Volume Overload Cleanup: An Approach for On-Line SPE-GC, GPC

A new concept for cleanup, based on volume overloading of the cleanup column, has been developed for on-line coupling of gel permeation chromatography...
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Anal. Chem. 2007, 79, 7975-7983

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Volume Overload Cleanup: An Approach for On-Line SPE-GC, GPC-GC, and GPC-SPE-GC Henk Kerkdijk,*,† Hans G. J. Mol,‡ and Bart van der Nagel

Department of Analytical Research, TNO Quality of Life, Utrechtseweg 48, 3704 HE Zeist, The Netherlands

A new concept for cleanup, based on volume overloading of the cleanup column, has been developed for on-line coupling of gel permeation chromatography (GPC), solidphase extraction (SPE), or both, to gas chromatography (GC). The principle is outlined and the applicability demonstrated by the determination of pesticide residues in food matrixes using integrated and automated cleanupGC-MS. Compared to conventional approaches for online cleanup-GC, the new technique involves introduction of much smaller volumes (e.g., 2-20 µL) into the GC without sacrificing method LODs. The much smaller injection volumes involved greatly simplify on-line coupling, improve robustness, and increase attractiveness for implementation in routine laboratories. Analytical methods for trace analysis which are based on gas chromatography usually involve a cleanup step. This is required for two reasons. The first is that GC systems tolerate only small amounts of nonvolatile material to be introduced. Repetitive injection of such material would result in a rapid deterioration of the GC performance. Second, the resolving power of the GC system is not always sufficient to allow adequate determination of the analytes. Cleanup during sample preparation solves the above issues but can be tedious. It may also be a limiting factor in sample throughput and adversely affect sample(extract) integrity and method accuracy. For this reason, there is an ongoing interest in the automation of cleanup procedures, preferable in closed systems, i.e., on-line coupling of cleanup with GC. Since the mid eighties, scientists have been investigating ways to couple liquid chromatography (e.g., LC, GPC, SPE) on-line to gas chromatography. The main problem which needs to be overcome is a proper and quantitative introduction of the relatively large liquid fraction from the LC system into the GC. Typical fraction volumes range between a few hundred microliters for LC up to several milliliters for GPC columns. The analytes are nonhomogeneously distributed within this volume. This is why the entire fraction needs to be transferred into the GC when accurate quantitative performance is required. Attempts have been * To whom correspondence should be addressed. E-mail: Hendrikkerkdijk@ hotmail.com. † Current address: NFI, P.O. Box 24044, 2490 AA, The Hague, The Netherlands. ‡ Current address: Rikilt Institute of Food Safety, P.O. Box 230, 6700 AE, Wageningen, The Netherlands. 10.1021/ac0701536 CCC: $37.00 Published on Web 09/27/2007

© 2007 American Chemical Society

made to diminish the volume to GC compatible sizes by using micro LC1,2 instead of conventional LC for on-line configurations. However, the attempts suffered from low sample capacity and volume loadability and lacked reproducibility and robustness.3 Consequently, development of on-line LC-GC heavily relies on robust techniques for introduction of large volumes into the GC. Several approaches for this exist which were mainly developed and fine-tuned in the late 1980s and early 1990s. For a detailed description the reader is referred to an overview by Teske et al.4 and reviews on LC-GC.5,6 Two techniques for large volume injection currently remain: on-column injection and PTV (programmed temperature vaporizer). With both techniques, the solvent is injected as a liquid and evaporated at relatively low temperature. The resulting vapors are discharged through a split exit. After elimination of the solvent, the split exit is closed and the retained analytes are transferred to the analytical column. This turns out to work well for the introduction of 10-100 µL volumes, provided that a suitable solvent is selected. Injection of larger volumes is much more complex. It is less robust and not very suitable for use in a routine environment. Despite the difficulties related to large volume injection in GC when coupling LC to GC, quite a number of papers have appeared on the subject. Examples include on-line SPE-GC for water analysis7-11 and on-line NPLC-GC for food analysis12-18 and (1) Cortes, H. J.; Richter, B. E.; Pfeiffer, C. D.; Jensen, D. E. J. Chromatogr. 1985, 349, 55-61. (2) Grob, K. In On-Line Coupled LC-GC; Huthig: Heidelberg, Germany, 1991; pp 27-69. (3) David, F.; Hoffmann, A.; Sandra, P. LC‚GC Eur. 1999, (September), 550558. (4) Teske, J.; Engewald, W. Trends Anal. Chem. 2002, 21, 584-593. (5) Hyotylainen, T.; Riekola, M.-L. J. Chromatogr., A 2003, 1000, 357-384. (6) Grob, K. J. Chromatogr., A 2000, 892, 407-420. (7) Vreuls, J. J.; Cuppen, W. J. G. M.; de Jong, G. J.; Brinkmann, U. A. Th. J. High Resolut. Chromatogr. 1990, 13, 157-161. (8) Pocurull, E.; Aguilar, C.; Borrull, F.; Marce´, R. M. J. Chromatogr., A 1998, 818, 85-93. (9) de Koning, S.; van Lieshout, M.; Janssen, H. G.; Brinkmann, U. A. Th. J. Microcolumn Sep. 2000, 12 (3), 153-159. (10) Brossa, L.; Marce´, R. M.; Borrull, F.; Pocurull, E. J. Chromatogr., A 2002, 963, 287-294. (11) Louter, A. J. H.; Ramalho, S.; Vreuls, R. J. J.; Jahr, D.; Brinkman, U. Th. J. Microcolumn Sep. 1996, 8 (7), 469-477. (12) Grob, K.; Vass, M.; Biedermann, M.; Neukom, H.-P. Food Addit. Contam. 2001, 18, 1-10. (13) Fankhauser-Noti, A.; Fiselier, K.; Biedermann-Brem, S.; Grob, K. J. Chromatogr., A 2005, 1082, 214-219.

