university of groningen on-line coupling of sample ...plasma concentrations of 250 pg/ml can be...

University of Groningen

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

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33INTEGRATION OF SOLID-PHASE

EXTRACTION AND GASCHROMATOGRAPHY

The pieces have

Evaluation of the PTV for LVI of biological samples in GC

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3.1

Evaluation of the programmed temperaturevaporiser for large-volume injection of biologicalsamples in gas chromatography*

Summary

The use of a programmed temperature vaporiser (PTV) with a packed liner wasevaluated for the injection of large volumes (up to 100 µl) of plasma extracts ina gas chromatograph. Solvent purity, which is essential when large volumes areinjected into the GC system, was determined. Special attention was paid to thepurity of the solvents used for the solid-phase extraction (SPE) procedure. Forthis SPE method, ethyl acetate was used as the extraction and reconstitutionsolvent, and thus the purity of the ethyl acetate was critical, especially when anon-selective GC detector was applied. The liquid capacity and inertness ofdifferent packed liners were investigated. The liner packed with ATAS "A"(modified Chromosorb-based material with special treatment) was found to bethe most suitable for the analysis of the tested drugs. Good linearity in responsefor variations in volume and concentration was observed. A comparison wasmade between the applicability of flame-ionisation detection (FID) and massselective detection (MSD). When 50-µl volumes of plasma extracts wereinjected with the PTV, the detection limits for secobarbital, lidocaine,phenobarbital, and diazepam were about 50 times lower than when 1-µlvolumes were injected. The detection limits of the tested compounds in plasmafor injection of 50-100 µl plasma extract are 5-10 ng/ml for GC-FID whereasplasma concentrations of 250 pg/ml can be detected using the selected ionmonitoring (SIM) mode of an MSD. For non-selective GC-FID, the backgroundfrom a 50-µl injection was substantially larger than with 1-µl injection as aresult of co-injected plasma matrix components and solvent impurities. Thesebackground effects were less with GC-MSD in the total ion current (TIC) modeand virtually absent with GC-MSD in the SIM mode.

*: M.W.J. van Hout, R.A. de Zeeuw, J.P. Franke, G.J. de Jong. J. Chromatogr. B 729 (1999) 199-210.

Chapter 3 – Integration of solid-phase extraction and gas chromatography

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3.1.1 Introduction

Increasing knowledge of the working mechanism of biologically activesubstances has led to the development of potent drugs. Hence, lower dosagescan be administered to produce a therapeutic effect and, consequently, drugconcentrations in biological samples often are much lower than before. For thedetermination of these lower levels in biological samples, analytical techniqueswith much higher sensitivity are needed. A way to increase the sensitivity is toincrease the amount of sample injected into the analytical system.

In gas chromatography (GC) several techniques are available to performlarge-volume injections (LVIs) [1]. On-column injection with the use ofso-called retention gaps is currently the most common technique [1]. A secondpossibility for LVI is the loop-type interface [2], originally designed for thecoupling of liquid chromatography (LC) and GC. The main advantage of thesetechniques is that the complete sample is introduced into the GC column.However, this may also become a disadvantage since all impurities areintroduced into the GC system as well. A third option to allow LVI in GC is touse a programmed temperature vaporiser (PTV). Despite good results obtainedby Vogt and co-workers [3,4] in the late seventies, only recently PTV injectionwas applied as a routine technique for environmental analysis [1].

Besides conventional split/splitless injection, the PTV can be used forseveral modes of LVI. The coupling of LC and GC using the PTV was reviewedby Grob [5], and recently interesting publications appeared on the same subject[6,7]. The PTV is often applied for this purpose because the packed linergenerally has a larger liquid storage capacity than a retention gap. In addition,wetability is not very critical for the liquid retention and packing materials aremore water-resistant than retention gaps with a silica backbone. The packing ismore easily and rapidly heated than a retention gap [5]. Main reasons to coupleLC with GC are that LC provides better resolution than more conventionaltechniques of sample preparation, and secondly, the possibility of automationthrough on-line coupling, which reduces or eliminates manual samplepreparation work and, therefore, reduces analysis time and improves accuracyand precision [5,7]. The use of a PTV as the interface between LC and GC hasbeen demonstrated for the analysis of olive oil and for environmental analysis[6,7]. The PTV is also used for thermal desorption-pyrolysis of solidgeochemical samples (characterisation of oil and kerogens in source rocks) [8],and for on-line solid-phase extraction-thermal desorption (SPETD) of methylesters of the C10-C26 carboxylic acids, pesticides, chlorobenzenes andchlorophenols in aqueous samples [9-11].

Most applications of LVI are in the analysis of environmental aqueoussamples [1,9-13]. Pesticides were determined in aqueous samples after SPE ofsamples of 200 ml with concentrations between 0.2 and 5 ng/l by Steen et al.


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[12], whereas Teske et al. [13] determined triazines like atrazine, propazine,ametryne and simazine in water after in-vial liquid-liquid extraction and directinjection of the extracts with detection limits as low as 0.01 µg/l, andpolychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons(PAHs) at the ppt-level [13]. Another application of the PTV is the residueanalysis of 385 pesticides down to the 0.01-ppm concentration level in plantfoodstuff [14].

The purpose of the present work is to investigate the possibilities of thePTV coupled to GC for the analysis of plasma extracts to provide lowerdetection limits for drugs. Special attention was paid to the impact of solventimpurities in view of the larger solvent volumes injected, to the liquid capacityand inertness of the PTV liners, and to the degree of selectivity provided byflame-ionisation detection (FID) and mass selective detection (MSD).

