.25.2 shows an analysis example of sugar in yogurt.

[Analysis of yogurt] 1. Add perchloric acid to the yogurt, and mix to deproteinize. 2. Centrifugally separate, and filter upper layer through a membrane filter. 3. Inject 10 µL of filtered liquid. ■ Analytical Conditions [Pickle liquid] Column

References Shimadzu LC Application Report No. 11 (C196-E036) Shimadzu HPLC Food Analysis Applications Data Book (C190-E047) ■ Pretreatment [Analysis of Japanese pickle liquid] 1. Filter the pickle liquid through a membrane filter. 2. Inject 10 µL of filtered liquid.

: Shim-pack SCR-101C (300mmL.×7.9mm I.D.) Mobile Phase : Water Temperature : 80 : 0.8mL/min Flow Rate : Refractive Index Detector Detection [Yogurt] Column

: Shim-pack SCR-101C (300mmL.×7.9mm I.D.) Mobile Phase : Water Temperature : 85 : 1.0mL/min Flow Rate : Refractive Index Detector Detection

■ Peaks

g Peaks

1

1. Lactose 2. Glucose 3. Galactose 4. Fructose

1. Sucrose 2. Glucose 3. Fructose 4. Sorbitol

4

1

2

4

3

2

0

3

10

20

Fig. 1.245.1 Analysis of pickle liquid

37

(min)

0

5

10

15

(min)

Fig. 1.25.2 Analysis of yogurt

1.26 Analysis of Alliin in Garlic - LC ■ Explanation The active ingredient alliin in garlic quickly changes to allicin by the action of the enzyme alliinase. As the efficacy of these substances has been elucidated, they are often marketed as health food products. Since alliin is a type of amino acid, it can be analyzed by the post-column derivatization method with fluorescence detection using OPA (o-phthalaldehyde) as the reaction reagent. Here we introduce the analysis of alliine in garlic using the Shimadzu amino acid analysis system.

■ Pretreatment Grated garlic 700 mg

■ Analytical Conditions : Shim-pack Amino-Na Column (100 mmL. × 6.0 mm I.D.) : A: 0.1M Sodium Citrate Buffer Mobile Phase Solution (pH 3.2) B: 0.2M Sodium HydroxideAqueous Solution Step Gradient Elution Method 0 – 23 (min) A 100% 23 – 33 (min) B 100% 33 – 50 (min) A 100% Mobile Phase Flow Rate : 0.4 mL/min Column Temperature : 60˚C Reaction Reagent : Amino Acid Reagent Kit (Solution B) Reaction Reagent : 0.4 mL/min Flow Rate Reaction Temperature : 60˚C : RF-10AXL Ex : 350 nm Em : 450 nm Detection

or processed garlic 100 mg

5% trifluoro acetic acid

Injection Volume

aqueous solution 50 mL

: 10 µL

NH2

Mix together

CH

Filter

CH2

COOH

S

CH2

CH2

CH

O

Inject 10 µL

Fig. 1.26.1 Alliin structural formula

■ Peak 1.Alliin

■ Peak 1.Alliin

1

1

0

10

20

Fig. 1.26.2 Analysis of garlic

30

min

0

10

20

30

Fig. 1.26.3 Analysis of processed garlic (tablet)

min

38

Food Product Components

C H O

1.27 Analysis of Catechins in Green Tea - LC ■ Explanation The efficacy of catechins, including antioxidative and cancer inhibition effects have been reported in various literature, and today catechins are receiving attention because several commercially sold beverages are now using them. Five types of catechins are known, and they are epigallocatechin, catechin, epigallocatechin gallate, epicatechin and epicatechin gallate. These components can be easily analyzed by HPLC.

■ Analytical Conditions : Shim-pack FC-ODS (75mmL. × 4.6mmI.D.) Column Mobile Phase : A: 10 mM Sodium Phosphate Buffer Solution (pH 2.6) B: Acetonitrile Gradient Elution Method : 1.0 mL/min Flow Rate Column Temperature : 40˚C : UV-VIS Detector 270 nm Detection Injection Volume : 5 µL (Gradient Program) Func B.Conc B.Conc B.Conc B.Conc B.Conc B.Conc STOP

Time(min) 0 6 20 20.01 25 25.01 35 OH

(+)-C HO

OH

(-)-EC

OH

HO

O

OH

O

OH

OH

OH HO

O

O

O

OH

O

OH OH

OH OH

OH O

OH

(-)-ECG

OH

HO OH

OH HO

OH

(-)-EGC

OH

OH OH

Value(%) 7 7 20 50 50 7

OH O OH

OH

(-)-EGCG

OH O OH

Fig. 1.27.1 Structural formulas of catechins mAU

mAU ■ Peaks 1.EGC 2.C 3.Caffeine 4.EC 5.EGCG 6.ECG

3

15

3 15

10

10

5

5

5 6

1

1 2 2

5

6

4 0

0

0

39

4

10

20 min

Fig. 1.27.2 Chromatogram of green tea beverage

0

10

20 min

Fig. 1.27.3 Chromatogram of oolong tea beverage

1.28 Analysis of Chlorogenic Acid in Coffee - LC ■ Explanation Chlorogenic acid (3-caffeoylquinic acid) is a type of polyphenol compound widely distributed in higher plants, and is found in large quantities in coffee, potatoes and sweet potatoes. The antioxidative effects of chlorogenic acid are now receiving attention, and various types of research on the efficacy of chlorogenic acid are being advanced. We present here the analysis of three components of processed coffee. They are chlorogenic acid, caffeine and caffeic acid (3,4- dihydroxycinnaminic acid) which is a structural component of chlorogenic acid. * It is sometimes referred to as 5-caffeoylquinic acid. However, here we use the term 3-caffeoylquinic acid as used in “The Merck Index 13th Edition”.

■ Analytical Conditions : Shim-pack VP-ODS (150 mmL. × 4.6 mm I.D.) Column Mobile Phase : A:10 mM Sodium Phosphate Buffer Solution (pH 2.6) B: Acetonitrile Low Pressure Gradient Elution Method : 1.0 mL/min Flow Rate Column Temperature : 40˚C : SPD-M10AVP 270 nm 325 nm Detection (Gradient Program) Time(min) 0 5 15 15.01 18 18.01 25

B Conc(%) 10 10 30 70 70 10 STOP Spectrum of Standard

100

OH

Spectrum of Sample (Canned Coffee)

OH

O

mAU

HOOC

50

C HO

O

C

OH

C

OH 0 200

Fig. 1.28.1 Structural formula of chlorogenic acid 100

■ Peaks 1. Chlorogenic Acid 2. Caffeine

2

at 270nm

100

nm

at 270nm 2

mAU

mAU

200

300

Fig.1.28.2 Spectra of chlorogenic acid

■ Peaks 1. Chlorogenic Acid 2. Caffeine

50 1

1

0

0 0

5

min

10

15

0

15

1

15

at 325nm 1

10

■ Peaks 1. Chlorogenic Acid 3. Caffeic Acid

mAU

mAU

10 min

■ Peaks 1. Chlorogenic Acid 3. Caffeic Acid

at 325nm

100

5

50 5 3

3

0

0 0

5

10

15

min

Fig. 1.28.3 Chromatograms of canned coffee

0

5

10

15

min

Fig. 1.28.4 Chromatograms of coffee beans

40

Food Product Components

C H O

1.29 Analysis of Lycopene and ß-Carotene in Tomato - LC ■ Explanation Lycopene is a type of carotenoid, and is found in large quantities as a red pigment in red tomatoes, etc. The antioxidative effects of lycopene are said to be 100 times stronger than that of vitamin E and more than twice stronger than that of ß-carotene, and lycopene is receiving attention for its effects to prevent lifestyle diseases such as cancer and arteriosclerosis, and to slow aging. Though lycopene and ß-carotene have similar structures, they can be easily separated by reversed phase chromatography using a non-aqueous mobile phase. CH3

CH3

H 3C

CH3

■ Analytical Conditions : Shim-pack VP-ODS (150 mmL. × 4.6 mm I.D.) Column Mobile Phase : Acetonitrile / Ethanol = 4/1 (v/v) : 1.0 mL/min Flow Rate Column Temperature : 50˚C : SPD-M20A 450 nm Detection Slit width: 8 nm, Bandwidth: 8 nm Cell Temperature : 50˚C Injection Volume : 5 µL H3C

CH3

CH3

H 3C

CH3

H 3C CH3 CH3

CH3

CH3

CH3

CH3

CH3

CH3

H 3C

CH3

β -Carotene

Lycopene

Fig. 1.29.1 Structural formulas for lycopene and ß-carotene

■ Pretreatment mAU 100

1

Tomato 5 g ■ Peaks 1:Lycopene 2:β -Carotene

Chloroform 40 mL Homogenization 1 min Shaking 5 min

75

Aqueous phase (upper layer) 50

Discard

Organic phase (lower layer)

Evaporative drying Chloroform 5 mL Inject 5 µL

25

2

0 0

5

10

15

20 min

mAU

Fig. 1.29.2 Chromatogram of tomato

100 80 60 40

Standard Tomato

20 0 min

200 15

300 10

400 5

500 0

600 nm

Fig. 1.29.3 Spectra of lycopene

41

Fig.1.29.4 3-D Chromatogram of tomato

1.30 Melting of Fats and Oils - TA ■ Explanation The melting process of various edible fats and oils is measured by DSC. Six types of crystals exist in cocoa oil, a component of chocolate. Of these, V type crystal is said to possess good thermal stability. Since V type crystal melts at about 34˚C, DSC measurement can be used to know the condition of V type crystal contained in a particular sample of chocolate. Figure 1.30.1 shows a DSC curve of the chocolate sample heated at 3˚C/min. Figure 1.30.2 shows a DSC curve of the same chocolate sample, reheated after cooling the melted sample to -50˚C to harden it. It is evident that the peak at 30.4˚C has completely disappeared.

■ Analytical Conditions : DSC-60 Instrument : Chocolate Sample Sample Amount : 22.87 mg Atmospheric Gas : Nitrogen : 30 mL/min Gas Flow Rate [Temperature Program] : 3˚C/min Heating Rate

DSC mW

–36.25J/g

0.00

3.39˚C 13.99˚C –2.00

–4.00

30.44˚C

–6.00

0.00 Temp [˚C]

50.00

Fig. 1.30.1 Chocolate measurement (1st time) DSC mW 2.00

–23.45J/g

0.00

–11.71˚C –2.00

7.92˚C 16.30˚C 19.60˚C

–4.00

–6.00 0.00 Temp [˚C]

50.00

Fig. 1.30.2 Chocolate measurement (2nd time)

42

C

Food Product Components

H O

1.31 Gelatinization of Starch - TA ●Flour

■ Analytical Conditions : DSC-60 Instrument : Flour Sample Sample Amount : 4.21 mg Atmospheric Gas : Nitrogen : 30 mL/min Gas Flow Rate

■ Explanation Starches gelatinize when heated with water. The gelatinization reaction can be analyzed by DSC because it is accompanied by endothermic reaction. Here we conducted measurement of flour starch (17.4%).

[Temperature Program] : 5˚C/min Heating Rate DSC mW 1.00

0.00 59.10˚C

–1.00 20.00

40.00

60.00

Temp [˚C]

80.00

100.00

Fig. 1.31.1 Gelatinization temperature of flour (17.4%)

●Corn ■ Explanation Here we conducted measurement of corn starch (19.9%). It is known that when sucrose and salt are added to starch, the gelatinization temperature changes.

■ Analytical Conditions : DSC-60 Instrument : Corn Sample Sample Amount : 4.97 mg Atmospheric Gas : Nitrogen : 30 mL/min Gas Flow Rate [Temperature Program] : 5˚C/min Heating Rate

DSC mW 1.00

0.00

68.90˚C

–1.00 40.00

43

60.00 Temp [˚C]

80.00

Fig. 1.31.2 Gelatinization temperature of corn (19.9%)

2. Food Additives 2.1 Propionic Acid in Cookies and Bread - GC ■ Explanation Propionic acid is one of the components that form flavor and fragrance, included in fermented products such as miso, soy sauce and cheese as a microbial metabolite. It is also used as a preservative in cookies and bread because of its low toxicity and minimal effect on bread yeast. When propionic acid is analyzed using GC with FID, the total calculation of the natural propionic acid, which is inherently included in the food, and the added propionic acid is obtained as the quantitative value. References 1) Standard Methods of Analysis for Hygienic Chemists (annotation) 455 (1990), edited by the Pharmaceutical Society of Japan 2) Ministry of Health and Welfare (currently Ministry of Health, Labour and Welfare), Environmental Health Bureau, Food Sanitation Testing Policy, 33-35 (1989)

■ Pretreatment Propionic acid was extracted using steam distillation method.

■ Analytical Conditions : 10% PEG6000 on shimalite TPA 1m × 3mm I.D.(glass) : 150°C : 230°C : 200°C(FID) : N2

Column Col. Temp. Inj. Temp. Det. Temp. Carrier Gas

■ Peaks 1. Propionic acid 2. Crotonic acid (I.S.)

1

2

S

10min

Fig. 2.1.1 Analysis of propionic acid

44

Food Additives

2.2 Saccharine and Sodium Saccharine - GC ■ Explanation Saccharine and sodium saccharine are used as artificial sweeteners. Saccharine is only used in chewing gum because it does not dissolve easily in water whereas sodium saccharine does and is widely used in pickles and jams. Saccharine and sodium saccharine are extracted from food products and refined, and after being methylated, they are analyzed by GC with FID or FPD. Here, a GC with FID analysis example will be introduced.

■ Pretreatment 1. Extract and refine sample by dialysis extraction or direct extraction. 2. Produce a derivative (methylate) of saccharine using diazomethane, etc. 3. Dissolve in ethyl acetate, etc. and use this liquid as the sample. ■ Analytical Conditions : SE-30 5% on chromosorb W Column 1.5m × 3mm I.D. (glass) : 190°C Col. Temp. : 250°C Inj. Temp. : 230°C (FID) Det. Temp. : N2 Carrier Gas

Reference Standard Methods of Analysis for Hygienic Chemists (annotation) 493 to 495 (1990), edited by the Pharmaceutical Society of Japan.

■ Peaks 1. Saccharine 2. (IS) trans-stilbene

1

2

S

5min

Fig. 2.2.1 Analysis of saccharine

45

2.3 Ethylene Glycols in Wine - GC ■ Explanation Normally wine does not contain ethylene glycol but there have been reports of temporary errors where diethylene glycol was mixed into wine. Here, ethylene glycol and diethylene glycol have been added to wine and directly analyzed by GC. Analysis was possible without any interference from impurities in the wine. Reference Shimadzu Application News No. G110

■ Analytical Conditions Column Col. Temp. Inj. Temp. Det. Temp. Carrier Gas Injection

: ULBON HR-20M (25m × 0.25mmI.D. df = 0.25µm) : 150°C : 200°C : 200°C (FID) : He 2mL/min : Split 1:30

■ Pretreatment Ethylene glycol and diethylene glycol were added to a shop-sold wine for direct analysis.

1

2

■ Peaks 1.Ethylene glycol 2.Diethylene glycol

■ Peaks 1.Propylene glycol 2.Ethylene glycol 3.Dipropylene glycol 4.Diethylene glycol

1 3

4 2

START

2

4

6

8 min

Fig. 2.3.1 Analysis of glycols (standard products)

START

2

4

6

8 min

Fig. 2.3.2 Analysis of shop-sold wine with glycols added

46

Food Additives

2.4 Sorbic Acid, Dehydroacetic Acid and Benzoic Acid - GC ■ Explanation The preservatives sorbic acid, dehydroacetic acid and benzoic acid are analyzed by UV absorption spectrum method or GC method. The UV method is fast and efficient but can be affected by coexisting substances such as fragrances, whereas GC has the advantage of being able to easily separate out such substances. Here, these preservatives were extracted from a food product by direct extraction or steam distillation and refined to be analyzed by GC with FID.

ether. Reversely extract the ether layer using sodium hydrogen carbonate solution, re-extract using ethyl ether, and concentrate. GC analyze the final liquid as an acetone. 2. Steam distillation Pulverize the sample, add water, and neutralize pH. Add tartaric acid solution and salt and perform steam distillation. Extract residue using ethyl ether as previously described.

5

5

trans-Stilbene(I.S.)

Dehydroacetic Acid

Sorbic Acid

■ Pretreatment 1. Direct extraction Add saturated saline solution and sulfuric acid, homogenize with strong acidity and extract with ethyl

Benzoic Acid

■ Analytical Conditions : 5% DEGS+1%H3PO4 on chromosorb W Column 2m × 3mm I.D.(glass) : 185°C Col. Temp. : 230°C Inj. Temp. : 250°C (FID) Det. Temp. : N2 Carrier Gas

Reference Standard Methods of Analysis for Hygienic Chemists (annotation) 445 to 451 (1990), edited by the Pharmaceutical Society of Japan

10min

Fig. 2.4.1 Analysis of preservatives

47

2.5 Analysis of Preservatives in Food Products with Absorption Photometry (1) - UV ■ Explanation Various preservatives are added to preservative and processed foods to prevent putrefaction and to keep freshness. The use of these food additives is strictly governed by the Food Sanitation Law to ensure that concentrations do not exceed the permitted safe concentrations for human consumption. Here, preservatives in food products regulated by the Food Sanitation Law were analyzed with a Shimadzu double-beam spectrophotometer after pretreatment in accordance with the law.

■ Pretreatment - Sodium nitrite in a food product The sodium nitrite preservative in meat was separated by distillation, and sulfamic acid was diazotized using nitrite acid under acidity of hydrochloric acid, and colored with naphthylethylenediamine for measurement. - Benzoic acid in a food product

■ Analytical Conditions Instrument : UV Spectrophotometer Reference : blank : H2O Solvent : 10mm Cell : 0~2Abs Range

0.5µg

0.7

0.8

0.6

0.4µg

0.5 Absorbance

0.3µg

0.4

Sample 0.3

0.6

Sample

0.4

0.2µg

0.2

0.25µg

Absorbance

The benzoic acid preservative was separated and extracted from soy sauce using steam distillation in readiness for UV absorption measurement. - Sorbic acid in a food product The sorbic acid preservative was separated and extracted from boiled fish paste using steam distillation in readiness for UV absorption measurement. - Dehydroacetic acid in a food product The dehydroacetic acid preservative was separated and extracted from bean jam using steam distillation in readiness for UV absorption measurement.

