Anal Bioanal Chem (2002) 373 : 31–45 DOI 10.1007/s00216-002-1269-z
S P E C I A L I S S U E PA P E R
Hiroyuki Kataoka
Automated sample preparation using in-tube solid-phase microextraction and its application – a review
Received: 29 October 2001 / Revised: 13 February 2002 / Accepted: 14 February 2002 / Published online: 3 April 2002 © Springer-Verlag 2002
Abstract Sample preparation, such as extraction, concentration, and isolation of analytes, greatly influences their reliable and accurate analysis. In-tube solid-phase microextraction (SPME) is a new effective sample preparation technique using an open tubular fused-silica capillary column as an extraction device. Organic compounds in aqueous samples are directly extracted and concentrated into the stationary phase of capillary columns by repeated draw/eject cycles of sample solution, and they can be directly transferred to the liquid chromatographic column. In-tube SPME is an ideal sample preparation technique because it is fast to operate, easy to automate, solvent-free, and inexpensive. On-line in-tube SPME-performed continuous extraction, concentration, desorption, and injection using an autosampler, is usually used in combination with high performance liquid chromatography and liquid chromatography-mass spectrometry. This technique has successfully been applied to the determination of various compounds such as pesticides, drugs, environmental pollutants, and food contaminants. In this review, an overview of the development of in-tube SPME technique and its applications to environmental, clinical, forensic, and food analyses are described.
GC-MS HPLC IC-CD IQ LC-MS MA MDA MDEA MDMA MeIQx MIP MS MSD PAHs PEEK PhIP PPY PTFE SIM SPME Trp-P-1
gas chromatography-mass spectrometry high performance liquid chromatography ion chromatography-conductivity detection 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline liquid chromatography-mass spectrometry methamphetamine methylenedioxyamphetamine methylenedioxyethylamphetamine methylenedioxymethamphetamine 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline molecularly imprinted polymer mass spectrometry mass selective detector polycyclic aromatic hydrocarbones polyether ether ketone 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine polypyrrole polytetrafluoroethylene selected ion monitoring solid-phase microextraction 3-amino-1,4-dimethyl-5H-pyrido[3,4-b]indole
Introduction Keywords In-tube solid-phase microextraction · Automated sample preparation · Capillary column · On-line analysis · Hyphenated technique Abbreviations AM BPA CE ESI GC
amphetamine bisphenol A capillary electrophoresis electrospray ionization gas chromatography
H. Kataoka (✉) Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700–8530, Japan e-mail:
[email protected]
In recent years, toxicant poisoning and environmental pollution by chemical substances have received great attention. The appropriate control of these problems and prevention of related health hazards has become an important issue. Therefore, there is a need for an accurate and precise method for determining these compounds in various matrices such as organism, atmosphere, water, and soil to grasp the dynamics of toxic compounds in various environments. In addition, a convenient, rapid, and automated method for routine analysis has been required with increase in the sample number. Though performance enhancement and improvement of sensitivity and specificity of the analytical instrument have been attempted to satisfy this demand, until now, most analytical instruments cannot directly handle the complex matrices such as biological, food, and environmental samples. In gen-
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eral, analytical methods involve various processes such as sampling, sample preparation, separation, detection, and data analysis. Over 80% of analysis time is spent on the sampling and sample preparation steps such as extraction, concentration, fractionation, and isolation of analytes. Therefore, it is not an exaggeration to say that choice of appropriate sample preparation method greatly influences the reliable and accurate analysis. Conditions for carrying out an efficient sample preparation are as follows: 1) the sample loss is minimum and analyte can be recovered reproducibly, 2) the coexistence component can be efficiently removed, 3) an adverse effect is not present in the chromatography system, 4) the operation can be performed conveniently and quickly, 5) analytical cost is low, etc. However, standard sample preparation techniques involve various problems such as complicated and timeconsuming operations, requiring large amounts of sample and organic solvent, and are difficult to automate. For example, long sample preparation time limits the number of samples, and multi-step procedures are prone to loss of analytes. Furthermore, use of harmful chemicals and a large amount of solvent causes environmental pollution, health hazards to laboratory personnel, and extra operational costs for waste treatment. Ideally, sample preparation techniques should be fast, easy to use, inexpensive, and compatible with a range of analytical instruments. Solid-phase microextraction (SPME) developed by Pawliszyn and co-workers in 1990 [1, 2], is a new sample preparation technique using a fused-silica fiber that is coated on the outside with an appropriate stationary phase. Sample analyte is directly extracted and concentrated by the fiber coating and then introduced to the chromatography. The extraction is carried out by the exposure
Fig. 1 Procedure of extraction by fiber SPME and desorption for GC and HPLC analyses
of the fused-silica fiber in the head-space or in the sample solution (Fig. 1A). The analyte is extracted to the stationary phase and is thermally desorbed in the gas chromatography (GC) injection port and then introduced into the GC column by the carrier gas (Fig. 1B). The method saves preparation time, solvent purchase, and disposal cost, and can improve the detection limits. It has been used routinely in combination with GC and GC-mass spectrometry (MS) and successfully applied to a wide variety of compounds in the gas, liquid, and solid phase, especially for the extraction of volatile and semi-volatile organic compounds from environmental, biological, and food samples [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]. Since the fiber SPME kit became commercially available from Supelco in 1993, it has been widely applied to fields such as environmental, forensic, food, and industrial chemistry, biochemistry, pharmacology, etc. However, application to less volatile and unstable compounds in SPME-GC or SPME-GC-MS method was difficult. As a method for overcoming these difficult points, SPME-GC and SPME-GC-MS methods combined with derivatization have been reported. The SPME technique was also introduced for direct coupling with high performance liquid chromatography (HPLC) and LC-MS to analyze weakly volatile or thermally labile compounds not amenable to GC or GC-MS [7, 9, 10, 11, 12, 19, 27]. An SPME-HPLC interface equipped with a special desorption chamber is utilized for solvent desorption prior to HPLC analysis instead of thermal desorption in the injection port of the GC. The analytes extracted in the fiber are desorbed in the desorption chamber by external addition of solvent or mobile phase, and then introduced to the HPLC column (Fig. 1C). By combination
33
with HPLC [29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42] and LC-MS [43, 43, 44, 45, 46, 47, 48, 49], it has been applied to the analysis of various polar compounds such as drugs and pesticides. These SPME methods are based on the adsorption of compounds by the liquid phase coated on the surface of the fiber. Moreover, a new SPME-HPLC system known as in-tube SPME, was recently developed using an open tubular fused-silica capillary column as the SPME device instead of SPME fiber [7, 9, 10, 11, 12, 19, 25, 26, 27]. The technique using a GC capillary tube is also known as open-tubular trapping, and can be coupled on-line with GC [50, 51, 52, 53]. Intube SPME is suitable for automation, and automated sample handling procedures not only shorten the total analysis time but also usually provide better accuracy and precision relative to manual techniques. In-tube SPME technique has been applied to the determination of pesticides, environmental pollutants, drugs, and food contaminants by hyphenation with HPLC, LC-MS, ion chromatography, and capillary electrophoresis (CE) [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]. This article reviews recent advances in in-tube SPME techniques coupled with various analytical instruments, the design of new capillary coatings for selective extraction, and their applications to various samples. The details of SPME and its applications are also summarized in books on SPME [27, 28] and well-documented reviews [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].
