University of Groningen On-line coupling of sample ... · GC transfer line, into the mass spectrometer [20,21]. This particular set-up was applied to determine volatiles of cheese

University of Groningen

On-line coupling of sample pretreatment with chromatography or mass spectrometry for high-throughput analysis of biological samplesHout, Mischa Willem Johannes van

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55COUPLING OF

SOLID-PHASE MICROEXTRACTIONAND MASS SPECTROMETRY

The pieces have been put

Non-equilibrium SPME coupled directly to MS2 for rapid bioanalysis

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5.1

Non-equilibrium solid-phase microextractioncoupled directly to ion-trap mass spectrometry forrapid analysis of biological samples*

Summary

To determine sub-ppb levels of drugs in biological samples selective, sensitiveand rapid analytical techniques are required. The present work shows thepossibilities for high-throughput analysis of solid-phase microextraction(SPME) directly coupled to an ion-trap mass spectrometer equipped with anatmospheric pressure chemical ionisation source. As no chromatographicseparation is performed, the SPME procedure is the time-limiting step.Direct-immersion SPME under non-equilibrium conditions permits thedetermination of lidocaine in urine within 10 min. After a 5-min sorption timewith a 100-µm polydimethylsiloxane-coated fiber, the extraction yield oflidocaine from urine is about 7%. When applying 4 min desorption, using amixture of ammonium acetate buffer (pH 4.5) and acetonitrile (85+15 v/v),about 10% of the analyte is retained on the fiber. An extra cleaning step of thefiber is therefore used to prevent carry-over. By use of tandem MS, no matrixinterference is observed. The detection limit for lidocaine is about 0.4 ng/ml,and intraday repeatability and interday reproducibility are within 14% over aconcentration range of 2-45 ng/ml.

*: M.W.J. van Hout, V. Jas, H.A.G. Niederländer, R.A. de Zeeuw, G.J. de Jong. Analyst 127 (2002) 355-359.

Chapter 5 – Coupling of solid-phase microextraction and mass spectrometry

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5.1.1 Introduction

High-throughput analysis is becoming increasingly important owing tothe large numbers of samples that have to be analysed. However, throughputshould not negatively affect the sensitivity and selectivity of the systems. As theconcentrations of interest are often in the (sub) ppb range, the systems mustallow fast, selective and sensitive analysis of samples. Most systems that offerthese possibilities combine sample preparation on-line with a separationtechnique and mass spectrometry. A commonly used coupled technique issolid-phase extraction (SPE) combined with liquid chromatography (LC) andmass spectrometry (MS) 1,2]. Sensitivity is obtained by the mass spectrometerand the large sample volumes in the on-line SPE procedure. The SPE proceduremight take too much time, but this can be counteracted by the use of highflow-rates and small volumes during the various steps of the procedure.Moreover, using a system that offers the possibility to perform dual SPE [3,4]may also increase the throughput. The second limiting factor is the time neededfor the separation step, which may be influenced by using short LC columns[1,5,6] or even by eliminating the separation step, i.e. direct coupling of SPE toMS [3,7-11].

Solid-phase microextraction (SPME), based on extraction by a coatedfiber, was originally designed to be combined with gas chromatography, but canalso be combined with LC. Direct immersion SPME is carried out, followed bydesorption in a desorption chamber filled with a suitable solvent to remove theanalytes from the fiber. Until now, SPME has hardly ever been used inhigh-throughput systems owing to the seemingly disadvantageouscharacteristics of SPME for such systems. Despite the various factors thatpositively influence the sorption [12-16], it is usually time consuming becauseof the slow equilibrium process. The maximum extraction yield is achievedafter equilibrium is reached between the sample and the fiber [15,17,18].Although desorption can be performed faster, this step is also an equilibriumprocess. Other limiting factors of SPME are the relatively low extraction yieldsand possible carry-over [12]. Furthermore, not as many stationary phases as forSPE are available yet, thereby limiting the choice for selectivity. However,SPME has some clear advantages [12] such as easy handling, little use ofsolvents, and little requirements regarding instruments.

Some reports [13-19] have been published concerning the use ofnon-equilibrium sorption conditions aiming for reduced analysis times.Reproducible GC analysis under non-equilibrium conditions using eitherheadspace or direct-immersion SPME has proved to be possible, provided thatthe agitation conditions and sorption time were held constant [19]. An increasein the throughput may also be achieved by direct coupling of SPME with MS,for example by using thermal desorption and direct introduction, across a short


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GC transfer line, into the mass spectrometer [20,21]. This particular set-up wasapplied to determine volatiles of cheese with headspace SPME and MS analysis[20]. In another study the desorption profiles of aromatic hydrocarbons wereinvestigated [21].

No reports are available concerning the direct coupling of SPME with MSusing an LC interface for relatively involatile compounds. The goal of thepresent work was to develop such a system that can meet the requirements ofhigh-throughput analysis. The potential of direct coupling of SPME with anion-trap mass spectrometer, equipped with an LC interface, was investigated.The required selectivity can be obtained by using multiple-stage MS (MSn withn≥2). Since the sorption and desorption processes are key factors in thethroughput, these parameters were studied and optimised for a rapid analysis ofa model substance (lidocaine) in urine.

5.1.2 Experimental

5.1.2.1 Chemicals and Instrumentation

Polydimethylsiloxane (PDMS) coated (7 and 100 µm) SPME fibers wereobtained from Supelco (Bellefonte, PA, USA). The SPME holder and fiberwere compatible with the SPME-LC desorption chamber (Supelco) [17]. AHewlett-Packard (Waldbronn, Germany) HPLC gradient pump series 1100 wasused for desorption and transport of the solvent to the mass spectrometer. Themass spectrometer was an LCQ Classic ion-trap (Thermoquest, San Jose, CA,USA) and it was equipped with an atmospheric pressure chemical ionisation(APCI) or electrospray ionisation (ESI) source. Ultrapure water was obtainedusing an Elga Maxima ultrapure water purification system (Salm & Kipp,Breukelen, The Netherlands).