3.1.2 Experimental

3.1.2.1 Instrumentation

Gas chromatographic analyses were performed with a Hewlett-PackardHP 5890 series II with FID or a GC-MSD system (HP 5971 series). A HP-530 m×0.32 mm capillary column with 0.25 µm film thickness was used for theanalyses with FID, whereas analyses with MSD were performed using a HP-5MS 30 m×0.25 mm column with 0.25 µm film thickness. The PTV injectionsystem was an OPTIC 2 (ATAS International, Veldhoven, The Netherlands),which was equipped with 80 mm×3.4 mm i.d. liners obtained from ATASInternational. The liners were packed with either ATAS "A" packing (amodified Chromosorb-based material with special treatment, ATASInternational), silanised glass wool (research grade, Serva, Feinbiochemica,Heidelberg, Germany), or disposable capillaries for thin-layer chromatography(TLC) (nine capillaries of 10 µl and two of 2 µl, cut at a length of 2 cm).

Plasma extractions were performed using Bond Elut Certify cartridges(Varian, Harbor City, CA, USA), column type LRC of 10 ml with 130 mgsorbent. A Visiprep system (Supelco, Bellefonte, PA, USA) was used to applyvacuum during the extraction.

3.1.2.2 Chemicals

Acetonitrile and methanol (Lab Scan, Dublin, Ireland) were of HPLCquality. Acetone, hexane, acetic acid glacial 100% (v/v), ammonia solution25%, and KH2PO4 were all of analytical-reagent quality (Merck, Darmstadt,Germany). Ethyl acetate (Reinst and Suprasolv - for organic residue analysis)


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was obtained from Merck (Darmstadt, Germany). Ethyl acetate Ultraresi-analysed (for organic residue analysis) was purchased from MallinckrodtBaker (Deventer, The Netherlands). Water used during SPE was ultra pure(Elgastat maxima, Salm en Kipp, Breukelen, The Netherlands). Secobarbital,phenobarbital (both BP quality, Siegfried, Zofingen, Switzerland), lidocaine(Eur. Ph., Holland Pharmaceutical Supply, Alphen A/D Rijn, The Netherlands),and diazepam (Centrafarm, Etten-Leur, The Netherlands) were used as testcompounds (Fig. 1) and dissolved in ethyl acetate (for organic residue analysis,Mallinckrodt Baker). Stock solutions of 1 mg/ml were stored in the dark at 4°C.Stock solutions were mixed and then diluted with ethyl acetate (for organicresidue analysis, Mallinckrodt Baker). The compounds of the referenceRI-mixture [15] were dissolved in ethyl acetate:methanol (1:1) (1 mg/ml).

Fig. 1. Structures of the test compounds: (A) secobarbital, (B) lidocaine,(C) phenobarbital, (D) diazepam.

3.1.2.3 Methods

The carrier gas for GC-FID and GC-MSD was helium. The sametemperature program was used for both methods. The starting temperature was40°C, and after 3 min the temperature was raised at 20°C/min to 215°C,followed by an increase at 5°C/min to 230°C and a final increase at 25°C/min to290°C. This final temperature was maintained for 5-10 min. The detectortemperature was 300°C. A column flow of 1.35 ml/min was used duringanalysis with GC-FID and 0.48 ml/min with GC-MSD. The injector was set at40°C and 10 s after the evaporation of the solvent (delay time) the temperaturewas raised with 5°C/s to 290°C. The end time was set at a time equal to the totalrun time of one analysis. Other PTV settings are presented in Table 1.

During analysis performed with GC-MSD in the total ion current (TIC)mode an m/z range of 50-300 was monitored. Using the selected ion monitoring(SIM) mode, the monitored m/z values were 86.0, 167.0, 204.0, and 256.0,which were corresponding to the most intense fragment of lidocaine,secobarbital, phenobarbital and diazepam, respectively.


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SPE was performed as described previously [16] with some minormodifications. The SPE column was activated with 2 ml methanol (2 ml/min),followed by conditioning of the SPE column with 2 ml 0.1 M of K2HPO4 bufferpH 6 (2 ml/min). Subsequently, 1 ml plasma, diluted with 4 ml K2HPO4 buffer,was brought on the column during approximately 1 min. Then the SPE columnwas washed with 1 ml water and 0.5 ml 1 M acetic acid (1.5 ml/min). Thecolumn was dried under vacuum for 4 min, after which 50 µl of methanol werepassed through to remove remaining traces of water. The column was driedunder vacuum for 1 min. The tips of the Visiprep system were dried and tubeswere inserted for the collection of the eluate. The acidic fraction was elutedwith 1 ml ethyl acetate-acetone (1:1) (0.8 ml/min), followed by the elution ofthe alkaline fraction with 0.5 ml acetonitrile-ammonia (98:2) (0.5 ml/min). Thefractions were evaporated until almost dry and reconstituted in 100 µl ethylacetate (for organic residue analysis, Mallinckrodt Baker). Finally, 50-100 µl ofthese extracts were injected into the GC system.

Table 1: PTV settings.