Comparison of fiber SPME and in-tube SPME techniques coupled with HPLC or LC-MS Analytes extracted by SPME must be desorbed into a suitable receiving solvent prior to HPLC analysis. There are Fig. 2 Schematic diagrams of in-tube SPME-LC-MS systems
two modes and some minor variations for interfacing the SPME technique with analytical instruments. Either conventional fiber coupling, or the newer in-tube SPME may be incorporated by placing the interface in the position of either the sample loop or a transfer line. Fiber extractions for LC applications are completely analogous to those for GC applications. An SPME-HPLC interface constructed with a special desorption chamber and switching valve was developed to couple the fiber SPME technique and HPLC (Fig. 1C). For conventional fiber coupling, the desorption chamber is essentially a chromatographic tee, with two of the ports connected in the position of the sample loop. In the third position, a fitting seals the fiber, and either static or dynamic desorption is used. In static desorption, a suitable desorption solvent is introduced to the tee before the fiber is introduced. After a predetermined desorption time, the six-port valve is switched and the mobile phase sweeps the desorbed analytes to the column. Where desorption into the mobile phase is efficient, dynamic desorption may be accomplished by introducing the fiber into the desorption chamber, and then immediately switching the valve to desorb analytes as the mobile phase flows over the fiber. With conventional fiber coupling, analysts are currently limited to performing manual extractions and desorptions. For automated extraction and analysis, in-tube SPME is relatively simple to implement. It can continuously perform extraction, desorption, and injection using a conventional autosampler. In-tube SPME is carried out using mainly the Agilent 1100 Series LC system without modification of the autosampler itself. The combination between in-tube SPME and HPLC or LC-MS can be done easily by fixing the capillary column as the SPME device between the injection loop and injection needle of the HPLC autosampler. A schematic diagram of the automated in-tube SPME-LC-MS system using LC-MS equipment, Agilent 1100 Series LC-MSD is illustrated in Fig. 2. This equipment, with the autosam-
34 Fig. 3A, B Extraction of analytes by fiber SPME (A) and in-tube SPME (B)
pler adopted on-line injection system in the standard way, is suitable for the construction of an on-line in-tube SPME-LC-MS system. As shown in Fig. 2A, while the injection syringe repeatedly draws and ejects sample from the vial under computer control, the analytes partition from the sample matrix into the stationary phase until equilibrium is almost reached. Subsequently, the extracted analytes are directly desorbed from the capillary coating by mobile phase flow or by aspirating desorption solvent after switching the six-port valve (Fig. 2B). The desorbed analytes are transported to the HPLC column for separation, and then detected with UV or mass selective detector (MSD). Therefore, the in-tube SPME technique does not need a special SPME-HPLC interface for the desorption of analytes. The injection loop is installed to prevent the pollution of the metering pump by the sample. As shown in Fig. 2, capillary connections are facilitated by the use of a 2.5 cm sleeve of 1/16 inch polyether ether ketone (PEEK) tubing at each end of the capillary, and fixed by 1/16 inch SS union (0.25 mm bore stainless steel nuts) and ferrules. By building in UV, diode array or fluorescence detectors between the HPLC and MSD, multi-dimensional and simultaneous multi-detections are also possible, and positive identification and fixed quantity can be achieved. Drawing/ejection of the sample solution, switching of the valve, control of peripheral equipment such as HPLC and MSD, and analytical datum processing are all controlled by the workstation. Therefore, labor saving and high precision can be achieved by extending the in-tube SPME-LC-MS system to extraction, concentration, elimination, separation, detection, and data processing by setting the sample to the autosampler. Furthermore, the in-tube SPME technique can be applied from low to high molecular weight compounds and from the more to less volatile compounds by using LC-MS. In addition, au-
tomatic processing of large sample numbers is possible by the autosampler without carryover, because the injection needle and capillary column are washed in methanol and the mobile phase before the sample is extracted. Figure 3 shows the transfer of the analytes in the extraction process of the fiber SPME and in-tube SPME, and each feature is summarized in Table 1. Although the theories of fiber and in-tube SPME methods are similar, the significant difference between these methods is that the extraction of analytes is performed on the outer surface of the fiber for fiber SPME by agitation and on the inner surface of the capillary column for in-tube SPME by flow of the sample solution. Therefore, it is necessary to prevent plugging of the capillary column and flow lines during extraction with the in-tube SPME, and particles must be removed from samples by filtration before extraction. For the fiber SPME, it is not necessary to remove particles before extraction, because they can be removed by washing the fiber with water before insertion into the desorption chamber of the SPME-HPLC interface. However, the fibers should be carefully handled, because they are fragile and can be easily broken, and the fiber coating can be damaged during insertion and agitation. Furthermore, high molecular weight compounds such as proteins can be irreversibly adsorbed by the fiber, thus changing the properties of the stationary phase and rendering it unusable. On the other hand, open-tubular GC capillary columns are very stable and useful as an SPME device for in-tube SPME coupled with HPLC or LC-MS. The capillary sections selected have coatings similar to common commercially available SPME fibers. Although applications of GC capillary columns for in-tube SPME are not sufficiently investigated yet, their properties are considered to be similar to those in use for GC analysis. Another significant difference between in-tube SPME and manual
35 Table 1 Comparison of fiber and in-tube SPME techniques for combination with HPLC or LC-MS Fiber SPME
In-tube SPME
SPME device
Commercially available SPME fibers are limited.
Field Extraction
Outer surface of fiber Immerse fiber in sample solution under agitation
Equilibration time Desorption
30–60 min (depending on compounds) Expose fiber in desorption chamber filled with mobile phase or additional solvent ~10% (depending on compounds) Clear and cloudy samples Manual Fiber must be carefully handled because the coating is prone to strip off from the needle during insertion and removal from desorption chamber.