0.2 0.1µg

0.1 0 450

500

550

600

650

0.1

0.2

0.3

0.4

0.5

Sodium nitrite concentration(µg/mL)

Wavelength (nm)

Fig. 2.5.1 Absorption spectrum for sodium nitrite

Fig. 2.5.2 Calibration curve for sodium nitrite

48

Food Additives

2.5 Analysis of Preservatives in Food Products with Absorption Photometry (2) - UV 0.7

Absorption spectrum for benzoic acid in a food product

Absorption spectrum for benzoic acid standard liquids

0.8 Sample 0.6

Absorbance

0.5 Absorbance

7.2µg

0.6

0.4 0.3 0.2

0.4

0.2

0.1

200

250 Wavelength (nm)

300

200

250 Wavelength (nm)

4

6

8

Fig. 2.5.4 Calibration curve for sorbic acid

Absorption spectrum for sorbic acid standard liquids

Absorption spectrum for sorbic acid in a food product

0.6

0.4 0.3

0.4

No.2 No.1 210

Absorbance

0.5

194

0.6 Absorbance

2

Sorbic acid concentration(µg/mL)

Fig. 2.5.3 Absorption spectrum for benzoic acid

0.7

0

300

0.2 0.2 0.1 0 200

250

300 Wavelength (nm)

350

250

300 Wavelength (nm)

350

80

160

240

320

Dehydroacetic acid concentration(µg/mL)

2.5.2

Fig. 2.5.5 Absorption spectrum for sorbic acid

0.6

Fig. 2.5.6 Calibration curve for dehydroacetic acid

Absorption spectrum for sample solutions 0.6

320µg/20mL

0.5 240µg/20mL

Sample No. 2

0.4

Sample No. 1 0.3 0.2

Absorbance

Absorbance

0.5

Absorption spectrum for dehydroacetic acid standard liquids

0.4 160µg/20mL

0.3 0.2

80µg/20mL 0.1

200

0.1

250 300 Wavelength (nm)

350

200

Fig. 2.5.7 Absorption spectrum for dehydroacetic acid

49

250 300 Wavelength (nm)

350

2.6 Color Control of Food Products (1) - UV ■ Explanation Color control is an important factor in quality control, as colors have large psychological effect and consumer image of products largely depends on the color of their coating resin or paint. Thus, colorimeters, which determine color of objects, are widely used in various fields. Colorimetry methods are largely divided into two: one is spectral colorimetry in which a spectrophotometer is used to measure reflectance or transmittance spectrum, and the tristimulus values X, Y and Z are determined by calculation; the other is the direct reading of the tristimulus values where a photoelectric photometer is used to directly measure the tristimulus values. Here, a measurement example using the color measurement software with the spectrophotometer UV3100PC will be introduced. Color Measurement of Processed Food Products available in consumer market The colors of processed foods can greatly enhance their appearance for marketing purposes, which makes color control an important facet of the food industry. Here, color measurement was performed on shop-sold flavoring products. 1. Vinegar 2. Ketchup 3. Sauce 4. Mayonnaise

Vinegar was analyzed using transmittance measurement and the other products by reflective measurement. Fig. 2.6.1 shows the spectra for the products. Next, based on these spectrum data, the x, y, Y stimulus values and L*, a*, b* values were calculated under the conditions of C illuminant and 2* field of view. Fig. 2.6.2 shows the printout of the results. Also, under the same conditions, CIE (xy) and UCS chromaticity diagrams were drawn up (see Fig. 2.6.3 and Fig. 2.6.4). The xy chromaticity diagram shows chromaticity (hue and saturation) using the x and y chromaticity coordinates. The closer to the center, the lower the saturation. Color differences can be discerned at a glance. In the Lab chromaticity diagram, the left-side L* displays brightness between zero and 100 while a* and b* on the right denote chromaticity. Plus a* is the red direction, minus a* the green direction, plus b* the yellow direction and minus b* the blue direction. The closer to the center, the lower the saturation and the closer to the edge, the higher the saturation. This is the chromaticity diagram most widely used.

■ Analytical Conditions Instrument : UV-3101PC with color measurement software : Vinegar, ketchup, sauce, Sample and mayonnaise Reference : MgO : 0 to 100% Range

1. Vinegar 2. Ketchup 3. Sauce

100.00 1

Reflective ratio

4. Mayonnaise

50.00

4

3 2

0.00 380.0

580.00 Wavelength (nm)

780.00

Fig. 2.6.1 Transmittance and reflectance spectra

50

Food Additives

2.6 Color Control of Food Products (2) - UV

Measurement results of x, y, Y and L*,a*,b* values

Title

: COLOR MEASUREMENT

Comment : UV-3100PC+ISR-3100 Illuminant

: C

Field of view (degree) : 2

Reference value : Sample ID 1 2 3 4

0.00

0.00

0.00

0.00

0.0000 0.0000

L*

a*

b*

Y

x

y

97.44 20.59 9.46 75.63

-2.67 21.05 2.88 -2.77

12.39 17.83 3.37 20.77

93.51 3.14 1.06 49.29

0.3286 0.4985 0.3546 0.3511

0.3406 0.3491 0.3345 0.3657

Vinegar Ketchup Sauce Mayonnaise

Fig. 2.6.2 Measurement results

1.000

60.000

100.0 1

4

4 2 1

L *

y 0.500

b *

50.0

3

0.000

4 1 3

2 2 3 0.0

0.000 0.000

0.400 x

0.800

Fig. 2.6.3 CIE (x,y) chromaticity diagram

51

-60.000 -50.000

0.000 a*

Fig. 2.6.4 UCS (Lab) chromaticity diagram

60.000

2.7 Analysis of Sweetener in Soft Drink - LC ■ Explanation This is an example of simultaneous analysis of the sweeteners aspartame, saccharine, benzoic acid, sorbic acid and glycyrrhizic acid.

Reference Shimadzu HPLC Food Analysis Applications Data Book (C190-E047)

■ Analytical Conditions : STR ODS-M(150mmL. × 4.6mmI.D.) Column Mobile Phase : 40mM Sodium Acetate Buffer (pH 4.0)/ Methanol = 3/1 (v/v) Temperature : 40˚C Flow Rate : 1.0mL/min : UV 250nm Detection

■ Pretreatment A soft drink was directly injected without pretreatment.

■ Peaks 1. Saccharine 2. Aspartame 3. Benzoic acid 4. Sorbic acid

3 1

4

2

0

8

16 (min)

Fig. 2.7.1 Analysis of sweetener in soft drink

52

Food Additives

2.8 Analysis of Fungicide in Oranges - LC ■ Explanation In Japan the use of o-phenylphenol (OPP), thiabendazole (TBZ) and diphenyl is permitted for preventing mold in citrus. Here, the simultaneous analysis of these components using fluorescent detection will be introduced. Reference Shimadzu HPLC Food Analysis Applications Data Book (C190-E047)

■ Pretreatment 1. Add 0.5g of anhydrous sodium acetate, 15g of anhydrous sodium sulfate and 40mL of ethyl acetate to 10g of orange, and homogenize twice. 2. Filter using glass filter.

3. Add 2.5mL of butanol to the acquired ethyl acetate layer. 4. Concentrate at 40˚C until 2.5mL is obtained. 5. Add methanol to dilute to 10mL and filter through membrane filter. 6. Inject 5µL of filtrate. ■ Analytical Conditions : Shim-pack CLC-ODS(150mmL.× 6.0mmI.D.) Column Mobile Phase : Acetonitrile/Methanol/Water = 30/35/35 (v/v/v) Prepare it to pH 2.4 with phosphoric acid containing 10mM sodium dodecyl acetate. Temperature : 40˚C : 1.0mL/min Flow Rate : Fluorescence Detector Detection Ex : 285nm Em : 325nm

■ Peaks 1. O-phenylphenol (OPP) 2. Thiabendazole (TBZ) 3. Diphenyl (DP) 2

3 1

0

53

10

Fig. 2.8.1 Analysis of fungicide in orange

20 (min)

2.9 Analysis of Phenol Antioxidant in Foods - LC ■ Explanation The exposure of food constituents to oxygen in the air leads to the creation of oxidation products and deterioration in quality. To prevent this, various antioxidants are used as food additives. Here, we will be looking at how phenol antioxidants, which are used particularly often in oil products, can be analyzed with HPLC. There are four types of phenol antioxidant that are approved as food additives in Japan: BHT (butylated hydroxytoluene), BHA (butylated hydroxyanisole), NDGA (nordihydroguaiaretic acid), and PG (propyl gallate). They are authorized for use in oil, fat, and butter, as well as frozen and dried seafood products.

■ Analytical Conditions : Shim-pack FC-ODS (75mmL. × 4.6mm I.D.) Column Mobile Phase : A: 5% Acetic Acid Solution B: Methanol/Acetonitrile = 1/1 (v/v) B 40% → 80% /15min Linear Gradient Flow Rate : 1.0mL/min Temperature : 40˚C : SPD-10AVP 280nm Detection

■ Pretreatment 0.5g of butter

The analysis of nine phenol antioxidants, the four mentioned above and five that are used in other countries, is described here as an example.

1g of sodium sulfate 5mL of acetonitrile/isopropanol/ethanol = 2/1/1 (v/v/v)

Mixing

Left for 1 hour at -20˚C Centrifugation at 3,000rpm for 5min Supernatant

■ Analysis of Butter The results of analyzing butter after performing the pretreatment described on the right are shown in Fig. 2.9.1. The lower line represents the result of analyzing butter and the upper line represents the result of analyzing butter after adding 20mg/L of the nine phenol antioxidants at the pretreatment stage.

mV

Filtration 10µL injected

■ Peaks 1.PG 2.THBP 7 3.TBHQ 4.NDGA

1 2

50

5.BHA 6.HMBP 7.OG 8.BHT 9.DG

9

4

5

3

6

8

0 0

10

20 min

Fig. 2.9.1 Analysis of butter

54

Food Additives

2.10 Analysis of L-Ascorbic Acid 2-Glucoside - LC ■ Explanation L-ascorbic acid 2-glucoside (2-o-α-D-glucopyranosyl Lascorbic acid) is a type of vitamin C derivative that can exist stably when exposed to heat and light and even in water, and in the body it attains vitamin C activity through the action of glucosidase. L-ascorbic acid 2glucoside is specified as a food additive (January 20, 2004, Ministry of Health, Labour and Welfare; Department of Food Safety, No. 0120001), and notification has been made that its analysis in food products is to be conducted by HPLC (May 13, 2004, Ministry of Health, Labour and Welfare; Department of Food Safety, No. 0513003). Though L-ascorbic acid (vitamin C) is currently used in various food products on the market as an antioxidant and nutrition enrichment agent, it is believed that L-ascorbic acid 2-glucoside will also be widely used. We introduce here an analysis of L-ascorbic acid 2glucoside in accordance with the Department of Food Safety notification, No. 0513003.

■ Analytical Conditions : Shim-pack VP-ODS (250 mmL. × 4.6mm I.D.) Column Mobile Phase : A : Water 800 mL + KH2PO4 1.4 g + TBAH* (10%) 26 mL → pH=5.2 using Phosphoric Acid → 1000 mL using Water (*Tetrapbutyl Ammonium Hydroxide) B : Acetonitrile A/B=9/1 (v/v) Flow Rate : 0.8mL/min Temperature : 40˚C : SPD-20A 260 nm Detection Cell Temperature: 40˚C

■ Pretreatment 12 11

Soft drink beverage 5 g

10 9

OH

100 mg/L standard product 10 mL

8

H

7

H H

5

O H H OH

H

HO

O

4

O

Filter (0.45 µm)

3

O H

Add mobile phase to 50 mL

6

mAU

OH

HO

HO

2

OH

1

Inject 10µL

0 -1 200

250

300 nm

350

Fig. 2.10.1 Structural formula for L-ascorbic acid 2-glucoside and absorption spectrum 125

3

■ Peaks 1. Nicotinamide 2. L-Ascorbic Acid 2-Glucoside 3. L-Ascorbic Acid(Vitamin C) 4. Riboflavin(Vitamin B2)

100

mAU

75

2 50

25

1

4

0 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

min

55

Fig. 2.10.2 Chromatogram of soft drink beverage (spiked with L-ascorbic acid 2-glucoside)

2.11 Analysis of EDTA in Mayonnaise - LC ■ Explanation EDTA in mayonnaise was analyzed after chelation of Fe ion. Reversed-phase ion pair chromatography with tetrabutylammonium ions was used for separation. In this analysis, a polymer column (ODP), instead of a silica column (ODS), was used because of the high pH of the mobile phase and the basicity of the tetrabutylammonium. The following chromatogram shows the measurement of marketed mayonnaise with PDTA (the internal standard substance) and EDTA added. References Shimadzu HPLC Food Analysis Applications Data Book (C190-E047) Shimadzu Application News No. L214 (C190-E050)

■ Pretreatment 1. Add chloroform to sample, mix together, and centrifugally separate (12000 r.p.m for 2 min, twice). 2. Add 0.01M FeCl3 solution to water layer and mix together. 3. Inject 20µL of sample. ■ Analytical Conditions : Asahipak ODP-50 (150mmL. × 6.0mmI.D.) Column Mobile Phase : 20mM Sodium Phosphate Buffer (pH 6.9) containing 10mM Tetrabutylammonium Hydrogensulfate (adjust to pH 7.5 with 4M of NaOH) Temperature : 40˚C : 0.8mL/min Flow Rate : UV 255nm Detection

■ Peaks 1. EDTA-Fe 2

2. PDTA-Fe (internal standard substance)

1

0

8

16

(min)

Fig. 2.11.1 Analysis of EDTA in mayonnaise

56

Food Additives

2.12 Analysis of Benzoyl Peroxide in Food Product - LC ■ Explanation In Japan, benzoyl peroxide is a food additive authorized for use only in flour as a processing agent, and its amount for use as diluted benzoyl peroxide is prescribed to be “0.30 g/kg or less” by the Ministry of Health, Labour and Welfare in “Standards for Foods and Food Additives”. In addition, the Ministry of Health, Labour and Welfare (May 13, 2004, Department of Food Safety; No.0513003) revised the testing method so that the analytical method was changed from gas chromatography to HPLC. Here we introduce an analysis of benzoyl peroxide in accordance with the Department of Food Safety notification No. 0513003.

■ Analytical Conditions : Shim-pack VP-ODS (250 mmL. × 4.6 mm I.D.) Column Mobile Phase : Water/Acetonitrile = 45/55 (v/v) : 1.0 mL/min Flow Rate Temperature : 40˚C : UV 235 nm Detection

O

O

C O O

C

Fig. 2.12.1 Structural formula for benzoyl peroxide

■ Pretreatment

■ Analysis of Flour Figure 2.12.2 shows the chromatogram of a pretreated sample of domestically produced flour, and the chromatogram of the same pretreated sample with the additional 1.0 mg/L (equivalent to flour 5.0 mg/kg) of benzoyl peroxide (indicated by the dotted arrow in Sample Pretreatment).

Flour 10 g 5.0 g/L standard product 10 µL 50 mL acetonitrile added Mix using stirrer for 15 min Filter (0.45 µm) Inject 20 µL

■ Peak 1.Benzoyl Peroxide

1 5.0

mAU

4.0

3.0

2.0

1.0

0 0

5

10

15

20

min

25

30

35

40

45

Fig. 2.12.2 Analysis of flour : (above) spiked with 1.0 mg/L of standard benzoyl peroxide (below) not spiked (each with 20 µL injection)

57

2.13 Analysis of p-Hydroxybenzoates in Soy Sauce - LC ■ Explanation LC is a great force in the analysis of preservatives used in food products. In particular, LC is useful for simultaneous analysis of such components. Here, an analysis example for p-hydroxybenzoates added to soy sauce will be introduced.

■ Pretreatment 1. Add pure water to soy sauce until diluted by 10 fold. 2. Filter through membrane filter. 3. Inject 10µL of filtrate. ■ Analytical Conditions Column : STR ODS-2 (150mmL. × 4.6mm I.D.) Mobile Phase : 10mM Sodium Phosphate Buffer

Reference Shimadzu Application News No. L222 (C190-E032)

(pH 2.6)/Methanol = 1/1 (v/v) Temperature : 40°C Flow Rate : 1.5mL/min Detection : UV 270nm

■ Peaks 1. Methyl p-Hydroxybenzoate 2. Ethyl p-Hydroxybenzoate 3. Isopropyl p-Hydroxybenzoate 4. Propyl p-Hydroxybenzoate 5. Iso butyl p-Hydroxybenzoate 6. Butyl p-Hydroxybenzoate

1

2

3 4 5

0

10

6

20 (min)

Fig. 2.13.1 Analysis of p-hydroxybenzoates in soy sauce

58

Food Additives

2.14 Analysis of Potassium Bromate in Bread - LC ■ Explanation The use of potassium bromate as an additive is allowed in bread production to make the bread-making process more effective. However, to ensure safety, it must not remain in the final product. Therefore, it is necessary to verify that there is no potassium bromate left in the bread. Chemical Hygiene No. 119 issued from the Japanese Fig. 2.14.1 shows the chromatogram obtained by analyzing commercially sold bread after the pretreatment procedure shown in Fig. 2.14.2 that conforms to Chemical Hygiene No. 119 (lower chromatogram), and the chromatogram obtained when 326 µg of potassium bromate standard (equivalent to 250 µg of bromate ions) was added to 10 grams of bread before pretreatment (upper chromatogram). No bromate ions were detected when analyzing bread alone. When analyzing the bread with potassium bromate added, 325.4 µg of potassium bromate (249.4 µg of bromate ions) was quantified in 10 grams of bread, demonstrating approximately 100% recovery rate.

Ministry of Health, Labour and Welfare on September 11, 1997 stipulates the post-column derivatization HPLC method using o-dianisidine as a reaction reagent to analyze potassium bromate in bread. This section shows an example of analyzing potassium bromate contained in bread.

1

■ Peak 1. Bromate

0

4

8

12

16

20 (min)

Fig. 2.14.1 Analysis of potassium bromate in bread

Bread 10.0g 50mL of Water  Stirred in Room Temperature for 30 min.  Left for 5 min  Centrifugation for 30 min (5˚C, 10000G)  Filtration (Filter Paper No.5A)  Filtration (0.45µm membrane filter)  Filtration (C18(ODS) mini cartridge column)  Filtration (Ion exchange mini cartridge column (Ag form) )  Filtration (Ultarfiltration)  Filtration (Ion exchange mini cartridge column (H form) )  Injection (200µL)

Fig. 2.14.2 Pretreatment Table 2.14.1 Analytical conditions : Shimadzu LC-VP Bromate Analysis System : Shim-pack VP-ODS (250mmL. × 4.6mmI.D.) : 100mL of Methanol, 2.0g of Acetic Acid and 19g of Tetrabutylammonium Hydroxide were added to 700mL of Water, and pH of Solution was adjusted to 6.3 - 6.5. And then, this solution was diluted to 1000 mL with Water. : 1.0mL/min Flow Rate : 40˚C Temperature Injection Volume : 200µL Reaction Regent : A ; 60mL of Nitric Acid (70%), 10.0g of Potassium Bromide was added to 700mL of Water. B ; 500mg of o-Dianisidine Dihydrochloride was added to 200mL of Methanol. Solution A and B were mixed, and diluted to 1000 mL with Water. : Piping Kit for Bromate Analysis Reaction Unit : 60˚C Temperature : UV-VIS Detector (450nm) Detection Instrument Column Mobile Phase

59

2.15 Simultaneous Analysis of Water-soluble Tar Pigments - LC ■ Explanation Synthetic and natural compounds are used as food pigments, and HPLC is a powerful tool for analyzing such compounds. The photodiode array analysis, which allows simultaneous analysis at multiple wavelengths and spectrum display, further facilitates the analysis and identification of unknown components. Here, a simultaneous analysis example for water-soluble tar pigments will be introduced showing multi chromatograms for each absorption wavelength using a photodiode array detector.

■ Analytical Conditions Column : STR ODS-2 (150mmL. × 4.6mm I.D.) Mobile Phase : A: 20mM Ammonium Phosphate Buffer (pH 6.8)/Isopropanol = 25/1 (v/v) B: Acetonitrile Gradient Elution Temperature : 40˚C Flow Rate : 1.0mL/min Detection : Photodiode Array Detection from 220nm to 700nm

Reference Masaaki Ishikawa et al; Summary of the 31st Annual Conference of the Japan Hygienic Chemistry Council (1994)

■ Gradient Conditions B concentration Time 0.00 min (initial condition) 0% 20% 15:00 min 45.00 min 55.00 min 55.01 min 65.00 min

mAbs 100

40% 70% 0% 0% ■ Peaks

Ch1 250nm 19

50

1

0 100

1. Yellow No. 4 2. Red No. 2 3. Blue No. 2 4. Yellow No. 203a 5. Red No. 102 6. Yellow No. 403 7. Yellow No. 5 8. Yellow No. 203b 9. Red No. 227 10. Red No. 40 11. Yellow No. 202 12. Orange No. 207 13. Red No. 503 14. Red No. 504 15. Green No. 401 16. Red No. 230 17. Orange No. 402 18. Blue No. 1 19. Red No. 502 20. Red No. 3 21. Black No. 401 22. Orange No. 201 23. Red No. 106 24. Green No. 201 25. Red No. 104 26. Yellow No. 407 27. Yellow No. 406 28. Red No. 105 29. Red No. 506 30. Brown No. 201 31. Violet No. 401 32. Red No. 401 33. Red No. 213 34. Yellow No. 402

31 24

3

Ch2 410nm

50

26

27

34

0 100

Ch3 430nm

50

6

4

8

0 100

Ch4 470nm

50

17

7

30

22

0 100

Ch5 510nm 11 12 10

50

5

0 100

13 14

Ch6 520nm

29

16 20

50

9

24

2

32

0 100

Ch7 550nm

23

33 25

50

28

0 100

Ch8 620nm

19

18 21

50 0 0

20

40

60

Fig. 2.15.1 Simultaneous analysis of water-soluble tar pigments

(min)

60

3. Residual Pesticides 3.1 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) - GC Analysis Based on Standards for Foods and Additives Specified in Japan's Food Sanitation Law (Notice 370 Issued by Japan's Ministry of Health and Welfare) ■ Explanation As a result of the diversification of foods and improvements in the ways foods are transported and stored, a wide variety of food products are imported from all over the world and eaten as part of our daily diet. There are, however, many reported cases of excessive levels of pesticide residue being found in imported foods, particularly imported vegetables, and the safety of imported vegetables has become an issue of some concern. The analysis of organophosphorus pesticide based on the standards for foods and additives specified

■ Pretreatment (Fruits, vegetables, green powdered tea, and hops)

Sample

(Fruits, vegetables, green powdered tea, or hops)

in Japan's Food Sanitation Law (Japan's Ministry of Health,Labour and Welfare: Notice 370, D, item (6)) is described here as an example. The analysis method varies with the sample; samples are categorized into three groups that each have different analysis methods: fruits, vegetables, green powdered tea, and hops; grain, beans, nuts, and seeds; and teas other than green powdered tea. The sample processing methods for the first two groups are described below.

