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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11INTRODUCTION

The

General remarks

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1.1 General remarks

The bioanalysis of drugs and related substances has always been aspecialism dealing with rather complex samples. The most frequently analysedbiological samples are urine and blood, plasma or serum, but analyses of saliva,hair, sweat, cerebrospinal fluid, vitreous humour and tissue homogenates arealso performed. Even though urine is mainly an aqueous sample, it can varybetween individuals in viscosity, salt contents and presence of other compoundsdue to diseases, drink and food consumption as well as by administered drugs.As urine is considered to be a major waste disposal of the body, one can expectmany substances to be present in this fluid. Blood is the transporter of manyvital substances and nutrients for the entire body and thus contains manyendogenous and exogenous compounds in different concentrations. An extraproblem posed by blood samples is the presence of proteins, which can lead toprotein binding of the analyte.

The importance of bioanalysis is most appreciated in various fields ofpharmaceutical sciences. During the development of new drugs, extensivestudies are performed in the pre-clinical and clinical stages. At the pre-clinicalstage, fluids from animals are analysed. In the clinical stage, human samplesneed to be examined. Virtually all aspects of a new drug will be investigated.The toxicological and therapeutic concentrations of the parent drug and itsmetabolites must be determined, in combination with the pharmacodynamic andpharmacokinetic properties of the potential drug. Finally, the formulation of thedrug must be optimised, which also relies to a large extent on bioanalyticalevaluation. The sooner a drug can be placed on the market, the better for allinvolved, thus rapid development is preferred. This means that many samplesshould be analysed in a rather short time. Once a drug is on the market,therapeutic drug monitoring can be a vital aspect. Other important applicationsof bioanalysis are, amongst others, the control of residues in food andfood-producing animals, drug abuse testing, clinical and forensic toxicology andenvironmental control. Thus, in various application fields many biologicalsamples need to be analysed. Furthermore, the knowledge of workingmechanisms of drugs is increasing. Consequently, more potent andendogenous-like drugs are developed, allowing the administration of lowerdosages of drugs, which results in lower concentrations of the compound and/orits metabolites in blood and/or urine samples. Thus, the number of complexsamples is increasing, whereas the concentrations of the analyte(s) aredecreasing. As a result, there is a strong demand for highly sensitive andselective systems that can be used in high-throughput analysis.

Biological samples can normally not be injected directly into theanalysing system without sample preparation. Sample pretreatment is thus ofutmost importance for the adequate analysis of drugs. However, as sample

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pretreatment can be a time-consuming process, this can limit the samplethroughput. The proper selectivity can be obtained during the samplepreparation, the separation and the detection. A major differentiation betweenthe analyte(s) of interest and the other compounds is often made during the firststep. Sensitivity is to a large extent obtained by the detector. However,pre-concentration of the analytes during sample preparation will also add tothis. Thus, sample pretreatment is required for achieving sufficient sensitivityand selectivity, whereas the time should be kept to a minimum in order to obtainadequate speed. Therefore, there is a clear trend towards integration of samplepretreatment with the separation and the detection.

Numerous sample preparation techniques have been developed forbioanalytical purposes. The most classical sample preparation technique isliquid-liquid extraction (LLE) [1]. The broad polarity range of solvents and itsgeneral applicability made this technique popular. However, from anenvironmental point of view, the use of large amounts of organic and oftenchlorinated solvents is unfavourable. Furthermore, LLE has often limitedseparation efficiency. Various techniques, like protein precipitation,membrane-based techniques (dialysis, ultrafiltration, supported liquidmembrane extraction), supercritical fluid extraction (SFE), solid-phaseextraction (SPE) and solid-phase microextraction (SPME), have been developedfor sample pretreatment in order to replace LLE or to introduce new approachesto sample preparation. Nowadays, in particular SPE is replacing LLE due to thevariety of available stationary phases, allowing either non-selective extractionor selective extraction of only a single compound or class of compounds.Generally less solvent is required for SPE than for LLE [1,2]. Despite thesedevelopments, the sample pretreatment was often considered a weak andtime-consuming link in a bioanalytical system up to ten years ago. Thus, mucheffort was put in exploring the possibilities of miniaturisation and automation ofthe extraction procedures to minimise or eliminate the limitations of the samplepreparation.