GC-FID GC-MSDVent flow (ml/min) 150 150Split flow (ml/min) 57.4 57.4Purge flow (ml/min) 2.32 2.32Purge press (p.s.i.*) 8.0 4.0Transfer press (p.s.i.*) 14.0 4.0Transfer time (min:s) 2:45 2:45Initial press (p.s.i.*) 8.0 2.0Final press (p.s.i.*) 8.0 2.0Vent mode AUTO AUTOSplit open time (min:s) 2:30 2:30Threshold 20 20

*: 1 p.s.i. = 6894.76 Pa

3.1.3 Results and discussion

The basic set-up of a PTV injector strongly resembles a conventionalsplit/splitless injector. The main difference is that a (packed) liner system isapplied which is temperature-controlled. Injections up to about 150 µl can occurat once, whereas larger volumes must be injected at a controlled rate. In theinjection mode the temperature of the liner is set at 30-40°C below the boilingpoint of the used solvent. A high vent flow ensures selective evaporation ofsolvent via the split line, whereas less volatile solutes are retained in the liner.After evaporation of almost all the solvent, rapid transfer of the lattercomponents to the column is performed, using the splitless mode, by rapidlyheating the liner, or, optionally, by a high transfer pressure. During the transfer


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of the components, the GC column is maintained at a low temperature (samestarting temperature as the injector), thus leading to a refocusing of componentsat the front of the column. Further analysis is performed with normaltemperature-programmed GC.

3.1.3.1 Purity of solvents and chemicals

Since much larger solvent volumes are injected into the PTV/GC system,the impact of solvent impurities was checked. In the plasma extractionprocedure, ethyl acetate is being used as extraction and reconstitution solvent[16]. Various qualities of ethyl acetate were tested, and some analytical resultsare presented in Fig. 2. Fig. 2A shows that a brand, which was found acceptablefor 1-µl injections, contained far too many impurities when 100 µl was injectedinto the PTV/GC system. The best results were obtained when using 100-µlinjections with the quality “for organic residue analysis” (Mallinckrodt Baker)as shown in Fig. 2B. Therefore, this latter quality was used in furtherexperiments. The purity of methanol, acetonitrile, ammonia and KH2PO4 werefound to be acceptable in that the quantities used in the present procedure didnot introduce major impurities. Attempts to purify ethyl acetate by a C18-LCcolumn or a C18 cartridge were not successful.

Fig. 2. GC-FID chromatogram of injection of 100 µl ethyl acetate (A) Reinst (Merck),(B) For organic residue analysis (Baker).


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3.1.3.2 Liquid capacity of the liners

A liner must have a relatively large liquid capacity (Vmax) to allowinjection of 50-100 µl sample at once (no speed-controlled injection). The liquidcapacity of a liner can be easily determined by removing the column from theinjector without turning off the carrier gas. Then, 200 µl solvent are injectedrapidly and the injector outlet is checked for solvent droplets. The amount ofsolvent injected is reduced until no droplets are observed which reflects Vmax. InTable 2 the measured Vmax for ethyl acetate is given for several liners. Allpackings were of similar dimensions (height 2.5 cm, 1.8 cm from the columnside).

Table 2: Liquid capacity of liners.

Liner packing Vmax (µl)ATAS-1* 150Glass wool** 65Glass capillaries*** 40

*: Total amount of 80-85 mg ATAS ‘A’ packing in fritted liner.**: Total amount of 144 mg glass wool was inserted into a open liner.***: 9 TLC capillaries of 10 µl and 2 TLC capillaries of 2 µl were cut at a length of 2 cm, andinserted into a fritted liner. A small plug of glass wool was placed under and above the capillaries.

The glass wool and the ATAS "A" liner appeared to be best suitable forthe injection of large volumes ethyl acetate (>50 µl), and the ATAS "A" linercan even be used for samples larger than 100 µl. Mol et al. [17] found a Vmax of115 µl for a liner packed with glass wool instead of 65 µl. This difference isprobably due to the major problem with glass wool, that is, inserting thepacking into the liner in a reproducible way [17,18].

3.1.3.3 Inertness of the liner packings

Comparison of packingsIt was necessary to investigate the inertness of liner packing materials for

biological samples since a high recovery of the analytes must be obtainedduring consecutive sample injections. Several ATAS "A" liners (Nos. 1-5), aglass wool packed liner (No. 6) and a liner packed with open capillaries (No. 7)were tested. Cutting glass capillaries can create active places at the cutting site,and glass wool is known to have a limited inertness [17]. The ATAS "A" linerwas originally designed for the analysis of pesticides and mineral oils. Theinertness of the ATAS "A" liner can be influenced by a high temperature sincethe packing has a Tmax of 325°C. Higher temperatures will degrade the packing,


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which will have a negative effect on the inertness of the liner. Analysis of highboiling compounds (>325°C) is therefore not possible.

Prior to testing the inertness, liners 1 and 2 were used for many injectionsof standard solution and plasma extracts. The colour of liner 1 had changedfrom white to completely brown, and the upper half of the packing of liner 2had changed to brown with the lower half still white. Liners 3-7 were not usedbefore. Injection of 1 µl of 250 µg/ml test compounds (secobarbital, lidocaine,phenobarbital and diazepam) into a liner with a glass frit without packingproduced the reference chromatogram, that is, since no active packing waspresent, the response of the compounds was set at 100%. Injections of 1 µl of250 µg/ml or 100 µl 2.5 µg/ml test compounds into liners 3, 4, and 5 producedthe same responses, thus the ATAS "A" liner can be considered to be inert if theliner packing is not previously used for analysis.