(Grain, beans, nuts, and seeds) Sample

(Grain, beans, nuts, or seeds) Pulverization; 420µm standard sieve

Approx. 1kg of sample homogenized 20g of sample weighed out

10g; 20mL of water; left for 2 hours Extraction Addition of 100mL of acetone; homogenization for 3min; suction filtration through layer of diatomaceous earth Homogenization of residue and 50mL of acetone; suction filtration; vacuum concentration at 40˚C max. 300mL separating funnel; 100mL of saturated NaCl solution Extracted twice with 1:4 mixture of ethyl acetate and n-hexane solution (first time: 100mL; second time: 50mL) Dehydration of organic solvent layer; removal of organic solvent at 40˚C max. 20mL of n-hexane added to residue

Extraction Addition of 100mL of acetone; homogenization for 3min; suction filtration through layer of diatomaceous earth Homogenization of residue and 50mL of acetone; suction filtration; vacuum concentration at 40˚C max. 300mL separating funnel; 100mL of saturated NaCl solution Extracted twice with 1:4-mixture of ethyl acetate and n-hexane solution (first time: 100mL; second time: 50mL) Dehydration of organic solvent layer; vacuum concentration at 40˚C max.

Degreasing

Residue dissolved with 1:1 mixture of acetone and n-hexane (approx. 5mL)

100mL separating funnel; 30mL of n-hexane saturated with acetonitrile; shaken for 5min; extracted another 2 times (3 times in total) Vacuum concentration of acetonitrile layer at 40˚C max.

Purification Column tube of inner diameter 15mm and length 300mm filled with 5g of silica gel and 5g of Na2SO4 Extract injected

Purification

Elution with 100mL mixture of acetone and n-hexane (1:1)

Quantitative analysis

Vacuum concentration of eluate at 40ºC max.; acetone added to residue to make 5mL solution GC-FTD, FPD Quantitative analysis

61

Residue dissolved with a 1:1-mixture of acetone and n-hexane (approx. 5mL) Column tube of inner diameter 15mm and length 300mm filled with 5g of silica gel and 5g of Na2SO4 Extract injected Elution with 100mL mixture of acetone and n-hexane (1:1) Vacuum concentration of eluate at 40˚C max.; acetone added to residue to make 5mL solution GC-FTD, FPD

3.1 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) - GC Fig. 3.1.1 shows a chromatogram of 0.1mg/L standard pesticide solution. organophosphorus pesticide solution. Analysis was Fig. 3.1.2 to 3.1.4 show the chromatograms of apples, performed after adding standard organophosphorus spinach, and soybeans. µV (×100,000) 4.0

7.0

µV (x100,000) ■ Peaks 1 : DDVP 2 : Dimethoate 3 : Diazinon 4 : IBP 5 : Methylparathion

3.5 3.0 2.5

6 : MEP 7 : Malathion 8 : Chlorpyrifos 9 : Prothiofos 10: EPN each 0.1mg/L

6.0 5.0

3

2

1

5 6 7

8

4

4.0 9

2

2.0 1.5

3 5 67 4 8

1

1.0

10

3.0 9

10

2.0

0.5

1.0

0.0 0.0 5.0

10.0

15.0

20.0

min 5.0

Fig. 3.1.1 Chromatogram of standard organophosphorus

10.0

15.0

20.0

min

Fig. 3.1.2 Chromatogram of apples

pesticide solution (0.1mg/L)

(0.05µg/g standard pesticide solution added)

µV (×100,000)

µV (×100,000) 6.0 6.0 5.0

9

5.0 2

4.0

1 3.0 2 2.0

4

4.0

3

1

7

3 10

7

4

5 6

3.0

8

5 6 8

9

2.0

10

1.0

1.0

0.0

0.0 5.0

10.0

15.0

20.0

min

Fig. 3.1.3 Chromatogram of spinach

(0.05µg/g standard pesticide solution added) ■ Analytical Conditions Instrument : GC-2010AF, FPD-2010, AOC-20i : Rtx-1 (15m × 0.53mm I.D., df = 1.5µm) Column Column Temp. : 80˚C(1min)-8˚C/min-250˚C(5min) Carrier Gas : He, 46kPa (16.5mL/min, 120cm/s, constant-velocity mode) : FPD-2010 (P Filter) Detector : 230˚C Inj. Temp. Det. Temp : 280˚C Injection Method : Splitless (1min) Injection Volume : 1µL

5.0

10.0

15.0

20.0

min

Fig. 3.1.4 Chromatogram of soybeans

(0.1µg/g standard pesticide solution added)

62

Residual Pesticides

3.2 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) - GC Analysis Based on the Rapid Analysis Method Specified in Japan's Food Sanitation Law (Notices 43, 44, and 45 Issued by the Environmental Health Bureau's Food Chemistry Division in 1997) ■ Explanation In 1997, Japan's Ministry of Health, Labour and Welfare issued notification of a pesticide-residue rapid analysis method as a simple and quick way for screening many pesticide residues. With this method, the same analysis method can be used for many different agricultural products and pesticides and some of the pretreatment operations can be automated using the GPC method. The pesticides are analyzed in groups, such as chlorine, phosphorus, and nitrogen, using GC-ECD, GC-FPD, and GC-FTD. If, however, pesticide residue with a concentration exceeding approximately 50% of the regulated value is detected using the rapid analysis method, quantitative measurement must be carried out as stipulated by the corresponding notification. The analysis of organophosphorus pesticide is described as an example.

Sample

■ Pretreatment Extraction with acetone is carried out on the sample and, after redissolving in ethyl acetate with a diatomaceousearth column, GPC clean-up is performed and the sample is purified with a silica-gel mini-column. Organophosphorus pesticides are analyzed with GC-FPD or GC-FTD after concentration. Carbamate pesticides are analyzed by taking a sample of the solution after performing GPC clean-up and analyzing the sample in this state or after diluting with hydrochloric acid. Organochlorine or pyrethroid pesticides are investigated by performing analysis after refining first with silica gel and then with a Florisil mini-column.

Vegetables, fruits, green powdered tea, hops: 20g Grain, beans, nuts, seeds: 10g of sample passed through 420µm standard sieve; 20mL of water added; left for 2 hours

Extraction 100mL of acetone added; homogenization and extraction for 3min; suction filtration through layer of diatomaceous earth Filtrate residue and 50mL of acetone Concentrated to 20mL max. at 40˚C max.; 6g of NaCl added Total volume injected into diatomaceous-earth column; left for 10min Container used for concentration washed with 150mL of ethyl acetate; wash liquid injected into column

GPC clean-up

2mL of extract injected into GPC clean-up apparatus

Eluted with 1:1 mixture of ethyl acetate and cyclohexane Separate out fraction corresponding to elution times between fluvalinate and quinomethionate Concentrated at 40˚C max.; dissolved into a 4mL (1:1) mixture of acetone and hexane (extract A)

Mini-column purification 2mL of extract A injected into silica-gel mini-column Eluted with 20mL of acetone and hexane (1:1)

Quantitative analysis

1mL of extract A collected; solvent removed; dissolved in 1mL of methanol; HPLC measurement (pirimicarb) Dilute hydrochloric acid added to 0.3mL of remaining sample to make solution of 3mL; filtration; HPLC measurement (N-methylcarbamate) HPLC (carbamates)

Solvent removed; 3:17 mixture of ether and hexane added to residue to make 4mL solution (extract B)

2mL of extract B collected; solvent removed; residue dissolved in acetone to make 2mL (1mL for grain or beans) solution

Quantitative analysis

63

GC-FTD, FPD (organophosphates, organonitrogens)

Quantitative analysis

2mL of extract B collected; solvent removed; residue dissolved in 2mL (3:17) mixture of ether and hexane 2mL of extract B injected into Florisil mini-column; eluted with 18mL (3:17) mixture of ether and hexane; eluted with 15mL (3:17) mixture of acetone and hexane; each outflow liquid concentrated; residue dissolved in hexane to make 2mL (1mL for grain or beans) solution GC-ECD (organochlorines, pyrethroids)

3.2 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) - GC µV(x100,000)

µV(×100,000)

4.0 ■ Peaks 1 : DDVP 2 : Dimethoate 3 : Diazinon 4 : IBP 5 : Methylparathion 6 : MEP 7 : Malathion 8 : Chloropyrifos 9 : Prothiofos 10 : EPN each 0.1mg/l

3.5 3.0 2.5

2

2.0

3 5 67

1 1.5

6.0

5 9

4

1

10

8

10

2.0

0.5

1.0

0.0

0.0 10.0

6

3

2

4.0

1.0

5.0

7

5.0

3.0

9

8

4

7.0

15.0

20.0

min

0.0

Fig. 3.2.1 Chromatogram of standard organophosphorus

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

min

Fig. 3.2.2 Chromatogram of processed spinach liquid with

pesticides solution (0.1mg/L)

0.05µg/g standard pesticide solution added

µV(×100,000)

µV(×100,000)

7.0 4

2

6.0

4

5.0

3

7 2

4.0

8

1

5

8 6

5.0

3 5 6

3.0

7

7.5

2.0

9

10 1

10

9

2.5 1.0 0.0 0.0 1.0 0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

min

Fig. 3.2.3 Chromatogram of processed soybean liquid with

0.1µg/g standard pesticide solution added

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

min

Fig. 3.2.4 Chromatogram of processed rice liquid with

0.1µg/g standard pesticide solution added

Reference Handbook for Food Sanitation Laws, 2003 Edition, Shinnippon-hoki Publishing Co., Ltd., (2002)

■ Analytical Conditions : GC-2010AF, FPD-2010, AOC-20i Instrument : Rtx-1 (15m × 0.53mm I.D., df = 1.5µm) Column Column Temp. : 80˚C(1min)-8˚C/min-250˚C(5min) Carrier Gas : He, 46kPa (16.5mL/min, 120cm/s, constant-velocity mode) : FPD-2010 (P Filter) Detector : 230˚C Inj. Temp. : 280˚C Det. Temp. Injection Method : Splitless (1min) Injection Volume : 1µL

64

Residual Pesticides

3.3 Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (1) - GC ■ Explanation The analysis of organonitrogen and pyrethroid pesticides using the rapid analysis method is described here. Fig. 3.3.1 and 3.3.2 show chromatograms of standard organophosphorus and standard organonitrogen pesticide solutions analyzed under the same conditions. Both organophosphorus and organonitrogen pesticides can be detected with GC-FTD. Also, in the pretreatment for the rapid analysis method, both pesticides are eluted into the same fraction. Fig. 3.3.3 shows a chromatogram of processed soy bean liquid with 13 organophosphorus and 14 organonitrogen pesticides added.

Fig. 3.3.4 shows a chromatogram of a standard pyrethroid pesticide solution. Separation and quantitative analysis can be difficult for pyrethroid pesticides as there are often many isomers within a standard product. Consideration is also required for substances that convert their forms at the GC injector inlet (for example, deltamethrin changes to tralomethrin). Fig. 3.3.5 shows a chromatogram of processed spinach liquid with a standard pyrethroid pesticide added.

■ Pretreatment (Pretreatment for Pesticide-residue Rapid Analysis Method)

Sample

Vegetables, fruits, green powdered tea, hops: 20g Grain, beans, nuts, seeds: 10g of sample passed through 420µm standard sieve; 20mL of water added; left for 2 hours

Extraction 100mL of acetone added; homogenization and extraction for 3min; suction filtration through layer of diatomaceous earth Filtrate residue and 50mL of acetone Concentrated to 20mL max. at 40˚C max.; 6g of NaCl added Total volume injected into diatomaceous-earth column; left for 10min Container used for concentration washed with 150mL of ethyl acetate; wash liquid injected into column Vacuum concentration at 40˚C max.; dissolved in 4mL (1:1) mixture of ethyl acetate and cyclohexane GPC clean-up

2mL of extract injected into GPC clean-up apparatus Eluted with 1:1 mixture of ethyl acetate and cyclohexane Separate out fraction corresponding to elution times between fluvalinate and quinomethionate Concentrated at 40˚C max.; dissolved into a 4mL (1:1) mixture of acetone and hexane (extract A)

Mini-column purification 2mL of extract A injected into silica-gel mini-column Eluted with 20mL of acetone and hexane (1:1)

Quantitative analysis After removal of solvent; 3:17 mixture of ether and hexane added to residue to make 4mL solution (extract B)

2mL of extract B collected; solvent removed; residue dissolved in acetone to make 2mL (1mL for grain or beans) solution

Quantitative analysis

65

GC-FTD, FPD (organophosphates, organonitrogens)

Quantitative analysis

1mL of extract A collected; solvent removed; dissolved in 1mL of methanol; HPLC measurement (pirimicarb) Dilute hydrochloric acid added to 0.3mL of remaining sample to make solution of 3mL; filtration; HPLC measurement (N-methylcarbamate) HPLC (carbamates)

2mL of extract B injected into Florisil mini-column; eluted with 18mL (3:17) mixture of ether and hexane (first fraction); eluted with 15mL (3:17) mixture of acetone and hexane (second fraction); each outflow liquid concentrated; residue dissolved in hexane to make 2mL (1mL for grain or beans) solution GC-ECD (organochlorines, pyrethroids)

3.3 Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (2) - GC 4.0 3.5 3.0 2.5 2.0

µV (x10,000)

µV (x100,000)

10.0

P1 DDVP P2 Ethoprophos P3 Dimethoate P4 Diazinon P5 IBP P6 Parathion-methyl P7 Pirimiphos-methyl P8 MEP P9 Malathion P10 Chlorpyrifos P1 P11 Parathion P12 Prothiofos P13 EPN

N1 Isoprocarb N2 Alachlor N3 Diethofencarb N4 Paclobutrazol N5 Flutolanil N6 Pretilachlor N7 Mepronil N8 Lenacil N9 Thenylchlor N10 Tebufenpyrad N11 Pyriproxyfen N12 Mefenacet N13 Fenarimol N14 Bitertanol 1 N15 Bitertanol 2

9.0

P2

8.0

P7 P9 P10 P8

P3 P4

7.0

P5 P6

P12

6.0

P13 P11

5.0

1.5

4.0

1.0

N1 N4

N10 N12 N13 N14

3.0 N2 2.0

0.5

N8 N9

N5 N6 N3

N11

N7

N15

1.0

0.0 2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

2.5

min

Fig. 3.3.1 Chromatogram of standard organophosphate pesticide

5.0

7.5

10.0

12.5

15.0

17.5

20.0

min

Fig. 3.3.2 Chromatogram of standard organonitrogen pesticide

solution obtained using GC-FTD

solution obtained using GC-FTD

µV (x10,000)

µV (x1,000,000)

10.0

1.25 9.0

P2 P3

8.0

P9

P7

1.00

P4

7.0

P5 6.0

N3+P10

P8

P6

0.75

P11 N6+P12

5.0

P13

P1

0.50

4.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Pyrethrine 1 Pyrethrine 2 Pyrethrine 3 Pyrethrine 4 Bifenthrin Pyrethrine 5 Cyhalothrin 1 Acrinathrin 1 Cyhalothrin 2 Acrinathrin 2 Acrinathrin 3 Acrinathrin 4 Permethrin 1 Permethrin 2 Cyfruthrin 1 Cyfruthrin 2

17 18 19 20 21 22 23 24 25 26 27

Cypermethrin 1 Cypermethrin 2 Flucythrinate 1 Flucythrinate 2 Fenbalerate 1 Fenbalerate 2 Fluvalinate Tralomethrin 1 Deltamethrin 1 Tralomethrin 2 Deltamethrin 2

26(27) 23 9+10+11

20

0.25 N10 N11

N8 N7

1

N14 N13

N9

N15

12.0 5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

min

Fig. 3.3.3 Chromatogram of processed soy bean solution

obtained using GC-FTD (13 organophosphates and 14 organonitrogens: 0.2 to 0.5µg/g added) µV (x100,000) 4.5 4.0

24(25)

26(27)

9+10+11

3.5

18 3.0 2.5

5

15

12

21 16 17

2.0 1.5

23

19 20

22

14

1.0

7+8

1 2

6

3

13

0.5 0.0 12.0

13.0

14.0

15.0

16.0

17.0

13 2

3

4

6

0.00

1.0

2.5

22

4 7+8

N5 N4 N2

21

16 19 17 1

N12

2.0

15

12

5

N1

3.0

24(25)

18

18.0

min

Fig. 3.3.5 Chromatogram of processed spinach liquid obtained

using GC-ECD (first fraction, 0.1µg/g pesticide added)

13.0

14.0

15.0

16.0

17.0

18.0

min

Fig. 3.3.4 Chromatogram of standard pyrethroid pesticide

solution obtained using GC-ECD (1mg/L) ■ Analytical Conditions 1 : GC-2010AF, FPD-2010, AOC-20i, GCsolution Instrument : BPX5 (30m × 0.25mm I.D., df = 0.25µm) Column Column Temp. : 80˚C(1min)-20˚C/min-190˚C-5˚C/min-280˚C(5min) Carrier Gas : He, 143kPa (2.4mL/min, 45cm/s, constant-velocity mode) : FTD-2010 Detector : 250˚C Inj. Temp. Det. Temp. : 280˚C Injection Method : High-pressure, splitless (300kPa, 1min) Injection Volume : 1µL ■ Analytical Conditions 2 : GC-17A, ECD-17, AOC-20i, GCsolution Instrument : ZB-1 (30m × 0.25mm I.D., df = 0.25µm) Column Column Temp. : 50˚C(1min)-25˚C/min-175˚C-10˚C/min-300˚C(4min) Carrier Gas : He, 150kPa (1.7mL/min, constant-pressure mode) : ECD-17 Detector : 280˚C Inj. Temp. Det. Temp. : 310˚C Injection Method : High-pressure, splitless (300kPa, 1min) Injection Volume : 1µL 66

Residual Pesticides

3.4 Simultaneous Analysis of Pesticides (1) - GC/MS ■Explanation Residual Pesticides on vegetables and fruits are a matter of concern. There are various kinds of pesticides used, among which approximately 240 are subjected to regulations in Japan. A good way of analyzing these pesticides is simultaneous GC/MS measurement. Here, an example of a simultaneous analysis of 86 pesticides using GC/MS is shown.