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petrochemical analysis.19,20 A very attractive application is the online coupling of GPC to GC which is typically used to remove fats and pigments (chlorophyll). This was already recognized in the early 1990s.21,22 Coupling of GPC to GC is very challenging because of the very large volume fractions involved. After quite extensive optimization, one robust application was presented (organophosphorus pesticides in olive oil,23,24). So far, despite the advantages mentioned above, on-line coupling of SPE, GPC, or HPLC has not found widespread acceptance and implementation in routine laboratories. The main reason for this is the complexity of the introduction of large volumes of LC eluent into the GC, which forces the analytical chemist to make compromises with regard to the selection of the LC column, column dimensions (i.e., capacity), flow rates, choice of solvents, volatility range of the analytes, and system robustness. In this work, a new concept25 for cleanup coupled on-line to GC is introduced. This eliminates the need for injection of a fixed large fraction into the GC without sacrificing quantitative performance. In principle, it is even possible to perform on-line cleanupGC without large volume injection. This enables the development of more optimal on-line couplings between LC and GC. In this paper, the principle and potential are described and experimentally verified. The applicability in practice is demonstrated by the determination of pesticides in food matrixes using SPE, GPC, and combined GPC-SPE cleanup. EXPERIMENTAL SECTION Chemicals and Reagents. Stock solutions of mixtures of pesticides in ethyl acetate were obtained from C. N. Schmidt (Amsterdam, The Netherlands). Ethyl acetate and cyclohexane were pesticide grade and purchased from J. T. Baker (Deventer, The Netherlands). Sodium sulfate (p.A.) was from Merck (Amsterdam, The Netherlands). Instrumentation. To enable different cleanup configurations (SPE-GC, GPC-GC, and GPC-SPE-GC), a system was set up consisting of the following units: (i) an advanced autosampler (Climex, Separations, Hendrik-Ido-Ambacht, The Netherlands). This unit was equipped with three six-port switching valves and a tray cooler to keep the sample extracts at 5-10 °C. (ii) Sample loops of 1-7 mL, (iii) an HPLC pump (Gynkotek, model 480, Separations, The Netherlands), (iv) GPC columns in series (PLgel 5 µm, 50 Å, two 30 cm × 7.8 mm i.d., Varian, Middelburg, The Netherlands), (v) a device (PROSPEKT, Spark, Emmen, The Netherlands) for automatic exchange of SPE cartridges (10 mm (14) van der Hoff, G. R.; Hoogerbrugge, R.; Bauman, R. A.; Brinkman, U. A. T.; van Zoonen, P. Chromatographia 2000, 53, 433-438. (15) Senorans, F. J.; Villen, J.; Tabera, J.; Herraiz, M. J. Agric. Food Chem. 1998, 46, 1022-1026. (16) Boselli, E.; Grob, K.; Lercker, G. J. Agric. Food Chem. 2000, 48, 28682873. (17) van der Hoff, G. R.; Baumann, R. A.; van Zoonen, P.; Brinkman, U. A. Th. J. High Resolut. Chromatogr. 1997, 20, 222-226. (18) Perez, M.; Alario, J.; Vazquez, A.; Villen, J. J. Microcolumn Sep. 1999, 11, 582-589. (19) Beens, J.; Tijssen, R. J. High Resolut. Chromatogr. 1997, 20, 131-137. (20) Jiang, T.; Guan, Y. J. Chromatogr. Sci. 1999, 37, 255-262. (21) van Rhijn, J. A.; Tuinstra, L. G. M. Th. J. Chromatogr. 1991, 552, 517-526. (22) Grob, K.; Ka¨lin, I. J. Agric. Food Chem. 1991, 39, 1950-1953. (23) Jongenotter, G. A.; Kerkhoff, M. A. T.; van der Knaap, H. C. M.; Vandeginste, B. G. M. J. High Resolut. Chromatogr. 1999, 22, 17-23. (24) Jongenotter, G. A.; Janssen, H. G. LC‚GC Eur. 2002, (June), 338-357. (25) Kerkdijk, H. PCT Int. Appl. WO 2005/111600 A1, 24.11.2005.