Acetonitrile (Lab-Scan, Dublin, Ireland) was of HPLC quality. Aceticacid, boric acid, and sodium chloride (all of analytical-reagent grade) wereobtained from Merck (Darmstadt, Germany) and ammonium acetate (99.99%)was from Sigma-Aldrich (St. Louis, MO, USA). Stock standard solutions oflidocaine hydrochloride (USP, Holland Pharmaceutical Supply, Alphen a/dRijn, The Netherlands) were prepared in acetonitrile. Ammonium acetate buffer(10 mM) was prepared by dissolving ammonium acetate in ultrapure water andadjusting the pH to 4.5 with acetic acid (10% v/v). Buffer solutions pH 10(0.2 M) were prepared by dissolving boric acid in ultrapure water and adjustingthe pH with 1 M sodium hydroxide. Triplicate injections (50 µl) of controlsamples [24.81 ng/ml lidocaine in buffer (pH 4.5)-acetonitrile (85+15 v/v)]were used to correct for day-to-day variations in the sensitivity of the mass


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spectrometer. Analyses performed to investigate the reproducibility weresubjected to two-way analysis of variance (ANOVA) with replications.

5.1.2.2 SPME procedure

A previously optimised SPME procedure for the determination oflidocaine in urine [15] was used as a starting point for the present work. Minormodifications to the system included the change from phosphate buffer(25 mM, pH 4.0) to ammonium acetate buffer (10 mM, pH 4.5) to make thesystem compatible with MS. Furthermore, the pH of the extraction buffer wasincreased from 9.5 to 10.

Samples were prepared by diluting calf urine with 0.2 M borate buffer(pH 10) in a ratio of 1:1. Sodium chloride was added to the urine-buffer solutionto give a final salt concentration of 0.3 g/ml. The pH was readjusted to pH 10with 10% sodium hydroxide. The coated part of the fiber was completelyimmersed into 1.25 ml of sample and subsequent extraction was performedwhile continuously stirring the sample using a 7×2 mm stirring rod and amagnetic stirrer (Metrohm, Herisau, Switzerland). Desorption was performedby placing the fiber into the desorption chamber (Supelco), which was filledwith 70 µl ammonium acetate buffer (10 mM, pH 4.5)-acetonitrile (85+15 v/v).After a certain time of (static) desorption the chamber was flushed to the MSwith the same solvent at a flow-rate of 0.5 ml/min.

5.1.2.3 Mass spectrometry

Using the ion-trap mass spectrometer with an APCI source, the vaporisertemperature was set at 400ºC. During analysis with the APCI source the sheathgas and auxiliary gas (both nitrogen) were 48 and 3 (arbitrary units),respectively, and the corona discharge current was set at 5.0 µA. If ESI wasused the sheath gas and auxiliary gas were 90 and 5 (arbitrary units),respectively, and a spray voltage of 5.0 kV was used. With both sources theheated capillary was set at 140ºC and a capillary voltage of 46.0 V was applied.The tube lens offset was 45.0 V. The first octapole offset, the inter-octapoleoffset, and the second octapole offset were -2.0 V, -26.0 V, and -6.0 V,respectively. All scans were recorded in the full-scan mode with threemicroscans over the range m/z 75-300, using the positive-ion mode. Themaximum injection time was set at 300 ms. Helium was applied as cooling andcollision gas. Extracted ion chromatograms in single-MS mode were obtainedfor [M+H]+ (m/z 235 ± 0.5). For MS/MS the fragment ion m/z 86 ± 0.5 wasmonitored. The isolation width during MS/MS experiments was 2.0 Th, using acollision energy of 34%.


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5.1.3 Results and Discussion

5.1.3.1 Setting up the SPME-MS system

A fast method, based on direct-immersion SPME-MS, was obtained byconnecting the desorption chamber to the LC-MS interface. After staticdesorption a pump was used to flush the contents of the desorption chamber tothe MS. Sorption was done at pH 10, which ensured that lidocaine is in itsneutral form, thereby increasing the yield [15]. A further increase of the yieldwas obtained by the addition of 0.3 g/ml NaCl. Equilibrium was more rapidlyreached by stirring of the sample [15,16]. The reproducibility of the extractionwas increased by dilution of urine with buffer (pH 10) and addition of saltalmost to saturation. An existing method [15] for lidocaine in urine involvingSPME-LC and diode-array detection used phosphate buffer (pH 4) duringdesorption (2×10 min). Since this buffer is not compatible with MS, a morevolatile ammonium acetate buffer at a similar pH was used. Desorption atpH 4.5 allowed lidocaine to become redissolved as the protonated base, sincethe pKa value of lidocaine is 7.9. To enhance the desorption of lidocaine fromthe fiber, 15% acetonitrile was added to the desorption solvent [15]. Moreacetonitrile can be advantageous for analysis by MS, since the desolvatationwill be more efficient [23,24]. However, the amount of acetonitrile is limited,since too much acetonitrile implies the possibility of stripping the fiber uponwithdrawal from the desorption chamber.

Both APCI and ESI are suitable for the analysis of lidocaine. ESI behavesas a concentration-sensitive detector, thus lowering the flow-rate will lead tolarger peak areas. Decreasing the flow-rate from 0.7 ml/min to 0.1 ml/minresulted in larger peak areas. However, as expected, no increase in peak heightswas observed, and there was no gain in sensitivity (signal-to-noise ratio, S/N).Moreover, the peaks were very broad at low flow-rates. Use of APCI provided afactor 4-5 better sensitivity (S/N) than ESI. Therefore, further experiments wereperformed with APCI. The minimum flow-rate for this type of source is0.5 ml/min. Although narrower peaks were obtained at higher flow-rates, theexpected increase in sensitivity (S/N) was not observed, as the noise increasedproportionally with peak height. Therefore, a flow-rate of 0.5 ml/min wasmaintained to minimise contamination of the ion source owing to involatilesubstances in the eluate [11].

5.1.3.2 Selectivity of the system

As no chromatographic separation was performed, the required selectivityof the system had to be obtained by the extraction procedure and by the massspectrometer. With respect to the extraction, the advantages and disadvantages


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of SPE and SPME must be taken in consideration. SPE is an exhaustiveextraction, whereas SPME has non-exhaustive properties. Despite the fact thatSPE has the advantage that it allows some chromatographic separation ofcompounds, severe matrix interference can be obtained with an SPE-MS system[11]. With SPE the aim is to eliminate breakthrough, thus the sorbent must havea high capacity. With SPME breakthrough is not an issue, thus allowing a betterchoice for selectivity of the sorbent. In SPME the amount extracted by the fibercoating is determined by the ratio of sample volume and coating volume owingto the non-exhaustive character of SPME. The sample volume (≥1 ml) is usuallymuch larger than the coating (0.628 µl for the 100-µm PDMS coating), so largepartition constants (between matrix and sorbent) are required for extraction[14,15]. In exhaustive extractions, i.e. SPE, the sample is forced through thesorbent, and thus the ratio of void volume and sorbent volume is decisive. Sincethese volumes are usually similar, only small partition constants are required fortrapping of compounds [15]. Hence, matrix compounds will also be trappedmore easily with SPE, thus decreasing the selectivity. Even more, especiallyunder non-equilibrium sorption conditions in SPME, diffusion plays a moreprominent role in the sorption process than with SPE. The analyte of interest,which is usually a small molecule, has a diffusion coefficient towards PDMSthat is commonly higher than the diffusion coefficient of the larger matrixcomponents [24]. This may also increase the selectivity of the extractionmethod.

