Development and Validation of a Method for

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Development and Validation of a Method for Simultaneous Quantification of Seven Water-Soluble Vitamins in Pediatrics Syrup by UPLC-MS/MS. Harmita a.
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RESEARCH ARTICLE

Development and Validation of a Method for Simultaneous Quantification of Seven Water-Soluble Vitamins in Pediatrics Syrup by UPLC-MS/MS Harmitaa, Herman Suryadia, Ati Setiawati1 and Dwi Damayantia,1* a

Faculty of Pharmacy, Universitas Indonesia, Depok-Jawa Barat, Indonesia 16424; 1National Quality Control Laboratory of Drug and Food, National Agency of Drug and Food Control, Jl. Percetakan Negara No 23 Jakarta Pusat Abstract: Baground: Vitamin has an important function in biochemical processes and plays an important role in the main process of the human body. Vitamin products are used to maintain health, restore vitality, control weight and help in disease prevention. To comply with regulatory requirements and to establish nutrient intake in some populations, the quality of vitamins in food and nutritional supplements needs to be ensured.

ARTICLE HISTORY Received: July 19, 2016 Revised: December 15, 2016 Accepted: December 20, 2016 DOI: 10.2174/15734129136661612281420 29

Methode: This study aims to obtain a simultaneous method for the quantification of thiamine hydrochloride, riboflavin, nicotinamide, dexpanthenol, pyridoxine hydrochloride, cyanocobalamin and ascorbic acid in pediatric syrup by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLCMS/MS). ACQUITY UPLC® BEH Shield C8 column (2.1 mm x 50 mm, 1.7 m), mobile phase is a mixture of 10 mM ammonium formate in 0.2% formic acid pH 2.8 with methanol by gradient elution at a flow rate of 0.4 mL/min achieved in 6.5 minutes was used for separation. Ionization method was positive electrospray ionization (ESI +) with Multiple Reaction Monitoring (MRM). Result: Confirmation result showed 7 water-soluble vitamin parent ions and daughter ion of thiamine HCl at m/z 265.15> 122; riboflavin at m/z 377.20> 243.10; nicotinamide at m/z 123> 80.10; pyridoxine HCl at m/z 170.10> 152.00; cyanocobalamin at m/z 678.45> 147.05; dexpanthenol at m/z 206.05> 76.00 and ascorbic acid at m/z 177.00> 94.90. The results of method validation showed that the method fulfills the acceptance criteria of precision, recovery and linearity Conclution: The method can be used for the routine analysis of determination of seven water soluble vitamins in pediatric syrup.

Keywords: Water-soluble vitamins, UPLC-MS/MS, Reversed-phase, development, validation, pediatric syrup, C8 column. 1. INTRODUCTION Vitamin has an important function in biochemical processes and plays an important role in the main processes of the human body, including metabolism, maintain blood glucose levels, regulation of cell growth and differentiation [1]. Vitamins are classified into water-soluble vitamins and fatsoluble vitamins. The human body needs 13 essential vitamins, namely: four fat-soluble vitamins (A, D, E and K) and 9 water-soluble vitamin (Ascorbic Acid, thiamin, Riboflavin, Pyridoxine, Cyanocobalamine, Nicotinamide, Biotin, Folic Acid and Pantothenic Acid and derivate) [2, 3]. Growth of food and beverage products that are fortified with vitamin [2] and use of vitamins in pharmaceutical *Address correspondence to this author at National Quality Control Laboratory of Drug and Food, National Agency of Drug and Food Control, Jl. PercetakanNegara No 23 Jakarta Pusat; Tel: +6221 4245075; +6281281914529; Fax: +62214245150; E-mails: [email protected]; [email protected]; [email protected]