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× 2 mm i.d.). The cartridges were packed with ENVI-carb 120/ 400 (Supelco, Zwijndrecht, The Netherlands) or PSA (primary secondary amine, Varian, Middelburg, The Netherlands). For transfer of 2 µL volumes into the GC, an additional nanovalve with a 2 µL internal loop was used (Valco, Switzerland). For transfer of 20 µL volumes into the GC, one of the standard six-port valves of the Climex with a 20 µL loop was used. In Figure 1 three different set ups, for on-line SPE-GC, GPC-GC, and GPC-SPE-GC are schematically shown. GC/MS Analysis. For GC/MS analysis, a model 8000-top GC equipped with a Best PTV injector, an AS800 autosampler, and a Voyager mass spectrometer (Interscience, Breda, The Netherlands) was used. The instrument was controlled by Xcaliber software. The injector was equipped with a 1 mm i.d. liner with porous sintered glass along the inner surface. Two microliter injections were performed in the cold splitless mode. Twenty microliter injections were performed in the solvent vent mode. Since the porous glass bed can retain up to 25-30 µL of liquid, the injection speed is not a critical parameter when transferring volumes of 20 µL or less. The GC was equipped with a 30 m × 0.25 mm i.d., 0.25 µm HP-5-MS column (Agilent, Amstelveen, The Netherlands). The conditions during GC/MS analysis are described below. PTV (Solvent Vent, 20 µL). The solvent was vented at 50 °C for 0.67 min using a split flow of 100 mL/min following the injection. Then the split valve was closed, and the analytes retained in the liner were transferred to the GC column by ramping the temperature at 10 °C/s to 300 °C. Total transfer time was 2.5 min after which the split was opened again. PTV (2 µL, Cold Splitless). This procedure was described above for transfer of the analytes to the GC column. GC. Helium was used as the carrier gas at a constant flow (1.5 mL/min). The temperature program was as follows: 90 °C (2 min) at 10 °C/min f 300 °C (hold 24 min). The transfer line into the MS was maintained at 300 °C. MS. Analytes were ionized by electron impact ionization (70 eV). Data acquisition was done in full scan mode (m/z 60400), after a solvent delay of 5.5 min, for 28 min. Scan time and interscan delay were 0.4 and 0.1 s, respectively, resulting in 2 scans/s. Data Handling. Masslab (Interscience, Breda, The Netherlands) and an in-house developed Excel macro were used for data handling and quantitative data evaluation. Sample Preparation. Lettuce, baby food (ready-to-eat mixture of pulses/apple/beef), wheat flour, and peanuts were purchased from local shops. Lettuce was homogenized using a Stephan food cutter. Peanuts were ground with liquid nitrogen using a Grindomix. The baby food and wheat flour were used as is. Subsamples of 25 g (12.5 g for peanut) were extracted with 50 mL of organic solvent (ethyl acetate in the case of lettuce, ethyl acetate/cyclohexane 1/1 for the other samples), after addition of 25 g of anhydrous sodium sulfate, using an UltraTurrax. After centrifugation, a 10 mL aliquot of the clear extract was transferred into a 10 mL autosampler vial. The amount of matrix equivalent in the sample extract was 0.5 g/mL (0.25 g/mL in the case of peanut). Automated Cleanup. Extracts were placed in the autosampler. The sample loop (1 or 7 mL) was filled using the syringe of

Figure 1. Schematic setup of system configurations used in this work: (A) on-line SPE-GC-MS with 2 µL injection into GC, (B) on-line GPC-GC-MS with 20 µL injection into GC, (C) on-line GPC-SPE-GC-MS with 20 µL injection into GC.