With the integration of sample pretreatment, separation and detection,coupled techniques were developed, which were linked by hyphens in writtenlanguage. Consequently, the term ‘hyphenated technique’ was introduced.Nowadays, hyphenation refers to systems that combine separation and detectionoffering structural information. The most important and routinely applieddetection in such systems in biopharmaceutical analysis is mass spectrometry(MS). Initially, MS was mainly used for identification of compounds. With theintroduction of atmospheric pressure ionisation interfaces like electrospray andatmospheric pressure chemical ionisation, (LC-) MS has gained tremendousinterest, and has shown good potential for quantitation as well as identification.Furthermore, multi-stage MS (MSn with n≥2) increased the potential forselective detection. Currently, the interest for bioanalysis using MS is somewhat

General remarks

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dualistic. On the one hand, MS can offer extreme selectivity, hereby enhancingthe possibilities for more rapid and simple sample pretreatment and separation.Incomplete separation of the analytes from interfering compounds can beovercome if the components have different m/z values. However, the presenceof such compounds may still lead to interferences during ionisation, e.g., by ionsuppression or enhancement [3,4], when the interfering compounds and theanalyte of interest co-elute. Furthermore, elution of substances that do notoverlap with the analyte may also pose problems due to contamination ofvarious parts of the MS. Thus, careful considerations should still be made aboutreducing sample preparation and separation. On the other hand, MS can offerhigh sensitivity, especially if the selectivity is enhanced as well. This is ofparticular importance for extractions in which little pre-concentration takesplace and/or in which the recoveries or yields are low, e.g., in SPME. Theoverall effect of the application of MS in the biopharmaceutical field is fasterseparation. Consequently, the sample preparation has become critical again.Good selectivity is required to minimise the risk of detection problems, andrapid extraction should be performed in order to maintain high-throughputanalysis. Thus, the sample pretreatment needs to be further accelerated andautomated.

To minimise the required volumes of solvent as well as sample, SPE isavailable in a miniaturised format such as the SPE disk and the SPE pipette tip(SPEPT) [5,6]. Generally, SPE disks contain a smaller bed with a morehomogeneous particle size distribution than conventional cartridges. The disk istypically a membrane-like bed with a thickness of about 0.5 mm and a diameterof down to 4 mm, which can be easily adapted to an at-line 96-well approach,thus decreasing the handling time per sample. The SPEPTs are modified pipettetips with a small disk (about 4 mg, 4 mm diameter, thickness including fritsabout 1.5 mm) of stationary phase positioned in the point of the tip. The formatsallow more rapid flushing of sample and smaller volumes of various solventsthan in conventional SPE [5]. However, even though the miniaturisationenhances the sample handling speed, the various steps in the SPE procedure,that is activation, conditioning, sampling, washing, drying and elution, are stillnecessary [2,6]. Generally, a disadvantage of miniaturisation is that smallersample volumes have to be used, hereby possibly decreasing the sensitivity if asufficient amount of sample is available. On the other hand, the miniaturisationof SPE enhances the potential for automation and on-line coupling with variousseparation systems. SPE has been primarily coupled on-line with liquidchromatography (LC) [7,8], since aqueous solvents may be flushed directly to areversed phase (RP) LC-column. The simplest on-line arrangement forSPE-RPLC is the use of switching valves and commercial pre-columns [7,9,10].On-line SPE-LC is now a well-established technique. With the automation ofthe various steps of the SPE procedure, the LC may now become the

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time-limiting step, but this could be counteracted by the use of MS. Thesimultaneous increase in selectivity and sensitivity of MS allowed the use ofshort-column LC [11-13], hereby reducing the time-limitations. Even the directcoupling of SPE with MS has been applied [14-17]. However, as already statedabove, systems based on SPE-MS will put extra pressure on the speed of theextraction as well as on the selectivity of the extraction and the detection, sinceno real separation is performed. Thus, the possibilities and limitations of bothsteps must be carefully explored and possibly modified with regard to those ofSPE-LC-MS systems.