1573-4129/17 $58.00+.00

industries have recently been increased [4]. Vitamin products are used to maintain health, restore vitality, control weight and help in disease prevention [2]. To ensure the quality, compliance with regulatory requirements and to establish nutrient intake in some populations, there is a need to determine the quality of vitamins in food and nutritional supplements [5]. Liquid chromatography is commonly used to separate water-soluble vitamins. Studies have proven that there is no method that can simultaneously separate all water soluble vitamins simultaneously. Only chromatography coupled with electrospray ionization mass spectrometry is capable of separating all water soluble vitamins [2]. Several methods using reversed-phase liquid chromatography with ion pairs were developed to improve the retention of water-soluble vitamins which are very polar [6-8]. This method has several limitations including that at the time of the analysis, the presence of an ascorbic acid is also detected which is likely to interfere with the analysis of nicotinamide, which has the earliest retention time. HPLC with ion pair © 2017 Bentham Science Publishers

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Harmita et al.

cannot detect the impurities or degradation of vitamins in pediatric syrup preparation like riboflavin and nicotinamide [8].

2. MATERIALS AND METHOD

Several studies have developed simultaneous methods for the quantification of water soluble vitamins using UPLCMS/MS [1, 2, 9-14]. UPLC-MS/MS with positive mode can analyze all of the water-soluble vitamins in pediatric syrup and separate lysine with other vitamins. Another advantage of UPLC-SM/SM is that it does not require complex samples cleanup procedures. Water soluble vitamins have a diverse structure and chemical properties and most of them are polar compounds with less retention in reversed-phase chromatography [11, 14]. UPLC-MS/MS is very selective and sensitive method which can determine samples with low concentration [14].

Thiamine Hydrochloride (Indonesian Pharmacopoeia Reference Standards); Riboflavin from Indonesian Reference Standards; Nicotinamide (Indonesian Pharmacopoeia Reference Standards); Dexpanthenol (DSM Nutritional Product, UK Ltd); Pyridoxine Hydrochloride (Indonesian Pharmacopoeia Reference Standards), Cyanocobalamin (Indonesian Pharmacopoeia Reference Standards) and Ascorbic Acid (Indonesian Pharmacopoeia Reference Standards). Methanol LC-MS grade (Merck, Darmstadt, Germany); acetonitrile LC-MS grade (Merck, Darmstadt, Germany); ammonium formate (Sigma-Aldrich, St. Louis, MO, USA); formic acid (Merck, Darmstadt, Germany); phosphate buffer pH 4.0 (Merck, Darmstadt Germany); citrate buffer pH 2.0 (Merck, Darmstadt, Germany); and purified water with conductivity 18.2 M.cm. filtered by Aerium Water Purification System (Sartorius, Gottingen Germany).

UPLC-MS/MS methods have been used for simultaneous method development to determine 14 water-soluble vitamins (thiamine, riboflavin, pantothenic acid, nicotinic acid, nicotinamide, pyridoxine, pyridoxal, biotin, folic acid, cyanocobalamin, ascorbic acid, carnitine, choline and taurine) in infant formula using ACQUITY UPLC® BEH Shield C18 column (2.1 mm  100 mm; 1.7 μm, Waters) with a mobile phase of methanol and 10 mM ammonium acetate. Identification and quantification were done using Multiple Reaction Monitoring (MRM) with ESI positive ionization mode [14]. Another method developed for simultaneous determination of thiamine, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk use ACQUITY UPLC® HSS T3 column (2.1 x 50 mm; 1.8 m, Waters) [15]. Vitamin pediatric syrups are vitamin preparations intended for children in which consumption does not exceed the recommended dose. To ensure the consumption of appropriate dose in children, the quality of vitamins needs to guaranteed through quality monitoring. Vitamin syrup is a complex preparation containing a lot of vitamins and ingredients that could influence analysis by HPLC, therefore, developing a method for the analysis determination of watersoluble vitamin in pediatric syrup by UPLC-MS/MS is needed. This research was aimed to develop an analytical method to determine simultaneously seven water soluble vitamins (thiamine hydrochloride, riboflavin, nicotinamide, pyridoxine, cyanocobalamin, dexpanthenol and ascorbic acid) in pediatric syrup by UPLC-MS/MS using ACQUITY UPLC® BEH Shield C8 column (2.1 mm  50 mm, 1.7 μm, Waters). Column length of 150 mm, 100 mm and 250 mm [2, 9, 14] was used in the previous studies. Type columns used in this study are the ACQUITY UPLC® BEH shield C8 column whereas C18 column [2, 14] and ACQUITY UPLC®HSS T3 [15] were used in the previous studies. C8 columns have shorter alkyl chains than C18, making it more suitable for the separation of polar compounds [16]. A column with a particle size of 5 μm, 3 μm and 1.8 μm was used in the previous studies whereas in the study, a smaller particle size of 1.7 μm was used which increased the efficiency of the column based on the Van Demeter equation [17]. The mobile phase used in this research was methanol-10 mM ammonium format as an elution gradient. The valid methods were then used to define the content of 7 water soluble vitamins in the pediatric syrup by 2 different brands.