■Analytical Conditions Instrument : GCMS-QP5000 : DB-1 30m × 0.25mmI.D. df=0.25µm Column Col.Temp. : 50°C(2min)-20°C/min-130°C -3°C/min-300°C(7min) : 280°C Inj.Temp. : 280°C I/F Temp. Carrier Gas : He 120kPa(2min)-2kPa/min-250kPa

Table 3.4.1 List of pesticides and molecular weights 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

67

Component Methamidophos Dichlorvos Propamocarb Acephate Isoprocarb Fenobucarb Ethoprophos Chlorproham Bendaiocarb Dimethipin α-BHC Dimethoate Thiometon β-BHC γ-BHC σ-BHC Terbufos Diazinon Ethiofencarb Etrimfos Pirimicarb Metribuzin Bentazone Parathion-methyl Carbaryl Heptachlor Fenitrothion Methiocarb Dichlofluanid Esprocarb Pirimifos-methyl Thiobencarb Malathion Aldrin Fenthion Parathion Chlorpyrifos Diethofencarb Captan Heptachlor epoxide Pendimethalin α-Chlorfenvinphos Pyrifenox

Molecular weight 141 220 188 183 193 207 242 213 223 210 288 229 246 288 288 288 288 304 225 292 238 214 254 263 201 370 277 225 332 265 305 257 330 362 278 291 349 267 299 386 281 358 294

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86

Component Chinomethionat β-Chlorfenvinphos Quinalphos Phenthoate Triadimenol Vamidothion Trichlamide Methoprene Flutolanil Dieldrin Prothiofos Myclobutanil p,p'-DDE Pretilachlor Endrin Fensulfothion Chlorobenzilate p,p'-DDD o,p'-DDT Mepronil Lenacil Edifenphos Captafol p,p'-DDT Propiconazole EPN Dicofol Phosalone Mefenacet Amitraz Cyhalothrin Bitertanol Pyridaben Inabenfide Permethrin Cyfluthrin Cypermethrin Flucythrinate Fenvalerate Fluvalinate Pyrazoxyfen Deltamethrin Tralomethrin

Molecular weight 234 358 298 320 295 287 339 310 323 378 344 288 316 311 378 308 324 318 352 269 234 310 347 352 341 323 370 367 298 293 449 337 364 338 390 363 415 451 419 502 437 503 661

3.4 Simultaneous Analysis of Pesticides (2) - GC/MS

40 18466908

TIC

11 5

7

53

54

26 14

61 34

89

56

46

66

67

15 16 62

17 21

32

22 2 19

3 6

1

18

27 28

48 57 85+86

24 25

50 51

73 74

59 58

75 78 81

4

43

69

71

84

76

72 71

68

80 81 82

83 83

82 10

15

20

25

30

35

40

45

50

Fig. 3.4.1 Analysis of 86 pesticides using DB-1

68

Residual Pesticides

3.5 Analysis of Pesticides Using NCI (1) - GC/MS ■Explanation Trace analysis is required for the measurement of residual pesticides in vegetables and fruits, but it is difficult to extract only pesticides, even after a cleanup pretreatment. NCI is an effective method for this analysis. Generally, positive ions are detected in mass spectrometry, but negative-ion analysis may be used depending on the compound. The negative ions of such compounds allow microanalysis with minimal interference from the matrix. Trace amount of pesticides that cannot be detected using the conventional EI method can be detected by this method.

■Analytical Conditions Instrument : GCMS-QP5050A : DB-1 30m × 0.25mmI.D. df=0.25µm Column Col.Temp. : 50°C(2min)-20°C/min-130°C -3°C/min-300°C(7min) : 280°C Inj.Temp. : 280°C I/F Temp. Carrier Gas : He 120kPa(2min)-2kPa/min-250kPa

181

219

109 51

50

85

38

75

145 96

121

0 200

100

35

71

14,284

Cl Cl

Cl 50 Cl

Cl Cl

0 100

200

Fig. 3.5.1 α-BHC mass spectrum (upper: EI, lower: NCI)

69

3.5 Analysis of Pesticides Using NCI (2) - GC/MS

Component : δ-BHC

11.908

Component : δ-BHC

11.867

13.905 11.863 35.00 * 1.0

183.00 * 1.0 181.00 * 1.0 11.867

71.00 * 1.0 217.00 * 1.0

11.75

13.5

12

Fig. 3.5.2 SIM chromatogram using EI

14

Fig. 3.5.3 SIM chromatogram using NCI

α–BHC TIC * 1.0 183.00 * 100.0 219.00 * 100.0 125

10

15

43

δ–BHC β–BHC γ–BHC

125

35

TIC * 1.0 35.00 * 3.0 71.00 * 3.0

15

71 73

50 69

50

113

95

122

150 167 183

0

262 273 289

221 200

100

318

343

57

0

300

100

126 147160 180 197

100

Fig. 3.5.4 MC and mass spectrum using EI

224

253

280 306 318 331 344

200

372 393

300

400

Fig. 3.5.5 MC and mass spectrum using NCI

cypermethrin

TIC * 1.0

TIC* 1.0

163.00 * 100.0 21

22

23

207.05* 3.0 205.10* 3.0 167.10* 3.0

24 10

45

50

20

207

207 281

69

35 50

96 109

0 100

121 143

157

191 181

215 227 245 265

200

288

317

171

344 360373 393

300

Fig. 3.5.6 MC and mass spectrum using EI

106 0

55

71

137 97 112 126

100

156

197 200

223 235

260 278

305 318

343

300

379 392 400

Fig. 3.5.7 MC and mass spectrum using NCI

70

Residual Pesticides

3.6 Analysis of Pesticide Residue in Foods Using GC/MS (1) - GC/MS ■ Explanation The measurement of pesticide using the NCI (negative chemical ionization) method is described here as an example. NCI enables detection with a higher degree of sensitivity than EI (electron ionization) for some chemical compounds. NCI is particularly effective for pesticides that contain chlorine but the analysis of pesticides not containing chlorine is also described here as an example. Fig. 3.6.1.1 to 3.6.3.4 show EI and NCI mass spectra and 10ppb SIM chromatograms for isofenphos, pyributicarb, and fenvalerate. Isofenphos and pyributicarb do not contain chlorine.

■ Analytical Instrument [GC] Column Column Temp. Inj. Temp. I/F Temp. Carrier Gas Injection Method [MS] Scan Range Reaction Gas

Conditions : GCMS-QP5050A : DB-5 (30m × 0.25mm I.D. df = 0.25µm) : 50˚C(1min)-20˚C/min-100˚C-5˚C/min-300˚C(1.5min) : 300˚C : 300˚C : He 100kPa (2min) – 3kPa/min – 220kPa (3min) : Splitless (2min) : EI: m/z 35 to 550; NCI: m/z 10 to 550 : Isobutane

182

58 125000

S (CH3)2CHNH P

100000

125000

O 100000

OC2H5 O

75000

OCH(CH3)2

50000

43 92 25000

0

50000

213 121 185 96 255 138 200 286 100

200

75000

25000

95 0

300

400

500

Fig. 3.6.1 EI mass spectrum of isofenphos

213.15 (1.22) 255.10 (2.22) 121.00 (1.00)

136

216 256 302 344 200

300

400

500

Fig. 3.6.2 NCI mass spectrum of isofenphos

8000 600

100

182.05 (1.00)

7000 6000

500

5000 400 4000 300

3000

200

2000 1000

100 21.4

21.5

21.6

21.7

21.8

Fig. 3.6.3 EI 10ppb-SIM chromatogram of isofenphos

71

21.4

21.5

21.6

21.7

21.8

Fig. 3.6.4 NCI 10ppb-SIM chromatogram of isofenphos

3.6 Analysis of Pesticide Residue in Foods Using GC/MS (2) - GC/MS 149

800e3

165

125000

108

700e3

O

100000

N

N

O

S

600e3 500e3

75000

400e3

166

50000

300e3

181

150

200e3

41 93

25000

100e3

330

0 100

200

42 71

0e3

300

400

Fig. 3.6.5 EI mass spectrum of pyributicarb

181.15 (3.00) 165.15 (1.00)

181 223 100

500

200

300

400

500

Fig. 3.6.6 NCI mass spectrum of pyributicarb

149.10 (1.00) 100000

1000

75000

750

50000

500

25000

250

27.5

27.6

27.7

27.8

27.9

27.6

Fig. 3.6.7 EI 10ppb-SIM chromatogram of pyributicarb

17500

125

27.9

28.0

167 O

O

15000

27.8

Fig. 3.6.8 NCI 10ppb-SIM chromatogram of pyributicarb

CN

O

Cl

27.7

400e3 350e3

167

12500

300e3

10000

209

152 7500 5000 2500

77 51 115

250e3

225

200e3

197

100e3

419

103

50e3

73

100

200

300

400

100

500

Fig. 3.6.9 EI mass spectrum of fenvalerate

200

300

400

500

Fig. 3.6.10 NCI mass spectrum of fenvalerate

30000

163.15 (1.00) 226.00 (3.00)

391

127

0e3

0

350

211

150e3

167.05 (1.00) 211.05 (2.00)

25000 300 20000 250 15000 200 10000 150 5000

100 33.25

33.50

33.75

34.00

Fig. 3.6.11 EI 10ppb-SIM chromatogram of fenvalerate

35.00

35.25

35.50

Fig. 3.6.12 NCI 10ppb-SIM chromatogram of fenvalerate

72

Residual Pesticides

3.7 Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (1) - GC/MS

Chrysene-d10

Fig. 3.7.1 TIC chromatogram of standard pesticides (1ppm each)

Chlorpyrifos

Fig. 3.7.2 TIC chromatogram (enlarged) and mass chromatogram of vegetable juice

73

Fenvalerate

Amitraz

Mepronil

■ Analytical Conditions : GCMS-QP2010 Instrument : ZB1 30m × 0.32mm I.D. df = 0.25µm) Column : 70˚C(1min)-20˚C/min-120˚C/minColumn Temp. 10˚C/min-270˚C(4min) : 270˚C Inj. Temp. : 250˚C I/F Temp. Ion Source Temperature : 200˚C

α-endosulfan

Pyrene-d10

Chlorpyrifos

Malathon

Chlorpyrifos-methyl

Pirimicarb Phenanthrene-d10

Simazine (CAT)

■ Explanation After extracting Vegetable juice with n-hexane (concentration factor of 10), standard pesticide products were added to concentrations of 20ng/mL and then analyzed with GC/MS. Library searches and quantitative analysis were performed on the three representative components for which the elution positions are indicated with arrows in Fig. 3.7.2. Clean-up was not performed for the sample.

3.7 Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (2) - GC/MS ■ Results of Library Searches and Quantitative Analysis α-BHC Library search results Target Line #: 1 Retention time: 8.400 (scan #: 109) Mass peak #: 56 Base peak Raw mode: Single 8.400 (109) Background mode: 8.425 (112)

Compound: a-BHC m/z: 181.00 Calibration curve: Quadratic Origin: Through origin Internal standard method f(x)=-0.000000*x^2+0.001749*x+0.000000 Correlation coefficient (R) = 0.999982 Contribution (R'2): 1.000000 Concentration (ppb) Area ratio

Hit #: 1 Entry #: 67749 Library: NIST 107.LIB SI: 74 Molecular formula: C6H6Cl6 CAS: 319-84-6 Molecular weight: 288 Retention index: 0 Compound:alpha.-Lindane $$ Cyclohexane, 1,2,3,4,5,6-hexachloro-,(1.

Compound: a-BHC ID #: 1 m/z: 181.00 Type: Target Retention time: 8.405 Area: 9821 Concentration: 26.201ppb

Chlorpyrifos Library search results Target Line #: 4 Retention time: 10.450 (scan #: 355) Mass peak #: 46 Base peak Raw mode: Single 10.450 (355) Background mode: 10.425 (352)

Compound: Chlorpyrifos m/z: 314.00 Calibration curve: Quadratic Origin: Through origin Internal standard method f(x)=-0.000002*x^2+0.001070*x+0.000000 Correlation coefficient (R) = 0.998496 Contribution (R'2): 0.999639 Concentration (ppb) Area ratio

Hit #: 1 Entry #: 19366 Library: NIST 21.LIB SI: 66 Molecular formula: C9H11Cl3NO3PS CAS: 2921-88-2 Molecular weight: 349 Retention index: 0 Compound: Chlorpyrifos

p,p’-DDT Library search results Target Line #: 5 Retention time: 12.592 (scan #: 612) Mass peak #: 69 Base peak Raw mode: Average 12.592-12.600 (612-613) Background mode: Average 1

Compound: p,p'-DDT m/z: 235.00 Calibration curve: Quadratic Origin: Through origin Internal standard method f(x)=-0.000006*x^2+0.004688*x+0.000000 Correlation coefficient (R) = 0.998956 Contribution (R'2): 0.999738 Concentration (ppb) Area ratio

Hit #: 1 Entry #: 84517 Library: NIST 107.LIB SI: 62 Molecular formula: C14H9Cl5 CAS: 50-29-3 Molecular weight: 352 Retention index: 0 Compound:Chlorophenothane $$ p,p’-DDT $$ Benzene, 1,1’-(2,2,2-tricl

Compound: p,p'-DDT ID #: 12 m/z: 235.00 Type: Target Retention time: 12.602 Area: 14105 Concentration: 30.207ppb

74

Residual Pesticides

3.8 Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (1) - GC/MS ■Explanation In recent years, there has been an increase in regulations regarding residual pesticides in food products and more agricultural products are subject to such regulations. In response to this trend, there has been an increased demand for automating and speeding up the pretreatment procedures for the analysis of residual pesticides. The Japnese Ministry of Health, Labour and Welfare has issued a notice about the rapid method for analyzing residual pesticides. This method employs GPC clean-up for part of pretreatment in order to simultaneously analyze multiple pesticide components (1997 Chemical Hygiene No. 43, 44 and 45). ■Equipment Overview Samples extracted from food contain large quantities of oils and pigments that will interfere with pesticide analysis. The GPC column separates the fat and pigment substances from the pesticides in the extracted samples in accordance with their molecular size. By switching the valve, fat and pigment substances which elute more quickly are discharged and the target pesticides are taken into the trapping loop. The pesticides trapped in the loop are injected into the GC, separated in the GC column and then detected in the MS section. ■Analytical Conditions : Prep-Q Systems GPC : LC-VP Series GC/MS : GCMS-QP5050A GPC Column : CLNpak EV-200 (Shodex 150mmL. × 2mm I.D.) GC Column : J&W uncoated : deactivated silica tubing (5m × 0.53mm I.D.) pre-column : DB-5 (5m × 0.25mm I.D. df=0.25µm) analysis : DB-5 (30m × 0.25mm I.D. df=0.25µm) Table 1.12.1 -GPCMobile Phase Flow Rate Injection Volume Fraction -GCPTV Column Temperature Carrier Gas -MSInterface Temperature Scan Range Interval

75

: : : : : : : : : :

In order to further improve the rapid analysis method, Shimadzu has developed a system that connects the GC/MS and GPC clean-up systems online. By completely automating the GPC and GC/MS processes, the Online GPC-GC/MS (Prep-Q) system realizes simpler and quicker analysis of residual pesticides. Prep-Q was developed under the proposal and directives of the Osaka Prefectural Public Health Laboratory. 1) 2) This section shows an analysis example where pesticides were added to an actual sample (potato) and analyzed by Prep-Q. Fats Pigments Pesticides (1, 2 and 3)

Pesticides (1, 2 and 3) Injection Trapping Loop

GC/MS

GCMS-QP5050A

LC-10A

GPC Column

sample Fats

Exhaust

Pigments

Data

1

2 3

Fats Pigments

Pesticides (1, 2 and 3)

m/z

Fig. 3.8.1 Outline of Prep-Q

■Sample Extraction and Pretreatment Sample pretreatment is performed in accordance with the rapid analysis method for residual pesticides.

Solvent extraction

Homogenize 20 g of sample ↓ Extract with 100 mL acetone ↓ Filter extract and wash with 50 mL acetone ↓ Vacuum concentrate and add 6 g Chart salt

Dehydration

Load to diatom earth column ↓ Elute with 150 mL ethyl acetate

Re-extract

After vacuum concentrating, dissolve in 10 mL acetone : cyclohexane (3:7)

GPC-GC/MS

Inject 20µL sample into Prep-Q

Fig. 3.8.2 Flow of pretreatment

3.8 Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (2) - GC/MS ■Example of Actual Sample Analysis In this case, a standard solution of pesticides was added to potato extract and the mixture was analyzed with Prep-Q. The mass chromatograms and mass spectra for four pesticide substances (fenobucarb, BHC, diazinon and permethrin) are provided as an example in Fig. 3.8.3. Intensity

I ) Fenobucarb 0.05 ppm

II) α-BHC 0.025 ppm

I)

Fenobucarb

100000000

TIC *1.00 121.00 *50.00 150.00 *50.00

50000000

0 10

11

12

13

0 12

91

120

130

140

150

m/z

219

145 158

90

Intensity

III) Diazinon 0.05 ppm

min

181

86

110

14

109

107 100

13

II)

150 90

TIC *1.00 183.00 *150.00 181.00 *400.00

α-BHC

#1 Retention time: 13.317 (scan #: 903) Base peak: 183.00 (118466)

121

I)

II)

50000000

14 min

#1 Retention time: 12.033 (scan #: 749) Base peak: 121.20 (1927587)

Intensity

110

130

150

205 170

190

210

252 230

250

m/z

Intensity

IV) Permethrin 0.05 ppm

III) 40000000 35000000 30000000 25000000 20000000 15000000 10000000

Diazinon

13

14

15

16

88 90

137 124 152

50000000 0

min

24

25

179

120

150

183 180

26 min

#1 Retention time: 25.333 (scan #: 2345) Base peak: 183.15 (832676)

183

IV)

199 163

111

TIC *1.00 183.00 *150.00 184.00 *300.00

Permethrin1

#1 Retention time: 14.683 (scan #: 1067) Base peak: 137.25 (354011)

III) 93

Permethrin2

100000000

TIC *1.00 304.00 *300.00 179.00 *100.00

0

IV) 150000000

216 210

248 240

276 270

304

89

300 m/z

115

90

100

110

129 130

147

163 168

150

170

180 m/z

Fig. 3.8.3 Mass Chromatograms and mass spectra for pesticides in potato extract

■Comparison to Rapid Analysis Method Prep-Q employs a small GPC column in the clean-up GPC section in order to reduce the time necessary for the cleanup procedure. In addition, by injecting a large quantity of sample into the GC, the concentration process of the rapid analysis method can be eliminated. As a result, the analysis time per sample was reduced to about one half compared to the conventional rapid analysis method. The amount of solvent used for clean-up GPC was also reduced from 200 mL to 1 mL per sample. Prep-Q enables environmentally friendly and economical analysis of residual pesticides.

Concentration

Rapid Analysis Method Extraction

Dehydration

Re-extraction

30min

Simultaneous multi-substance GC/MS measurement Pesticide A, B, C, D ...

GPC + (concentration) (solid phase column) 60min + 30min

10 min

40min

170min

Online GPC-GC/MS Method Extraction

Dehydration

Re-extraction

30min

GPC

10 min

Simultaneous multi-substance GC/MS measurement Pesticide A, B, C, D ...

40min

Can be operated at night.

80min

Fig. 3.8.4 Comparison of analysis times for rapid analysis and on-line GPC-GC/MS methods

■Conclusion The Prep-Q system, developed specifically for analyzing residual pesticides in food products, fully automates all procedures from pretreatment and reduces analysis time and solvent consumption. Therefore, residual pesticide analysis can be accomplished more simply and quickly than the conventional rapid analysis method. In addition, since the system is automated, improvements in analytical accuracy and ease of validation (for both equipment and method) can be expected, enabling even more reliable analysis. ■References 1) Study for making the GPC-GC/MS process of analyzing residual pesticides in foods online - large volume injections to GC Osaka Prefectural Public Health Laboratory: Mikiya Kitagawa, Shinjiro Hori, et al. Food Hygienic Society of Japan, 73rd Technical Symposium 2) Analysis of Residual Pesticides in Foods Using Online GPC-GC/MS Osaka Prefectural Public Health Laboratory: Mikiya Kitagawa, Shinjiro Hori, et al. Food Hygienic Society of Japan, 77th Technical Symposium 76

Residual Pesticides

3.9 Analysis of Pesticides with Specific Threshold Levels in Foods (1) - LC ■ Explanation The regulations on pesticide residue in foods based on the Food Sanitation Law have undergone many revisions since October 1992 and, as of January 2005, regulated values were specified for 244 of pesticides. The analysis of some standard pesticides for which HPLC is used is described here as an example.

■ Analysis of Fenpyroximate The fenpyroximate content can be obtained from the sum of fenpyroximate-E and fenpyroximate-Z. 600

■ Peaks 1:FenpyroximateZ 2:FenpyroximateE

400

2 1

mAU

■ Analytical Conditions : Shim-pack VP-ODS (250mmL. × 4.6mm I.D.) Column Mobile Phase : Water/Acetonitrile = 1/4 (v/v) : 1.0mL/min Flow Rate Temperature : 40˚C : SPD-10AVVP 254nm Detection

200

0 0

2

4

6 min

8

10

12

Fig. 3.9.1 Chromatogram of fenpyroximate

■ Analysis of Cyromazine Because cyromazine has a high polarity, there is insufficient retaining power with an ODS column and so an aminopropyl column is used.