the Climex autosampler. The content of the loop was transferred to the cleanup column(s) by eluent from the HPLC pump. In the case of combined GPC-SPE cleanup, the appropriate GPC fraction of the eluent was directed to the SPE cartridge for further cleanup and then directed to the GC injection loop. The syringe of the autosampler transferred the content of the GC injection loop into the GC via a fused silica capillary mounted in the PTV injector through the septum by switching the appropriate valves. During the GC run, the tubing was rinsed by clean eluent. At the end of the GC run, at high PTV and GC temperature and in split mode, three dummy injections were performed in order to clean the fused silica transfer tube mounted into the GC. Schematic setups of the three system configurations used in this work are given in Figure 1. Quantification. For quantification purposes, the response (peak area) of one diagnostic ion for each pesticide was used. Samples were injected between matrix-matched standards, and the concentrations were calculated using the mean of the bracketing standards (concentration corresponding to 50 µg/kg, one-point

calibration). Matrix-matched standards were prepared by addition of 100 µL of concentrated mix standard solution to 10 mL of raw extract. Validation. As part of the evaluation of the system performance, a direct comparison in response of pesticides was made between a direct 20 µL injection using the standard GC autosampler and a 20 µL transfer of the appropriate fraction from the cleanup system. In the subsequent validation of the system, both standards and samples were injected through the cleanup system since in routine practice it was considered unpractical to switch between a fixed fused silica transfer line setup and normal autosampler. The GPC-SPE system was validated according to EU guideline SANCO/825.26 Five portions of homogenized sample (× 2) were spiked with a mixture of 140 pesticides, at the 10 µg/kg and 50 µg/kg levels, respectively. Together with two unfortified control (26) SANCO/825/rev. 7 17.03.2004 Guidance Document on Residue Analytical Methods http://ec.europa.eu/food/plant/protection/resources/publications_en.htm (accessed January 2007).

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Figure 2. Comparison of peak profiles of two compounds (e.g., analyte and interference) without (upper trace) and with (lower trace) volume overloading of the LC column. The block between the analyte and interference represents the preferential zone, where the analyte concentration is undiluted while the interference is not yet eluting.

portions of the sample, they were extracted, automatically cleaned, and analyzed as described above. At each level, the average recovery and repeatability (as relative standard deviation, RSD) were determined. CONCEPT OF VOLUME OVERLOAD BASED CLEANUP Analytical chromatography focuses on completely resolving the analyte(s) of interest from other compounds. Method development usually strives for obtaining the highest selectivity and plate number, i.e., well-resolved narrow peaks. The outcome is chromatograms where compounds elute as separate narrow Gaussian shaped peaks with nonhomogeneous concentration profiles (Figure 2, upper trace). A consequence of the nonhomogeneity is that in an on-line LC-GC configuration, the complete LC analyte fraction needs to be transferred into the GC to enable quantification. In essence, the newly developed technique releases this concept. With the use of optimized conditions from the analytical LC separation, the injection volume is excessively increased. This results in volume overloading of the column. In itself, this is a well-known approach used in preparative chromatography to isolate and collect a pure substance. The use here as an on-line analytical cleanup step is new but follows the same basic principles.27 The injection volume into the LC is increased until the elution profile of the analyte loses its Gaussian shape and changes into a frontal peak with a horizontal plateau (Figure 2, lower trace). In the ideal situation, the increase in sample volume does not affect the distribution coefficient and the diffusion characteristics of compounds. This supposes that no mass overloading27 of the column occurs. The increased sample volume can also be seen as a number of consecutive analytical injections. Adding up the resulting Gaussian peaks gives the integrated frontal peak with its horizontal plateau. From this it can be derived that it takes approximately one analytical peak width to reach the horizontal plateau. The main advantage of a volume overload separation is that at the horizontal plateau, the analyte concentration is undiluted, (27) Scott, R. P. W.; Kucera, P. J. Chromatogr. 1976, 119, 467.

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constant, and equal to the concentration of the injected sample (visualized in Figure 2). Because of this, any fraction of the plateau can be transferred into the GC while still allowing a quantitative determination of the analyte concentration in the sample. At first glance, a serious drawback of the increase in sample volume seems to be the loss in resolution and therefore cleanup potential. In practice, this is usually not an issue since the retention time start of every compound and the absolute differences in retention (start and end) between compounds remain the same when shifting from analytical to volume overload chromatography. For baseline separated compounds, a shift from analytical to volume overload results in a separation which contains a zone where one of the compounds (i.e., analyte) is at its horizontal plateau while the other (interfering matrix) is not yet present. This undiluted cleaned zone is the fraction of interest that is partially transferred into the GC. We call this the preferential zone. The main advantage is that, within this zone, the volume that needs to be transferred into the GC is not critical with respect to quantitative performance. This gives us maximum freedom in selection of GC inlet parameters. The mandatory injection of very large volumes in conventional on-line cleanup-GC approaches has been eliminated this way. Within the preferential zone, any volume can be selected for transfer into the GC. In practice, the volume will be determined by the concentration of the analyte in the raw extract, the sensitivity of the detector, and the required limit of detection. To allow on-line cleanup-GC analysis based on volume overload, the following conditions need to be met: (i) The composition of the extract solvent and the mobile phase of the cleanup-LC system should be the same. (ii) With the use of the above solvent composition and an appropriate stationary phase (SPE, LC, or GPC), the analyte(s) of interest should have different retention behavior than the interfering compound(s). Furthermore, the analyte(s) of interest should elute within a practical time. The latter depends on retention, column dimensions, and flow rate. (iii) The sample must be introduced, and the analytes must elute under isocratic conditions. Optionally, a gradient elution step can be used after transfer of the analytes into the GC. (iv) Mass overloading of the column by analytes or matrix constituents should be avoided. This could affect the retention behavior of the compounds on the column. In that case, a direct translation from analytical to overload conditions cannot always be made, and a reoptimization of retention windows may be required. Mass overload is more likely to occur in adsorption chromatography (NPLC) and less likely in GPC. EXAMPLE APPLICATIONS The concept and potential are further illustrated by a number of theoretical examples. In Figure 3 (top figure) an analytical chromatogram is shown for an analyte and two interfering peaks, one of them just baseline resolved. The objective is to determine the analyte by on-line LCGC after elimination of the interference. This can be achieved by changing to a volume overload situation. Here the preferential zone starts at the end of the first eluting interference peak. The maximum volume of the preferential zone is the difference between the end of the interference peak and start of the analyte peak in the conventional chromatogram. A fraction of this zone can be injected into the GC and used to determine the analyte concentration in the sample.