However, the PDMS-coated fiber did not show sufficient selectivity forthe analysis of urine. Applying 45 min sorption and 10 min desorption withsingle-MS detection resulted in a significantly interfering peak (Fig. 1A) atm/z 235, which is also the m/z value of [M+H]+ of lidocaine. Despite the matrixinterference no ion suppression was observed after extraction of blank urine,using continuous infusion of analyte into the desorption solvent in front of theMS interface [25,26]. The absence of ion suppression indicates that SPME mayindeed be more selective than SPE, which resulted in significant ionsuppression if directly coupled to MS for urine analysis [11]. Other matrixinterferences could be avoided by performing MS/MS. If MS/MS is applied,protonated lidocaine will be fragmented to the product ion at m/z 86 (Fig. 2)with a fragmentation efficiency of about 61%. The chromatogram of blankurine analysed with SPME-MS/MS showed no interference of the urine matrix(Fig. 1B).


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Fig. 1: Extracted ion chromatograms of blank urine samples using SPME (45 minsorption, 10 min desorption) and analysed (A) in the MS mode and (B) in theMS/MS mode.

Fig. 2: Fragmentation pathway of lidocaine.


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5.1.3.3 Sorption and desorption conditions

A characteristic feature of SPME is the time-sorption curve. Such curvesshow the equilibrium time and also the maximum yield under the experimentalconditions [27]. Since this work combined SPME with the sensitivity of the MSlow yields may not be critical. A 7-µm and a 100-µm PDMS coating were usedin order to investigate the possibilities of an SPME-MS system for thedetermination of the model substance lidocaine. The data points were fittedaccording to time-sorption equations described by Ai [19]. The equilibriumtime, i.e. the time required to reach 95% of the maximum extraction yield, wasabout 1.1 min for the 7-µm coating (Fig. 3A), whereas the 100-µm coating hadan equilibrium time of about 31 min (Fig. 3B). The maximum extraction yieldfor the 7-µm coated fiber (0.15%) is significantly less than predicted [13,14](0.7%) based on the difference in volume (i.e. capacity) of the stationary phaseand the yield obtained with the 100-µm coated fiber (19%). This may beexplained by the fact that the 7-µm coating is highly cross-linked PDMS,whereas the 100-µm coating is only partially cross-linked [28]. As a result,though both fibers are chemically similar (i.e., build up from the same monomerand cross-linker), the physico-chemical properties governing extractionthermodynamics may differ considerably.

Fig. 3: Time-sorption curves using (A) a 7-µm and (B) a 100-µm PDMS-coated fiber.Urine samples (diluted 1+1 with buffer) spiked with 50 ng and 10 ng lidocainein (A) and (B), respectively.

Applying equilibrium extraction, the sensitivity of the SPME-MS systemwill be higher using the 100-µm coating, but the analysis time will be muchshorter with the 7-µm coating. However, the yield after 1 min sorption with the100-µm coating (about 1.7%) is still higher than the yield of the 7-µm coatingunder equilibrium conditions. Therefore, further experiments were done usingthe 100-µm coated fiber. Nevertheless, the 7-µm coated fiber may still be usefulif the reproducibility of the system is more important and the sensitivity is notthe main issue.


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Desorption is also an equilibrium process. Changes in pH and solventcomposition are used to desorb the analytes rapidly from the fiber. After asorption time of 5 min at pH 10, which resulted in a yield of 7.2%, staticdesorption was performed by applying a pH change and adding an organicmodifier to the desorption solvent [85% buffer (pH 4.5)-15% acetonitrile].Using a desorption time of 4 min, about 10% of the analyte was still retained bythe fiber. Therefore, after 4 min desorption, an extra 4 min clean-up step with afresh portion of the desorption solvent was required, to remove the remaininglidocaine form the fiber prior to using it for the next analysis. The solvent fromthe first desorption was introduced into the mass spectrometer with an analysistime of about 1 min.

5.1.3.4 Analytical data

The present SPME-MS/MS system was developed to obtain a shortanalysis time per sample by use of non-equilibrium sorption conditions. Therelatively low yield was compensated for by the sensitivity of the MS. The limitof detection (LOD, S/N = 3) of this set-up, using a 100-µm PDMS-coated fiberand a 5 min sorption time, was about 0.4 ng/ml lidocaine in urine. Arepresentative chromatogram is shown in Fig. 4.

Fig. 4: Extracted ion chromatograms at m/z 86 using SPME-MS/MS (5 min sorption,4 min desorption) for the determination of 0.4 ng/ml lidocaine in urine.

Since the urine volume was 0.625 ml, diluted to 1.25 ml with buffer, a totalyield of 6.5% (sorption yield of 7.2% and desorption efficiency of 90%) wasobtained. This corresponds to an absolute amount of about 16 pg (or 70 fmol)lidocaine. A limit of quantitation (RSD = 15%) of about 2 ng/ml was obtained.


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Good linearity (correlation coefficient (R) = 0.9968, weighted regression 1/x,n=8) was observed over the concentration range 0.4-80 ng/ml.

The reproducibility of the non-equilibrium system was investigated atthree concentration levels (Table 1), using the control samples to correct forday-to-day variation in the sensitivity of the mass spectrometer. The intraday(repeatability) and interday RSD (reproducibility) were always <14%, even forthe lowest concentration. At the highest concentration, the interday RSD wasabout 5%. It should be noted that the interday RSD of the system underequilibrium SPME extraction conditions is 4.8% for a lidocaine concentrationof 6.2 ng/ml. It can therefore be concluded that the RSD is slightly increasedowing to non-equilibrium sorption conditions. This increase in RSD withnon-equilibrium SPME can be expected as the sorption is carried out at thesteep part of the time-sorption curve. Slight variations in experimentalconditions may thus have a larger impact on the reproducibility and the amountof analyte being trapped in the PDMS coating. Nonetheless, the reproducibilitywith non-equilibrium SPME is still very acceptable.