Commercial capillary columns with a vast array of stationary phases are available. Inner surface of capillary column Repeatedly draw and eject sample solution into capillary column 10–15 min (depending on compounds) Draw desorption solvent or mobile phase into capillary column Negligible Clear sample only Automatic Sample solution must be miscible with mobile phase and not contain insoluble matters because the flow-line is prone to stop.
Carryover Applicable sample Operation Precaution
fiber SPME-HPLC is the possible decoupling of desorption and injection with in-tube SPME. In fiber SPME, analytes are desorbed during injection as the mobile phase passes over the fiber. In in-tube SPME, analytes are desorbed either by mobile phase flow or by aspirating desorption solvent from a second vial, which is then transferred to the HPLC column by mobile phase flow. The fiber SPME-HPLC method also has the advantage of eliminating the solvent front peak from the chromatogram, but peak broadening is sometimes observed because analytes can be slowly desorbed from the fiber. With in-tube SPME, peak broadening is comparatively small, because analytes are completely desorbed before injection. The coating thickness is only in the order of 0.1 µm, so desorption is fast. If analytes are sufficiently solvated by the mobile phase, there is no need to use additional solvent for desorption. On the other hand, in-tube SPME can be easily automated because extraction and subsequent desorption can be continuously carried out without detaching the capillary column. Furthermore, carryover in in-tube SPME is diminished or eliminated in comparison with fiber SPME. The extraction of analyte in fiber SPME and in-tube SPME are based on the distribution coefficient between sample solution phase and SPME stationary phase. Thus, the time in which the analyte reaches distribution equilibrium between two phases becomes the extraction time. Although SPME has a maximum sensitivity at the partition equilibrium, a proportional relationship is obtained between the amount of analyte extracted by SPME and its initial concentration in the sample matrix before reaching partition equilibrium. Therefore, full equilibration is not necessary for quantitative analysis by SPME. Generally, SPME is affected by various factors described in the following section. For example, fiber SPME is influenced by types of fiber coating, pH of the sample solution, salt level, warming, agitation, etc., and in-tube SPME is influenced by the type of capillary coating, pH of the sample solution, length of the capillary column, draw/eject volume of the sample, and their cycles and speeds, etc. The
detailed theory of SPME including these factors is reviewed by Lord and Pawliszyn [10, 12, 19, 22, 27].
Optimization of in-tube SPME In-tube SPME is an extraction method based on the transfer of analyte, which follows the distribution coefficient as for fiber SPME, and it is important to increase the distribution factor of the stationary phase to obtain rapid and high extraction efficiency. For in-tube SPME, the amount of analyte extracted into the stationary phase of the capillary column depends on the polarity of capillary coating, number and volume of draw/eject cycles, and sample pH, etc. The optimization factor of in-tube SPME is described in the following section. Selection of the capillary coating For in-tube SPME, several commercially available capillary columns are generally used as the SPME device. Each column has different selectivity in the type of stationary phase, internal diameter, length, and film thickness as is the case for GC analysis. For example, in the low polarity column with a methyl silicon liquid phase, the hydrophobic compounds are selectively retained (extracted) relative to hydrophilic compounds, and in the high polarity column with a polyethylene glycol liquid phase, hydrophilic compounds are selectively extracted relative to hydrophobic compounds. The extraction efficiency can be defined as the extraction yield of a compound from the sample solution. The extraction yields of various compounds by in-tube SPME with several commercial capillary columns were calculated by comparison with the corresponding direct injection of the sample solution into the LC column. As shown in Fig. 4, it was proven that Omegawax with a polyethylene glycol system was suitable for the extraction of relatively highly polar compounds. Although the extraction yields are low, it is
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Fig. 4 Evaluation of capillary columns for in-tube SPME of several compounds. Extraction yield was calculated by comparison of the amount of compound extracted onto the capillary column with the initial amount of the compound in the sample before extraction. Capillary column: 60 cm × 0.25 mm i.d., 0.25 µm film thickness. SPME conditions: compounds, 0.5–1.0 µg mL–1; sample pH, 8.5 (50 mM Tris-HCl); draw/eject cycle, 15; draw/eject volume, 30 µL; draw/eject rate, 100 µL min–1. HPLC conditions are as follows. For the analysis of propranolol and acebutolol: column, Hypersil BDS C18 (5.0 cm × 2.1 mm i.d., 3 µm particle size); mobile phase, acetonitrile/methanol/water/acetic acid (15:15:70:1); flowrate, program from 0.2 to 0.45 mL min–1 for a 25 min run. ESI+MS conditions: nebulizer gas N2, 40 psi; drying gas N2, 10 L min–1 at 350 °C; fragmentor voltage, 70 V; capillary voltage, 3500 V. For the analysis of methamphetamine and MDMA: column, Supelcosil LC-CN (3.3 cm × 4.6 mm i.d., 3 µm particle size); mobile phase, acetonitrile/50 mM ammonium acetate (15:85); flow-rate, 0.4 mL min–1 for a 25 min run. ESI+-MS conditions: nebulizer gas N2, 40 psi; drying gas N2, 10 L min–1 at 350 °C; fragmentor voltage, 40 V; capillary voltage, 3500 V. For the analysis of heterocyclic amines: column, Supelcosil LC-CN (3.3 cm × 4.6 mm i.d., 3 µm particle size); mobile phase, 15% acetonitrile/methanol (4:1) + 85% 0.1 M ammonium acetate (pH 7.0); flow-rate, program from 0.2 to 0.8 mL min–1 for a 20 min run. ESI+-MS conditions: nebulizer gas N2, 40 psi; drying gas N2, 10 L min–1 at 350 °C; fragmentor voltage, 90 V; capillary voltage, 3500 V. Selected ion monitoring (SIM) of the [M+H]+ ions: propranolol (m/z 260), acebutolol (m/z 337), amphetamine (m/z 136), MDMA (methylenedioxy methamphetamine) (m/z 194), Trp-P-1 (3-amino-1,4-dimethyl-5Hpyrido[3,4-b]indole) (m/z 212), IQ (2-amino-3-methyl-3H-imidazo[4,5-f]quinoline) (m/z 199), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) (m/z 225)