In Table 3 the responses are tabulated for liners 1, 2, 6 and 7, as comparedto liners 3, 4 and 5. Liners 1, 2, 6 and 7 showed adsorption activity forphenobarbital, liner 1 and 6 being the most active. Injection of 1 µgphenobarbital into liner 1 produced even no peak. For secobarbital, liner 1 isvery active whereas liners 2, 6 and 7 are much less active. For lidocaine, liners1 and 2 showed limited activity and the same was observed for diazepam. Fromthese results, it appears that adsorption losses are more pronounced in the orderdiazepam/lidocaine<secobarbital<phenobarbital. The change in colour seems agood parameter to indicate adsorption activity of the ATAS "A" liner forbarbiturates whereas it has hardly any effect on the response of lidocaine anddiazepam.

Table 3: Recovery (%) of 250 ng of compounds compared with average response ofliner 3, 4, and 5 (set at 100%). Liners: 1-5 = ATAS ‘A’ (No.1 completelybrown, No. 2 upper half brown, No. 3-5 white), 6 = silanized glass wool,7 = glass capillaries.

Liner Secobarbital Lidocaine Phenobarbital Diazepam1 3 86 0 872 96 92 59 963,4,5 100 100 100 1006 81 99 17 1007 88 97 49 100

Glass wool packed liners were shown to adsorb fatty acids from 42 to100% [17]. In this work glass wool also showed adsorption activity for theweakly acidic compounds secobarbital and phenobarbital, but the materialappears to be better suitable for weakly basic drugs. Thus, a glass wool liner canbe used for some of the drugs investigated in this work whereas the glass woolliner used by Mol et al. [17] was unsuitable for the analysis of all tested


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compounds. Biedermann et al. [19] pointed out that the deactivation ofvaporising chambers can also play an important role. Deactivating of the glasssurface was performed before the packing material was inserted into the liner.In order to investigate if there are other classes of drugs for which theATAS "A" packing is not inert, a reference mixture [15] was analysed usingliner 1, 2 and 5. The results are presented in Fig. 3.

Fig. 3: Effect of packing inertness on response of compounds of RI-mix [15].¨ = liner 1, = liner 2, n = liner 5. Compounds: 1 = amphetamine,2 = ephedrine, 3 = benzocaine, 4 = methylphenidate, 5 = diphenhydramine,6 = tripelenamine, 7 = methaqualone, 8 = trimipramine, 9 = codeine,10 = nordazepam, 11 = prazepam, 12 = papaverine, 13 = haloperidol,14 = strychnine.

The percentages given below are recoveries with the response of the compoundduring analysis with liner 5 set at 100%. No effect on the response was foundfor amphetamine, ephedrine, diphenhydramine, tripelenamine and trimipramine.Liner 1 showed a small decrease in recovery for benzocaine (90%),methaqualone (96%), nordazepam (95%), prazepam (85%) and haloperidol(80%). Liner 2 was not active for these substances. The same was observed withliner 2 for strychnine, but liner 1 was active for this compound (46%). Theeffect of liner activity for methylphenidate (liner 2 and 1, 82 and 5%,respectively), codeine (90 and 28%), and papaverine (38 and 5%) wascomparable with that for phenobarbital. Thus, the ATAS "A" liner can becomeless inert to substances due to injection of plasma extracts and/or standardsolution. The results confirm the suggestion that a beginning brown colouration


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can be used as an indication that the liner is starting to loose its inertness andthat it needs replacement. From the results with both the test compounds as wellas the compounds of the RI-mixture, it can be concluded that barbiturates andopium alkaloids are particularly prone to recovery losses. On the other hand,even a completely brown ATAS "A" liner has little effects on the recoveries ofbenzodiazepines.

Long-term useThe ATAS "A" liner and the glass wool liner have a sufficient liquid

capacity to allow injections up to 50 µl. However, the glass wool liner appearedto be less inert for some type of compounds. Therefore, only the inertness of theATAS "A" liners as a function of the number of injections was alsoinvestigated. Two new ATAS "A" liners were used. For one liner only astandard solution (50 µl of 2.5 µg/ml) was injected 35 times (Fig. 4). The linerappeared to remain inert under these conditions since no loss in response wasobserved for all test compounds. For the second liner, 50 µl of 2.5 µg/mlstandard solution with two injections of plasma extract (one alkaline and oneacidic fraction) between subsequent injections of the standard solution wereanalysed. The response of the test compounds (secobarbital, phenobarbital,lidocaine and diazepam) in the standard solution is plotted as a function of thenumber of plasma injections (Fig. 4).

Since up to 35 injections of standard solution had no influence on thestability of the liner packing, a decrease in response must have been caused bythe plasma extracts influencing the inertness of the packing material. Injectionof plasma extracts introduces a large decrease in inertness of the packing forphenobarbital starting with a slightly variable response at 14 injections ofplasma extracts, and a definite loss in response after 20 injections. Forsecobarbital, after 20 injections of plasma extracts only a small increase inactivity of the ATAS "A" packing is observed. Injection of 32 plasma extractshas no effect on the response of diazepam and lidocaine (lidocaine not shown).An increase in activity of the packing was again found to correlate with thecolour of the packing material. Inert ATAS "A" material is white but thisbecomes brown with increasing activity. After some 10-15 injections of plasmaextracts the colour of the ATAS "A" packing started to change from white tobrown, and the brown colour became more apparent on continued analysis ofplasma extracts. The decrease in inertness and change in colour of theATAS "A" packing when plasma extracts are injected is probably due to thedegradation of matrix components that are not desorbed from the liner onheating.