2.1. Materials

2.2. Instrumentation Ultra Performance Liquid Chromatography (UPLCMS/MS) ACQUITY UPLC (Waters corp., Milford, MA,USA) coupled with Binary Solvent Manager, auto sampler and Triple quadrupole (TQD) detector; ultrasonic (Branson); 0.2 m membrane filter (Millipore, MA, USA); vacuum (Buchi); 5 mL, 10 mL, 20 mL, 25 mL, 50 mL, 100 mL brown volumetric flask, 2000 mL; 1,0 mL volume pipette; 1000 μL, 250 μL, 10 μL micropipette; Sartorius AW 220 analytical balance; microbalance (Sartorius ME 36S); ACQUITY UPLC® BEH Shield C8 column (2.1 mm  50 mm; 1.7 μm, Waters). Ionization method: ESI positive, capillary voltage : 3.0 KV, cone voltage: 40 volts, source temperature 150 ºC, desolvation temperature 500 ºC and collision gas flow rate 0.2 mL/min. Collision gas: Argon (0.5 bar). Fragmentation of transition ion was monitored by the Multiple Reaction Monitor (MRM). Needle wash was performed after every injection with 200 L acetonitrile 50% in formic acid 1% (strong wash) and 200 L acetonitrile 10% (weak wash). 2.3. Samples Two different pediatrics syrup consisting of vitamin A, vitamin D, vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinamide, vitamin C, dexpanthenol, lysine and glutamic acid. 2.4. Preparation of Standard and Samples Solutions Standard Stock Solution The standard of thiamine hydrochloride, riboflavin, nicotinamide, pyridoxine hydrochloride, cyanocobalamin and dexpanthenol was accurately weighed and diluted with water to produce concentration in a series of 0.25 mg/mL; 0.05 mg/mL; 0.25 mg/mL; 0.25 mg/mL; 0. 625 mg/mL and 0.625 mg/mL, respectively. The standard of ascorbic acid was added directly to standard working solution because of the instability in solution. Ascorbic acid was prepared daily. Intermediate Mixed Standard Solution Each standard stock solution was taken in a series of thiamine hydrochloride, riboflavin, nicotinamide, pyridoxine

Development and Validation of a Method for Simultaneous Quantification

Table 1.