tively desorb the compound from the capillary column. In our experience, a 50–60 cm length of capillary column and/or narrow bore column, in which the film thickness is comparatively small, proved to be suitable to increase the extracted amount as much as possible, to reduce the peak tailing to a minimum, and to prevent carryover with intube SPME. Although the capillary column with a chemically bonded or cross-linked liquid phase is very stable for water and organic solvent, it can easily be deteriorated by inorganic strong acids or strong alkali. Therefore, it is important to monitor the coating durability to exposure of mobile phases and any desorption solvents required. This is more difficult than monitoring a fiber coating, as one cannot directly visualize the coating on the inside of the tube. Thus, it is necessary to monitor the loss in response of known analytes. However, the capillary column is generally stable for the mobile phase usually used in the HPLC. Actually, a reduction of the extraction efficiency was not observed, even if the Omegawax column was repeatedly used over 500 times [66, 69, 70]. As a conventional commercial capillary column, Omegawax 250 was used for the extraction of pesticides, drugs, and heterocyclic amines [54, 55, 56, 57, 66, 69, 70, 72, 76], and Supel-Q PLOT coated with the porous a divinylbenzene polymer was used for the extraction of organometallic compounds, benzodiazepines, and endocrine disruptors [62, 63, 75, 77]. On the other hand, Saito et al. used a modified capillary column inserted with stainless steel wire [73] and a polyether ether ketone (PEEK) tube packed with fibrous rigid-rod heterocyclic polymer [61] to increase extraction efficiency. Furthermore, Wu et al. developed a new capillary column, which was coated with polypyrrole (PPY) polymer in the inner wall of a commercial fused-silica capillary, in order to increase the extraction efficiency and selectivity, and this was applied to the effective extraction of various compounds [59, 64, 65, 67, 71, 78]. Recently, Mullet et al. developed a new in-tube SPME technique using a PEEK tube packed with the particles of molecularly imprinted polymer (MIP) for propranolol [68]. Kataoka et al. also developed a new capillary column coated with MIP on the inner wall of a commercial fused-silica capillary, and applied it to in-tube SPME. The MIP-coated capillary imprinted for β-estradiol selectively recognized some estrogens, but did not recognize androgens or corticosteroids. Effect of sample solution
possible to extract the compounds reproducibly by using an autosampler, and to introduce all amounts of the extracts into the LC column after in-tube SPME. Internal diameters, length, film thickness of the column, etc., are related to sample load and the amount of compounds extracted. If these increase, the load and amount extracted increase, the extension of sample bandwidth causes peak broadening and tailing. In addition, if the film thickness of the stationary phase is large, large amounts of compound can be extracted, but it may not be possible to quantita-
Generally, it is possible to increase extraction efficiency of analyte-to-stationary phase in SPME by changing the pH and salt level of the sample solution. The extractions of acidic and basic compounds are effective from sample solutions under acidic and alkaline conditions, respectively. However, the stability of the compound in the sample solution must be verified beforehand. As shown in Fig. 5, the extraction efficiency of some basic drugs was highest at pH 8.5 (Tris-HCl buffer), and the optimum concentration of buffer solution was 50–100 mM (data not
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Fig. 5 Effect of sample pH on the in-tube SPME of several compounds with Omegawax. Buffer: pH 5.5 (sodium acetate buffer); pH 7.0 (sodium phosphate buffer); pH 8.5 (Tris-HCl buffer); pH 10 (sodium carbonate buffer). Other conditions are the same as in Fig. 4
shown). Although the salting out increases extraction efficiency for fiber SPME, it causes blockage of the column by salt deposits with in-tube SPME. In addition, hydrophilic solvents such as methanol in the sample decrease the extraction efficiency, because the solubility of compound in the sample increases. However, the extraction efficiency is almost uninfluenced by methanol concentrations of 5% or less. The amount of compound extracted by the stationary phase is dependent on the concentration of the compound in the sample, and over 0.5 mL is necessary for the injection needle soak using a 2 mL autosampler vial. Effect of sample solution draw/eject Draw/eject volume and number of the sample solution are related to the extracted amount, and they are dependent on the capacity of the column. However, complete equilibrium extraction is generally not obtained for any of the analytes, because the analytes are partially desorbed into the mobile phase during each ejection step. Although the amount extracted increases independently on the increase of draw/ eject volume, the bandwidth extends and the peak becomes broad. The column capacity is 29.4 µL for the capillary column of 60 cm length and 0.25 mm internal diameters, and the capacity from the injection needle (10 µL) to the tip of the column becomes 39.4 µL. In our experiments, the optimum draw/eject volume was 30–40 µL for tested drugs, and the extraction efficiency did not increase even with high volumes. Although an increase in number of draw/eject cycles can enhance the extraction efficiency, peak broadening is often observed in this case. In addi-
Fig. 6 Effect of draw/eject cycle on the in-tube SPME of several compounds with Omegawax. Further conditions are the same as in Fig. 4
tion, the draw/eject speed corresponds to the agitation speed of fiber SPME, and the extraction efficiency increases with speed. However, the optimal flow-rate of draw/ eject cycles was 50–100 µL min–1 in our experience. Below this level, extraction required an inconveniently long time, and above this level, bubbles form inside the capillary and extraction efficiency was reduced. Ideally, the sample solution draw/eject should be carried out once the compound has reached distribution equilibrium, in order to obtain the maximum extraction amount. However, it is possible to end the extraction before equilibrium to reduce the analysis time, if sufficient sensitivity is obtained. The extraction time depends on volume, speed, and cycle of the draw/eject, and these conditions must be fixed to obtain a quantitative reproducibility. As shown in Fig. 6, the extraction equilibrium can nearly be reached with a draw/eject cycle over 10–15 times at 100 µL min–1 for a 30 µL sample, although it depended on the compound. Desorption of compounds from the capillary There are two methods for the desorption of the compound adsorbed to the capillary column: (i) the dynamic method, which desorbs into the flow of the mobile phase, and (ii) the static method, which desorbs into the solvent aspirated from the outside. Static desorption is preferably used when the analytes are more strongly adsorbed to the capillary coating. In either case, it is necessary to carry out the quick and perfect desorption with a minimum volume of solvent. For the capillary column of 60 cm length and 0.25 mm internal diameters, the desorption is usually carried out by aspirating 40 µL considering the capacity of the column. For the static method, it is also necessary to consider the compound’s solubility and miscibility with
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the mobile phase. For the dynamic method, it is possible to directly desorb to the mobile phase flow after switching the six-port valve. Although carryover may be observed after the analysis of highly concentrated samples, it is possible to wash the injection needle and the capillary column by rinsing with methanol and the mobile phase several times prior to the next analysis via the draw/eject mode. Therefore, carryover with in-tube SPME is lower or eliminated in comparison with fiber SPME. Furthermore, it is possible that these conditioning, extraction, and desorption operations are automatically carried out by the overall injection program.