■ Peak

1

1:Cyromazine

mAU

■ Analytical Conditions : Shodex Asahipak NH2P-50 4E (250mmL. × 4.6mm I.D.) Column Mobile Phase : Water/Acetonitrile = 7/93 (v/v) : 0.8mL/min Flow Rate Temperature : 40˚C : SPD-10AVVP 215nm Detection

100

50

0 0

2

4

6

8

10

min

Fig. 3.9.2 Chromatogram of cyromazine

77

12

3.9 Analysis of Pesticides with Specific Threshold Levels in Foods (2) - LC

■ Analytical Conditions : Shim-pack VP-ODS (150mmL. × 4.6mm I.D.) Column Mobile Phase : 50mM KH2PO4/Methanol = 17/3 (v/v) : 0.8mL/min Flow Rate Temperature : 40˚C : SPD-10AVVP 270nm Detection

■ Peak

2000

1

1:Nitenpyram

mAU

■ Analysis of Nitenpyram In the nitenpyram testing method, the nitenpyram content is obtained by analyzing nitenpyram with HPLC and analyzing its CPF metabolite with GC.

1000

0 0

2

4

6

8 min

10

12

14

Fig. 3.9.3 Chromatogram of nitenpyram

■ Analytical Conditions : Shim-pack VP-ODS (250mmL. × 4.6mm I.D.) Column Mobile Phase : Water/Acetonitrile = 3/7 (v/v) : 0.8mL/min Flow Rate Temperature : 40˚C : SPD-10AVVP 250nm Detection

100

■ Peaks 1 2

mAU

■ Analysis of Chlorfluazuron Seven pesticides, including chlorfluazuron, can be analyzed simultaneously.

3

50

4

5

1:Diflubenzuron 2:Tebufenozide 3:Hexaflumuron 4:Teflubenzuron 5:Lufenuron 6:Flufenoxuron 7:Chlorfluazuron

7

6 0 0.0

2.5

5.0

7.5

10.0

12.5 min

15.0

17.5

20.0

22.5

25.0

Fig. 3.9.4 Chromatogram of 7 pesticides including chlorfluazuron

78

Residual Pesticides

3.10 GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (1) - GC/MS ■ Explanation In the analysis of pesticide residue in agricultural products, fats and pigments in the sample can cause contamination of the GC or GC/MS injection port and peaks that interfere with the target components and so they must be removed as part of the pretreatment process. The conventional solvent extraction method requires considerable time and effort and so difficulties arise when processing large numbers of samples. GPC (gel permeation chromatography) is a technique that separates the sample components by molecular size. Using this technique, the pesticide components can be easily separated from the fats and pigments, which have relatively large molecular weights, and clean-up can be automated. For this reason, GPC is adopted as one of the clean-up methods in the pesticide-residue rapid analysis method prescribed by Japan's Ministry of Health, Labour and Welfare (Notice 43 issued by the Environmental Health Bureau's Food Chemistry Division on 8 April 1997). The principle of the GPC clean-up method and an application example of Shimadzu's GPC Clean-up System are described here.

■ Principle of GPC Clean-up Method Fig. 3.10.1 shows the principle of the GPC clean-up method. There are small holes (pores) of a fixed size in the packing material of the GPC column. Components in the sample with a small molecular size (e.g., pesticides: gray sections in the figure) can permeate deep into the pores while constituents with a large molecular size (e.g., fats and pigments: striped sections in the figure) cannot. For this reason, fats and pigments are eluted from the column sooner than pesticides*) and so the sample can be purified by fractionating this pesticide eluate. *)In practice, the separation process is not only affected by the molecular size but also by the adsorption onto the packing material.

Packing material

[References] 1) Committee for Studying and Developing the Pesticideresidue Rapid Analysis Method: Food Hygiene Research, Vol. 47, P35 (1997) 2) Isao Saito: LCtalk, Vol. 35, P3 (1995) Fig. 3.10.1 Principle of GPC clean-up method

■ Pretreatment for the Pesticide-

residue Rapid Analysis Method Fig. 3.10.2 shows the different stages in the pesticideresidue rapid analysis method. With the notified method (individual analysis method), fats and pigments were removed using liquid-liquid extraction and solid-phase extraction, whereas with the pesticide-residue rapid

Acetone extraction

Diatomaceous-earth column solid-phase extraction

GPC clean-up

analysis method, they are removed using GPC. The pesticide-residue rapid analysis method makes it possible to perform pretreatment for all the pesticides together in almost the same amount of time required by the notified (individual analysis) method.

Silica-gel column purification

Hydrochloric acid processing

Florisil column purification

Organochlorines, pyrethroids

Organophosphates, organonitrogens N-methylcarbamates

Pirimicarb Fig. 3.10.2 Stages of pretreatment using the rapid analysis method

79

3.10 GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (2) - LC ■ Fractionation Conditions The fractionation conditions for extracting a rice sample in accordance with the rapid analysis method and purifying it with Shimadzu's GPC Clean-up System are given in Table 3.10.1. The corresponding chromatogram is shown in Fig. 3.10.3.

Rice sample Standard sample

Chinomethionat Fluvalinate

Fig. 3.10.3 also shows the chromatogram for two pesticides, fluvalinate and quinomethionate, obtained with GPC. 0

5

In general, the pesticides that are analyzed with the rapid analysis method are eluted between fluvalinate and quinomethionat and so fractionation is performed for the interval between the elution times of these two constituents.

10

15

20 min

25

30

35

40

Fig. 3.10.3 GPC chromatogram of rice extract

Table 1.14.1 Fractionation conditions

Instrument : Shimadzu GPC Clean-up System : CLNpak EV-G+CLNpak EV-2000 Column Mobile Phase : A: Ethyl Acetate B: Cyclohexane A/B = 1/4 (v/v) : 4.0mL/min Flow Rate : SPD-10AVP 254nm Detection ■ Analysis Example for Organophosphorus Pesticides Fig. 3.10.4 shows the result obtained by purifying soybean, to which organophosphorus pesticides are added, using Shimadzu's GPC Clean-up System, purifying with a silica-gel mini-column, redissolving with acetone, and then analyzing the sample solution using GC. Fig. 3.10.5 shows the result obtained by processing soybean, to which organophosphorus pesticides are added, in accordance with the notified (individual analysis) method, and analyzing with GC. It can be seen that almost identical results are obtained with both methods.

■ Analytical Conditions : GC-2010 Instrument : Rtx-1 (15m × 0.53mm I.D. df = 1.5µm) Column Column Temp. : 80˚C(1min)-8˚C/min - 250˚C(10min) : 230˚C Inj. Temp. : 280˚C Det. Temp. : He, 16.5mL/min Carrier Gas : FPD-2010 Detection Injection Method : Splitless (1min)

FIG.5

0.50

2.0 1.0

EPN

0.75

Prothiofos

Dimethoate

3.0

Diazinon IBP Parathion-methyl MEP Malathion Chlorpyrifos

4.0

1.00

DDVP

DDVP

5.0

EPN

6.0

1.25

Prothiofos

Dimethoate

7.0

Diazinon IBP Parathion-methyl MEP Malathion Chlorpyrifos

FIG.4

0.25

0.0 1.0 0.0

0.00 2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

min

Fig. 3.10.4 Chromatogram of organophosphorus pesticides obtained

using the rapid analysis method

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

min

Fig. 3.10.5 Chromatogram of organophosphorus pesticides obtained

using the notified (individual analysis) method

80

Residual Pesticides

3.11 Analysis of Carbamate Pesticides - LC ■ Explanation N-methylcarbamate pesticides are used widely as insecticides and herbicides. In a publication issued by the Japanese government in 1994 (Ministry of Health, Labour and Welfare, Notice 199), post-column fluorescent derivatization using HPLC was adopted as the method for analyzing N-methylcarbamate pesticides. N-methylcarbamate pesticides undergo hydrolysis in alkaline conditions and generate methylamine, which is a primary amine that can be analyzed by fluorescent detection after fluorescent derivatization.

■ Rapid Analysis of N-methylcarbamate Pesticides Fig. 3.11.1 shows the result of analyzing nine standard substances, including the N-methylcarbamate pesticides mentioned in Notice 199 issued by Japan's Ministry of Health, Labour and Welfare. By using the high-separation FC-ODS column as the analysis column, and optimizing the gradient program, methiocarbs, which are the slowest

■ Analytical Conditions [Separation] : Shim-pack FC-ODS (75mm × 4.6mm I.D.) Column : Water/Methanol (Gradient Elution Method) Mobile Phase : 1.0mL/min Flow Rate : 50˚C Temperature [Detection] Reaction Reagent 1 : 50mM NaOH : 0.5mL/min Flow Rate : 100˚C Temperature Reaction Reagent 2 : OPA Solution : 0.5mL/min Flow Rate : 50˚C Temperature Detection : RF-10AXL Ex: 340nm Em: 445nm

to elute, can be eluted in about 25 minutes. Because the column is shorter than those employed in conventional methods, gradient re-equilibration time and column cleaning time are also reduced. The time for one analysis cycle can be reduced to 32 minutes. ■ Peaks

1 4 6

2

5

3

0

5

8 7

10

15

20

9

1. Oxamyl 2. Methiocarb-sulfoxide 3. Methiocarb-sulfone 4. Aldicarb 5. Bendiocarb 6. Carbaryl 7. Ethiofencarb 8. Fenobucarb 9. Methiocarb

25

min

Fig. 3.11.1 Analysis of standard N-methylcarbamate pesticides (1ppm each, 10µL injected)

■ High-sensitivity Analysis Example for

N-methylcarbamate Pesticides Fig. 3.11.2 shows the results of injecting 10µL of a sample with a concentration of 5ppb and performing

high-sensitivity analysis. ■ Peaks

89 4 2

1

0

5

6 5

3

10

15

7

20

1. Oxamyl 2. Methiocarb-sulfoxide 3. Methiocarb-sulfone 4. Aldicarb 5. Bendiocarb 6. Carbaryl 7. Ethiofencarb 8. Fenobucarb 9. Methiocarb

25

min

81

Fig. 3.11.2 Analysis of standard N-methylcarbamate pesticides (5ppm each, 10µL injected)

3.12 Analysis of Imazalil in Oranges - LC ■Explanation Fungicide imazalil is mostly contained in imported oranges and bananas imported to Japan. Here, analysis of imported oranges will be introduced. The target component was confirmed by comparison with UV spectrum of standard Sample using a photodiode array UV-VIS detector.

■Pretreatment Performed in accordance with Standard Methods of Analysis for Hygienic Chemists, annotation (supplement 1995)

■Analytical Conditions : STR ODS-II (150mmL. × 4.6mm I.D.) Column Mobile Phase : 5mM (Sodium) Phosphate Buffer(pH6.9) /Acetonitrile=45/55 (v/v) : 1.0mL/min Flow Rate Temperature : 40°C : Photodiode Array Detection Detection λ=210nm to 300nm

Reference Shimadzu Application News No. L246 (C190-E068)

■ Peaks

Peak spectrum

1. imazalil

3

mAbs 0 mAbs 10

1

Ch1 220nm

250 Wavelength (nm)

300

250 Wavelength (nm)

300

5 0 0

5

10

min

16

Peak spectrum

mAbs 0

Fig. 3.12.1 Chromatogram of imazalil in imported orange sample (220nm)

Fig. 3.12.2 Spectra of imazalil (upper: standard sample, lower: sample)

82

Residual Pesticides

3.13 Analysis of Pesticide Residue in Agricultural Products Using LC/MS (1) - LC/MS ■ Explanation Under Japan's Food Sanitation Law, the levels of pesticide residue in agricultural products are strictly regulated, and at present there are 244 pesticides for 262 types of agricultural products. Out of the regulated pesticides, LC is used to analyze non-volatile pesticides or pesticides that are easily decomposed by heating. Because there are many impurities in food extracts, qualitative determination using LC/MS, which uses a mass spectrometer for detection and thereby offers greater selectivity, is increasingly employed for the purpose of verification. Int. 2000e3 1750e3 1500e3 1250e3 1000e3 750e3

1:201.85(1.00) 1:221.90(2.44) 1:333.95(7.24) 1:296.85(1.00) 1:339.90(1.28) 1:268.95(1.00) 1:353.05(2.19) 1:402.90(10.53) 1:345.90(2.59) 1:328.90(2.46) 1:305.95(1.00) 1:422.00(11.36) 2:268.90(4.00) 2:337.00(4.00) 2:347.05(30.00) 2:327.90(40.00) 2:308.90(4.00) 2:458.85(4.00) 2:486.90(8.00) 2:537.80(8.00)

The batch analysis of 20 residual pesticides in agricultural products is described here as an example. Fig. 3.13.1 shows a mass chromatogram obtained in scan mode. There is no need to perform optimization for each of the pesticides and high-sensitivity analysis is possible under the conditions set with autotuning. Also, if multisequence mode is used, reliable qualitative and quantitative determination is possible with one analysis by performing mass chromatography with positive ions or negative ions using the mass numbers of each of the pesticides.

1

4 2

6

11

5 3

7

9 8

10 12

14

13

16 15 17 18

500e3

19

20

250e3

2.5

5.0

7.5

10.0

12.5

15.0

17.5

Fig. 3.13.1 Mass chromatogram of pesticides in agricultural products

■ Peaks ESI-Positive mode 1. thiabendazole MW 201 2. methabenzthiazuron MW 221 3. furametpyr MW 333 4. imazalil MW 296 5. etobenzanid MW 339 6. daimuron MW 268 7. tebufenozide MW 352 8. pyrazoxyfen MW 402 9. triflumizole MW 345 10. pencycuron MW 328 11. buprofezin MW 305 12. fenpyroximate MW 421

83

ESI-Negative mode 13. imibenconazole-debenzyl MW 270 14. inabenfide MW 338 15. myclobutanil MW 288 16. iprodione metabolite MW 329 17. diflubenzuron MW 310 18. hexaflumuron MW 460 19. flufenoxuron MW 488 20. chlorfluazuron MW 539

20.0

min

3.13 Analysis of Pesticide Residue in Agricultural Products Using LC/MS (2) - LC/MS Fig. 3.13.2 shows SIM chromatograms and calibration curves (n=5) for pencycuron (12.5pg) and hexaflumuron (25pg) and Table 3.13.1 and 3.13.2 give the reproducibility results for each substance. As shown in

this example, constituents with roughly the same retention times and different measurement modes (positive and negative ions) can be quantified at the same time.

328.90(1.00)

3500

Cl

14.470

Y = (5655.35133)X + (4034.05823) r2=0.99982 Int.

Area

CH2 N

500e3

CONH

3250

400e3

300e3

3000

200e3

C19H21ClN2O Exact Mass: 328.13 Mol. Wt.: 328.84

2750 100e3 2500 10

15

min

0e3

0

25

50

75

Conc

Int.

458.85(1.00)

2400

F

14.170

Y = (1766.53398)X + (-192.42839) r2=0.99998 Area 450e3 400e3

Cl

2300 350e3

CONHCONH

O

CF2CHF2

2200

300e3

2100

250e3 200e3

F

Cl

C16H8Cl2F6N2O3 Exact Mass: 459.98 Mol. Wt.: 461.14

2000 150e3 1900

100e3 50e3

1800 10

15

min

0e3

0

50

100

150

200

Conc

Fig. 3.13.2 SIM chromatogram and calibration curves for pencycuron and hexaflumuron Table 3.13.1 Repeatability for pencycuron 12.5pg 25pg 50pg 125pg 250pg 500pg

1 13917 33710 67238 150565 289289 586968

2 14526 33710 68996 145253 287762 581783

3 14018 30242 69932 144468 270265 560675

4 13948 30793 66772 152698 288482 575145

5 15251 31331 61325 140439 273127 551669

Average 14332.00 31957.20 66852.60 146684.60 281785.00 571248.00

Standard deviation 570.19163 1645.7532 3346.1268 4929.2189 9281.106 14734.456

CV 3.98 % 5.15 % 5.01 % 3.36 % 3.29 % 2.58 %

Standard devaition 883.07401 1001.644 1825.9509 3808.3029 2894.5713 10502.585

CV 9.05 % 5.29 % 4.24 % 4.33 % 1.65 % 2.38 %

Table 3.13.2 Repeatability for hexaflumuron 25pg 50pg 125pg 250pg 500pg 1250pg

1 11153 18690 42881 81842 174911 437000

2 8984 19229 43726 89280 177600 437627

3 9859 20473 40001 90536 180001 439882

4 9766 18580 44840 86530 174125 434111

5 9007 17762 43646 91154 172789 460184

Average 9753.80 18946.80 43018.80 87868.40 175885.20 441760.80

Table 3.13.3 Analytical conditions

: Shim-pack VP-ODS (150mmL. × 2.0mm I.D.) : A: Water B: Acetonitrile : 20% B → 60% B (0.03min) → 80% B (20min) → 100% B (20.01-30min) → 20% B (30.01-40min) : 0.2mL/min Flow Rate : 40˚C Column Temperature : 5µL Injection Volume : +4.5kV (ESI-Positive Mode), -3.0kV (ESI-Negative Mode) Probe Voltage : 200˚C CDL Temperature Block Heater Temperature : 200˚C Nebulizer Gas Flow Rate : 4.5L/min : +0V (ESI-Positive Mode), +0V (ESI-Negative Mode) CDL Voltage : Scan Mode Q-array DC Voltage : Scan Mode Q-array RF : m/z 50-650 (1.5sec/scan) Scan Range Column Mobile Phase Gradient Program

84

Residual Pesticides

3.14 Analysis of N-methylcarbamate Pesticides Using LC/MS (1) - LC/MS ■ Explanation N-methylcarbamate pesticides are used widely in insecticides and herbicides and their residue in agricultural products has become an issue of concern. The method of separating eight n-methylcarbamate pesticides with an HPLC column, performing on-line hydrolysis, and applying fluorescent derivatization to the resulting methylamine is adopted as the testing method in revisions to the standards for foods and additives made by Japan's Ministry of Health, Labour and Welfare. Here, however, Int.

1. Aldicarbsulfone M.W. 222

H3C

Fig. 3.14.1 shows the structures and mass spectra of nmethylcarbamate pesticides. The protonated molecules can be confirmed in each case.

Int. 5. Carbaryl M.W. 201 250e3

223.05

300e3

200e3

the analysis of n-methylcarbamate pesticides directly using LC/MS in order to increase the simplicity and sensitivity, without applying derivatization, is described as an example.

H3CHNOCO

CH3 O H S C C O CH3

200e3

O N O C N CH3 H

150e3 100e3

144.95

100e3 255.05

Int. 150e3

125

150

2. Methomyl M.W. 162

50e3

234.35

239.65 254.00

147.85 166.00

0e3 100

202.05

175

200

225

250

275

m/z

195.05

0e3 100

125

150

175

219.05

200

225

Int. 6. Ethiofencarb M.W. 225 300e3

163.00

H3C 100e3

C

NOCONHCH3

250

275

m/z

226.05

OCONHCH3

200e3

CH2SC2H5

H3CS 100e3

50e3

164.05 106.00

0e3 100

179.20195.15

125

150

175

200

282.75

225

Int. 3. Primicarb M.W. 238 1000e3

250

275

m/z

106.80

0e3 100

258.05

125

150

Int. 7. Fenobucarb M.W. 207 750e3

239.15

CH3 OCON

750e3 H3C

H3C

N

N

200

225

250

275

m/z

275

m/z

275

m/z

208.10

OCONHCH3

CH3

500e3

N

500e3

175

CHC2H5 H3C

CH3 CH3

250e3

250e3

125

150

175

240.05

240.30

168.30

0e3 100

200

Int. 4. Bendiocarb M.W. 223 200e3

225

250

275

m/z

224.05

200e3

183.90 167.30 184.65

125

150

175

200

250

226.05

OCONHCH3

175

200

H3C CH3S

CH3

100e3 226.05

225

258.20

255.65

250

168.90 275

m/z

0e3 100

125

150

175

207.95 200

Fig. 3.14.1 Structures and mass spectra of n-methylcarbamate pesticides

85

225

300e3 CH3

50e3 0e3 100

150

CH3 C

O

125

400e3

O

100e3

152.45

Int. 8. Methiocarb M.W. 225 500e3

OCONHCH3

150e3

0e3 100

225

250

3.14 Analysis of N-methylcarbamate Pesticides Using LC/MS (2) - LC/MS

3500e3

5/Carbaryl

4/Bendiocarb

2/Methomyl

1/Aldicarbsulfon

TIC 223.05 5000e3 163.00 239.15 224.05 4500e3 202.05 226.05 208.10 4000e3

3/Primicarb

Int.