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Concentration of Calibration Curve Solution. Concentration (ppb) Vitamine 1

2

3

4

5

6

7

8

9

B1

100

75

50

25

10

7.5

5

2.5

1

B2

1000

750

500

250

100

75

50

25

10

B3

100

75

50

25

10

7.5

5

2.5

1

B6

100

75

50

25

10

7.5

5

2.5

1

B12

1250

937.5

625

312.5

125

93.75

62.5

31.25

12.5

Dexpanthenol

1000

750

500

250

100

75

50

25

10

Ascorbic Acid

10000

7500

5000

2500

10000

750

500

250

100

hydrochloride, cyanocobalamin and dexpanthenol, in an amount of 1 mL; 50 mL; 1 mL; 1 mL; 5 mL and 4 mL to a 100 mL brown volumetric flask and 25 mg standard of ascorbic acid was added and diluted with water in order to obtain a concentration of 0.0025 mg/mL; 0.025 mg/mL; 0.0025 mg/mL; 0.0025 mg/mL; 0.0313 mg/mL and 0.025 mg/mL respectively. The concentration of ascorbic acid was 0.25 mg/mL. Calibration Curve Standard Solution 10 L; 25 L; 50 L; 75 L; 100 L; 250 L; 500 L; 750 L and 1000 L intermediate mixed standard solution was transferred to 25 mL brown volumetric flask and was diluted with ammonium formate 10 mM in 0.2 % formic acid at pH 2.8 to obtain nine concentration of vitamins (can be seen in Table 1). The calibration curve for each vitamin was consisted of minimum six standard solutions in linear range of 1–100 ng/mL of thiamin hydrochloride, nicotinamide, pyridoxine hydrochloride, 10-1000 ng/mL riboflavin and dexpanthenol, 100-10000 ng/mL of ascorbic acid, 12.5 312.5 ng/mL of cyanocobalamine. The calibration curves over a time period of six months showed a mean linear correlation coefficient (r) greater than 0.99 for all vitamins, indicating a strong positive correlation between the theoretical concentrations and the analyzed values. Preparation of Sample Solution Samples were transferred and measured equivalent to 0.5 mg of thiamine hydrochloride, to 100 mL-brown volumetric flasks added 50 mL water, sonicated for 5 minutes and diluted with water to volume. Solution diluted with ammonium formate 10 mM in 0.2% formic acid at pH 2.8 to obtain concentration of thiamine hydrochloride 50 ng/mL, riboflavin 480 ng/mL, nicotinamide 50 ng/mL, pyridoxine hydrochloride 50 ng/mL, cyanocobalamine 20 ng/mL, dexpanthenol 250 ng/mL and ascorbic acid 1.5 g/mL. The solution was filtered using a membrane filter with a porosity of 0.2 μm.

pounds except ascorbic acid which is an acidic compound. APCI is generally used for polar to nonpolar compounds with medium molecular weight up to 1500 Da [18]. Optimization ionization mode shows that ESI positive response gives greater energy than the APCI method so even smaller analytic concentrations result in a great response. In this research, ESI positive is used as an ionization mode. In addition, cone optimization and collision energy were required for the quantitative determination of the vitamin. Results of collision and cone energy optimization can be seen in Table 2. In this study, seven water-soluble vitamins were analyzed using ESI positive ionization method with capillary voltage 3,0 KV, cone voltage of 40 volts, source temperature of 150 º C, desolvation temperature of 500º C and collision gas flow rate of 0.2 mL / min. Collision gas used was Argon pressure of 0.5 bars. Ion fragmentation transition is observed with Multiple Reaction Monitored mode (MRM). 3.2. Chromatography Optimization In addition to MS/MS optimization, chromatography system optimization is performed. The parameters are optimized, among others: the column type, mobile phase composition (gradient system), pH of mobile phase, flow rate, column temperature and injection methods. Selected columns are a reversed-phase column. Previous research separates some water soluble vitamins in infant formula used ACQUITY UPLC® BEH shield C18 (2.1 mm x 100 mm; 1.7 m) column [14]. In this research, Reversed-phase ACQUITY UPLC® Shield C8 column (2.1 mm x 50 mm; 1.7 m) was used. C8 columns have shorter alkyl chains than C18 column. C18 column is more suitable for the separation of non-polar compounds [15]. The Gradient Elution System can be seen in Table 3.