Applications of the in-tube SPME technique Most of the on-line in-tube SPME methods reported previously were applied to the extraction and concentration of various polar and thermolabile compounds using com-
mercial autosamplers without modification of the autosampler itself. In the FAMOS autosampler from LC Packings, the sampling needle and the extraction capillary are permanently located externally to the normal flow path of the mobile phase through the system. In the Agilent 1100 autosampler, the sampling needle and the extraction capillary are alternatively located in the mobile phase flow path or externally to it, depending on the position of the six-port valve. In the current work, the technology is implemented on the Agilent type. On the other hand, Saito et al. [61, 73] constructed an in-tube SPME system using two Microfeeder MF-2 pumps equipped with MS-GAN microsyringes. To establish the in-tube SPME method, extraction and desorption parameters were optimized. These included capillary column stationary phase selection, sample pH, extraction flow-rate and number and volume of draw/eject steps, desorption solvent, and flow-rate. Both standard and capillary LC formats were also employed and separation conditions were optimized. These
Table 2 Applications of in-tube SPME technique for various samples Analyte (1) Environmental analysis Pesticides: Phenylureas and carbamates Carbamates Phenoxy acid herbicides Aromatic compounds: PAHs, aromatic amines BTEX, phenols Endocrine disruptors: Phthalates Phthalates and phenols Organometallic compounds: Trimethyllead and triethyllead Organiarsenic compounds Inorganic compounds: Inorganic anions
Matrix
Capillary
Detection
Reference
Water Tap water, surface water River water
Omegawax 250 Omegawax 250 DB-WAX
HPLC-UV HPLC-UV LC-MS
[54] [54, 56, 57] [58]
Tap water, lake water Water
PPY-coated BP-20 PEG
HPLC-UV GC-FID
[59] [60]
Waste water River water
Fiber-packed PEEK Supel-Q PLOT
HPLC-UV HPLC-UV
[61] –
Tap water Tap water
Supel-Q PLOT PPY-coated
LC-MS LC-MS
[62, 63] [64]
Tap water
PPY-coated
IC-CD
[65]
Urine, serum Urine, serum Serum
Omegawax 250 PPY-coated MIP-packed
LC-MS LC-MS HPLC-UV
[66, 69] [67] [68]
(2) Clinical and forensic analysis Antihypertensives: β-Blockers β-Blockers Propranolol Stimulants: Amphetamines and derivatives Amphetamines and derivatives Other drugs: Ranitidine (antihistaminics) Tricyclic antidepressants Tricyclic antidepressants Benzodiazepines
Urine Urine, hair
Omegawax 250 PPY-coated
LC-MS LC-MS
[69, 70] [71]
Urine Urine Urine Serum
Omegawax 250 Wire-packed DB-1 Fiber-packed DB-5 Supel-Q PLOT
LC-MS HPLC-UV CE-UV LC-MS
[72] [73] [74] [75]
(3) Food analysis Heterocyclic amines Phthalates and phenols Catechins and caffeine Isoflavones
Meat Packaged foods Tea, juice, wine Beans
Omegawax 250 Supel-Q PLOT PPY-coated Supel-Q PLOT
LC-MS HPLC-UV LC-MS HPLC-UV
[76] [77] [78] –
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in-tube SPME techniques were mainly coupled with HPLC-UV and LC-MS. In addition, the coupling with ion chromatography with conductivity detection (IC-CD) [65] and GC with flame ionization detection (GC-FID) [60] were reported. Applications of in-tube SPME methods for the analysis of various samples are listed in Table 2 , according to the compound type, sample matrix type, extraction capillary, and analytical technique. Application to environmental analysis Pesticides Pesticides are used in agriculture on a large scale worldwide, and their pollutants are spread throughout ecosystems by leaching and runoff from soil into groundwater and surface water. Therefore, simple and rapid methods for the analysis of these pesticides in environmental samples are required. Sample preparation is particularly important for the extraction of traces of these compounds from aqueous medium, because environmental water samples are too diluted and too complex. Eisert and Pawliszyn [54] first applied in-tube SPME technique for the effective sample preparation of six phenylureas and eight carbamates that are used as biocides for industrial and other applications, and in household products. They constructed an in-tube SPME system using a FAMOS autosampler and Omegawax 250 as the extraction device, and determined these pesticides by coupling with HPLC-UV. The precision of this method was below 6% RSD and good linearity was obtained over a concentration ranging of 10–1000 ng mL–1 for all investigated pesticides. The de-
Fig. 7a–d Chromatograms of extraction and analysis of six carbamates using the in-tube SPME-HPLC method with four concentrations: a 5, b 10, c 100, and d 1000 µg mL–1. In-tube SPME conditions: capillary, Omegawax 250 (60 cm × 0.25 mm i.d., 0.25 µm film thickness); draw/eject cycle, 15; draw/eject volume, 25 µL. HPLC conditions: column, Nova-Pak C18 (10 cm × 8 mm i.d., 4 µm particle size); column temperature, 25 °C; mobile phase, acetonitrile/water (50:50); flow-rate, 1.4 mL min–1; detection, 220 nm. (Reprinted with permission from Gou et al. [55]. Copyright 2000 Elsevier Science Ltd.)
tection limit was below 5 ng mL–1. Furthermore, Gou et al. modified this method using an Agilent autosampler, and applied it to the determination of six carbamates in tap water and surface water samples [55, 56, 57]. As shown in Fig. 7, these compounds were completely separated and gave good response at different concentrations. In addition, the detection limits were reduced by a factor of >20 by the introduction of capillary LC. Takino et al. [58] developed an in-tube SPME-LC-MS method for the determination of six chlorinated phenoxy acid herbicides, which have low mammalian toxicity and teratogenic effects in rodents due to their impurities and high dosages. The herbicides and their metabolites were detected in surface water and groundwater. These herbicides were analyzed by HPLC with negative ion mode electrospray ionization (ESI)-MS after extraction by in-
Fig. 8A–D Scanning electron micrographs of the PPY-coated capillary (A) and (C) and host silica capillary (B) and (D). A and B are cross-sectional views, C and D are enlargements of the inner surfaces. (Reprinted with permission from Wu and Pawliszyn [25]. Copyright 2001 Elsevier Science Ltd.)