7/Fenobucarb 8/Methiocarb

Fig. 3.14.3 shows the SIM chromatogram for fenobucarb at 40pg (8ppb, 5µL) and Fig. 3.14.4 shows the calibration curve (n = 5) between 40pg and 5ng. The CV values at different concentration are in the range 2% to 6%; highly precise results are obtained.

6/Ethiofencarb

Fig. 3.14.2 shows the chromatograms obtained when 5µL (5ng) of a 1ppm mixture of eight n-methylcarbamate pesticides is injected. The components that do not have clear peaks in the TIC chromatogram can be qualitatively analyzed easily by drawing mass chromatograms based on the characteristic mass numbers.

3000e3 2500e3 2000e3 1500e3 1000e3 500e3 0e3

9.0

10.0

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

min

Fig. 3.14.2 TIC and mass chromatograms for n-methylcarbamate pesticides Int.

ID#:7 Mass:208.10 Name:Fenobucarb F(×)=175642.62*×+0.00 r2=1.000000

19.400

208.10

Area

3000 800e3

2500 700e3 600e3

2000

500e3

1500 400e3

1000

300e3

No. 1 2 3 4

200e3

500 100e3

0 18.50

19.00

19.50

20.00

20.50

0e3 0.0

min

Fig. 3.14.3 SIM chromatogram of fenobucarb (40pg)

1.0

2.0

3.0

Conc. Area 0.040 8951.84 0.200 36587.88 1.000 176493.00 5.000 877969.22 4.0

Conc

Fig. 3.14.4 Calibration curve for fenobucarb

Table 3.14.1 Analytical conditions

Column Mobile Phase Gradient Program Flow Rate Column Temperature Injection Volume Probe Voltage CDL Temperature Probe Temperature Nebulizer Gas Flow Rate CDL Voltage Deflector Voltage Scan Range

: Shim-pack STR-ODS (150mmL. × 2.0mm I.D.) : A: 0.2% Acetic Acid Solution B: Acetonitrile containing 0.2% Acetic Acid : 0% B (0min) → 100% B (20min) : 0.2mL/min : 40˚C : 5µL : +4.5kV (APCI-Positive Mode) : 230˚C : 200˚C : 2.5L/min : -30V : +30V : m/z 100-400 (1.2sec/scan) 86

Residual Pesticides

3.15 Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (1) - LC/MS ■ Explanation Metribuzin, which is a triazine-group herbicide for annual weeds on potato, asparagus, and sugarcane fields, parks and roads, was included in an announcement made by the Japanese Environment Agency regarding the 67 types of chemical substances that are suspected of disrupting the endocrine system. Metribuzin has not yet been subjected to a thorough endocrinological investigation and so endocrine disruption has not been categorically established. It is, however, suspected of being an endocrine disruptor because of its reproductive toxicity and carcinogenicity.

are not registered or are invalid as pesticides in Japan. Metribuzin, however, is in active use and in order to investigate whether or not it is an endocrine disruptor, it is necessary to establish an easy analysis method and monitor metribuzin in the environment. Japan's Food Sanitation Law specifies residue threshold levels not only for metribuzin itself but also for the combined total including its metabolites, desamino (DA), diketo (DK, methylthio-based desorption oxidant), and desaminodiket (DADK). The analysis of metribuzin and its metabolites using atmospheric pressure chemical ionization (APCI) LC/MS is described here as an example. Fig. 3.15.1 shows the structures of metribuzin and its metabolites.

In the 67 types of chemical compounds suspected of being endocrine disruptors, 44 are herbicides, insecticides, or germicides. Of these 44, there are 22 that O (H3C)3C

N N

N

(H3C)3C

(H3C)3C

N

NH N

SCH3

N N

SCH3

Desaminometribuzin (DA) C8H13N3OS Exact Mass: 199.08 Mol. Wt. : 199.27

Metribuzin C8H14N4OS Exact Mass: 214.09 Mol. Wt.: 214.29

O

O

O NH2

N H

(H3C)3C

NH2

NH N

O

Diketometribuzin (DK) C7H12N4O2 Exact Mass: 184.10 Mol. Wt. : 184.20

APCI-positive mode

APCI-negative mode Int. 198

215 12.5e6

25000

Metribuzin

Metribuzin

183

10.0e6

20000

7.5e6

15000

5.0e6

10000 168 5000

2.5e6

152 0.0e6 58 50

79

94 107 100

131 146 150

218

172 187

237

200

288 305 322 340 360 300 350

250

395 400

422

451 450

55 69 0 50

469 485 m/z

Int.

92

122

213 228 243 258

141

100

150

200

280

294

250

314

300

339 352 368 350

407 396 400

430 445

464 478 496

450

m/z

Int. 200

198 700e3

DA

10.0e6

DA

600e3 500e3

7.5e6

400e3 5.0e6

300e3 200e3

2.5e6 100e3 0.0e6 57 67 50

84 95 107 100

222

131 143154 172 184 150 200

244 260 276 250

304 320 336 353 367 382 399 300 350 400

421

443 467 450

492

0e3 55 50

m/z

Int.

77

258 154 168 186 216 232 150 200 250

98 112 128 100

280 296 314 300

362 378

336 350

397 400

419

435

460 450

489 m/z

Int. 185

350e3

183

500e3

DK

300e3

DK 400e3

250e3 300e3

200e3 150e3

200e3

100e3 197 207

50e3 0e3 57 66 50

105

82

100

128 142 155 167 150

100e3 227

243 257268 280 295

200

250

317

300

341

369

391

350

404

425

400

453 469 482 497 450 m/z

Int.

0e350 50

71

113

89 100

141 153 168 150

193 207 200

228

243 250 265 281 297 250 300

322

356 368 350

405 416 435 461 400 450

484 499 m/z

Int. 274

15000

168 350e3

DADK

12500

DADK

300e3 250e3

10000

170 184

7500

200e3 150e3

393

216

5000

229

100e3

197 2500 65 0 50

91

239

128 151 104 117 139

100

150

266 299

200

250

300

313

369 385

408 432 428

340 355 350

400

50e3

445 459 474 450

500 m/z

0e350 65 50

228 84 97 111 100

131

153 150

183 200 200

250 266 283 250

300

318 332 348 363 378 392 350 400

424 441 456 474 489 450 m/z

Fig. 3.15.2 Positive and negative APCI mass spectra of metribuzin and its metabolites

87

O

Desaminodiketmetoribuzin (DADK) C7H11N3O2 Exact Mass: 169.09 Mol. Wt. : 169.18

Fig. 3.15.1 Structures of metribuzin and its metabolites

Int.

N H

3.15 Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (2) - LC/MS In positive APCI mass spectra for metribuzin, DA, and DK, protonated molecules can be observed as standard peaks. The ion intensity is low for DADK protonated molecules (m/z 170) and the m/z 274 (M-H + 2Na + AcOH)+ ion is observed instead. In negative APCI mass _ spectra, the (M-H) molecular ion type can be observed as standard peaks for DA, DK, and DADK; metribuzin itself is observed as fragment ions m/z 199, 180, and 169, but the m/z 213 molecular ion type can hardly be observed. These results are thought to reflect the differences in the proton affinity of the compounds. In order to analyze metribuzin and its metabolites, however,

the analysis of positive and negative ions is required. Fig. 1.19.3 shows the results of analyzing metribuzin and its metabolites using positive and negative ions. DK and DADK were detected with negative ions m/z 183 and 168, and metribuzin and DA were detected with positive ions m/z 215 and 200. The metribuzin, DA, DK, and DADK calibration curves (3.2 - 2,000 ppb, n = 5) showed good linearity at Y = 38312 X + 48246 (r2 = 0.9998), Y = 3146 X + 21011 (r2 = 0.9999), Y = 1527 X + 3320 (r2 = 0.9999), and Y = 1596 X + 1764 (r 2 = 0.9999) respectively, which means that highly accurate quantitative analysis is possible.

2000 ppb

3.2 ppb

Int.

Int.

250e3

8500

DK (m/z 183.2)

DK (m/z 183.2)

8250 200e3 8000 150e3 7750 100e3 7500 50e3 7250 0e3 2.5

5.0

7.5

10.0

12.5

2.5

min

Int.

5.0

7.5

10.0

12.5

min

7.5

10.0

12.5

min

5.0

7.5

10.0

12.5

min

5.0

7.5

10.0

12.5

min

Int.

250e3

DADK (m/z 168.2)

8500

200e3

DADK (m/z 168.2)

8250

150e3

8000

100e3 7750 50e3 7500

0e3 2.5

5.0

7.5

10.0

12.5

2.5

min

Int. 750e3

5.0

Int.

Metribuzin (m/z 215.2)

12000

Metribuzin (m/z 215.2)

11000

500e3

10000 250e3

9000 8000

0e3 2.5

5.0

7.5

10.0

12.5

2.5

min

Int.

Int. 12000

DA (m/z 200.2)

DA (m/z 200.2)

500e3

11000

10000 250e3 9000

8000

0e3 2.5

5.0

7.5

10.0

12.5

min

2.5

Fig. 3.15.3 Positive and negative SIM chromatograms of metribuzin and its metabolites (2,000ppb and 3.2ppb) Table 3.15.1 Analytical conditions

Column Mobile Phase Gradient Program Flow Rate Column Temperature Injection Volume Probe Voltage CDL Temperature Probe Temperature Nebulizer Gas Flow Rate CDL Voltage Deflector Voltage Scan Range

: Insertsil ODS-2 (150mmL. × 2.1mm I.D.) : A: 0.2% Acetic Acid Solution B: Methanol containing 0.2% Acetic Acid : 30% B (0min) → 90% B (13-15min) : 0.2mL/min : 40˚C : 50µL : +4.5kV (APCI-Positive Mode), -3.0kV (APCI-Negative Mode) : 230˚C : 400˚C : 2.5L/min : -30V (Positive), +30V (Negative) : +30V (Positive), -20V (Negative) : m/z 215.2, 200.2 (Positive), m/z 183.2, 168.2 (Negative) 88

Residual Pesticides

3.16 Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (1) - LC/MS ■ Explanation Fluazifop, quizalofop-butyl, and other phenoxypropionicacid herbicides are used widely throughout the world because they have strong herbicidal effects at low doses. The active substance is carboxylic acid-based and inhibits the biosynthesis of fatty acids by acetyl-CoA carboxylase inhibition. In Japan, the residue of these herbicides is an issue of concern and official testing methods for quizalofop-ethyl, cyhalofop-butyl, and fluazifop have been established. The total amount of quizalofop is measured using HPLC or LC/MS after hydrolysis of quizalofop-ethyl, and the total amount of fluazifop is measured by performing GC or GC/MS on the esters formed from butyl esterification after hydrolysis.

Int.

The batch analysis of fluazifop, fluazifop-butyl, quizalofop, and quizalofop-ethyl using electrospray ionization (ESI) is described here. The ESI method effectively ionizes carboxylic acid-based herbicides (fluazifop and quizalofop) with negative ions and the ester-based herbicides (fluazifop-butyl and quizalofopethyl) with positive ions. Fig. 3.16.1 shows the mass spectra of these compounds. The deprotonated molecules _ (M-H) of the carboxylic acid-based herbicides in negative ion mode and the protonated molecules (M+H)+ of the ester-based agricultural chemicals in positive ion mode can be confirmed.

326

15000

F3C

O

N

12500

CH3

O

10000

Fluazifop C15H12F3NO4 Exact Mass : 327.07 Mol. Wt. : 327.26

7500

ESI Negative

COOH

254

5000 588

2500 0

56

50

100

198

156

102 117 133

78

272

179

241

217

150

200

250

295

300

375

350

487

438 442

412 399 415

344 332

400

468

450

502

569

535

500

550

600

m/z

Int. 384

400e3 F3C

O

N

ESI Positive

425

COOC4H9 CH3

300e3

O

200e3

Fluazifop-butyl C19H20F3NO4 Exact Mass : 383.13 Mol. Wt. : 383.36

100e3 106

60

0e3 50

126

100

147

224

150

369 386 402

279

200

250

300

350

429

400

450

500

550

m/z

Int. Cl

75000

N

N

83

ESI Negative

373

COOH CH3

O

Quizalofop C17H13ClN2O4 Exact Mass : 344.06 Mol. Wt. : 344.75

50000

25000

O

391

60 94

114

0 50

190 209

146 163

74

100

150

259 279 262

238 226

200

250

306 320

300

414 415

349

428

350

400

455 470

450

508

Int. N

Cl

7500

N

O

O

345

546 467 476

235 59 67

89 105 123

100

151 170 185

150

200

200

320 220

254

250

364

293

300

350

403 388

400

428

503

525

496 536 555

444

450

Fig. 3.16.1 ESI mass spectra of phenoxypropionic-acid herbicides

89

m/z

381

271

2500 0

592

CH3

Quizalofop-ethyl C19H17ClN2O4 Exact Mass : 372.09 Mol. Wt. : 372.80

5000

559

550

ESI Positive

343

COOC2H5

537

500

500

550

584

m/z

3.16 Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (2) - LC/MS When using a reversed-phase column, the retention time of ester-based herbicides is longer than that of carboxylic acid-based herbicides. Therefore, it is possible to analyze both carboxylic acid-based and ester-based herbicides at the same time by first selecting negative ion detection, and then, after the elution of carboxylic acid-based

herbicides is complete, switching to positive ion detection (Fig. 3.16.2). Good calibration curves were produced for each substance at concentrations in the range 0.8ppb to 500ppb. Fig. 3.16.3 shows the calibration curve and SIM chromatogram at 0.8ppb for fluazifop.

Int. 326.00(4.00) 343.00(5.00)

384.00(1.00) 373.00(2.00)

ESI Negative (0-18.5min)

ESI Positive (18.5-30min)

450e3 400e3 350e3 300e3 250e3 200e3

Fluazifop

150e3

Fluazifop-butyl

100e3 50e3

each 500 ppb, 5 µL inj.

Quizalofop Quizalofop-ethyl

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

min

Fig. 3.16.2 ESI mass chromatograms of phenoxypropionic-acid herbicides Int.

Area

326.00(1.00) 750e3 3000

Fluazifop

500e3

2750 250e3 0.8ppb ~ 500ppb (n = 5) r2 = 0.99993 Y = (1786.70031)X + (16957.44868)

0.8 ppb 2500

0e3 0

100

200

300

400

Conc

15.0

min

Fig. 3.16.3 Calibration curve and SIM chromatogram of fluazifop Table 3.16.1 Analytical conditions

Column Mobile Phase Gradient Program Flow Rate Column Temperature Injection Volume Probe Voltage CDL Temperature Block Heater Temperature Nebulizer Gas Flow Rate Q-array DC Voltage Q-array RF Voltage Scan Range SIM

: Shim-pack VP-ODS (150mmL. × 2.0mm I.D.) : A: 0.1% Formic Acid Solution B: Acetonitrile containing 0.1% Formic Acid : 20% B (0min) → 90% B (20-30min) : 0.2mL/min : 40˚C : 5µL : -3.5kV (ESI- Negative Mode), +4.5kV (ESI-Positive Mode) : 200˚C : 200˚C : 4.5L/min : -30V, 10V : 150 : m/z 50-600 (1.0sec/scan) : m/z 326, 343, 384, 373 (0.5sec/ch) 90

4. Aromas and Odors 4.1 Aromatic Components of Alcohols - GC ■ Explanation The headspace method enables analysis of volatile components in solids and liquids without complicated pretreatment. The following are the advantages of the headspace GC. 1) Components with low boiling points can be analyzed at high sensitivity. 2) Induction of components with high boiling points into GC can be prevented, reducing the analysis time. 3) Contamination of GC injection port and column is minimized because non-volatile components are not inducted into the GC. Here, several analysis examples for volatile components in sake and whisky will be introduced.

■ Pretreatment Shop-sold sake and whisky were sealed in 5mL vials and kept at 100˚C for 60 min. ■ Analytical Conditions : GC-14BPFsc + HSS-2B Instrument : CBP20 (25m × 0.32mmI.D. df=0.5µm) Column Column Temp. : 50˚C(5min)-10˚C/min-200˚C : 230˚C Inj. Temp. : 230˚C(FID) Det. Temp. : He(1.35mL/min) Carrier Gas Injection Method : Split(1:16) Injection Volume : 0.4mL 4

4

4

■ Peaks 1 2 3 4 5 6 7 8 9 10 11

8 3

3 8 6 3

Acetaldehyde Acetone Ethyl Acetate Ethanol n-Propyl Alcohol Isobutyl Alcohol Isoamyl Acetate Isoamyl Alcohol Ethyl n-Caproate Ethyl n-Caprylate Ethyl n-Caprate

8 1

6

1

6 1

5

5 5 7 9(0.032ppm)

7

0

4

8

12

16(min)

Fig. 4.1.1 Analysis of brewage

91

7

10(0.637ppm)

0

4

8

12

16 (min)

Fig. 4.1.2 Analysis of shochu

11

2

10

2

9(1.477ppm) 10(7.353ppm)

9(0.587ppm)

0

4

8

12

16 (min)

Fig. 4.1.3 Analysis of whisky

4.2 Aromatic Components of Tea - GC ■ Explanation Volatile components in solid samples like tea can be easily analyzed using the headspace method. With this method, sample extraction by steam distillation is not required, as the sample is simply sealed to be analyzed.

■ Analytical Conditions : GC-14BPFsc+HSS-2B Instrument : CBP20 (25m × 0.32mmI.D. df=0.5µm) Column Column Temp. : 50˚C(5min)-10˚C/min-200˚C : 230˚C Inj. Temp. : 230˚C(FID) Det. Temp. : He(1.4mL/min) Carrier Gas Injection Method : Split(1:15) Injection Volume : 0.4mL

■ Pretreatment 3g of tea leaf in 10mL of distilled water was kept at 100˚C for 60 min.

■ Peaks 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1

1

8

2

9 13

5 14

45

Acetaldehyde Hexanal Methyl n-Caproate Isoamyl Alcohol Ethyl n-Caproate Hexyl Acetate cis-3-Hexenyl Acetate or Hexanol Methyl n-Caprylate cis-3-Hexenol trans-2-Hexenol Ethyl n-Caprylate Ethyl Acetoacetate Linalool Benzaldehyde Ethyl n-Caprate Geraniol

9 13 14 15

3 5 4

13 9 14 8 11

8

12

16 0

4

8

12

16 (min)

Fig. 4.2.1 Analysis of refined green tea

0

4

8

12

16 (min)

Fig. 4.2.2 Analysis of coarse green tea

0

4

16(min)

Fig. 4.2.3 Analysis of black tea

92

Aromas and Odors 4.3 Essential Oil (Headspace Analysis) - GC ■ Explanation This is an analysis example for essential oil used as flavors for food products.

■ Analytical Conditions : GC-14BPF+HSS-2B Instrument : CBP1 (25m × 0.53mmI.D. df=3.0µm) Column Column Temp. : 50˚C(15min)-5˚C/min-200˚C : 230˚C Inj. Temp. : 230˚C(FID) Det. Temp. : He(10.5mL/min) Carrier Gas Injection Method : Direct Injection Injection Volume : 0.8mL

■ Pretreatment Essential oils were sealed in 5µL vials and kept at 40˚C for 30 min.

0

10

20

30

40 (min)

Fig. 4.3.1 Analysis of orange oil

93

0

10

20

30

40 (min)

Fig. 4.3.2 Analysis of lavender oil

0

10

20

30

40 (min)

Fig. 4.3.3 Analysis of spearmint oil

4.4 Essential Oil (Direct Analysis) - GC ■ Explanation Here, direct GC analysis examples of peppermint oil and spearmint oil used as flavorings are introduced.