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Fig. 4: Stability of ATAS-1 packing. Response of 125 ng test compound in 50 µlstandard solution of (A) diazepam, (B) secobarbital, (C) phenobarbital;u = response for injections of only the standard solution, n = response forinjections of the standard solution with two plasma extract injections betweensubsequent injections of the standard solution (number of injectionscorresponds with the amount of plasma extract injections).


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Carry-overCarry-over was checked by injecting large amounts of the test compounds

or RI-mixture (up to 2.5 µg), followed by a second injection in which a blank,i.e. 100 µl ethyl acetate, was introduced. No carry-over was observed for eitherthe test compounds or the components of the RI-mixture when using anATAS "A" liner.

3.1.3.4 Linearity with PTV/GC-FID for standard solutions

Since the ATAS "A" liner has a relatively large liquid capacity, is inertfor the tested compounds and no carry-over occurs, the ATAS "A" liner issuitable for LVI of bioanalysis. Linearity in response for lidocaine, diazepam,secobarbital and phenobarbital was determined for variation in volume andconcentration. For the determination of the linearity in response when thevolume is varied, volumes varying from 20 to 100 µl of a 1.0 µg/ml standardsolution were injected on a new ATAS "A" liner. It was found that allcompounds showed a good linearity (Table 4).

The linearity of response versus concentration was determined byinjections of 100 µl standard solution over a concentration range of 5 to2000 ng/ml. As can be observed in Table 4, a good linearity was obtained. Oneshould note that no internal standard was applied to correct for injection volumeand signal drift.

Table 4: Linearity (coefficient of correlation (R)) of secobarbital, lidocaine,phenobarbital, and diazepam with variation of volume (20-100 µl of 1 µg/ml)and concentration (5-2000 ng/ml).

R (volume) R (concentration)Secobarbital 0.9989 0.9974Lidocaine 0.9937 0.9965Phenobarbital 0.9979 0.9923Diazepam 0.9976 0.9982

3.1.3.5 PTV/GC-FID and PTV/GC-MSD of plasma extracts

Linearities as well as the detection limits for lidocaine, diazepam,secobarbital, and phenobarbital in plasma extracts were determined using FIDand MSD. With LVI a large amount of matrix components and solventimpurities is injected. Therefore peak identification can become difficult whennon-selective detectors are used. Use of a mass selective detector may help toovercome this problem. With the mass selective detector analyses wereperformed in both the TIC and the SIM mode.


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Linearity was determined for concentrations ranging from the detectionlimit up to 100 ng using 1 ml of spiked plasma extract. The results are listed inTable 5. For all detectors linearity was found to be better for the alkalinefraction than the acidic fraction, except for diazepam. For this compound,coefficients of correlation are comparable for both fractions. The acidic fractionanalysed with MSD shows a relatively low linearity for phenobarbital. Thismight be due to an increase in activity of the GC system during analysis (seeSection 3.1.3.3).

Table 5: Linearity (coefficient of correlation) of secobarbital, lidocaine, phenobarbital,and diazepam in acidic and alkaline SPE fraction of plasma; range: detectionlimit (see text below) to 100 ng/ml.

RFID(acidic)

FID(alkaline)

TIC(acidic)

TIC(alkaline)

SIM(acidic)

SIM(alkaline)

Secobarbital 0.9963 0.9981 0.9940 0.9976 0.9997 0.9998Lidocaine 0.9936 0.9992 0.9986 0.9962 0.9991 0.9997Phenobarbital 0.9947 0.9996 0.9993 0.9958 0.9719 0.9992Diazepam 0.9975 0.9962 0.9969 0.9953 0.9991 0.9994

The detection limits were determined at a signal-to-noise ratio of 3 forboth FID and MSD. It should, however, be mentioned that when using FID andMSD in the TIC mode, interfering matrix compounds and solvent impuritiescan make the determination of the detection limit laborious as blank peaks havea negative influence. Blank plasma was extracted and the extracts were spikedwith the test compounds and detection limits for plasma extracts werecalculated assuming a 100% recovery of the test substances in the SPEprocedure. The actual recoveries using this SPE method were found to be80-100% [16].

Chromatograms of 40-45 ng compounds in the alkaline fraction analysedwith FID, TIC and SIM are presented in Fig. 5. Using FID, the detection limitusing 1 ml of plasma is 5-10 ng for all compounds in both the acidic andalkaline fraction. The detection limits observed using the TIC mode of the MSDare 4-5 ng. Therefore, a small gain in detection limit can be achieved. This ismainly due to the fact that with TIC a positive identification can be given forthe peaks present in a sample since reliable mass spectra are obtained with goodcorrespondence with library spectra. Acidic plasma extracts analysed in the SIMmode give a detection limit of 0.5 ng for each test compound whereas thealkaline fraction gives a detection limit of 0.25 ng.

The gain in sensitivity when compared with FID or TIC can be explainedby the enhanced selectivity since detection occurs only at four m/z values. If thecomplete sample is injected, plasma concentrations as low as 250 pg/ml could


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be detected in the alkaline fraction. With conventional GC, that is injection of1 µl plasma extract, also a detection limit of 0.25 ng for the test compounds isfound. As a consequence, the corresponding plasma concentration is 50 timeshigher than with injection of 50 µl, i.e. a concentration of 12.5 ng/ml can bedetected.