The on-line coupling of SPE with gas chromatography (GC) is importantfor toxicological and drug abuse analysis, since various recommended and/orlegally accepted procedures require the use of GC. However, integrating SPEwith GC is more complicated than SPE-LC, and care should be taken to avoidthe introduction of water and other polar solvents into the GC. This implies thatthe SPE stationary phase should be dried extensively prior to the elution. Theelution poses another key problem, since the elution volume is usually too largefor direct injection into the GC. To increase the compatibility of integrated SPEand GC, a special injection procedure allowing large-volume injection (LVI)must be applied, hereby possibly eliminating the error-prone evaporation/reconstitution processes. With LVI the entire eluate, or a major portion there of,is introduced into the GC, which can result in increased sensitivity. Yet, notonly a large amount of solvent is introduced, but also an equivalent amount ofimpurities that are co-extracted with the analytes. Thus, special attention shouldbe paid to the selectivity of the extraction and detection. A number of interfaceshave been proposed for LVI [18], i.e., on-column injection [19], loop-typeinjection [20] and the programmed temperature vaporiser (PTV). The PTVinjector was designed by Vogt et al. [21,22], and resembles a conventionalsplit/splitless injector. The main solvent is injected at temperatures 30-40ºCbelow its boiling point into a packed liner. After almost complete evaporation ofthe solvent, the analytes are thermally released from the packing and transferredto the GC column. Critical factors in the use of the PTV for LVI are themaximum injection volume, which depends on the flow-rate during theinjection, the injected solvent and the temperature of the injector [23].Furthermore, the inertness of the liner packing must be closely monitored.Besides in LVI, the PTV injector can also be used for solid-phase extraction –thermal desorption (SPETD), since the injector can be heated very rapidly (upto 16°C/s). After the extraction the analytes are thermally desorbed from thesorbent inside the PTV injector and, subsequently, conventional GC isperformed. In SPETD with the PTV, a suitable sorbent must be used inside theliner to obtain retention of the analytes. Furthermore, the sorbent must be cleanand thermostable. Obviously, regardless of the approach, the analytes must haveadequate thermostability to withstand the high temperatures in both the injector

Scope of the thesis

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and the GC. Generally, the use of MS is required for adequate selectivity inSPE-GC systems for bioanalysis. The investigation of the possibilities andlimitations of integrating SPE with GC will also be a major part of this thesis.

Another interesting approach of miniaturised sample preparation is SPME[1,24]. In contrast to the flow-through sampling of SPE, a passivediffusion-based process is used in SPME. The set-up is rather simple, as acoated fiber is used in a syringe-like device. Excellent papers can be found onthe principles, theory, practical aspects and applications of SPME [24-29]. Thelatter refer mainly to SPME combined with GC, as this type of extraction wasoriginally designed for GC analysis. Most applications are in the field ofenvironmental analysis, but more recently the utility of SPME for bioanalysishas also been demonstrated [25,26]. The number of SPME-LC applications isstill limited, but nowadays the possibilities of SPME followed by liquiddesorption and LC are being explored more and more in various areas, allowingthe analysis of thermolabile compounds. A simple valve-like interface with adesorption chamber is used for the desorption of the analytes using anappropriate solvent composition [30,31]. The three main limiting factors inSPME-based systems are correlated to the SPME principles. The extraction isdiffusion-driven and non-exhaustive, which can result in low yields, especiallyin comparison with SPE. The diffusion also limits the throughput due to thelong times required to reach equilibrium. The third factor, which is only validfor SPME-LC, is the static desorption, which is also based on an equilibriumprocess. Thus, incomplete desorption of the analyte from the coating may alsooccur, resulting in carry-over. Non-equilibrium SPME can be used to decreasethe extraction time, which also allows the direct coupling of SPME with MS.

1.2 Scope of the thesis

The objectives of this thesis are to develop fast, sensitive, integratedsystems for high-throughput analysis in the biopharmaceutical field. Since thepossibilities of integrated SPE-LC and SPME-GC are reasonably well known,also with regard to automation, our major focus was on SPE-GC and on thedirect coupling of SPE and SPME with MS. SPE-GC has hardly been applied inthe biopharmaceutical field so far. Generally, the limitations of SPE-GC are thedesorption volume and the time-consuming drying step. Therefore, thepossibilities and limitations of novel types of integration of SPE with GC havebeen investigated and the application for biological samples has beendemonstrated.

Though SPE-LC is a well-established technique, its use inhigh-throughput systems is limited by the time required for both the extractionand the separation. The direct coupling of SPE with MS (i.e. omitting the major

Chapter 1 – Introduction

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separation step) by a suitable interface is very interesting for more rapidbioanalysis. Investigations with regard to the potentials and pitfalls of SPE-MSwere performed.

Another appealing system is the direct coupling of SPME with MS. Theuse of SPME in high-throughput systems is more limited than for SPE-basedsystems due to the long sorption times. An approach based on non-equilibriumSPME was developed that allowed the use of SPME directly coupled with MSfor bioanalysis while high sample throughput and adequate sensitivity isobtained.