■ Analytical Conditions : ULBON HR-20M Column (50m × 0.25mmI.D. df=0.25µm) Column Temp. : 60˚C-3˚C/min-220˚C : 250˚C Inj. Temp. : 250˚C(FID) Det. Temp. : He(1.4mL/min) Carrier Gas Injection Method : Split(1:15) Injection Volume : 0.2µL

■ Peaks

Fig. 4.4.1 Analysis of peppermint oil

5

6

7

2. 3. 4. 5. 6. 7.

48

44

40

36

32

28

24

20

16

1,8-Cineole Menthone Terpinene-4-ol β-Caryophyllene Dihydrocarvone Carvone

56

4

Limonene 1,8-Cineole Menthone Terpinene-4-ol β-Caryophyllene Dihydrocarvone ■ Peaks 1. Limonene Carvone

56

52

48

44

40

36

32

28

24

20

16

12

8

4

0

3

1. 2. 3. 4. 5. 6. 7.

L-Menthone Menthoturan D-Isomenthone Neo-Menthol Terpinene-4-ol β-Caryophyllene Menthol

8

4. 5. 6. 7. 8. 9. 10.

1 2

12

7 8 9 10

4

3 4 5 6

2

Limonene 1,8-Cineole trans-Sabinenehydrate L-Menthone Menthoturan D-Isomenthone Neo-Menthol ■ Peaks Terpinene-4-ol 1. Limonene β-Caryophyllene 2. 1,8-Cineole Menthol3. trans-Sabinenehydrate

0

1

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

52

■ Peaks

Fig. 4.4.2 Analysis of spearmint oil

94

Aromas and Odors 4.5 Diketones - GC ■ Explanation This introduces analysis examples using a headspace system with ECD for diketones contained in brewed products such as sake.

■ Analytical Conditions : GC-14APE+HSS-2B Instrument : DB-WAX Column (60m × 0.25mmI.D. df=0.25µm) Column Temp. : 40˚C : 200˚C Inj. Temp. : 200˚C (ECD,Current 0.5nA) Det. Temp. : He(1.7mL/min) Carrier Gas Injection Method : Split(1:15)

■ Pretreatment 5mL of solution samples or 3g of solid samples were sealed in vials and kept at 60˚C for 40 min.

1

2

(519ppb)

(333ppb)

Injection Volume : 0.4mL

■ Peaks 1 Diacetyl 2 2,3-Pentanedione 3 2,3-Hexanedione (internal standard)

(217ppb)

1

1

(55ppb)

(43ppb) (7ppb)

2

3

2

3

0

10

20 (min)

Fig. 4.5.1 Analysis of strong soy sauce

95

0

10

20 (min)

Fig. 4.5.2 Analysis of Japanese sake

0

10

3

20 (min)

Fig. 4.5.3 Analysis of shochu

4.6 Fruit Fragrances - GC ■ Explanation This introduces several analysis examples using a headspace system for various fruits fragrances. The results show how lower alcohol and esters form distinctive fruit fragrances.

■ Analytical Conditions : GC-14BPFsc+HSS-2B Instrument : DB-WAX Column (60m × 0.25mmI.D. df=0.25µm) Column Temp. : 50˚C(5min)-10˚C/min-200˚C : 230˚C Inj. Temp. : 230˚C(FID) Det. Temp. : He(1.1mL/min) Carrier Gas

■ Pretreatment 10g of fruit samples were sealed in vials and kept at 60˚C for 30 min.

Injection Method : Split(1:18) Injection Volume : 0.8mL

■ Peaks

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

34 6

4 6

12 8

Acetaldehyde Acetone Methyl Acetate Ethyl Acetate Methanol Ethanol Methyl Butyrate Ethyl Butyrate 2-Methyl-3-Butene-2-ol or n-propyl Alcohol Hexanal Isobutyl Alcohol Isoamy Acetate n-Amyl Acetate Methyl n-Caproate Isoamyl Alcohol or trans-2-Hexenal Ethyl n-Caproate Hexyl Acetate cis-3-Hexenyl Acetate Hexanol Methly n-Caprylate or cis-3-Hexenol

4 6

12

3 8 17

5 15

1 1

17

14

5 2 16

11

2 7

11

9

1314

12 11

15

17 19

2

18

3 5

19

20

13

0

4

8

12

16

20

24 (min)

Fig. 4.6.1 Analysis of melon

0

4

8

12

16

20

24 (min)

Fig. 4.6.2 Analysis of strawberry

0

4

8

12

16

20

24 (min)

Fig. 4.6.3 Analysis of banana

96

Aromas and Odors 4.7 Vegetable Fragrances - GC ■ Explanation This introduces several analysis examples using a headspace system for many vegetable fragrances. The results show how terpene compounds are a main component in providing vegetables with earthy, fresh fragrances.

■ Analytical Conditions : GC-14BPF+HSS-2B Instrument : CBP1 (25m × 0.53mmI.D. df=3.00µm) Column Column Temp. : 50˚C(15min)-5˚C/min-200˚C : 230˚C Inj. Temp. : 230˚C(FID) Det. Temp. : He(10.5mL/min) Carrier Gas Injection Method : Direct Injection Volume : 0.8mL

■ Pretreatment Suitable amount of vegetable samples were sealed in vials and kept at 40˚C for 30 min.

■ Peaks 1 2 3 4

1 2 4

1 2 34

1 34

α-Pinene β-Pinene Myrcene Cineole/ Limonene 2

3

0

10

20

30

40 (min)

Fig. 4.7.1 Analysis of perilla (1.5g)

97

0

10

20

30

40 (min)

Fig. 4.7.2 Analysis of parsley (2g)

0

10

20

30

40 (min)

Fig. 4.7.3 Analysis of ginger (10g)

4.8 Flavor of Rice - GC ■ Explanation In order to analyze the flavor components of rice, the chromatograms of new rice and old rice (more than 3 years old) were compared. 1g of polished rice and 1mL of water were put into a vial and, after heating at 100ºC for 30min, the head space was analyzed. When quantitative analysis was performed for hexanal, which is said to be an off-flavor constituent of rice, the results for new rice and old rice differed significantly at 0.20ppm and 0.95ppm respectively.

■ Analytical Conditions : GC-17AAFW ver.3 + HSS-4A Instrument : DB-WAX (30m × 0.53mm I.D. df = 1µm) Column : 50˚C(5min)-10˚C/min-200˚C Column Temp. Injection method : Split 250˚C : WFID 250˚C Detector : He, 30cm/sec Carrier Gas : 1:5 Split Ratio Sample Heating Conditions : 100˚C, 60min

Note:Here, "new rice" refers to rice that was harvested in autumn and analyzed the following April. The brands of new rice and old rice used as samples here were different.

mV

mV

8

8

Hexanal (0.95ppm) 6

Hexanal (0.20ppm)

6

4

4

2

2

0 0

5

10

Fig. 4.8.1 Analysis of new rice

15 min

0 0

5

10

15 min

Fig. 4.8.2 Analysis of old rice

98

Aromas and Odors 4.9 Flavoring Agent for Food Product - GC ■ Explanation This introduces several analysis examples using a headspace system for flavoring agents that give sweet fragrances to cookies, etc. Examples of sweets are also given.

■ Pretreatment Suitable amount of standard flavoring agent and sweets were sealed in vials and kept at 130˚C for 40 min.

■ Analytical Conditions : GC-14BPF+HSS-2B Instrument : CBP1 (25m × 0.53mmI.D. df=3.0µm) Column Column Temp. : 90˚C-6˚C/min-230˚C : 300˚C Inj. Temp. : 300˚C(FID) Det. Temp. : He(4.3mL/min) Carrier Gas Injection Method : Direct Injection Volume : 0.8mL

■ Peaks 1 Ethyl n-Butyrate 2 Isoamyl Acetate 3 Ethyl Isovalerate 4 Benzaldehyde 5 Isoamyl n-Butyrate 6 Allyl n-Caproate 7 Ethyl n-Heptanoate 8 Isoamyl Isovalerate 9 R-Menthol 10 Ethyl n-Caprylate 11 γ-Nonalactone 12 Vanillin 13 Ethyl n-Caprate

2

5 68

1 3

7

13

10 9

12

3 1

1 4

12

12 11

0

4

8

12

16

20(min)

Fig. 4.9.1 Analysis of standard flavoring agent

99

0

4

8

12

16

20(min)

Fig. 4.9.2 Analysis of cookie (6.4g)

0

4

8

12

16

20(min)

Fig. 4.9.3 Analysis of chocolate (with nut cream) (3.9g)

4.10 Analysis of Fishy Smell in Water (1) - GC/MS ■ Explanation Fishy smells are attributed to unsaturated aldehyde in uroglene Americane and has become a problem in drinking water supplies along with musty smell ever since vast outbreaks of it occurred in Lake Biwa in 1995. The 4 compounds of unsaturated aldehyde with carbon number 7 or 10 trans, cis-2,4-heptadienal and trans, cis2,4-decadienal are the cause of this fishy smell. The purge & trap method is more effective than the headspace method to analyze these substances because of the low vapor pressure. The threshold values of these substances as odors are several 100ppb, and the lower detection limit of this method is several ppb.

■ Analytical Conditions : GCMS-QP5000 Instrument : DB-1701 (30m × 0.32mmI.D. df=1.0µm) Column Column Temp. Inj.Temp. I/F Temp. Carrier Gas -P&TInstrument Sample Size Trap Tube Purge Dry Purge Thermal Desorption

2,4-Heptadienal 9878391

2-1

1-1

: Tekmar 3000J : 5mL(35˚C) : Tenax GR : 11min : 3min : 225˚C, 8min

1-1, 1-2

*** CLASS-5000 *** report No. : 5 Data file : 970714.D11 Sample : 10ppb 1 Method file : AO.MET

TIC

: 40˚C(8min)-20˚C/min-200˚C(5min) : 230˚C : 230˚C : He(20kPa)

2-2 0 MW : 110

1-2

2-1, 2-2 2,4-decadienal

0

10

11

12

13

14

15

16

17

18

19

MW : 152

Fig. 4.10.1 TIC chromatogram of fishy smell components

100

Aromas and Odors 4.10 Analysis of Fishy Smell in Water (2) - GC/MS

81

81 41

39 67

55

53

67

110

95

95 60

40

80

100

120

140

160

180

200

40

60

80

Peak 1-1 mass spectrum

119 100

152

120

140

160

180

200

Peak 2-1 mass spectrum

Fig. 4.10.2 Mass spectra

Ion set No.: 1

Ion set No.: 1

2900782

2-1

466420

2-1 2-2

2-2

1-1 1-1 1-2 1-2

81.00 110.00 ✽ 3.00 152.00 ✽ 3.00 10

11

12

13

14

15

16

17

18

10

19

11

Fig. 4.10.3 SIM chromatogram of 100ppt

13

14

15

16

17

18

19

Fig. 4.10.4 SIM chromatogram of 1ppb

ID # 2 Mass No.: 152.00 Component: 2,4-decadienal Area = 142136✽Conc. + - 1729.78 Contribution rate: 0.999922

ID # 1 Mass No.: 110.00 Component: 2,4-heptadienal Area = 171214✽Conc. + 3258.56 Contribution rate: 0.999998 Area 106

Area 106

2.0

1 2 3

Conc. (ppb) 0.100 1.000 10.000

Area 19007 175983 1715263

1.0

2.0

1 2 3

Conc. (ppb) 0.100 1.000 10.000

Area 19016 133221 1420285

1.0

0

0.5 Conc

1.0 101

Fig. 4.10.5 Calibration curve for 2,4-heptadienal

101

12

81.00 110.00 ✽ 3.00 152.00 ✽ 3.00

0

0.5 Conc

1.0 101

Fig. 4.10.6 Calibration curve for 2,4-decadienal

4.11 Analysis of Alcohols (1) - GC/MS ■ Explanation There are two headspace methods: static headspace method and dynamic headspace method. Generally, the term headspace method refers to the static headspace method. The dynamic headspace method refers to a method where purge gas is continuously fed into the sample to purge out volatile elements, and then the volatile elements are concentrated onto the trapping agent. After concentration, target components are desorped and analyzed by GC/MS. This method enables microanalysis because it involves the concentration of the sample. Here, the difference between the static and dynamic headspace methods will be shown using Japanese sake and wine. A Chrompack CP4010 and a Tenax trapping set were used in the dynamic headspace analysis. Sensitivity was clearly higher in dynamic headspace analysis.

■ Analytical Conditions : GCMS-QP5000 Instrument : DB-1701 (30m × 0.32mmI.D. df=1.0µm) Column Column Temp. I/F Temp. Carrier Gas -HSInstrument Sample Size Sample Temp. Thermostat Injection -TCTInstrument Sample Size Purge Trap Tube Precool Thermal Desorption

: 40˚C(5min)-5˚C/min-250˚C(5min) : 250˚C : He(35kPa) : HSS-4A : 10mL : 60˚C : 30min : 0.8mL : CP4010+Tenax Trap Set : 20mL(Room Temp.) : 20mL/min(5min) : TenaxGR(0.1g) : -150˚C(3min) : 250˚C(5min)

Trap tube

Carrier in He Gas

Trap tube

Activated carbon tube Split vent Liquid nitrogen Back flush vent

Water bath Purge/desorption vent Detector

Fig. 4.11.1 CP4010 flow line diagram

Lab jack

Fig. 4.11.2 Schematic diagram of Tenax trapping set

102

Aromas and Odors 4.11 Analysis of Alcohols (2) - GC/MS TIC

7500000

1

■ Peaks 1 2 3 4 5 6 7 8

2

9

3 5

Ethanol Ethyl Acetate Isobutyl Alcohol 1,1-Diethoxyethane Ethyl Propanoate Propyl Acetate Ethyl Isobutyrate Isobutyl Acetate

13

10

15

20

25

30

Fig. 4.11.3 TIC chromatogram of Japanese sake (HS method)

TIC

1

9

2

40000000

13

3

14 15

4 5 6

14

10

15 TIC 68.00 138.00 89.00 99.00

8 7 5

17

10

15

18

19

20

3.00 3.00

20

25

30

Fig. 4.11.4 TIC chromatogram of Japanese sake (TCT method)

TIC

7500000 1

2

■ Peaks 9 10 11 12 13 14 15 16

9

3 7

10

13 15

5

10

15

1-Pentanol Ethyl Butanoate Ethyl 2-methylbutanoate Ethyl Isovalerate Isopentyl Acetate Limonene Ethyl Caproate Ethyl Caprylate 16

20

25

30

Fig. 4.11.5 TIC chromatogram of wine (HS method)

TIC

1

2

13

9

3

7

40000000

14

10

4 5 6

16

15

11 12 8

5

103

10

15

20

Fig. 4.11.6 TIC chromatogram of wine (TCT method)

25

30

4.12 Analysis of Strawberry Fragrances - GC/MS ■ Explanation Strawberry fragrance components consist of fatty acid methyl esters from C2 to C6. Old and new varieties of marketed strawberries were compared and the correlation between type and fragrance studied. Normally, steam distillation or the headspace method is used for pretreatment of fragrance components; however, sometimes problems occur with the heating process. In the case of strawberries, heat destroys cells and release large amounts of special esters that are sometimes mistaken for fragrance components. Here, the Chrompack CP4010 + GCMS system (TCT + GCMS) was used to dry-air purge the sample without heating to enable optimum measurement of strawberry fragrances.

TIC

■ Analytical Conditions : GCMS-QP5000 Instrument : DB-624 (60m × 0.25mmI.D. df=1.4µm) Column Column Temp. : 40˚C(5min)-5˚C/min-230˚C(5min) : 230˚C I/F Temp. : He(100kPa) Carrier Gas -TCT: CP4010(TCT Mode) Instrument Sample Amount : 10g (Room Temperature) : Tenax TA Trap Tube : -150˚C, 5min Pre Cool : 50˚C, 1min Pre Flush Thermal Desorption : 250˚C, 10min, 20mL/min

9

Old brand strawberry fragrances ■ Peaks 1 2 3 4 5 6

Ethanol Acetone Acetic acid, Methyl ester Ethanoic acid, Methyl ester Butanoic acid, Methyl ester Butanoic acid, Ethyl ester

7 8 9 10 11

Hexanal 2-Hexenal Hexanoic acid, Methyl ester Hexanoic acid, Ethyl ester C6H11OH

5

3

4

6

8

10

14

16

18

20

11 10

8

67 12

60000000

22

24

26

60000000

TIC

New brand strawberry fragrances

9

5

2 1 4

6

8

10

8

67

3

11

4 10

12

14

16

18

20

22

24

26

Fig. 4.12.1 TIC chromatogram of strawberry fragrance components (upper: old brand, lower: new brand)

104

Aromas and Odors 4.13 Analysis of Beverage Odors (1) - GC/MS ■ Explanation 2,4,6-trichloroanisole (2,4,6-TCA), a cause of musty odor, is contained in wood and paper-manufactured packing materials, and its transfer to food products and drinking water may cause problem. The perceptual threshold value of TCA in water is extremely low at the ppt level. Conventionally, 2,4,6-TCA was analyzed by solvent extraction or steam distillation method, but these methods require a lot of time and are extremely complicated; moreover, the poor collection rate would make ppt-level measurement difficult. Here, measurement was conducted using a combination of the Chrompack CP4010 and Tenax trapping set. In this method, the sample is purged to collect the target components in the trap tube. The trap tube is heated by the TCT mode of the CP4010, and the desorped components are analyzed by GC/MS. This system setup is an offline one, so the Tenax unit is easy to clean and there is no sample memory.

■ Analytical Conditions : GCMS-QP5000 Instrument : DB-1701 (30m × 0.32mmI.D. df=1.0µm) Column Column Temp. : 50˚C(2min)-30˚C/min-140˚C -10˚C/min-220˚C : 250˚C I/F Temp. : He(50kPa) Carrier Gas -TCTInstrument Sample Size Trap Tube Purge Thermal Desorption

: GP4010(TCT Mode) : 25mL(50˚C) : Tenax GR : 50˚C, 15min, 100mL/min : 250˚C, 5min

Trap tube

Carrier in

He Gas

Trap tube

Activated carbon tube

Split vent Liquid nitrogen Back flush vent

Water bath Lab jack

Fig. 4.13.1 Schematic diagram of Tenax trapping set

105

Purge/desorption vent Detector

Fig. 4.13.2 TCT main unit flow line diagram

4.13 Analysis of Beverage Odors (2) - GC/MS

Sample

: 1ng/L SIM

ID # 1 Mass No.: 195.00 Component: 2,4,6-TCA Area = 2424.61✽Conc. contribution rate = 0.999816 Conc. (ng/L) 1.000 5.000 10.000 20.000 50.000 100.000

Fig. 4.13.3 SIM chromatogram (1ng/L)

Area 2637 11856 25010 51111 122666 241154

Fig. 4.13.4 TCA calibration curve (1 to 100ng/L)

Sample: 3ng/L black tea

Sample: 4.5ng/L Japanese sake ID Mass No. Type Component

:1 : 195.00 : Target : 2,4,6-TCA

ID Mass No. Type Component

:1 : 195.00 : Target : 2,4,6-TCA

Retention time : 10.097 Area : 11079 Conc. : 4.550ng/L

Retention time : 10.093 Area : 7859 Conc. : 3.020ng/L

Mass No. 1 210.00

Mass No. 1 210.00

Area Intensity ratio 7624 69

Fig. 4.13.5 Analysis of Japanese sake (4.5ng/L added)

Area Intensity ratio 5544 71

Fig. 4.13.6 Analysis of black tea (3ng/L added)

106

Aromas and Odors 4.14 Analysis of Fragrant Material (1) - GC/MS ■ Explanation Many fragrant components are contained in food products. These components are compounds of alcohols, esters, aldehydes, ketones, terpenes and others. The amount and mixture ratio of these components determine the aroma, and any aroma can be artificially synthesized by mixing these components. Here, some 100-aroma components were mixed together and analyzed by GC/MS.