Fig. 5. Chromatograms of 50 µl of the alkaline fraction of plasma extracts analysedwith (A) GC-FID (41.7 ng), (B) GC-MSD; TIC mode (45.5 ng), (C) GC-MSD;SIM mode (45.5 ng). The monitored m/z values were 86.0, 167.0, 204.0 and256.0 for lidocaine (L), secobarbital (S), phenobarbital (P) and diazepam (D),respectively. Note: the peak (*) eluting just after the diazepam peak (D) iscaused by an impurity with an m/z value similar to phenobarbital.


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3.1.4 Conclusions

It was demonstrated that the PTV is potentially suitable to inject largevolumes of extracts of biological samples in GC. In this way detection limitscan be improved considerably. In order to be able to apply LVI in GC it isnecessary to select an appropriate liner packing. As for the liner packings testedin this work the ATAS "A" packing was found to be the most suitable since thismaterial has a large liquid capacity and is relatively inert. However, the packingcan become active after a number of injections of plasma extracts for certaintypes of compounds. It is therefore recommended to carefully monitor theinertness of the packing material by injection of a few suitable compounds andto monitor the colour of the liner packing.

Injection of large volumes implies that an equivalent amount of impuritiesis injected. Therefore, it is essential to use very pure solvents and chemicalsduring the work-up procedure. However, not only solvent impurities areinjected. Also matrix components that are co-extracted with the analytes makeidentification and quantitation difficult when a non-selective detector is used.The use of a selective detector is essential to overcome this problem. Using amass selective detector, a 100 times gain in concentration-sensitivity can beachieved if 100 µl of a plasma extract instead of 1 µl is injected.

The present system can be used as a routine technique in research andclinical laboratories. However, further evaluation of the system for variouspurposes (including other matrices) and different types of compounds is needed.The on-line coupling of SPE and GC for bioanalysis will also be investigated inthe near future in our laboratory.

Acknowledgements

Jan Henk Marsman and Ronald Veenhuis (Department of ChemicalEngineering, University of Groningen) are gratefully acknowledged for the useof the GC-MSD system and their assistance. This research was supported by theTechnology Foundation STW, applied science division of NWO and thetechnology programme of the Ministry of Economic Affairs.

3.1.5 References

[1] H.G.J. Mol, H.-G. M. Janssen, C.A. Cramers, J.J. Vreuls, U.A.Th. Brinkman.J. Chromatogr. A 703 (1995) 277.

[2] K. Grob, J.-M. Stoll. J. High Resolut. Chromatogr. Commun. 9 (1986) 518.[3] W. Vogt, K. Jacob, H.W. Obwexer. J. Chromatogr. 174 (1979) 437.


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[4] W. Vogt, K. Jacob, A.-B. Ohnesorge, H.W. Obwexer. J. Chromatogr. 186 (1979)197.

[5] K. Grob. J. Chromatogr. A 703 (1995) 265.[6] P. Van Zoonen, G.R. van der Hoff. LC-GC Int. 16 (1998) 240.[7] T. Hyötyläinen, M.-L. Riekkola. J. Chromatogr. A 819 (1998) 13.[8] M.P.M. van Lieshout, H.-G. Janssen, C.A. Cramers, G.A. van den Bos.

J. Chromatogr. A 764 (1997) 73.[9] J.J. Vreuls, U.A.Th. Brinkman, G.J. de Jong, K. Grob, A. Artho. J. High Resolut.

Chromatogr. 14 (1991) 455.[10] J.J. Vreuls, G.J. de Jong, R.T. Ghijsen, U.A.Th. Brinkman. J. Microcol. Sep. 5

(1993) 317.[11] A.J.H. Louter, J. Van Doornmalen, J.J. Vreuls, U.A.Th. Brinkman. J. High

Resolut. Chromatogr. 19 (1996) 679.[12] R.J.C.A Steen, I.L. Freriks, W.P.Cofino, U.A.Th. Brinkman. Anal. Chim. Acta

353 (1997) 153.[13] J. Teske, J. Efer, W. Engewald. Chromatographia 47 (1998) 35.[14] H.J. Stan, M. Linkerhagner. J. Chromatogr. A 750 (1996) 369.[15] R.A. de Zeeuw, J.P. Franke, H.H. Maurer, K. Pfleger. Gas chromatographic

retention indices of toxicologically relevant substances on packed and capillarycolumns with dimethylsilicone stationary phases, third edition, VCH, Weinheim,1992.

[16] X.-H. Chen, J. Wijsbeek, J.P. Franke, R.A. de Zeeuw. J. Forensic Sci. 37 (1992)61.

[17] H.G.J. Mol, P.J.M. Hendriks, H.-G. Janssen, C.A. Cramers, U.A.Th. Brinkman.J. High Resolut. Chromatogr. 18 (1995) 124.

[18] H.G.J. Mol, H.-G. Janssen, C.A. Cramers, U.A.Th. Brinkman. J. High Resolut.Chromatogr. 18 (1995) 19.

[19] M. Biedermann, K. Grob, M. Wiedmer. J. Chromatogr. A 764 (1997) 65.