Figure 3. Three different possibilities where volume overload injection allows generation of cleaned undiluted zones in the elution profiles. In each figure the upper trace is the conventional situation and the lower trace the volume overload situation. For detailed explanation, see text.

In Figure 3 (middle figure), two closely eluting analytes need to be quantified simultaneously while eliminating a later eluting interference. In this case, the sample volume is increased until the horizontal plateaus of both analytes overlap. The resulting preferential zone lies after the end of the second analyte in the conventional chromatogram. The maximum volume of this zone is the difference between the end of the second analyte and the start of the interfering peak. Again any fraction from this zone can be injected into the GC and used for determination of both analytes. The final example represents a multianalyte method in which a number of analytes, eluting between the first and last analyte, need to be quantified while eliminating an earlier eluting interference. This is typical for, for example, GPC. Now the sample volume needs to be increased until the horizontal plateaus of the first and last analyte, and consequently all other analytes in between, overlap. The preferential zone lies after the end of the interference. The maximum volume of the preferential zone is the difference between the analytical end of the interference peak and the start of the first analyte peak. A limitation of the overload cleanup approach is that it is not possible to remove interferences that elute in between multiple target analytes or an analyte which is sandwiched in between two barely baseline resolved interferences. The examples show that the optimal cleanup parameters depend on the objective and achieved LC separation.

RESULTS AND DISCUSSION Experimental Verification of Applicability of Volume Overload Based Cleanup. The new approach was experimentally verified. Below, three illustrative multianalyte examples for application of the technique are described. On-Line SPE-GC, Removal of Chlorophyll from Extracts of Leafy Vegetables. Extraction of leafy vegetables using ethyl acetate results in coextraction of chlorophyll. Repeated injection of the nonvolatile chlorophyll results in contamination of the GC inlet. One possibility for removal of chlorophyll is an SPE cleanup with graphitized carbon black (GCB). GCB retains compounds with a planar structure that fit into the carbon skeleton, such as chlorophyll and cartenoids. Dissolved in ethyl acetate, most pesticides do not interact with GCB. There are, however, several exceptions such as pesticides with a planar structure component.28 Examples are hexachlorobenzene (HCB), chlorothalonil, pyrimethanil, mepanipyrim, cyprodinil, and quintozene. Our aim was to use the volume overload approach for the removal of chlorophyll but not the “planar” pesticides. This would allow GCB to be used as a generic cleanup SPE phase in pesticide residue analysis. To study the retention behavior of the “nonplanar” pesticides, “planar” pesticides and chlorophyll and standard solutions and lettuce extracts were injected onto SPE cartridges dry-packed with 120/400 mesh GCB. The elution profiles were measured by connecting a UV detector to the SPE cartridge. Various flow rates, injection volumes, and mobile phase compositions were investigated. The final selected conditions were a 1 mL injection volume at a flow rate of 70 µL/min. The resulting elution profiles are presented in Figure 4. The 1 mL volume was not critical, a smaller volume would have sufficed to reach the minimum overlap requirement for planar and nonplanar pesticides. The nonplanar pesticides are virtually unretained. The retention behavior of HCB and the other planar pesticides mentioned above were found to be very similar. They are retained but less than chlorophyll. Under the conditions applied, a zone in the elution profile was indeed observed where both nonplanar and planar pesticides are at their maximum concentration (or at least >70% of that) while the chlorophyll concentration is still very low. In this zone, the extract eluting from the cartridge contains all pesticides at 70-100% of the initial concentration while chlorophyll has been removed for over 90%, demonstrating the feasibility of the approach. For the planar pesticides, the cleaned undiluted zone was approximately 100 µL in volume (1.5 min in time), indicating that transfer to the GC will be a noncritical event. Next, the cartridge was mounted in the on-line SPE-GC-MS system (see Figure 1a). The eluent was directed through a nanovalve with a 2 µL internal loop which could be switched toward either waste or the GC inlet. In the preferential zone, as established above, the content of the loop was transferred into the GC through a fused silica capillary mounted into the GC inlet. Reproducible injection of only 2 µL volumes via a loop was a challenge on its own but will not be discussed in detail here. In essence, the loop content was automatically bracketed with air plugs thereby ensuring that the entire 2 µL volume ended up in the liner of the GC inlet. Rinsing procedures of tubing ensured that carry-over between subsequent injections was less than 0.3%. (28) Mol, H. G. J.; Rooseboom, A.; Van Dam, R.; Roding, M.; Arondeus, K.; Sunarto, S. J. Anal. Bioanal. Chem. [Online early access].DOI: 10.1007/ s00216-007-1357-1.