Table 1: Repeatability and reproducibility [relative standard deviation (RSD)] of theSPME-MS/MS system for the determination of lidocaine in urine using a5-min sorption and a 4-min desorption time (n=6)

Concentration(ng/ml)

Intraday RSD(%)

Interday RSD(%)

2.3 13.7 12.88.7 3.9 12.3

43.7 2.5 5.7

5.1.4 Conclusions

The developed method shows the potential for high-throughput analysis,using SPME directly coupled to MS. The present work was carried out with anon-selective coating on the fiber, but MS/MS provided clean chromatograms.The speed of analysis of an SPME-MS/MS system is determined by the SPMEprocedure. However, using non-equilibrium SPME drastically reduced theanalysis time to about 10 min per sample. For lidocaine as a model substance inurine, an LOD of 0.4 ng/ml can be obtained with 5 min sorption and 4 mindesorption. A simple clean-up step of the fiber was applied to eliminatecarry-over. The reproducibility of the non-equilibrium SPME system wassatisfactory over the entire concentration range investigated. Although internalstandards are often used to improve reproducibility, it should be noted thatnon-equilibrium SPME requires standards with diffusion coefficients that areclosely comparable to that of the analyte [13]. It may be interesting to perform


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non-equilibrium SPME using a deuterated internal standard, thereby alsoensuring that the time-sorption curves of the analyte of interest and the internalstandard are equal.

The use of elevated temperatures during sorption and desorption is apossibility to enhance the sensitivity while maintaining a short extraction time.This also allows further reduction of sorption and desorption times as will bedemonstrated in a subsequent paper. An understanding of the fundamentalprocesses at the fiber-sample boundary and inside the fiber might lead to bettercontrol of the processes, so improving the development of a reproducible,sensitive, and fast SPME procedure. The effects of coating thickness,temperature during sorption and desorption and limiting factors in the sorptionkinetics are currently being investigated in our laboratory. The throughput canfurthermore be enhanced by the use of multiple fibers simultaneously forsorption, desorption and cleaning of the fiber. Since the mass spectrometer willbe the cost-limiting part of an SPME-MS system, the set-up can easily beexpanded by using multiple fibers and desorption chambers. Automation ofSPME-MS can be achieved by use of robotics.

Acknowledgements

Jan Brands from Sigma-Aldrich is gratefully acknowledged for supplyingthe SPME fibers. This research was supported by the Technology FoundationSTW, applied science division of NWO and the technology programme of theMinistry of Economic Affairs.

5.1.5 References

[1] D.A. McLoughlin, T.V. Olah, J.D. Gilbert. J. Pharm. Biomed. Anal 15 (1997)1893.

[2] M. Jemal, D. Teitz, Z. Ouyang, S. Khan. J. Chromatogr. B 732 (1999) 501.[3] A. Schellen, B. Ooms, M. van Gils, O. Halmingh, E. van der Vlis, D. van de

Lagemaat, E. Verheij. Rapid Commun. Mass Spectrom. 14 (2000) 230.[4] A.C. Hogenboom, M.P. Hofman, D.A. Jolly, W.M.A. Niessen, U.A. Th.

Brinkman. J. Chromatogr. A 885 (2000) 377.[5] M.L. Constanzer, C.M. Chavez, B.K. Matuszewski, J. Carlin, D. Graham.

J. Chromatogr. B 693 (1997) 117.[6] N.C. van de Merbel, A.P. Tinke, W.D. van Dongen, B. Oosterhuis,

J.H.G. Jonkman, Ph. Ladure, C. Puozzo. J. Chromatogr. B 708 (1998) 113.[7] A.C. Hogenboom, P. Speksnijder, R.J. Vreeken, W.M.A. Niessen,

U.A.Th. Brinkman. J. Chromatogr. A 77 (1997) 81.[8] A.C. Hogenboom, W.M.A. Niessen, U.A.Th. Brinkman. J. Chromatogr. A 794

(1998) 201.


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[9] J. Ding, U.D. Neue. Rapid Commun. Mass Spectrom. 13 (1999) 2151.[10] W.A. Minnaard, A.C. Hogenboom, U.K. Malmqvist, P. Manini, W.M.A.

Niessen, U.A.Th. Brinkman. Rapid Commun. Mass Spectrom. 10 (1996) 1569.[11] M.W.J. van Hout, C.M. Hofland, H.A.G. Niederländer, G.J. de Jong. Rapid

Commun. Mass Spectrom. 14 (2000) 2103.[12] S. Ulrich. J. Chromatogr. A 902 (2000) 167.[13] H. Lord, J. Pawliszyn. J. Chromatogr. A 902 (2000) 17.[14] H. Lord, J. Pawliszyn. J. Chromatogr. A 885 (2000) 153.[15] E.H.M. Koster, N.S.K. Hofman, G.J. de Jong. Chromatographia 47 (1998) 678.[16] N.H. Snow. J. Chromatogr. A 885 (2000) 445.[17] A.A. Boyd-Boland, J. Pawliszyn. Anal. Chem. 64 (1996) 1521.[18] B.J. Hall, M. Satterfield-Doerr, A.R. Parikh, J.S. Brodbelt. Anal. Chem. 70

(1998) 1788.[19] J. Ai. Anal. Chem. 69 (1997) 1230.[20] C. Pérès, C. Viallon, J.-L. Berdagué. Anal. Chem 73 (2000) 1030.[21] J.J. Langenfeld, S.B. Hawthorne, D.J. Miller. J. Chromatogr. A 740 (1996) 139.[22] A.P. Bruins. J. Chromatogr. A 794 (1998) 345.[23] M.G. Ikonomou, A.T. Blades, P. Kebarle. Anal. Chem. 62 (1990) 957.[24] E.L. Cussler. Diffusion, Mass Transfer in Fluid Systems, Cambrige University

Press, Cambrigde, 1984.[25] R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle. Rapid Commun. Mass Spectrom.

13 (1999) 1175.[26] R. King, R. Bonfiglio, C. Fernandez-Metzler, C. Miller-Stein, T. Olah. J. Am.

Soc. Mass Spectrom. 11 (2000) 942.[27] D. Louch, S. Motlagh, J. Pawliszyn. Anal. Chem. 64 (1992) 1187.[28] Supelco, personal communications.