3.1. Optimization of MS/MS Parameters

Optimization of column temperature at 38° C, 40° C, and 42° C shows that 40° C was the optimal column temperature where the chromatogram all of the vitamins is better than other temperatures. The flow rate used was 0.4 mL/min. Optimization of pH of ammonium format shows that 2.8 was optimal pH to separate all vitamins.

Optimization of ionization mode used is ESI positive and APCI. The ESI positive method was used because most of the compounds (B1, B2, B3, B6, and B12) are basic com-

The injection system used in this study was partial loop with needle overfill with 5 mL volume injection. The autosampler temperature maintains at 4ºC. The strong wash sol-

3. RESULTS AND DISCUSSIONS

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Table 2.

Harmita et al.

LC-MS/MS Condition with MRM. Vitamine

Parent Ion

Daughter Ion

Dwell

Cone ()

Collision energy (eV)

B1

265.15

122.00

0.100

20.00

20.00

B2

377.20

243.10

0.100

40.00

28.00

B3

123.00

80.10

0.100

35.00

20.00

B6

170.10

152.00

0.100

25.00

17.00

B12

678.45

147.05

0.100

33.00

45.00

Dexpanthenol

268.05

76.00

0.100

26.00

20.00

Ascorbic Acid

177.0

141.00

0.100

21.00

17.00

Table 3.

Gradient Elution System. Time (minutes)

A

B

Initial

95.0

5.0

3.0

65.0

35.0

4.00

5.0

95.0

4.20

95.0

5.0

6.50

95.0

50

vent use was 50% acetonitrile in 0.1% formic acid. The week washes solvent use was 10% acetonitrile in water. Optimization result of column temperature 40ºC and flow rate 0.4 mL/ min for separate 7 water soluble vitamins can be seen in Figs. (1-7).

Table 4.

Vitamine

Matrix Effect (%) n=3

Thiamine HCl

91.41

Riboflavin

97.94

Nicotinamide

92.92

Pyridoxine HCl

91.61

Dexpanthenol

81.15

Cyanocobalamin

93.14

Ascorbic acid

92.67

3.3. Validation 3.3.1. Matrix Effect (ME) The matrix effect was evaluated by comparing the response of vitamins in standard solutions (A) with the response of those in a sample which were spiked at concentrations approximately equivalent to the estimated contents (B). Because no blank samples were used, the responses of the compounds in the samples were calculated by subtracting the responses of the non-spiked samples from those of the spiked samples. ME =

B x100% A [19]

Matrix effect.

Table 5.

Matrix effect from Recovery. Vitamine

Matrix Effect (%)

Based on matrix effects study, ion suppression effects were found in 8.59% thiamine HCl; 2.06% riboflavin; 7.08% nicotinamide; 8.39% pyridoxine HCl; 9.74% dexpathenol; 7.33% ascorbic acid and 6.86% cyanocobalamin.

Thiamine HCl

97.84

Riboflavin

98.53

Nicotinamide

100.21

In addition, matrix effect was determined by spiking sample solution with a standard solution of known concentration. The matrix effect was obtained from the recovery study. The response of compound was calculated by subtracting the response of spiked samples with the response of unspike samples. The results of matrix effect from recovery can be seen in Table 5.

Pyridoxine HCl

99.95

Dexpanthenol

99.09

Cyanocobalamin

97.01

Ascorbic acid

101.20

The result of Matrix effect can be seen in Table 4.

Development and Validation of a Method for Simultaneous Quantification

Fig. (1). Mass Spectrum of Thiamine Hydrochloride.

Fig. (2). Mass Spectrum of Riboflavin.

Fig. (3). Mass Spectrum of Cyanocobalamin.

Current Pharmaceutical Analysis, 2017, Vol. 13, No. 00

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Fig. (4). Mass Spectrum of Ascorbic Acid.

Fig. (5). Mass Spectrum of Dexpanthenol.

Fig. (6). Mass Spectrum of Nicotinamide and Pyridoxine HCl.