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Figure 4. Elution profiles obtained using SPE-UV after separate injections of (i) mixture of nonplanar pesticides in ethyl acetate, (ii) hexachlorobenzene (as an example, a planar pesticides) in ethyl acetate, (iii) chlorophyll in lettuce extract in ethyl acetate. Injection volume (loop) ) 1 mL. Eluent in pump: ethyl acetate. SPE cartridge: 10 × 2 mm packed with GCB (graphitized carbon black). Flow rate: 70 µL/min. Table 1. Recoveries Obtained after On-Line SPE-GC-MS Analysis (2 µL Transfer) of Pesticides in Lettuce Extracts (0.25 µg Pesticide/mL Extract)a pesticide ethoprofos lambda-cyhalothrin HCB cyprodinil mepanipyrim pyrimethanil

% recovery (%)

RSD% (n ) 4)

Nonplanar 106 102

1 5

Planar 76 86 95 82

12 10 14 2

a Recoveries are relative to a direct 2 µL injection of a matrixmatched standard.

In Table 1, the recoveries and relative standard deviation (RSD) are given for several pesticides in lettuce extracts after on-line SPE-GC-MS analysis with 2 µL injections into the GC. Recoveries and relative standard deviations (RSDs) for all pesticides, including the planar ones, are acceptable for quantitative analysis according to EU criteria.26 On-Line GPC-GC, Removal of High Molecular Weight Compounds. A second example is the use of volume overload in GPC cleanup. GPC is a very common sample preparation procedure for GC analysis. It is used to separate high molecular weight interferences, such as fat, from the low molecular weight target analytes. As mentioned in the introduction, on-line GPC-GC using the conventional approach involves transfer of very large fraction volumes (>1 mL) into the GC which is far from straightforward. Here the new approach makes a real difference. First the proof of principle was experimentally verified by measuring elution profiles of fish oil and pesticides from the GPC system. The fish oil was diluted to 5% in the GPC mobile phase 7980

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(ethyl acetate/cyclohexane 1/1), and 5 mL was injected onto a single 30 cm × 7.8 mm GPC column. Initially, the in-line DAD detector was used to monitor the elution profiles of the fish oil and pesticides. Since the response ratios of the fish oil constituents were unknown, it was not possible to derive the percent removal of fat from this. Therefore, fractions of 1 mL were also collected. The oil content in each fraction was gravimetrically determined after evaporation of the mobile phase. For the pesticides, standard solutions were injected and the 1 mL fractions were measured by GC/MS. The reconstructed elution profiles are presented in Figure 5. An adequate separation was achieved between the large triglyceride molecules (MW typically around 800) and the small pesticide molecules like etridiazole (MW 246). The cleaned undiluted zone is easily recognized. For larger size pesticides such as deltamethrin (MW 503), the separation is more critical. This is also well-known from conventional GPC cleanup. At first sight, the deltamethrin profile seems to be very steep which potentially could make the transfer timing critical. However, the expanded section shows that up to volumes of at least 100 µL, the concentration profile is sufficiently flat to enable transfer to the GC with good repeatability. The fact that the transfer can be accurately controlled in time will also contribute to this. This was verified during validation of the on-line GPC-SPE cleanup setup (see appropriate section below). To further improve separation, two GPC columns were coupled in series. This does not only improve separation between fat and pesticides but also results in extended separation of the individual pesticides from each other. In order to maintain overlapping plateaus of the first and last target pesticide, the injection volume had to be increased to 7 mL. If the availability of extract is limited, the same optimum overlap conditions can be realized for lower injection volumes by using smaller i.d. GPC columns. Here