Ultra-rapid non-equilibrium SPME at elevated temperatures

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5.2

Ultra-rapid non-equilibriumsolid-phase microextraction at elevatedtemperatures and direct coupling to ion-trap massspectrometry for the analysis of biological samples*

Summary

Solid-phase microextraction (SPME) has been directly coupled to an ion-trapmass spectrometer (MS) for the determination of lidocaine in urine. Thethroughput of samples has been increased using non-equilibrium SPME withpolydimethylsiloxane (PDMS) fibers. The effect of the temperature on thesorption and the desorption was studied. Elevated temperatures during sorption(65ºC) and desorption (55ºC) had a considerable influence on the speed of theextraction. The desorption was carried out with home-made desorption chamberallowing thermostating. Only 1 min sorption and 1 min desorption wereperformed, after which MS detection took place, resulting in a total analysistime of 3 min. Detection limits below 1 ng/ml could be obtained despite yieldsof only 2.1 and 1.5% for a 100 and a 30-µm PDMS-coated fiber, respectively.Furthermore, the determination of lidocaine in urine had acceptablereproducibilities, i.e. relative standard deviations (RSDs) below 10%. A limit ofquantitation (RSD<20%) of about 1 ng/ml was obtained. No extra wash step ofthe extraction fiber was required after desorption if a 30-µm coating was used,whereas not all the analyte was desorbed from the 100-µm coating in a singledesorption. Therefore, the SPME-MS/MS system with a 30-µm PDMS-coatedfiber for rapid non-equilibrium SPME at elevated temperatures is the mostsuitable for high-throughput analysis of biological samples.

*: M.W.J. van Hout, V. Jas, H.A.G. Niederländer, R.A. de Zeeuw, G.J. de Jong. Submitted to Anal. Chem.


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5.2.1 Introduction

In many areas of bioanalysis there is a strong call for high-throughputsystems. Besides fast analysis, the systems must also be selective and sensitiveas the analytes of interest should be determined in the sub-ng/ml range in acomplex matrix. Traditionally, solid-phase extraction (SPE) was performedoff-line [1-4], after which the eluate could be analysed by liquidchromatography (LC) or gas chromatography (GC). In these approaches, theSPE procedure was often the time-limiting step. Automation and coupling ofSPE and LC, which was more easily established than SPE-GC, allowed fasterextraction prior to separation [5,6]. By these means, systems were created inwhich the separation was the time-limiting step. To overcome the latterproblem, short LC columns were used [6-8], or the real separation step waseven omitted, thus SPE was directly coupled with mass spectrometry (MS)[9-14]. These systems make the extraction time more critical. This may beproblematic due to the various steps in the SPE procedure, i.e., activation,conditioning, sampling, washing and elution. Increasing the extraction speedmay result in less selectivity during the extraction, which subsequently canresult in detection problems, e.g., ion suppression [14-18]. Reduction of thematrix influence may be obtained by performing multiple-stage MS (MSn, withn≥2).

Solid-phase microextraction (SPME) is a simple technique. AlthoughSPME was originally designed for GC analysis [19-22], it is now also combinedwith LC. Usually, immersion of a coated fiber into the sample is carried out,after which the fiber is withdrawn from the sample and transferred to thedesorption chamber. Here, the analyte is released from the coating using a pHchange and/or an organic solvent. The main disadvantage of SPME is the slowsorption process, due to diffusion limitations. Fundamental studies [23,24] ofthe sorption processes pointed out that in an agitated sample mainly a staticwater layer around the fiber is limiting the sorption. Besides the time factor, theextraction yields with SPME are relatively low [21]. Normally, the extraction isstopped when equilibrium is established between analyte concentrations in thesample and in the coating of the fiber, that is, if 95% of the maximum yield isestablished. In some studies, non-equilibrium SPME has been performed[25,26], in which the extraction was stopped before equilibrium was reached.

Möder et al. [27] coupled SPME directly to electrospray (ESI)/MS usingthe selected ion monitoring mode. A suitable desorption chamber was used. A60-min sorption time was applied after which direct detection was performed,i.e. no separation step was used. This study clearly showed the time-limitationof the SPME procedure. Moreover, despite the use of MS in the selected ionmonitoring mode, severe matrix interference due to poor selectivity deterioratedthe reproducibility of the system (relative standard deviations (RSDs) >15% at


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ng/ml-levels). SPME was also directly coupled to ESI-high field asymmetricwaveform ion mobility spectrometry-MS for the determination ofamphetamines in urine [28]. High sensitivity (down to 200 pg/ml) andselectivity were obtained. However, the sorption time was still 12 min (with ayield of about 1%), and the static desorption took about 3 min. Furthermore, a2-min cleaning of the coating (at 250°C) was performed to prevent carry-over.Thus, the SPME procedure still hampered the sample throughput. In a previousstudy of our group [29] lidocaine was determined in urine by SPME directlycoupled to MS, thus eliminating the separation step. Good selectivity wasobtained by the SPME procedure and by the use of MS/MS. A 100-µmpolydimethylsiloxane (PDMS) coated fiber was used for extraction at roomtemperature. A 0.5 ml/min flow-rate after desorption proved to be optimal fordetection and peak shape. The sample throughput was enhanced by performingnon-equilibrium SPME, applying only 5 min sorption and 4 min desorption.However, despite the short SPME procedure, this remained the time-limitingstep, as the MS detection took only about 1 min. Furthermore, a washing of thefiber was required after desorption to eliminate remaining amounts of analyte inthe coating (about 10%) after a single desorption. The washing took anadditional 4 min. The limit of detection (LOD) was about 0.4 ng/ml and theRSDs were below 14%. This system showed the potential of non-equilibriumSPME, and the results of this study were the starting point for the currentinvestigation of the applicability of ultra-rapid non-equilibrium SPME at anelevated sorption and desorption temperature.

5.2.2 Experimental section

5.2.2.1 Chemicals and Instrumentation

Acetic acid, boric acid, and sodium chloride (all of analytical-reagentgrade) were obtained from Merck (Darmstadt, Germany) and ammoniumacetate (99.99%) was from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile(Lab-Scan, Dublin, Ireland) was of HPLC quality. Stock solutions of lidocainehydrochloride (USP, Holland Pharmaceutical Supply, Alphen a/d Rijn, TheNetherlands) were prepared in acetonitrile. Ammonium acetate buffer (10 mM)was prepared by dissolving ammonium acetate in ultrapure water and adjustingthe pH to 4.5 with acetic acid (10% v/v). Buffer solutions pH 10 (0.2 M) wereprepared by dissolving boric acid in ultrapure water and adjusting the pH with1 M sodium hydroxide. Water was obtained using an Elga Maxima ultrapurewater purification system (Salm & Kipp, Breukelen, The Netherlands).

PDMS-coated (30 and 100 µm) SPME fibers were obtained from Supelco(Bellefonte, PA, USA). The SPME holder and fiber were compatible with a


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modified desorption chamber. Heating during sorption was performed using amagnetic heater (Metrohm, Herisau, Switzerland). A Hewlett-Packard(Waldbronn, Germany) HPLC gradient pump series 1100 was used fordesorption and transport of the solvent to the mass spectrometer. The massspectrometer was an LCQ Classic ion trap (Thermoquest, San Jose, CA, USA)equipped with an atmospheric pressure chemical ionisation (APCI) source.