Harmita et al.

Development and Validation of a Method for Simultaneous Quantification

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Fig. (7). Chromatogram of seven water soluble vitamins with ACQUITY UPLC®BEH Shield C8 column (2.1 mm  50 mm; 1.7 μm, Waters), column temperature was 40ºC and pH of mobile phase was 2.8. The chromatogram in Fig. (7) shows the separation of seven water-soluble vitamins. Retention time of cyanocobalamin was 2,41; riboflavin was 2,44; dexpanthenol 1,39; pyridoxine was 0,53; ascorbic acid was 0,41; thiamine hydrochloride was 0,35 and nicotinamide was 0,5.

3.3.2. Precision Precision was investigated by analyzing six consecutive injections of an analytical sample solution and the % R.S.D. values (n = 6) of peak areas were used for evaluation. For the evaluation of method precision (repeatability and intermediate precision), six independent sample solutions were prepared and analyzed in different days and then the mean value, standard deviation (sd) and Coefficient Variance (CV) were calculated. The acceptance criteria was according to Horwitz equation [20-22]. Precision results can be seen in Table 6 and the acceptance criteria is shown in Table 7. From these results obtained precision for vitamin B1, B2, B3, B6, B12 and dexpanthenol meets the acceptance criteria while ascorbic acid providing coefficient variance more than 2.7%. Ascorbic acid is an unstable compound in solution, causing differences in the results on different days and also provides a great deviation. In solution, ascorbic acid is oxidized to dehydroascorbic acid. Reversible hydrolysis will

cause the L-dehydroascorbic acid react into an inactive 2,3 diketo-1-gulonat acid [23]. 3.3.3. Recovery Accuracy is a measure of the closeness of test results or the average value of a set of data against the true value. Recovery consists of recovery of the value of certain analyte where the addition of a number of known levels into a product or samples which have been assayed [24]. In this study, recovery comes with a range of 3 concentration are 80%, 100% and 120%. Results of recovery methods can be seen in Table 8. The recovery method shows that this method meets the acceptance criteria of recovery. 3.3.4. Linearity and Range Linearity Determination used to observe the relationship that the test results are directly or mathematically proportional to the analytic concentration of a sample in a given range. Linearity meets the acceptance criteria if the correla-

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Table 6.

Harmita et al.

Precision. Ripitability

Reproducibility

Vitamine

Table 7.

Concentration from lebel amount (%)

KV (%)

Concentration from lebel amount (%)

KV (%)

Thiamine HCl

99.03

1.67

97.69

1.60

Riboflavin

98.68

1.54

99.64

1.31

Nicotinamide

111.19

1.66

109.16

0.79

Pyridoxine HCl

103.52

0.82

102.42

1.35

Dexpanthenol

105.24

1.07

103.99

0.38

Cyanocobalamin

98.09

1.51

99.19

1.17

Ascorbic acid

98.16

1.30

79.68

3.38

Acceptance Criteria (CV Horwitz). Vitamine

Analyst (%)

2/3 CV Horwitz

Thiamin HCl

0.16667

3.7

Riboflavin

0.20000

3.7

Nicotinamide

1.66667

2.7

Pyridoxine HCl

0.16667

3.7

Dexpanthenol

0.83333

3.7

Cyanocobalamin

0.00033

11

Ascorbic acid

5.00000

2.7

tion (r) 0.99. Sy/x value and Vx0 (relative residual standard deviation) calculated using the formula given below:

 ( y  yi)

Sy =

2

n2

x

The acceptance criteria of Vx0 is  5.0%. In this study, the linearity and Vx0 of each vitamin can be seen in Table 9. Range analysis determination of 7 water soluble vitamins can be seen in Table 10. 3.3.5. LOD and LOQ

Vx0 =

Sx0 x100% Qxx

Limit of Detection and limit of quantification determined used instrumental method based on signal-to-noise ratio and regression equation approach can be seen in Table 10.