Figure 5. Elution profiles of triglycerides from fish oil, a high molecular weight pesticide (deltamethrin) and a medium molecular weight pesticide (etridiazole) after a 5 mL injection into a single 30 cm × 7.5 mm i.d. GPC column at 1 mL/min.

conventional 7.8 mm i.d. columns were preferred over 3.0 mm i.d. columns because they are more rugged. Combined On-Line GPC and SPE Cleanup. With the use of the on-line GPC-GC-MS system, over 50 injections were performed of a range of commodities (including cereals, baby food, peanuts,

vegetable oil) without deterioration of the performance. However, while GPC effectively removes large molecular weight compounds, smaller matrix constituents like free fatty acids remain in the eluate. They can interfere with analysis of the target compounds (see for example the upper trace in Figure 6). In off-line methods, an additional SPE cleanup is frequently used to remove such interferences. Here the possibility of SPE cleanup after the GPC cleanup was investigated. To this end, a 100 µL volume of the preferential zone leaving the GPC columns was directed to a 10 × 2 mm i.d. cartridge packed with PSA by switching the appropriate valves and decreasing the flow rate to 70 µL/min. A schematic drawing of the configuration used is given in Figure 1c. The SPE cartridge was located in a cartridge exchanger device (PROSPEKT). This allowed for the automatic exchange of cartridges for each new sample and was preferred over regeneration of one fixed SPE cartridge. While the pesticides hardly interact with PSA, free fatty acids are effectively retained and much cleaner TIC chromatograms are obtained (see Figure 6, compare lower and upper traces). For optimization of the cleanup system, elution profiles of fat and pesticides were established separately. On the basis of this, the preferential zone and valve switching times were set. With real samples, matrix and analytes are mixed and the matrix might affect the elution profiles (e.g., in the case of mass overload). In such case, there may also be differences in elution profiles between matrixes. This was verified by validating the GPC-SPE cleanup system for different matrixes. During the validation, it was not possible to verify the occurrence of any losses during the automated cleanup process because calibration standards were also analyzed using the cleanup system. To gain insight into the losses of target compounds, an additional experiment was conducted to establish absolute system recoveries. The results of this

Figure 6. GC/MS TIC chromatogram after transfer of 20 µL from the preferential zone of a peanut extract (0.25 g/mL). Upper trace: GPCGC-MS. Lower trace: GPC-SPE-GC-MS. For details, see text.

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Table 2. Recoveries of Pesticides after On-Line GPC and SPE Cleanup (20 µL Transfer into GC) Relative to a Direct 20 µL Injection by the GC Autosamplera pesticide

recovery (%)

pesticide

recovery (%)

pesticide

acrinathrin azinphos-methyl azoxystrobin benalaxyl bifenthrin biphenyl bitertanol bromopropylate bromuconazole bupirimate buprofezin badusafos captan carbaryl carbofuran chlorfenvinphos chlorothalonil chlorpropham chlorpyrifos chlorpyrifos-methyl chlorthal-dimethyl cinerin-1 cyfluthrin cyhalothrin, lambdacypermethrin cypermethrin cyproconazole cyprodinil DDE, p,p′ DDT, o,p′ DDT, p,p′ deltamethrin diazinon dichlofluanid dichlorvos dicloran

8 78 91 101 89 6 101 93 89 105 100 98 86 37 64 95 65 100 95 88 102 98 81 79 91 90 96 109 102 108 95 89 108 29 70 81

dieldrin diethofencarb difenoconazole dimethoate dimethomorph diniconazole diphenylamine DMSA DMST endosulfan-R endosulfan-β endosulfan-sulfate EPN epoxiconazole esfenvalerate ethion ethoprophos etofenprox etridiazole famoxadone fenamiphos fenarimol fenazaquin fenbuconazole fenhexamid fenitrothion fenoxycarb fenpiclonil fenpropathrin fenpropimorph fenthion fenvalerate fipronil flucythrinate fludioxonil flusilazole

114 81 85 102 97 94 103 89 82 97 108 100 77 99 97 104 92 100 78 94 87 106 101 95 13 76 93 108 110 102 96 96 36 77 110 93

flutolanil fluvalinate, τfolpet furalaxyl heptenophos hexaconazole iprodione jasmolin-1 kresoxim-methyl lindane malathion mecarbam mepanipyrim mepronil metalaxyl methidathion mevinphos mevinphos myclobutanil nitrothal-isopropyl nuarimol oxadixyl paclobutrazol parathion parathion-methyl penconazole permethrin-cis permethrin-trans phenylphenol,2phorate phosalone phosmet phosphamidon piperonyl-butoxide pirimicarb pirimicarb, desmethyl-

a

recovery (%) 96 68 44 102 95 91 87 114 103 90 93 95 99 100 87 84 82 100 84 106 98 88 77 66 97 104 97 114 102 91 78 79 85 94 104

pesticide

recovery (%)

pirimiphos-methyl procymidone profenofos propargite propiconazole propoxur propyzamide prothiofos pyrazophos pyrethrins pyridaben pyridaphenthion pyrifenox pyrimethanil pyriproxyfen quinoxyfen quintozene TDE, p,p′ tebuconazole tebufenpyrad tefluthrin tetraconazole tetradifon tolclofos-methyl tolylfluanid triadimefon triadimenol triazamate triazophos trifloxystrobin triflumizole trifluralin vinclozolin