Triplicate injections (50 µl) of control samples (24.81 ng/ml lidocaine inbuffer pH 4.5-acetonitrile (85:15 v/v)) were used to correct for day-to-dayvariations in the sensitivity of the mass spectrometer. Analyses performed toinvestigate the reproducibility were subjected to two-way analysis of variance(ANOVA) with replications.

5.2.2.2 SPME procedure

The method was similar to the one described previously [29]. Sampleswere prepared by diluting blank or spiked calf urine with 0.2 M borate buffer(pH 10) in a ratio of 1:1. Sodium chloride was added to the urine-buffer solutionto give a final salt concentration of 0.3 g/ml. The pH was readjusted to 10 with10% sodium hydroxide. The coated part of the fiber was completely immersedinto 1.25 ml sample and extraction was performed for a particular time, whilecontinuously stirring the sample using a 7×2 mm stirring rod and a magneticstirrer (Metrohm, Herisau, Switzerland). Desorption was performed by placingthe fiber into the desorption chamber, which was filled with 70 µl ammoniumacetate buffer (10 mM, pH 4.5):acetonitrile (85:15 v/v). After a certain time ofstatic desorption, the contents of the chamber were flushed to the MS with thesame solvent at a flow-rate of 0.5 ml/min.

Elevated sorption temperatures were obtained by placing the sample ontothe heater (Metrohm) and allowing the sample (about 5 min) to obtain thedesired temperature (about 65°C) prior to extraction under continuous stirring.Elevated temperatures during desorption (about 55°C) could be achieved bymodifying a standard SPME desorption chamber (Supelco). Four heatingelements were inserted into the stainless steel housing of the unit (Fig. 1). Byusing a thermocouple, the temperature of the desorption solvent could becontrolled during desorption. A PTFE housing was used for isolation of thedesorption unit.

5.2.2.3 Mass spectrometry

Using the ion-trap mass spectrometer with an APCI source, the vaporisertemperature was set at 400ºC. During analysis the sheath gas and auxiliary gas(both nitrogen) were 48 and 3 (arbitrary units), respectively, and the coronadischarge current was set at 5.0 µA. The heated capillary was set at 140ºC and a


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capillary voltage of 46.0 V was applied. The tube lens offset was 45.0 V. Thefirst octapole offset, the inter-octapole offset, and the second octapole offsetwere - 2.0 V, - 26.0 V, and - 6.0 V, respectively. All scans were recorded in thefull-scan mode with three microscans over the range m/z 75-300, using thepositive-ion mode. The maximum injection time of the ion-trap was set at300 ms. Helium was applied as dampening and collision gas. Extracted ionchromatograms in the single-MS mode were obtained for [M+H]+

(m/z 235 ± 0.5) and for MS/MS the fragment ion m/z 86 ± 0.5 was monitored.The isolation width during MS/MS experiments was 2.0 Th using a collisionenergy of 34%.

Fig. 1: Schematic presentation of (A) the modified desorption chamber withpossibility to heat the system, (B) the bottom view of modified desorptionchamber. 1 = SPME fiber holder, 2 = needle guide, 3 = needle,4 = compression unit, 5 = ferrule, 6 = SPME fiber, 7 = solvent desorptionchamber, 8 = thermocouple, 9 = PTFE housing (isolation), 10a-d = heatingelements, 11 = temperature control unit (not to scale).


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5.2.3 Results and discussion

5.2.3.1 Effect of temperature on sorption

In our previous study [29], all experiments were carried out at 25ºC andwith the 100-µm PDMS coating. In order to further enhance the sensitivity, thespeed of the sorption process, i.e. the diffusion, can be increased by usingelevated temperatures during extraction. Increasing the temperature from 25ºCto about 65ºC during sorption resulted in a decrease of the equilibrium time, thetime at which 95% of the maximum yield is reached, from about 90 min(Fig. 2A) to 28 min (Fig. 2B) for the 100-µm coating. Both time-sorption curvesin Fig. 2 were fitted according to equations described by Ai [26]. It should benoted that the equilibrium time in this study is longer than in our previousstudies [29,30] due to a different geometry of the experimental set-up, whichwas necessary to allow temperature control. Typical yields after 1 and 5 minwith sorption at 25ºC are 0.7 and 3.3%, respectively. At these sorption times,but with 65ºC, the yields change into 2.1 and 8.7%. Thus, in non-equilibriumsituations the yield is significantly higher at 65ºC, whereas at equilibrium onlyminor differences in the yield are observed at the temperatures investigated.

Fig. 2: Effect of sorption temperature (Ts) on yield and equilibrium time.Time-sorption curve of (A) 100-µm coating, Ts = 25ºC; (B) 100-µm coating,Ts = 65ºC. Concentration of 16 ng/ml lidocaine in urine.

The decrease in viscosity of the sample leads to more rapid diffusion [24,31],resulting in a shorter equilibrium time. In non-equilibrium situations the yield ishigher at elevated temperatures due to the increased diffusion through a static


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water layer around the fiber. Based on thermodynamics, higher temperatureswould result in a decrease in the partition constant of the analyte and thus inlower yields [25]. However, this effect seems to be restricted, which is inaccordance with the results of others [31-34]. This may be explained by the factthat the sorption yield is the overall result of a series of processes, where anincrease in the sorption temperature may lead to opposite effects. Physicalchanges of the polymeric phase, as suggested by the results from differentialscanning calorimetry of PDMS [32], may have a positive effect on the partitionconstant.

Decreasing the volume of the coating implies a more rapid establishmentof the equilibrium. If the coating thickness decreases by a factor of three (i.e.,volume and equilibrium extracted amount decrease a factor of five), the timerequired to establish equilibrium is thought to decrease similarly [23]. However,the surface area of the coating also decreases, hereby reducing the effect of thesmaller volume. This decrease in area is counteracted due to a thinner staticwater layer around a smaller coating [32]. Thus, although various factorsinfluence the equilibrium time, the overall effect is a considerable decrease inequilibrium time with a smaller coating [24,32]. For the 30-µm coating, anequilibrium time of 3.5 min was achieved at 65ºC with a yield of 2.2%. So, theequilibrium time with the 30-µm coating is shorter than predicted using thedifference in volume between the 100-µm and the 30-µm coating. After 1 minsorption at 65ºC, a yield of 1.5% was obtained, which is already about 65% ofthe maximum yield.