Sx0 =

Sy Qxy m= m Qxx

3.3.6. Robustness

Sy =

Qyy  Qxy / Qxx n2

Qxy =  Xi.Yi.

Qxx =  xi 2 

2

 ( yi.xi) n

( xi) 2

Qyy =  yi 2  [26, 27]

n

( yi) 2 n

This study examined the effect of change in mobile phase pH (2.7; 2.8 and 2.9), and the effects of changes in column temperature (38ºC, 40 ºC and 42ºC) on the concentration of the analyte. The data were processed using one-way ANOVA with 95% confidence interval ( 0:05). Based on the Tables 11 and 12, H1 rejected and H0 accepted with the conclusion there was no effect of temperature changes (38ºC, 40 ºC and 42ºC) and mobile phase pH changes (2.7, 2.8 and 2.9) of the vitamin content. 3.3.7. Determination of Sample from Market The validated method, used for the determination of two samples from the market with different batches (Table 13). Requirement based on USP 37, 2014.

Development and Validation of a Method for Simultaneous Quantification

Table 8.

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Recovery of seven water soluble vitamins. Recovery (%) n=3

Acceptance Criteria

Vitamine

Table 9.

80%

100%

120%

Thiamine HCl

101.66

96.13

95.73

95-105

Riboflavin

98.28

99.24

98.07

95-105

Nicotinamide

100.62

101.18

98.82

97-103

Pyridoxine HCl

102.30

102.12

95.42

95-105

Dexpanthenol

98.72

98.91

99.66

95-105

Cyanocobalamin

102.23

93.24

95.57

80-110

Ascorbic acid

101.38

100.94

101.30

97-103

Linearity results for 7 water soluble vitamins. b Vitamine

a (Intercept)

r

Vx0

(Slope) Thiamin HCl

229.9241

134.9960

0.999741

0.7

Riboflavin

9.0112

33.2030

0.999942

0.4

Nicotinamide

248.3881

76.1331

0.999881

0.5

Pyridoxine HCl

3929.1654

251.2412

0.997098

2.6

Dexpanthenol

1414.9013

234.1954

0.999572

1.0

Cyanocobalamin

4.1120

1.0485

0.998866

1.7

Ascorbic acid

-81.2812

11.7124

0.999996

0.1

Table 10. Range analysis determination of 7 water soluble vitamins. Vitamine

Range (ppb)

Thiamin HCl

33.2000-65.9633

Riboflavin

284.6656-664.2674

Nicotinamide

43.3213-97.4910

Pyridoxine HCl

30.3465-69.9492

Dexpanthenol

139.8749-343.9870

Cyanocobalamin

24.7519-55.2176

Ascorbic acid

963.3589-2086.3825

9

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Harmita et al.

Table 11. The results of a one-way ANOVA test to column temperature changes on levels of vitamin. Vitamine

F

P-Value

F Crit

Thiamin HCl

9.05577

0.05357

9.55209

Riboflavin

7.20626

0.07151

9.55209

Nicotinamide

2.76088

0.20888

9.55209

Pyridoxine HCl

0.09017

0.91616

9.55209

Dexpanthenol

2.29147

0.24884

9.55209

Cyanocobalamin

9.22200

0.05233

9.55209

Ascorbic acid

0.24013

0.80032

9.55209

 Table 12. The results of one-way ANOVA test to mobile phase pH changes on levels of vitamin. Vitamine