Coupling device for desorption of drugs from SPEPTs into GC

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3.2

Coupling device for desorption of drugs fromsolid-phase extraction – pipette tips and on-linegas chromatographic analysis*

Summary

Solid-phase extraction – pipette tips (SPEPTs) were used for micro solid-phaseextraction of lidocaine and diazepam. Off-line desorption was done after in-vialcollection for reference purposes, whereas with on-line desorption the eluatewas directly introduced in the gas chromatograph. With both methods the totaleluate (100 µl) was introduced into the GC, which was equipped with aprogrammed temperature vaporiser (PTV) for large-volume injection. Foron-line desorption a laboratory-made coupling device was developed to connectthe pipette tips with the injector of the PTV. The coupling device was appliedsuccessfully since no leakage occurred at the connection of the coupling deviceand the pipette tip. No significant differences in recovery of lidocaine anddiazepam and in presence of impurities were observed between chromatogramsobtained with either off-line or on-line desorption. Preliminary experimentswith standard solutions showed recoveries of about 75 % for a concentrationlevel of 1 µg/ml. The system seems particularly suitable for high-throughputanalysis.

*: M.W.J. van Hout, R.A. de Zeeuw, G.J. de Jong. J. Chromatogr. A 858 (1999) 117-122.


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3.2.1 Introduction

High sample throughput is becoming increasingly important in variousareas of bioanalysis. Therefore, it is essential to reduce analysis time includingsample pre-treatment. A very popular sample pre-treatment technique issolid-phase extraction (SPE), which was originally developed as an off-linesample clean-up and pre-concentration procedure [1-7]. In order to obtain highsample throughput, there is a growing interest in SPE with at-line and on-lineliquid chromatography [8-11] and, more recently, gas chromatography (GC)[12-19]. In addition, it has been claimed that the various steps in on-line SPEcan be carried out with greater precision than in off-line SPE, resulting in morereliable data. Another advantage of on-line SPE-GC is that the total eluate canbe analysed, which was not possible with off-line SPE. However, withlarge-volume injection (LVI) of extracts of biological samples in gaschromatography via a retention gap [13,19] or a programmed temperaturevaporiser (PTV) [20], it is possible to inject nearly the total eluate of off-lineextractions. Yet, with LVI, special attention must be given to solvent purity andselectivity of the extraction procedure [20], since with the injection of largevolumes an equivalent amount of impurities is also injected into the analysingequipment.

Another interesting development is the miniaturisation of analyticalsystems. The first attempts to miniaturise SPE were done using SPE disksinstead of conventional SPE cartridges [5,6,9-11,21-28]. Generally, SPE diskscontain a smaller bed with smaller particles and a more homogeneous particlesize distribution than conventional cartridges. An advantage of SPE disks overSPE cartridges is the possibility to use smaller desorption volumes (50-400 µlvs. 1-6 ml) [21,23]. Another benefit besides the use of less solvent is that ifLVI/GC is applied in combination with miniaturised SPE no evaporation andreconstitution of the extracts is required, which eliminates an error-prone step inthe extraction procedure, and thus increases reliability and reduces samplepreparation time. Moreover, solvent purity is less critical than when LVI isapplied in combination with conventional SPE, because less desorption solventis used.

Further miniaturisation has led to micro-SPE, which can be performedusing solid-phase extraction – pipette tips (SPEPTs) [7,29]. Extractions can becarried out more easily and rapidly than with conventional SPE or with SPEdisks simply by using a pipettor and pipette tips with extraction material insidethe tip [29]. An interesting aspect of extractions with pipette tips is thatbi-directional flow and cycling, that is aspirating and dispensing, can be applied[7,29]. However, micro-SPE may imply that smaller sample volumes have to beused which leads to higher concentration detection limits. This loss insensitivity may be overcome by applying LVI/GC, which may even lead to an


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increase in sensitivity when compared to conventional injection volumes of 1 to2 µl [20].

In order to reduce the analysis time and increase the reliability of theresults, an on-line coupling of micro-SPE with GC appears to be attractive. TheGC system must be equipped with a special injector, e.g., a PTV, so that LVI ispossible. In this study a coupling device for the connection of pipette tips andthe PTV injector was developed and evaluated for micro-SPE-GC, that is, theextraction was carried out off-line but the desorption and subsequent GCanalysis was done on-line.

3.2.2 Experimental

3.2.2.1 Apparatus and chromatographic conditions

Gas chromatographic analyses were performed with a Hewlett-PackardHP 5890 series II instrument with flame-ionisation detection (FID). Thecapillary column was a HP-5 30 m×0.32 mm with 0.25 µm film thickness.Helium was used as carrier gas. The following temperature program was usedfor the GC. The starting temperature was 40°C and after 3 min the temperaturewas raised at 20°C/min to 215°C, followed by a raise of 5°C/min to 230°C anda final raise of 25°C/min to 290°C. This final temperature was maintained for 5to 10 min. The detector temperature was set at 300°C, and a column flow of1.1 ml/min was used during analysis.

The PTV injection system was an OPTIC 2 (ATAS International,Veldhoven, The Netherlands), which was equipped with a 80 mm×3.4 mm i.d.liner obtained from ATAS International. The liner was packed with ATAS "A"packing (a modified Chromosorb-based material with special treatment). Theinjector was set at 40°C in the vent mode and evaporation of the solventoccurred using the “AUTO vent mode” with a vent flow of 150 ml/min. Afterthe evaporation of the solvent the valve was switched to the splitless mode andafter 10 s the temperature was raised with 5°C/s to 290°C. This finaltemperature was maintained during the analysis. The splitless mode was appliedfor 2.50 min and, subsequently, the valve was switched to the split mode. Theused split flow was 57.4 ml/min, whereas the purge flow and pressure were2.32 ml/min and 2.5 p.s.i., respectively (1 p.s.i.=6894.76 Pa). A transferpressure of 14.0 p.s.i. was applied for 2.75 min. During the analysis the initialand final pressure were maintained at 8.0 p.s.i.