5.2.3.2 Effect of temperature on desorption

The time-desorption curves presented in Fig. 3, using the 100-µm coating,have been obtained after 5 min sorption at 25ºC (¡ and o) or 65ºC (l and n),followed by desorption. Sorption at 25ºC and subsequent desorption at25ºC (¡) resulted in incomplete desorption, even after 10 min desorption. Afterabout 4 min, the desorption level had reached its plateau and no more analytecould be desorbed. This is due to the fact that the desorption is also anequilibrium process. A similar effect was observed for sorption at 65ºCcombined with desorption at 25ºC (l). The maximum desorption level wasreached after about 4 min. No more analyte was released from the coating, eventhough about 25% of the sorbed amount was still present in the coating.Lidocaine is protonated upon release from the coating. A change in propertiesof the PDMS coating during the high sorption temperature may result in astronger retention by the stationary phase.

A high desorption temperature (o and n) proved to be advantageous interms of completeness of the desorption regardless the sorption temperature.The overall effect of the higher desorption temperature is a shift of the


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equilibrium of the desorption process towards the desorption solvent. The bestdesorption was observed at an elevated temperature after sorption at roomtemperature (o). However, the low sorption yield is disadvantageous for theoverall sensitivity of the system. Desorption at an elevated temperature ensuredthat the desorption was not only more complete, but the process was also muchfaster at this temperature. This effect was most prominently observed whencomparing the results of experiments in which similar sorption and desorptiontemperatures, i.e., both at either low (¡) or at high temperatures (n) wereapplied. Despite the larger amount of analyte sorbed due to the higher sorptiontemperature (n), the desorption was more complete. Currently, we are stillinvestigating the fundamental aspects involved during the desorption.

Fig. 3: Time-desorption curves after 5 min sorption using a 100 µm PDMS-coatedfiber. Temperature during sorption (Ts) and desorption (Td): Ts = 25°C,Td = 25°C (¡); Ts = 65°C, Td = 25°C (l); Ts = 25°C, Td = 55°C (o); Ts = 65°C,Td = 55°C (n). Concentration of 16 ng/ml lidocaine in urine.

Using the 100-µm PDMS-coated fiber, after 4 min desorption at anelevated temperature about 5% of the sorbed analyte was still retained by thecoating. Thus, an additional wash step still remained to prevent carry-over, or alonger desorption time should be applied, since after 10 min desorption theamount of retained lidocaine is negligible. Both approaches are disadvantageous


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for high-throughput analysis. With the 30-µm coating, a negligible amount oflidocaine was retained after desorption at 55ºC for 1 min.

5.2.3.3 Application

The SPME-MS/MS system was applied to the determination of lidocainein urine. The 100 and 30-µm coatings were both used to investigate theirpotentials towards ultra-rapid non-equilibrium SPME. For the 100-µm coating,using a 5-min sorption time at 65ºC combined with 4 min desorption at 55ºCresulted in an LOD (signal-to-noise ratio of 3) of about 0.16 ng/ml and a goodlinearity was obtained (Table 1). A sorption yield of 8.7% was found underthese conditions. However, after the desorption about 5% of the extractedlidocaine was still retained by the fiber, which required removal by anadditional wash step. Shorter sorption and desorption times were also applied tothis fiber, i.e. both processes were performed for 1 min. A slightly higher LODis obtained due to the lower yield (about 2%). During the 1 min desorption alsoabout 95% of the extracted lidocaine was removed from the fiber. Thus, anextra washing step, with the same solvent, was still required to preventcarry-over. Since the incompleteness of the desorption was disadvantageous,only a small concentration range was tested.

Table 1. Conditions and corresponding analytical data of SPME-MS/MS applyingnon-equilibrium SPME with PDMS-coated fibers at elevated temperatures(sorption at 65°C; desorption at 55°C).

100 µm 100 µm 30 µmSorption time (min) 5 1 1Desorption time (min) 4 1 1Sorption yield (%) 8.7 2.1 1.5Desorption yield (%) 95 95 100Limit of detection (ng/ml) 0.16 0.40 0.50Correlation coefficient 0.9998 0.9960 0.9986Range (ng/ml) 0.16-400 0.40-16* 0.5-225

*: maximum concentration studied

To obtain an ultra-rapid system with good sensitivity and full desorptionof the analyte without an extra wash step, the 30-µm coating was used underextreme non-equilibrium conditions with elevated temperatures, i.e., 1 minsorption at 65ºC and 1 min desorption at 55ºC. Even though the yield was lowerthan with the 100-µm coating, the LOD (0.50 ng/ml; determined as three timesthe level of occasional spikes in the chromatogram) was still in the sub-ng/mlrange (Table 1). Since negligible amounts of analyte were retained by thecoating after the desorption, no extra wash step was required. Good linearitywas obtained over a large range. Chromatograms of blank and spiked urine are


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presented in Figs. 4A and 4B, respectively. Some slight tailing of the peaks wasobserved due to the direct coupling of SPME and MS.

Fig. 4: Extracted ion chromatograms (m/z 86) of SPME-MS/MS with the 30-µmPDMS-coated fiber after 1 min sorption at 65°C and 1 min desorption at 55°C:(A) blank urine; (B) 1.6 ng/ml lidocaine in urine.

The repeatabilities (intraday RSD) and reproducibilities (interday RSD)obtained with the 30-µm coating are presented in Table 2. Both the intraday andthe interday RSDs were less than 10%, except near the LOD. At high


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concentrations, i.e. 218 ng/ml, the reproducibilities were substantially better. Alimit of quantitation (LOQ; RSD <15%) of about 1 ng/ml could be obtained.The results show a slight improvement of the reproducibility in comparisonwith our previous study [29], in which rapid non-equilibrium SPME at roomtemperature was applied (intraday and interday RSD lower than 14%). The useof a deuterated standard or a homologue of lidocaine as internal standard mayhelp to obtain even better reproducibility.

Table 2. Repeatability and reproducibility of the SPME-MS/MS system with the 30-µmPDMS coating using elevated temperatures during sorption and desorption(both 1 min) for the determination of lidocaine in urine.

Concentration (ng/ml) 0.9 12 44 218Intraday RSD (%) 18.6 8.5 9.1 1.2Interday RSD (%) 18.5 8.1 9.2 2.5

Note: n = 3 for 0.9 ng/ml; n = 6 for other concentrations.