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tube SPME. The optimum extraction conditions were 25 draw/eject cycles of 30 µL of sample in 0.2% formic acid (pH 2) at a flow-rate of 200 µL min–1 using a DB-WAX capillary. The herbicides extracted by the capillary were easily desorbed with acetonitrile (10 µL) by a static desorption technique. Using this method, good linearity in the range 0.05–50 ng mL–1 and high sensitivity with a detection limit of 0.02–0.06 ng mL–1 were obtained, and it was successfully applied to the analysis of river water samples without interferences by selected ion monitoring (SIM).
rently used commercial capillary coatings, especially for PAHs and polar aromatics due to the increasing π–π interactions, interactions by polar functional groups, and hydrophobic interactions between the polymer and the analytes. This method was successfully applied to the analysis of tap water and lake water samples. Tan et al. [60] reported a combination of an in-tube SPME technique and GC analysis. Although this approach may be referred to as an in-tube SPME, it is basically similar to the open-tubular trapping coupled on-line with GC [50, 51, 52, 53]. The technique was evaluated using a mixture of BTEX (benzene, toluene, ethylbenzene, and xylenes) in water.
Aromatic compounds Recently, Wu et al. developed an in-tube SPME technique using a new capillary column coated with polypyrrole (PPY) polymer on the inner wall of a fused-silica capillary [25]. PPY-coated capillary was easily prepared by an oxidative polymerization method. Figure 8 shows the scanning electron micrographs of inner surfaces of a PPYcoated capillary and host capillary. The porous structures significantly increase the effective surface areas of the films, and therefore higher extraction efficiencies can be expected in comparison with non-porous films. Using the PPY-coated capillary in-tube SPME-HPLC method, three groups of aromatic compounds were examined, which included a group of model compounds containing both polar and non-polar aromatics, a group of 16 polycyclic aromatic hydrocarbons (PAHs), and a group of six heterocyclic aromatic amines [59]. As shown in Fig. 9, PPY coating shows higher extraction efficiency than the cur-
Fig. 9 HPLC-UV chromatograms of polar and non-polar aromatic compounds by standard direct injection (10 µL), non-coated host silica capillary in-tube SPME, PPY-coated capillary in-tube SPME, and in-tube SPME with some commercial capillaries. Intube SPME conditions: draw/eject cycle, 15; draw/eject volume, 30 µL; flow-rate, 100 µL min–1. HPLC conditions: column, Hypersil BDS C18 (5.0 cm × 2.1 mm i.d., 3 µm particle size); column temperature, 25 °C; mobile phase, acetonitrile/water (40:60); flowrate, 0.2 mL min–1; detection, 200 nm for the first seven min and then changed to 219 nm for the rest of the run. Peaks: 1 phenol (400 ng mL–1), 2 dimethyl phthalate (200 ng mL–1), 3 benzene (1000 ng mL–1), 4 diethyl phthalate (200 ng mL–1), 5 toluene (1000 ng mL–1), 6 naphthalene (200 ng mL–1)
Endocrine disruptors Endocrine disruptors have been reported to contribute to the adverse health, reproduction, and developmental effects in humans and wildlife, such as abnormal reproductive function. Therefore, the existence of these compounds has become a serious social problem in recent years. Especially, the occurrence and toxicity of phthalate esters, bisphenol A (BPA), and alkylphenols have received par-
Fig. 10A–E HPLC-UV chromatograms of phthalate esters, BPA, and alkylphenols by in-tube SPME-HPLC. A Standard solution, B river water, C river water spiked with standard mixture, D lake water, E lake water spiked with standard mixture. In-tube SPME conditions: capillary, Supel-Q PLOT (60 cm × 0.32 mm i.d., 12 µm film thickness); draw/eject cycle, 20; draw/eject volume, 40 µL; flow-rate, 100 µL min–1. HPLC conditions: column, Hypersil ODS C18 (12.5 cm × 4.0 mm i.d., 5 µm particle size); column temperature, 40 °C; mobile phase and flow-rate, programmed by linear gradient of acetonitrile/water from 65% to 75% at 1.5 mL min–1 for a 5 min run, from 75% to 95% at 2.0 mL min–1 for a 5 min run and held 95% at 2.0 mL min–1 for 2 min; detection, 225 nm. Peak: 1 BPA (50 ng mL–1), 2 diethyl phthalate (50 ng mL–1), 3 di-n-propyl phthalate (50 ng mL), 4 benzyl n-butyl phthalate (50 ng mL–1), 5 di-n-butyl phthalate (50 ng mL–1), 6 octylphenol (50 ng mL–1), 7 nonylphenol (50 ng mL–1), 8 di-n-amyl phthalate (100 ng mL–1), 9 dicyclohexyl phthalate (100 ng mL–1), 10 di-n-hexyl phthalate (100 ng mL–1), 11 di-2-ethlyhexyl phthalate (500 ng mL–1), 12 di-n-octyl phthalate (500 ng mL–1)
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ticular attention, because these chemicals are widely used in our living environment. Recently, Saito et al. [61] reported a unique approach using an in-tube SPME technique with a PEEK tube packed with fibrous rigid-rod heterocyclic polymer, Zylon®. Using this fiber-in-tube SPME method coupled with HPLC-UV, n-butyl phthalate was analyzed in wastewater at sub-ng mL–1 levels. The pre-concentration factor was about 160 at 20 min extraction in comparison with direct injection of a wastewater sample, and no interference peak was observed in the chromatograms. Recently, Kataoka et al. developed a fully automated on-line in-tube SPME method for the simultaneous determination of phthalate esters, BPA, and alkylphenols in river and lake water. The optimum extraction conditions were 20 draw/eject cycles of 40 µL of sample at a flowrate of 100 µL min–1 using a Supel-Q PLOT capillary. As shown in Fig. 10A, phthalate esters, BPA, and alkylphenols concentrated by in-tube SPME were well separated within 12 min. The in-tube SPME-HPLC method from extraction to data analysis was automatically accomplished within 35 min, and about 40 samples per day were also possible by all-night operation. The in-tube SPME method gave 18–125 times higher sensitivity than the direct injection method. Using this method, good linearity in the range 1–500 ng mL–1 and high sensitivity