In Chapter 2 an overview is given over various sample pretreatmenttechniques in bioanalysis with a focus on integration of sample pretreatment andseparation and/or detection. New developments and less common combinationsare highlighted, whereas well-established techniques such as SPE-LC are notdiscussed in detail. Firstly, the principles of the techniques and special aspectsare presented. Secondly, bioanalytical applications are described. Finally, thepotentials and limitations of the techniques are discussed, with a focus on theobtained selectivity and sensitivity. The versatility, the ease of operation and thecomplexity of instrumentation and possible automation are also included in thediscussion.

The integration of SPE with GC for bioanalytical purposes is described inChapter 3, focusing on the miniaturisation of SPE and possible automation ofthe SPE-GC system. Integration of SPE with GC implies the injection of largevolumes of solvents into the GC. Section 3.1 shows the results of the evaluationof the PTV for LVI of extracts of biological samples. Special attention was paidto the solvent purity as well as the liner packing (maximum injection volumeand packing inertness). Also the selectivity obtained by the total procedureproved to be critical. Extra selectivity was obtained by the use of a massselective detector (MSD) in the selected ion monitoring (SIM) mode. The finalsystem allowed injection of the entire eluate of SPE (100 µl), and goodsensitivity was obtained, i.e. a limit of detection (LOD) down to 250 pg/ml forthe test drugs lidocaine, phenobarbital, secobarbital and diazepam in plasma.Section 3.2 deals with the use of SPEPTs, which allows smaller desorptionvolumes, and thus eliminates the need for evaporation and reconstitution of theeluate. An exterior coupling device was developed by which the SPEPTs couldbe coupled to the PTV injector. By these means, rapid off-line extraction wasfollowed by on-line desorption. Even further integration was performed byapplying a so-called internal coupling device (Section 3.3). A shortened packedliner and a shortened SPEPT were coupled inside the injector after off-lineextraction of 200 µl plasma. The desorption was performed in-line by theinjection of ethyl acetate on top of the disk of the SPEPT. This coupling deviceis an important step towards the integration of miniaturised and rapid SPE withPTV/GC. Another approach is that the extraction is followed by thermal

Scope of the thesis

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desorption. This SPETD approach is discussed in Section 3.4. Tenax wasinserted into a fritted liner after which off-line extraction and subsequentlyin-line thermal desorption was performed. A small amount of stationary phase(5 mg) allowed rapid extraction (8 min, including 5 min drying). With thisSPETD-GC-MSD system (SIM mode) and by extracting only 50 µl urine,LODs below 500 pg/ml were obtained for the test drugs lidocaine anddiazepam.

Chapter 4 deals with the direct coupling of SPE via an LC-MS interfacewith an ion-trap MS to obtain a rapid screening system. In Section 4.1,clenbuterol was determined in urine at a sub-ng/ml level applying SPE with apolymeric stationary phase and multiple-stage MS, that is MS3. Although cleanchromatograms were obtained, some ion suppression was observed. InSection 4.2, a post-cartridge continuous infusion system was applied todetermine ion suppression and an investigation was performed to check whichurine compounds were causing the ion suppression during the determination ofclenbuterol. A comparison was made between the selectivity and ionsuppression effects after extraction by the polymeric phase and a more selectivephase, that is a molecularly imprinted polymer (MIP). The former showedsignificant interferences of urine matrix compounds, whereas with MIPcartridges the bleeding of the template molecule caused the ion suppression.Section 4.3 describes the use of an SPE-MS2 system for the determination ofprednisolone in serum. Due to unfavourable fragmentation of the analyte a highdetection limit (5 ng/ml) was observed when using an ion-trap MS. Due tomatrix interference, no improvement was observed with a triple-quadrupole MSwith atmospheric pressure chemical ionisation or atmospheric pressure photoionisation. The total analysis time was less than 5 min.