5.2.4 Conclusions

The current study has clearly shown the potential of ultra-rapid SPME forhigh-throughput systems. Even though no chromatographic step wasincorporated, the use of MS/MS provided adequate selectivity. The sensitivityof the combined SPME-MS/MS system was acceptable with LODs in thesub-ng/ml range and an LOQ of about 1 ng/ml. To obtain goodreproducibilities, the conditions of ultra-rapid non-equilibrium SPME should becarefully controlled. Elevated temperatures during sorption and desorptionincreased the diffusion, and thus the sensitivity and/or speed of the system, andcontrolling the temperatures improved the reproducibility. A high desorptiontemperature is advantageous for complete desorption and should thus be used incombination with any sorption temperature. If a high sorption temperature isused, a high desorption temperature is even more required to overcomeretention of analyte in the coating, otherwise an additional wash step is needed.The 30-µm coated PDMS fiber was most suitable for the rapid determination oflidocaine in urine, since an acceptable yield was obtained with goodreproducibility, and a negligible amount of analyte was retained by the coatingafter a single desorption.

The throughput of the current system, applying 1 min sorption, 1 mindesorption and 1 min detection, can be increased by using two fibers and twodesorption chambers. When the first fiber is in the sorption phase, the secondfiber can be desorbed, while the eluate of a third sample can be analysed by MS.In this way, 3 analyses can be performed within 3 min, increasing thethroughput to 1 sample/min, provided that all samples have been brought to


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65°C prior to starting the sorption process. Obviously, when applyingultra-rapid SPME-MS/MS, attention must be paid to the performance of the MSover time, since matrix components may precipitate on parts of the MS.Possible memory effects by the fiber after repeated use should also bemonitored. Negligible amounts of analyte retained by the coating after onedesorption may build up to a larger amount, especially after analysis of highconcentrations of analyte, and may finally cause carry-over. The performanceand reproducibility of different batches of fibers should also be investigated.The latter aspect will be more important when multi-fiber systems are used.

Acknowledgements

Jan Brands from Sigma-Aldrich is gratefully acknowledged for supplyingthe SPME fibers. This research was supported by the Technology FoundationSTW, applied science division of NWO and the technology programme of theMinistry of Economic Affairs.

5.2.5 References

[1] M. Dressler. J. Chromatogr. 165 (1979) 167.[2] A. Lagana, B.M. Petronio, M. Rotatori. J. Chromatogr. 198 (1980) 143.[3] R.E. Majors. LC-GC Intern. May (1998) S8.[4] Z.E. Penton. Advances in Chromatogr. 37 (1997) 205.[5] M. Jemal, D. Teitz, Z. Ouyang, S. Khan. J. Chromatogr. B 732 (1999) 501.[6] D.A. McLoughlin, T.V. Olah, J.D. Gilbert. J. Pharm. Biomed. Anal. 15 (1997)

1893.[7] M.L. Constanzer, C.M. Chavez, B.K. Matuszewski, J. Carlin, D.Graham.

J. Chromatogr. B 693 (1997) 117.[8] N.C. van de Merbel, A.P. Tinke, W.D. van Dongen, B. Oosterhuis,

J.H.G. Jonkman, Ph. Ladure, C. Puozzo. J. Chromatogr. B 708 (1998) 113.[9] A. Schellen, B. Ooms, M. van Gils, O. Halmingh, E. van der Vlis, D. van de

Lagemaat, E. Verheij. Rapid Commun. Mass Spectrom. 14 (2000) 230.[10] A.C. Hogenboom, P. Speksnijder, R.J. Vreeken, W.M.A. Niessen,

U.A.Th. Brinkman. J. Chromatogr. A 77 (1997) 81.[11] A.C. Hogenboom, W.M.A. Niessen, U.A.Th. Brinkman. J. Chromatogr. A 794

(1998) 201.[12] J. Ding, U.D. Neue. Rapid Commun. Mass Spectrom. 13 (1999) 2151.[13] W.A. Minnaard, A.C. Hogenboom, U.K. Malmqvist, P. Manini, W.M.A.

Niessen, U.A.Th. Brinkman. Rapid Commun. Mass Spectrom. 10 (1996) 1569.[14] M.W.J. van Hout, C.M. Hofland, H.A.G. Niederländer, G.J. de Jong. Rapid

Commun. Mass Spectrom. 14 (2000) 2103.[15] K. Matuszewski K, M.L. Constanzer, C.M. Chavez-Eng. Anal. Chem. 70 (1998)

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[16] R. Bonfiglio, R.C. King, T.V. Olah, K. Merkle. Rapid Commun. Mass Spectrom.19 (1999) 1175.

[17] D.L. Buhrman, P.I. Price, P.J. Rudewicz. J. Am. Soc. Mass Spectrom. 7 (1996)1099.

[18] I. Fu, E.J. Woolf, B.K. Matuszewski. J. Pharm. Biomed. Anal. 18 (1998) 347.[19] S. Ulrich. J. Chromatogr. A 902 (2000) 167.[20] G. Theodoridis, E.H.M. Koster, G.J. de Jong. J. Chromatogr. B 745 (2000) 49.[21] N.H. Snow. J. Chromatogr. A 885 (2000) 445.[22] C.L. Arthur, J.B. Pawliszyn. Anal. Chem. 62 (1990) 2145.[23] D. Louch, S. Motlagh, J.B. Pawliszyn. Anal. Chem. 64 (1992) 1187.[24] J.B. Pawliszyn, Solid Phase Microextraction – Theory and Practice, Wiley, New

York, 1997.[25] H. Lord, J.B. Pawliszyn. J. Chromatogr. A 902 (2000) 17.[26] J. Ai. Anal. Chem. 69 (1997) 1230.[27] M. Möder, H. Löster, R. Herzschuh, P. Popp. J. Mass Spectrom. 32 (1997) 1195.[28] M.A. McCooeye, Z. Mester, B. Ells, D.A. Barnett, R.W. Purves, R. Guevremont.

Anal. Chem. 74 (2002) 3071.[29] M.W.J. van Hout, V. Jas, H.A.G. Niederländer, R.A. de Zeeuw, G.J. de Jong.

Analyst 127 (2002) 355.[30] E.H.M. Koster, N.S.K. Hofman, G.J. de Jong. Chromatographia 47 (1998) 678.[31] K. Jinno, M. Taniguchi, M. Hayashida. J. Pharm. Biomed. Anal. 17 (1998) 1081.[32] H.A.G. Niederländer, V. Jas, M.W.J. van Hout. Submitted to Anal. Chem.[33] K. Jinno, M. Kawazoe, M. Hayashida. Chromatographia 52 (2000) 309.[34] M. Satterfield, D.M. Black, J.S. Brodbelt. J. Chromatogr. B 759 (2001) 33.

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