with a detection limit of 0.1–4.0 ng mL–1 were obtained. To apply this method to environmental samples, surface waters from the Asahi River and the Kojima Lake in Okayama (Japan) were collected in cleaned glass bottles from the shoreline. After removal of insoluble materials such as clay and silt by centrifugation, the supernatant was directly used for the in-tube SPME-HPLC analysis. As shown in Fig. 10B–E, this was successfully applied to the analysis of river water samples without interferences. The recoveries of these compounds spiked to several environmental water samples were in the rage of 71–109%, and the relative standard deviations of three replicate analyses were in the range of 0.7–11.3%. Organometallic and inorganic compounds Mester et al. [62, 63] described an in-tube SPME system for the extraction of organoleads from aqueous samples and evaluated different coatings for extraction efficiency. Trimethyllead and triethyllead were analyzed by HPLC with positive ion mode ESI-MS (SIM) after extraction by in-tube SPME, and the detection limit was 11.3 and 12.6 ng mL–1, respectively. In-tube SPME has also been applied to the determination of selenomethionine and selenoethionine in several biological tissues [26]. Wu et al. [64] applied an in-tube SPME technique by using PPY-coated capillary to the determination of four organoarsenicals. The extraction efficiency of PPY to the four arsenic compounds followed the order of monomethylarsonic acid > dimethylarsonic acid > arsenobetaine > arsenocholine. These results demonstrated the selectivity of PPY for anionic compounds. The inherent an-
ion exchange property of PPY was also used for the extraction of inorganic anions such as arsenate, selenite, and selenate [65]. Chloride and sulfate contents in tap water were evaluated directly by coupling PPY-coated capillary in-tube SPME to ion chromatography with conductivity detection (IC-CD) [65]. Application to clinical and forensic analysis Antihypertensives β-Adrenoceptor blocking drugs (β-blockers) are therapeutic drugs for circulatory system disease such as hypertension, angina pectoris, and arrhythmia, and the use of these drugs as doping agents is banned by the International Olympic Committee. The development of a simple and rapid analysis method is desired for clinical control, doping inspection, and forensic chemistry, because the toxicity is strong and the threshold value between therapeutic dose and toxic dose is narrow. Kataoka et al. [66, 69] developed an automated in-tube SPME coupled with LCESI-MS (positive ion mode, SIM) for nine β-blockers. The optimum extraction conditions were 15 draw/eject cycles of 30 µL of sample in Tris-HCl buffer (pH 8.5) at a flow-rate of 100 µL min–1 using an Omegawax 250 capillary. The β-blockers extracted in the capillary were easily desorbed into the mobile phase by dynamic desorption technique. Using this method, the detection limit was 0.1–1.2 ng mL–1 (S/N=3), and the linearity was at the range 2–100 ng mL–1. This method can be directly applied to diluted urine and ultrafiltered serum without interferences. Recoveries of β-blockers added to urine and serum samples were higher than 71%, and gave reproducible determinations with a relative standard deviation at 7.6% or less. Furthermore, this method can be successfully applied to the determination of propranolol and its metabolites in serum of a patient administered with propranolol (Fig. 11). On the other hand, the development of extraction phases better suited to extraction of relatively polar compounds from aqueous samples will enhance the sensitivity and overall utility of the method. Initial steps in this direction have been shown by Wu et al. with the development of PPY polymers [67]. Their superior performance in the analysis of β-blockers, relative to the initial publication [66, 69] hold promise for the improved and broader application of the method in the future. PPY-coated capillary in-tube SPME coupled with LC-ESI-MS was successfully applied to the analysis of biological samples. Recently, Mullett et al. [68] synthesized a molecularly imprinted polymer (MIP) material for use as an in-tube SPME adsorbent. The inherent selectivity and chemical and physical robustness of the MIP material was demonstrated as an effective stationary phase material for intube SPME. Using a PEEK tube packed with MIP particles, an automated on-line in-tube SPME-HPLC system was developed for the selective analysis of propranolol in serum samples. Pre-concentration of the sample by the
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Stimulants
Fig. 11A, B Total ion and SIM chromatograms obtained from standard propranolol and its metabolites, and a clinical serum sample by in-tube SPME-LC-MS-SIM. A Standard solution containing 200 ng mL–1 propranolol, 50 ng mL–1 4-hydroxypropranolol and 7-hydroxypropranolol, 20 ng mL–1 5-hydroxypropranolol and N-desisopropylpropranolol. B Clinical serum sample (100 µL). Intube SPME conditions: capillary, Omegawax 250 (60 cm × 0.25 mm i.d., 0.25 µm film thickness); draw/eject cycle, 15; draw/eject volume, 30 µL; flow-rate, 100 µL min–1; sample pH, 8.5 (100 mM Tris-HCl). LC conditions: column, Hypersil BDS C18 (5.0 cm × 2.1 mm i.d., 3 µm particle size); mobile phase, acetonitrile/ methanol/water/acetic acid (15:15:70:1); flow-rate, program from 0.25 to 0.45 mL min–1 for a 20 min run. ESI+-MS conditions: nebulizer gas N2, 40 psi; drying gas N2, 10 L min–1 at 350 °C; fragmentor voltage, 70 V; capillary voltage, 3500 V. Peaks: 1 5-hydroxypropranolol (m/z 276), 2 4-hydroxypropranolol (m/z 276), 3 7-hydroxypropranolol (m/z 276), 4 N-desisopropylpropranolol (m/z 218), 5 propranolol (m/z 260). (Reprinted with permission from Kataoka et al.[66]. Copyright 1999 American Analytical Chemistry)
MIP adsorbent increased the sensitivity, yielding a limit of detection of 0.32 µg mL–1 by UV detection. Excellent method reproducibility (RSD 500 injections) were observed over a fairly wide linear dynamic range (0.5–100 µg mL–1) in serum samples.