Chapter 5 describes the direct coupling of SPME with MS as ahigh-throughput system for bioanalysis. Lidocaine was extracted from urine bynon-equilibrium SPME with a 100-µm polydimethylsiloxane (PDMS) coatedfiber at room temperature (Section 5.1). The total analysis time was about10 min (including desorption) and an LOD in the sub-ng/ml range was obtained,even though the total yield was only about 7%. Acceptable reproducibility(interday relative standard deviations < 14%) was observed. In an SPME-MSn

system the sorption is time-limiting. In order to speed up the system it is usefulto discern the limiting factors during the sorption. Using elevated temperaturesduring the sorption and desorption, the diffusion processes could besignificantly enhanced. In Section 5.2, the effect of the temperature on thesorption and the desorption is described, and an optimised SPME-MS2 system ispresented for determination of lidocaine in urine. When applying a 30-µmPDMS coated fiber with only 1 min sorption at 60°C, 1 min desorption at 55°Cand 1 min MS (i.e. a turn-around time of some 3 min), reproducible results were

Chapter 1 – Introduction

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obtained in the sub-ng/ml range. This system clearly shows the potential ofnon-equilibrium SPME at elevated temperatures coupled directly to MS.

In Chapter 6 some general conclusions and future perspectives arepresented. The potentials and limitations of the various systems with integratedsample preparation, which are presented in this thesis are discussed and, wherepossible, compared with each other.

1.3 References

[1] Z.E. Penton. Advances in Chromatogr. 37 (1997) 205.[2] J.P. Franke, R.A. de Zeeuw. J. Chromatogr. B 713 (1998) 51.[3] K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng. Anal. Chem. 70 (1998)

882.[4] D.L. Buhrman, P.I. Price, P.J. Rudewicz. J. Am. Soc. Mass Spectrom. 7 (1996)

1099.[5] R.E. Majors. LC-GC Intern. May, 1998, S8-S15.[6] J.S. Fritz, M. Macka. J. Chromatogr. A 902 (2000) 137.[7] M.C. Hennion. J. Chromatogr. A 856 (1999) 3.[8] D.T. Rossi, N. Zhang. J. Chromatogr. A 885 (2000) 97.[9] D.A. McLoughlin, T.V. Olah, J.D. Gilbert. J. Pharm. Biomed. Anal. 15 (1997)

1893.[10] M.W.F. Nielen, A.J. Valk, R.W. Frei, U.A.Th. Brinkman. J. Chromatogr. 393

(1987) 69.[11] A.C. Hogenboom, P. Speksnijder, R.J. Vreeken, W.M.A. Niessen, U.A.Th.

Brinkman. J. Chromatogr. A 777 (1997) 81.[12] A.C. Hogenboom, W.M.A. Niessen, U.A.Th. Brinkman. J. Chromatogr. A 794

(1998) 201.[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] 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.[15] J. Ding, U.D. Neue. Rapid Commun. Mass Spectrom. 13 (1999) 2151.[16] C.H.P. Bruins, C.M. Jeronimus-Stratingh, K. Ensing, W.D. van Dongen, G.J. de

Jong. J. Chromatogr. A 863 (1999) 115.[17] G.D. Bowers, C.P. Clegg, S.C. HughesC, A.J. Harker, S. Lambert. LC•GC 15

(1997) 48.[18] H.G.J. Mol, H.-G. Janssen, C.A. Cramers, J.J. Vreuls, U.A.Th. Brinkman.

J. Chromatogr. A 703 (1995) 277.[19] K. Grob, J.-M. Stoll. J. High Resolut. Chromatogr. 9 (1986) 518.[20] K. Grob Jr., G. Karrer, M.-L. Riekkola. J. Chromatogr. 334 (1985) 129.[21] W. Vogt, K. Jacob, H.W. Obwexer. J. Chromatogr. 174 (1979) 437.[22] W. Vogt, K. Jacob, A.-B. Ohnesorge, H.W. Obwexer. J. Chromatogr. 186 (1979)

179.[23] W. Engewald, J. Teske, J. Efer. J. Chromatogr. A 856 (1999) 259.[24] H.L. Lord, J.B. Pawliszyn. J. Chromatogr. A 885 (2000) 153.[25] G. Theodoridis, E.H.M. Koster, G.J. de Jong. J. Chromatogr. B 745 (2000) 49.[26] S. Ulrich. J. Chromatogr. A 902 (2000) 167.

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[27] N.H. Snow. J. Chromatogr. A 885 (2000) 455.[28] H.L. Lord, J.B. Pawliszyn. J. Chromatogr. A 902 (2000) 17.[29] R. Eisert, J. Pawliszyn. Crit. Rev. Anal. Chem. 27 (1997) 103.[30] J. Chen, J.B. Pawliszyn. Anal. Chem. 67 (1995) 2530.[31] A.A. Boyd-Boland, J.B. Pawliszyn. Anal. Chem. 68 (1996) 1521.