■ Analytical Conditions : GCMS-QP5000 Instrument : DB-WAX (60m × 0.25mmI.D. df=0.25µm) Column Column Temp. Inj.Temp. I/F Temp. Carrier Gas Injection

: 70˚C(5min)-3˚C/min-210˚C(30/min) : 250˚C : 230˚C : He(180kPa) : Split(100:1)

TIC 5

9000000 53

27

2

63

35 26

49

25 19

14 8 10 9

62

81

59

45

99

46

36

91

80 29

16 15

42 18

69

44

58

32 33

23

39

52 56 57 50

3 4

51

34

24

38

7 13 17

10

71 74

37 30

70 73

100

92

95

98

101

96

105 104

107

106

78 79 77

54

20

30

40

50

Fig. 4.14.1 TIC chromatogram of fragrant components

107

102 103

83

47

41

93

86

55 40

22 20

65

68

97

87 89 88 90

76 75

61 64 67 72

48

31 11

66 60

43

6 21

94

82 85

28

12

84

60

70

4.14 Analysis of Fragrant Material (2) - GC/MS Table 4.14.1 Component names 1 Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

Alcohol

Ester

Aldehyde

Ketone

Terepene

Others

Ethyl acetate Diethyl acetal Ethyl alcohol Ethyl propionate i-Butyl acetate Chloroform Ethyl n-butyrate Ethyl 2-methyl butyrate Ethyl i-valerate n-Butyl acetate n-Hexanal i-Butyl alcohol n-Amyl acetate n-Butyl alcohol Methyl i-amyl ketone n-Amyl propionate Limonene 2-Methyl butyl alcohol n-Amyl furmate c-2-Hexenal Ethyl caproate n-Amyl alcohol i-Amyl n-butyrate n-Hexyl acetate Methyl n-hexyl ketone i-Amyl i-valerate

Ethyl lactate n-Hexanol Ethyl n-hexyl ketone Allyl caproate Methyl n-heptyl ketone t-3-Hexenol Ethyl caprylate Acetic acid Furfural Methyl n-octyl ketone Tetrahydro furfuryl alcohol Benzaldehyde Ethyl nonanoate Linalool Diethyl malonate Methyl n-nonyl ketone Ethytl levulinate Methyl benzoate Ehtyl caprate l-Menthol

108

Aromas and Odors 4.14 Analysis of Fragrant Material (3) - GC/MS Table 4.14.2 Component names 2 Compound 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107

109

Furfuryl alcohol Ethyl benzoate Phenyl diethyl acetate Methyl n-decyl ketone Benzyl acetate Methyl phenyl acetate Dimethyl benzyl carbinyl acetate

Allyl caprate Ethyl phenyl acetate Allyl β-cyclohexyl propionate

Phenethyl acetate Anethol Caproic acid Ethyl laurate t-2-Decenal Benzyl n-butyrate Benzyl alcohol Phenetyl propionate i-Butyl phenyl acetate Dimethyl benzyl carbinylbutyrate

Phenyl ethyl alcohol

Phenyl ethyl propionate Phenethyl i-valerate Methyl n-tridecyl ketone Anisaldehyde γ-Nonalactone Ethyl myristate Triacetine Methyl cinnamate Benzylidene acetone Ethyl cinnamate γ-Decalactone Eugenol Phenethyl caproate δ-Decalactone Heliotropine γ-Undecalactone Anisalcohol Cinnamy alcohol Diethyl sebacate γ-Dodecalactone Phenethyl octanoate δ-Dodecalactone TEC Benzophenone Ethyl vanillin vanilline Benzyl benzoate

Alcohol

Ester

Aldehyde

Ketone

Terepene

Others

Na Mg

5. Inorganic Metals

Ca

5.1 Analysis of Inorganic Ions in Milk (1) - LC ■ Explanation Ion chromatography is the best method for analyzing inorganic ions in food products. In particular, use of a dual flow line system allows simultaneous analysis of anions and cations, which is useful in ion balance measurement. Here, an application example for analysis of inorganic ions in milk will be introduced. References Shimadzu HPLC Food Analysis Applications Data Book (C190-E047) Yagi, Funato, Ito; Analytical Chemistry, 38 (11), 655 (1989) ■ Pretreatment The sample was injected into column after deproteinization with ultrafiltration membrane.

■ Analytical Conditions ■ Anions : Shim-pack IC-A3(150mmL. × 4.6mmI.D.) Column Mobile Phase : 8.0mM p-Hydroxy Benzoic Acid 3.2mM Tris Hydroxy Aminomethane Temperature : 40˚C Flow Rate : 1.5mL/min : Conductivity Detector Detection ■ Cations : Shim-pack IC-C3(100mmL. × 4.6mmI.D.) Column Mobile Phase : 3.0mM Oxalic Acid Temperature : 40˚C Flow Rate : 1.2mL/min : Conductivity Detector Detection

■ Peaks

2

1

■ Peaks

2

1.H2PO42.CI-

1.Na+

3.SO42-

3.Mg2+

2.K+ 4.Ca2+

4 1 3 3

0

5

10

15 (min)

Fig. 5.1.1 Analysis of inorganic anions

0

5

10

15 (min)

Fig. 5.1.2 Analysis of inorganic cations

110

Na Mg

Inorganic Metals

Ca

5.1 Analysis of Inorganic Ions in Milk (2) - LC

SIL-10A 1st flow line Column (1)

CDD-6A (1) LC-10AD (1)

2nd flow line Column (2)

CDD-6A (2)

FCV-12AH

Drain

DGU-12A

LC-10AD (2)

CDD-6A (2)

Fig. 5.1.3 Diagram of dual flow line system

111

CDD-6A (1)

5.2 Analysis of Pb in Milk Using Atomic Absorption Spectrophotometry - AA ■ Explanation Lead is harmful to human body and stricter regulations are being applied to lead in food and pharmaceutical products. Lead can be effectively detected by electrothermal atomization with atomic absorption. Analysis of Pb in milk generally involves the flame method or electrothermal atomization where an acid is added and the sample is thermally decomposed. However, these methods require time-consuming pretreatment. With direct analysis using electrothermal atomization, oxygen is often added during incineration to enhance the decomposition of organic matter in milk. However, the oxygen causes the deterioration of the graphite tube. Here, the use of a platform tube, instead of the graphite tube, allowed accurate measurement without the addition of oxygen or air.

■ Analytical Conditions : AA Instrument : Pb 283.3nm Wavelength Lamp Current Low (mA) : 10 Lamp Current High (mA) : 0 : 0.5 Slit Width (nm) Background Correction : BGC-D2

Table 5.2.1 Heat program

Air not added Sample No.

lamp

Ar

0.20

30

lamp

Ar

0.50

3

400

20

lamp

Ar

0.50

4

500

10

lamp

Ar

1.00

5

700

10

step

Ar

1.00

6

700

3

step

Ar

0.0H

7

2400

3

step

Ar

0.0H

8

2600

2

step

Ar

1.00

Abs

3

120

Sec

70

2

5.00

1

Repeat No. Peak height (Abs) Area (Abs-sec) BKG height (Abs) CV value (%)

0.600

Inner gas flow rate

0.400

Gas

0.200

Heat mode

0.000

Time (sec)

-0.200

Temperature (˚C)

0.00

Stage

2

1

0.0776

0.0206

0.0802

2

2

0.0721

0.0238

0.1048

5.12

Sample No. Conc. (ppb) Peak height (Abs) BKG height (Abs) CV value (%)

2

10.3523

0.0749

0.0921

5.12

Furnace quantitative measurement/calibration curve method Calibration curve SDT. NO.

Table 5.2.2 Measurement results for Pb in milk

1 2 3

CONC. (ppb) 4.0000 8.0000 16.0000

ABS. 283.3nm 0.0260 0.0520 0.1194

ABS. 0.14 3

2 1

0.00 0.0 [ABS]-X1✽[C]+K0 K0=0.0000, K1=0.0072

Measurement results

20.0 CONC.(ppb)

Added amount

Air added

10.5ppb

10.0ppb

Air not added

10.4ppb

10.0ppb

Fig. 5.2.1 Peak profile and calibration curve of Pb in milk

112

Na Mg

Inorganic Metals

Ca

5.3 Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (1) - AA ■ Explanation Lead is harmful to human body and stricter regulations are being applied to lead in food and pharmaceutical products. Lead can be effectively detected by the electrothermal atomization with atomic absorption. The 13th revision of the Japanese Pharmacopoeia requires the measurement of lead, instead of heavy metal, using the electrothermal atomization method in purity tests for refined white sugar. Here, analysis was performed in accordance with the Pharmacopoeia, with pretreatment (see Table 5.4.1) and sample preparation using an autosampler for the standard addition method. Table 5.3.1 shows the measurement parameters and Fig. 5.3.2 shows the measurement results. Lead was not detected in the analyzed white sugar, but 1ppb of lead was clearly detected in the calibration curve. It can be said that this analysis method is effective for the detection of 0.5 ppm lead in white sugar (5ppb or less in processed solution), which is specified in the standard.

Accurately load 0.050g of sample into polytetrafluoroethylene container ↓ Add 0.5mL of nitric acid to dissolve sample ↓ Seal container and heat for 5 hr at 150˚C ↓ Cool, add water to accurately make 5mL of sample solution ↓ Analyze using standard addition method for AA (electrothermal)

Fig 5.3.1 Pretreatment for Pb in refined white sugar Table 5.3.1 Measurement parameters

Lighting Conditions Element : Pb Turret No. : 1 Lamp current Low (mA) : 10 Lamp current High (mA) : 0 Wavelength (nm) : 283.3

■ Analytical Conditions : AA Instrument : Pb 283.3nm Wavelength Lamp Current Low (mA) : 10 Lamp Current High (mA) : 0 : 0.5 Slit Width (nm) Background Correction : BGC-D2

Slit width (nm) : 0.5 Lighting mode : BGC-D2

Temperature Program Final stage No. of concentration in oven : 5 Concentration frequency : 1 Temperature Time (˚C) (sec)

Heat mode

Sensitivity

Gas

Inner gas Sampling Previous stage flow rate (sec)

1

110

30

Ramp

Regular

Gas #1

0.20

Off

0

2

250

10

Ramp

Regular

Gas #1

0.20

Off

0

3

600

20

Ramp

Regular

Gas #1

1.00

Off

0

4

600

20

Step

Regular

Gas #1

1.00

Off

0

5

600

5

Step

High

Gas #1

0.00

Off

0

6

2100

3

Step

High

Gas #1

0.00

On

2

7

2600

2

Step

Regular

Gas #1

1.00

Off

0

Autosampler Mixing Conditions Adding Conc. (ppb) Blank

Sample amount

R1 R2 (Pb: 10ppb standard solution) (pure water)

Total

0µL

0µL

200µL

200µL

0

100µL

0µL

100µL

200µL

1

100µL

20µL

80µL

200µL

2

100µL

40µL

60µL

200µL

3

100µL

60µL

40µL

200µL

Pb: 10ppb standard solution and pure water containing approximately 1.1mol/L of nitric acid Inj. Vol. : 20µL

113

Time

5.3 Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (2) - AA 83.0000 88.0000 -0.100

0.100

0.200

0.0070

-0.0043

0.0000

Absorption 0.0000 ppb

REP

0.0087

-0.0045

0.0000

0.0000 ppb

0.0067 0.0068 2.5254%

-0.0024 -0.0034

0.0000 0.0000

0.0000 ppb 0.0000 ppb

REP AVG CV = sugar Type Time (min)

0.000

BLK

Absorption

BG

(ppb)

Actual Conc. sugar

83.0000 88.0000 -0.100

0.000

0.100

0.200

-0.0017

0.0000

Absorption 0.0000 ppb sugar

REP

0.0008

-0.0007

0.0000

0.0000 ppb sugar

REP 0.0003 -0.0014 AVG 0.0003 -0.0015 CV = 24.9567% sugar Type Absorption BG

0,0000 0.0000

0.0000 ppb 0.0000 ppb

Time (min)

0.0002

88.0000 -0.100

REP AVG CV = sugar Type Time (min)

0.300

0.400

Actual Conc. sugar

0.300

0.400

0.300

0.400

0.300

0.400

Unit

83.0000 0.000

RSA

0.100

0.200

Absorption 200.0000 ppb sugar

0.0308

-0.0314

1.0000

0.0300 0.0303 1.3869%

0.0001 -0.0006

1.0000 200.0000 ppb 1.0000 200.0000 ppb

Absorption

BG

(ppb)

Actual Conc. sugar

Unit

83.0000 88.0000 -0.100

RSA

REP AVG CV = sugar Type Time (min)

0.400

Unit

NSA

(ppb)

0.300

0.000

0.0589

0.0597 0.0593 1.1471% Absorption

0.100

-0.0006

-0.0004 -0.0005 BG

2.0000

(ppb)

0.000

RSA

0.0883

Actual Conc. sugar

REP AVG CV = sugar Type RES

0.0888 0.0885 0.4023% Absorption

0.100

-0.0015

-0.0027 -0.0021 BG

0.200

2.0000 400.0000 ppb 2.0000 400.0003 ppb

83.0000 88.0000 -0.100

Absorption 400.0000 ppb sugar

3.0000

Absorption 600.0000 ppb sugar

Unit

0.200

3.0000 600.0000 ppb 3.0000 600.0000 ppb (ppb) 0.0185

Actual Conc. 3.6944 ppb

Effective Calibration Curve-MSA (sugar) 0.098

0.080

Absorption

0.060

0.040

0.020 Abs=0.0293 Conc+0.000542 Correlation coefficient (r) =0.99997 -0.001 -0.018

1.000

2.000

3.302

Conc. (ppb)

Fig. 5.3.2 Measurement results for Pb in refined white sugar

114

Na Mg

Inorganic Metals

Ca

5.4 Analysis of Cadmium in Rice (1) - AA ■ Explanation In Japan, the concentration of cadmium in rice is regulated in accordance with the Food Sanitation Law and directives issued by the Food Agency. Recently, however, as part of an international movement, a proposal to set a threshold level of 0.2ppm (mg/kg), which is lower than the level applied to circulation within Japan, has been discussed at the Codex Committee (joint FAO/WHO food standards committee). The analysis of cadmium in polished and unpolished rice with Shimadzu's AA-6300 Atomic Absorption Spectrophotometer using the flame and furnace methods is described here as an example. The AA-6300 uses an optical double-beam (with the flame method) or an electrical double-beam (with the furnace method) to ensure both a stable baseline and high sensitivity. It is also easy to switch between the flame method, which offers high speed, and the furnace method, which offers high sensitivity, without the need for any special tools. ■ Measurement Methods and Conditions Measurement was performed with the flame and furnace methods. Measurement was also performed using an atom booster with the flame method in order to attain

greater sensitivity. The main measurement conditions are given in Table 5.4.1 and 5.4.2.

Table 5.4.1 Spectrophotometer parameters

Table 5.4.2 Atomization parameters

Analysis wavelength

228.8nm

Slit width

2.0nm

Current

8mA

Lamp mode

115

■ Pretreatment A 5g sample was put into a beaker, sulfuric acid and nitric acid were added, thermal decomposition was carried out, and a solution of 50mL was prepared using the sample. Although acid digestion was used here, other methods that may be used include dry ashing, microwave decomposition, and acid extraction. Alternatively, it is possible to separate the alkaline elements and alkaline earth elements, which can cause interference, in the decomposed liquid, and, in order to concentrate the cadmium, carry out chelate organic-solvent extraction.

Flame method

Flame type

Air-C2H2

Burner height

7mm (without booster) 11mm (with booster)

Temperature program

Drying: 120˚C (20s), 250˚C (10s) Ashing: 400˚C (20s) Atomization: 1,800˚C (3s) Cleaning: 2,600˚C (2s)

Tube type

Pyro-covered tube

Injection volume

2 to 10µL (2µL here)

Total injection volume

15µL

BGC-D2

■ Measurement Results Fig. 5.4.1.1, 5.4.2.1 and 5.4.3.1 show the calibration curves for each analysis method. Table 5.4.3 provides a comparison of the 1% absorption values for each method. It can be seen that, compared to the standard flame method, using a booster with the flame method increases the sensitivity by a factor of 2.5 whereas using the furnace method increases the sensitivity by a factor of approx. 300. Table 5.4.4 shows the measurement results obtained for polished and unpolished rice using each of the methods. The results obtained with each method are roughly the same. The lower quantitative limits for cadmium in rice that can be estimated from these results are approx. 0.10ppm with the flame method, approx. 0.05ppm when using a booster with the flame method, and approx. 0.001ppm when using the furnace method with an injection volume of 10µL. This means that quantitative measurement at the 0.2ppm level is possible with any of the methods.

Furnace method

Interference-suppression agent 5µL of 100pm palladium nitrate

Table 5.4.3 Comparison of 1% absorption values for each method (in solution) Measurement method

1% absorption value

Flame method without booster

0.007 ppm

Flame method with booster

0.003 ppm

Furnace method

0.02 ppb

Table 5.4.4 Analysis results (in each type of rice) Flame method Flame method Furnace method without booster with booster Unpolished rice

0.073ppm

0.066ppm

0.070ppm

Polished rice

0.118ppm

0.127ppm

0.118ppm

5.4 Analysis of Cadmium in Rice (2) - AA

0.050ppm

Polished rice Unpolished rice Blank

Fig. 5.4.1.1 Calibration curve for flame method without booster

Fig. 5.4.1.2 Signal profile for flame method without booster

0.050ppm

Polished rice Unpolished rice Blank

Fig. 5.4.2.1 Calibration curve for flame method with booster

Fig. 5.4.2.2 Signal profile for flame method with booster

Polished rice 0.002ppm

Unpolished rice

Blank

Fig. 5.4.3.1 Calibration curve for furnace method

Fig. 5.4.3.2 Signal profile for furnace method

116

Na Mg

Inorganic Metals

Ca

5.5 Analysis of Inorganic Components in Powdered Milk (1) - ICP-AES ■ Explanation The microwave sample decomposition method is quicker than the conventional wet decomposition method and takes place in a sealed system to prevent external contamination and volatilization loss of components such as As and Se. It is an extremely useful method to decompose the sample when the sample amount is small, or when a micro-amount element is to be analyzed. Here, powdered milk was liquidized using a microwave decomposition unit and analyzed using ICP-AES. The ICP-AES, which causes little self-absorption and has a wide dynamic range, enables analysis of major components like sodium and calcium, as well as minor components such as cadmium and lead, in the same solution. Arsenic, selenium and antimony can be analyzed at higher sensitivity by using a hydride generator.

■ Pretreatment See Fig. 5.5.1 for details of the operation flow for microwave decomposition.

■ Analytical Conditions : ICPS-7500 Instrument : HVG-1 (Hydride Generator) High Frequency Output : 1.2kW : Ar 14.0L/min Cooling Gas : Ar 1.2L/min Plasma Gas : Ar 0.7L/min Carrier Gas Sample Introduction : Coaxial Nebulizer/Cyclone Chamber, Hydride Generator System Observation Method : Horizontal/Axial

Table 5.5.1 Powdered milk analysis results (µg/g) Element 0.5g sample (Teflon high-pressure container) 10mL nitric acid, 3mL hydrogen peroxide Preparative reaction (Approx. 4 hr)

Element

Measured value

Si

Mg

376

Ba

1.4

P

2238

Ni

0.21

Cooling (Approx. 1 hr)

K

4644

Sn

0.2

Microwave decomposition (pressure mode) (Approx. 30 min)

Ca

3960

Cr

0.04

Cooling (Approx. 2 hr) 5mL hydrochloric acid (1:5) Heating 190˚C (beaker) Heating 120˚C (evaporation to dryness) Evaporate liquid down to few mL 5mL hydrochloric 5mL hydrochloric acid (1:4) acid (1:4) KI (20%wt/v) 0.5mL Cooling Heating 120˚C (Approx. 10 min) Heating 70˚C (Approx. 4 min) 25mL measure up 10mL measure up 10mL measure up Coaxial nebulizer analysis Se As, Sb Na, Mg, Al, Si, P, K, Ca, Cr, Mn HVG analysis Ni, Cu, Zn, Cd, Fe, Sn, Ba, Pb

Fig. 5.5.1 Microwave decomposition flowchart

117

Measured value 1259

Microwave decomposition (pressure mode) (Approx. 30 min)

Na

Mn Fe Cu Zn Al

0.34 75 2.8 23 3.0

Cd Pb

23

0.022