Amphetamine (AM) and methamphetamine (MA) are powerful stimulants of the central nervous system and are frequently abused by athletes, drug addicts, and recreational users. Their methylenedioxy analogues, such as 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethamphetamine (MDEA), are also abused to enhance sociability and liberate inhibitions, allowing the user to experience feelings of euphoria. Overdose of these compounds often causes hallucination, paranoid delirium, seizures, coma, or even death. AM, MA, and their methylenedioxy analogues are classified as controlled or illicit drugs in many countries. Recently, the number of requests for routine tests has increased with the sudden increase in abuse of this drug, and a convenient and rapid method for the determination of these compounds is required. Kataoka et al. [69, 70] developed an automated in-tube SPME coupled with LC-ESI-MS (positive ion mode, SIM) of AM, MA, and their methylenedioxy analogues. The optimum extraction conditions were 15 draw/eject cycles of 35 µL of sample in Tris-HCl buffer (pH 8.5) at a flow-rate of 100 µL min–1 using a Omegawax 250 capillary. The stimulants extracted in the capillary were easily desorbed with mobile phase by a dynamic desorption technique. Using this method, the detection limit was 0.2–0.8 ng mL–1 (S/N=3), and linearity was obtained in the range of 2– 100 ng mL–1. This method can be directly applied to diluted urine samples without interferences. Recoveries of stimulants added to urine samples were over 80%, and it was possible to reproducibly analyze with a relative standard deviation of 7.9% or less. In addition, the within-day and between-day variations for analysis of spiked urine samples were 0.9–3.0 and 2.1–6.0%, respectively. Furthermore, Wu et al. [71] improved the extraction efficiency of this method by using a PPY-coated capillary. PPY-coated capillary in-tube SPME coupled with LC-ESI-MS was successfully applied for the analysis of urine and hair samples. Other drugs Kataoka et al. [72] developed an automated in-tube SPME coupled with LC-ESI-MS (positive ion mode, SIM) of ranitidine, which is a histamine H2-receptor blocker widely used for the treatment of stomach and duodenal ulcers and acute and chronic gastritis. The optimum extraction conditions were 10 draw/eject cycles of 30 µL of sample in Tris-HCl buffer (pH 8.5) at a flow-rate of 100 µL min–1 using a Omegawax 250 capillary. The stimulants extracted in the capillary were easily desorbed with methanol (40 µL) by a static desorption technique. Using this method, the detection limit was 1.4 ng mL–1 (S/N=3), and linearity was obtained at the range 5–1000 ng mL–1. This method was directly applied to tablet and urine samples without interferences. Recoveries of ranitidine added to the tablet and urine samples were over 92 and 58%, re-
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spectively, and analysis was reproducible with relative standard deviation of 0.7–3.4%. In addition, the withinday and between-day variations for analysis of spiked urine samples were 2.5 and 6.2% (n=5), respectively. Saito et al. [73] have recently described an interesting innovation for in-tube SPME, where a fine wire is incorporated in the lumen of the extraction capillary, to effectively increase the surface-to-volume ratio in the analysis, which is thought to limit the extraction efficiency. Using this wire-in-tube SPME method coupled with microcolumn HPLC, four tricyclic antidepressants (amitriptyline, imipramine, nortriptyline, and desipramine) in urine samples were determined in the 5–500 ng mL–1 range by UV detection. The pre-concentration factors for desipramine, nortriptyline, imipramine, and amitriptyline were about 14.7, 17.5, 52.5, and 110, respectively, in comparison with direct injection. More recently, Jinno et al [74] have developed an on-line interface between the fiber-in-tube SPME and capillary electrophoresis (CE), and the preconcentration and separation of the above four tricyclic antidepressant drugs were performed with the hyphenated system. Yuan et al. [76] reported an automated in-tube SPME coupled with LC-ESI-MS (positive ion mode, SIM) of seven benzodiazepines, which are frequently used in clinical practice as tranquilizers, sleep inducers, antiepileptic hypnotics, anticonvulsants, and muscle relaxants. The optimum extraction conditions were 10 draw/ eject cycles of 30 µL of sample in Tris-HCl buffer (pH 8.5) at a flow-rate of 300 µL min–1 using a Supel-Q PLOT capillary. The benzodiazepines extracted in the capillary were easily desorbed with mobile phase by the dynamic desorption technique. Using this method, the detection limits of these compounds were 0.02–2 ng mL–1 (S/N=3), and linearity was obtained over the range 0.5–500 ng mL–1. This method was directly applied to urine and serum samples without interferences. Recoveries of benzodiazepines added to urine and serum samples were over 75 and 35%, respectively, and reproducible analysis was performed with a relative standard deviation below 10%. Application to food analysis Food contaminants Kataoka et al. [76] developed an automated in-tube SPME method coupled with LC-ESI-MS (positive ion mode, SIM) for potent mutagenic and carcinogenic heterocyclic amines, which are detected in the atmosphere, rivers, food, etc. The optimum extraction conditions were 10 draw/ eject cycles of 30 µL of sample in Tris-HCl buffer (pH 8.5) at a flow-rate of 100 µL min–1 using a Omegawax 250 capillary. The heterocyclic amines extracted in the capillary were easily desorbed with the mobile phase by a dynamic desorption technique. Using this method, the detection limits of these compounds were 0.2–3.1 ng mL–1 (S/N=3), and linearity was obtained over the range 5–200 ng mL–1. This method was successfully applied to food samples without interferences. Figure 12 shows the
Fig. 12A–C SIM chromatograms obtained from grilled beefsteak sample by in-tube SPME-LC-MS-SIM. A m/z 214, B m/z 199, and C m/z 225. Grilled beefsteak was treated with Blue-rayon before in-tube SPME. In-tube SPME conditions: capillary, Omegawax 250 (60 cm × 0.25 mm i.d., 0.25 µm film thickness); draw/eject cycle, 10; draw/eject volume, 30 µL; flow-rate, 100 µL min–1; sample pH, 8.5 (100 mM Tris-HCl). LC conditions: column, Supelcosil LC-CN (3.3 cm × 4.6 mm i.d., 3 µm particle size); mobile phase, 15% acetonitrile/methanol (4:1) + 85% 0.1 M ammonium acetate (pH 7.0); flow-rate, program from 0.2 to 0.8 mL min–1 for a 20 min run. ESI+-MS conditions: fragmentor voltage, 90 V. Other LC-MS conditions are the same as in Fig. 11. (Reprinted with permission from Kataoka and Pawliszyn [76]. Copyright 1999 Vieweg Publishing)
chromatogram obtained from cooked beefsteak. Heterocyclic amines in the beefsteak were extracted in blue rayon (the rayon which fixed copper phthalocyanine trisulfonic acid from Funakoshi Pharmaceutical, Tokyo, Japan), and then applied to the in-tube SPME-LC-MS system. Although an unknown peak was detected in the SIM chromatogram, 2-amino-3,8-dimethylimidazo[4,5f]quinoxaline (MeIQx), 2-amino-3-methyl-3H-imidazo[4,5f]quinoline (IQ), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were selectively detected in grilled beefsteak at the 1.15, 0.25, and 1.18 ng g–1, respectively. In addition, Kataoka et al. [77] analyzed endocrine disruptors related to plastics by the in-tube SPME-HPLCUV method described above in the environmental analysis section. This method was applied to the migration test from plastics and the analysis of food samples. The results indicated that phthalate esters and alkylphenols migrated
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easily from wrap film made of poly(vinylidene chloride) and poly(vinyl chloride) glove to fatty foods, and the pollutants from these compounds can be spread in various foods. Food components Wu et al. [78] applied an in-tube SPME technique using PPY-coated capillaries to the determination of five catechins and caffeine. Catechins were determined in both negative and positive ion mode ESI-MS after separation by in-tube SPME-HPLC. Caffeine could only be determined under positive ion mode ESI-MS conditions. The detection limits of catechins and caffeine were