F

P-Value

F Crit

Thiamin HCl

9.05577

0.05357

9.55209

Riboflavin

7.20626

0.07151

9.55209

Nicotinamide

2.76088

0.20888

9.55209

Pyridoxine HCl

0.09017

0.91616

9.55209

Dexpanthenol

2.29147

0.24884

9.55209

Cyanocobalamin

9.22200

0.05233

9.55209

Ascorbic acid

0.24013

0.80032

9.55209

The assay of riboflavin was obtained at low concentrations, because the content of riboflavin in syrup preparation is riboflavin 5 phosphate sodium that has different molecular weights with riboflavin. Due to unavailability of reference standards riboflavin 5 phosphate sodium, the riboflavin 5 phosphate sodium cannot be validated and assayed by UPLC-MS/MS. In this study conducted identification contents of riboflavin 5 phosphate sodium in the sample. Parent ion of riboflavin is 377.2, with two daughter ions valued at 243.1 and 172.1, while the parent ion riboflavin 5 phosphate sodium is 457.2 with two daughter ion valued at 439.2 and 359.2. Identification of riboflavin 5 phosphate contents in the samples was done by injecting sample solution and comparing parent ions with daughter ion of riboflavin 5 phosphate sodium with riboflavin (Fig. 8) and also compared with previous research(10). Identification by UPLC-MS/MS concluded that vitamin B2 in the sample is riboflavin 5 phosphate sodium and also contains small amounts of riboflavin (10%). The content of riboflavin may be due to carrying over from the column, contamination during preparation or degradation of riboflavin 5 phosphate sodium. Quantification result of Pyridoxine hydrochloride obtained concentration on one type of syrup from the market

does not meet the requirements according to USP 37 2014, while the other types fulfill the requirements according to USP 37 in 2014 (Table 13) (27). Ascorbic acid on one type of syrup obtained low concentration below 100%, while other types obtained concentration 112.46%. It can be caused due to ionization mode used in the determination of ascorbic acid which was not suitable with ESI positive ionization mode. Ascorbic acid is an acid compound that is more suitable for ESI negative ionization mode. There is no requirement of ascorbic acid in syrup preparation in the USP 37 2014 due to the instability of ascorbic acid in a liquid preparation. So it cannot be determined whether ascorbic acid meets or does not meet the requirements. CONCLUSION Thiamine hydrochloride, riboflavin, nicotinamide, dexpanthenol, pyridoxine hydrochloride, cyanocobalamin and ascorbic acid in the pediatric syrup can be separated by Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS) using ACQUITY UPLC®BEH Shield C8 column with the size a length of 50 mm, an inner diameter of 2.1 mm and a particle size of 1.7 m. Validation of method determination of thiamin hydrochloride, riboflavin, nicotinamide, dexpanthenol, pyridoxine hydrochloride, cyanocobalamin and ascorbic acid in the pediatric syrup

Development and Validation of a Method for Simultaneous Quantification

Current Pharmaceutical Analysis, 2017, Vol. 13, No. 00

11

Table 13. Determination of water soluble vitamin in pediatric syrup from the market. Concentration of label amount Vitamine

Requirement (%) A1

A2

Thiamin HCl

98.95

91.67

90,0-250,0

Riboflavin

7.00

9.47

90,0-150,0

Nicotinamide

116.98

148.18

90,0-150,0

Pyridoxine HCl

102.10

198.78

90,0-150,0

Dexpanthenol

110.53

148.06

90,0-150,0

Cyanocobalamin

106.76

139.83

90,0-450,0

Ascorbic acid

54.59

112.46

-



Fig. (8). Chromatogram of seven water soluble vitamins with ACQUITY BEH Shield C8 column, column temperature 40ºC and pH of mobile phase 2.8. A: chromatogram of riboflavin, B: chromatogram riboflavin 5 Phosphate sodium. C: a mixture of riboflavin with riboflavin 5 phosphate.

meets the acceptance criteria of accuracy, precision, and linearity. This method can be used for the routine analysis on the determination of seven water soluble vitamins in pediatric syrup. CONFLICT OF INTEREST

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The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS This work was supported by the National Agency of Drug and Food Control Republic of Indonesia - National Quality Control Laboratory of Drug and Food and Faculty of Pharmacy, University of Indonesia.

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