95 94 89 97 102 79 102 102 93 65 91 87 96 106 101 92 51 104 100 99 85 93 99 100 97 96 106 92 86 93 97 90 103

Sample introduced into GPC-SPE-GC-MS system: 7 mL tomato extract in ethyl acetate/cyclohexane. Pesticide concentration was 25 ng/mL.

experiment, followed by the results of the validation, are described below. Establishment of Absolute Recoveries in On-Line GPC-SPE-GCMS. In order to establish absolute system recoveries of all the pesticides, a 20 µL injection of the preferential zone from the automated cleanup system was compared with a direct 20 µL injection into the GC/MS. For this purpose a fat-free extract (tomato) was used because direct injection of a fat-containing extract into the GC would contaminate the GC inlet and GC column. The results are presented in Table 2. For the majority of the pesticides, (131 out of 140 compounds) recoveries of 70% or better were obtained. For some of the pesticides, the concentration profiles deviated from most others. Consequently, at the conditions used, they could not be transferred at their optimum concentration. This occurred for the large molecular weight compounds acrinathrin and fipronil (plateau descending shortly after the triglycerides) and for biphenyl and quintozene (plateau concentrations not yet reached in the transfer zone due to secondary interaction, absorption, to the GPC stationary phase). Consequently, low recoveries were observed for those pesticides. For biphenyl and quintozene, this can be solved by increasing the 7982

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injection volume (or using smaller i.d. GPC columns), but this was not done here. Lower recoveries were also observed for a number of pesticides known to be less GC-amenable (i.e., carbaryl, carbofuran, folpet, dichlofluanide). This was attributed to matrix-effects, i.e., the solution introduced into the GC after cleanup was cleaner than the reference injection. In the cleaned extract, less shielding of active sites in the inlet occurred resulting in increased adsorption/ breakdown of labile compounds. This phenomenon was another reason to perform calibration based on matrix-matched standards analyzed using the automated cleanup system. Validation of On-Line GPC-SPE-GC-MS System. The on-line system combining both GPC and SPE (PSA) cleanup was considered a generic cleanup procedure for pesticide analysis in fat containing samples. The system was validated by spiking homogenized portions of several commodities, i.e., wheat flour, baby food (pulses/apple/beef mixture) and ground peanuts. This was done at two levels (10 and 50 µg/kg) in 5-fold. The samples were extracted using a mixture of ethyl acetate/cyclohexane (1/ 1). After centrifugation, 10 mL aliquots were transferred into autosampler vials and placed in the autosampler for automated cleanup and GC/MS analysis. The results are summarized in

Figure 7. Average recoveries (top) and repeatabilities (bottom) obtained after analysis of samples fortified with pesticides (see Table 2) at 10 and 50 µg/kg (n ) 5) using the on-line GPC-SPE-GC-MS system.

Figure 7. For the majority of the pesticides (same as in Table 2), adequate recoveries (70-110%) were obtained. Repeatabilities were generally below 10% for wheat and baby food and generally below 20% for peanut. CONCLUSION A new concept for cleanup, applied here for on-line combination of cleanup and chromatography, has been described. The approach was experimentally demonstrated for SPE, GPC, and combined GPC-SPE coupled to GC/MS and validated for analysis of pesticides in fat containing samples using on-line GPC-SPEGC-MS. The new approach has all the advantages of on-line systems such as automation, strong reduction of manual sample handling, and preservation of sample integrity but eliminates complexities associated with introduction of (very) large volumes into the GC. A key feature of the new approach is that cleaned undiluted fractions are obtained. Volumes that need to be

introduced into the GC can, in principle, be small and freely selected. This takes away one of the main obstacles for implementing on-line cleanup GC systems in routine laboratories. The method was successfully validated for determination of more than 100 pesticides in wheat flour, baby food, and peanuts. This first example using this technique focused on on-line cleanup and GC analysis, but there are many other applications to be explored. ACKNOWLEDGMENT We would like to acknowledge Spark Holland for their financial support and packing of the GCB and PSA cartridges. In addition, we acknowledge Valco Switzerland for donation of the nanovalve to allow small volume loop injection into the GC. Received for review January 26, 2007. Revised manuscript received June 22, 2007. Accepted July 2, 2007. AC0701536

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