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3.2.2.2 Chemicals

Methanol (Lab Scan, Dublin, Ireland) was of HPLC quality. KH2PO4 wasof analytical-reagent quality (Merck, Darmstadt, Germany). Ethyl acetate Ultraresi-analysed (for organic residue analysis) was purchased from MallinckrodtBaker (Deventer, The Netherlands). Water used during SPE was ultra pure(Elgastat maxima, Salm en Kipp, Breukelen, The Netherlands). Lidocaine(Eur. Ph., Holland Pharmaceutical Supply, Alphen A/D Rijn, The Netherlands)and diazepam (Centrafarm, Etten-Leur, The Netherlands) were used as testcompounds and dissolved in ethyl acetate (for organic residue analysis,Mallinckrodt Baker) or in phosphate buffer pH 8.0. Stock solutions of 1 mg/mlwere stored in the dark at 4°C.

3.2.2.3 Methods

Micro-SPE was performed using pipette tips (SPEC•PLUS•PT) with aC18-AR stationary phase (Ansys Diagnostics, Lake Forest, CA, USA). The SPEprocedure was carried out by connecting a 10-ml gas-tight plastic syringe(Omnifix syringe, B. Braun, Melsungen, Germany) (Fig. 1(A)) to the pipette tip(B). The SPE disk (C) in the pipette tip was activated with ca. 200 µl methanolfollowed by conditioning of the disk two times with ca. 100 µl of 0.1 MK2HPO4 buffer (pH 8.0). Subsequently, 200 µl phosphate buffer spiked with1 µg/ml lidocaine and diazepam were extracted on the disk. Then the disk waswashed with ca. 100 µl water and, subsequently, dried by pushing air throughthe disk (10×10 ml). For the desorption of the analytes from the disk 100 µlethyl acetate were used. The desorption occurred in-vial or on-line. The solventsand samples were drawn into the pipette tip until the fluid had gone through thedisk and then the fluid was completely pushed back out of the pipette tip(bi-directional flow). The fluids did not enter the plastic syringe, thus thesyringe could be used for subsequent extractions.

On-line desorption was possible by connecting the pipette tip to the PTVinjector via a laboratory-made coupling device as depicted in Fig. 1. Thecoupling device (D) was made by replacing the glass from a gas-tight GCsyringe by a piece of PTFE (E). All other parts (F-I) of the coupling device arealso present in conventional GC syringes with a removable needle (I). Thedimensions of the PTFE piece were 5.5 mm×9 mm o.d. (2.0 mm i.d.) (upperhalf) and 6 mm×6 mm o.d. (0.5 mm i.d.) (lower half, inside the screw thread).


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Fig. 1. Scheme of the coupling device of the solid-phase extraction-pipette tip and thePTV injector. Parts: A = plastic syringe, B = SPEC•PLUS•PT pipette tip,C = SPE disk, D = coupling device, E = PTFE piece, F = standard metal screwthread, G = vulcanised rubber, H = standard needle nut, I = needle.


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The use of pipette tips offered the possibility of bi-directional flow andcycling (aspirating and dispensing) [7,29]. Visualisation of these phenomenawas described by Blevins and Hall [29]. A plastic syringe was used in ourapproach instead of a conventional pipettor since the solvents and samplescould be better drawn into and pushed back out of the pipette tip with the plasticsyringe. However, bi-directional flow should not be applied with air, since arapid flow of air in upward direction may dislodge the disk. Therefore, upondrying the disk with air, only dispensing is possible. Generally, aspirating anddispensing solvents was carried out gently to make sure that not too much airwent through the disk with the exception of the final part of the dispension inthe elution step. Ethyl acetate was pushed back completely followed by air toremove as much ethyl acetate as possible from the disk.

During the development of the coupling device for on-line desorption thedimensions of the PTFE piece were chosen so that it ensured to fit just right inplace of the removed glass and that approximately half of the tip of the pipettetip could be inserted tightly into the coupling device. When desorption wascarried out on-line, the needle of the coupling device was completely insertedinto the PTV injector. Subsequently, ethyl acetate was drawn into the pipettetip, and then the pipette tip was immediately placed on top of the couplingdevice and the ethyl acetate was pushed back through the disk directly into thePTV injector. The coupling device and pipette tip were removed at the sametime, so that no leakage of carrier gas and solvent vapour occurred through theneedle of the coupling device.

In order to be able to inject the extract on-line into the GC systemsubstantial pressure must be applied during the push-back since a highback-pressure is present when the pipette tip and coupling device are attached tothe PTV injector. The back-pressure can be lowered slightly by decreasing thepurge pressure of the PTV injector. However, lowering the purge pressure leadsto an increase of vent time, and thus to an increase of analysis time. Theback-pressure is mainly caused by the small inner diameter of the needle(0.1 mm) of the coupling device. Despite the back-pressure no leakage occurredwhen desorption was carried out on-line. Due to the back-pressure the diskcould not be dried completely. With SPEPTs, bi-directional flow is appliedwhich means that the desorption solvent that flows through the disk first, leavesthe pipette tip last (“first in, last out-principle”). Hence, the ethyl acetate thatremains in the disk may contain a part of the analytes. Thus, if the disk cannotbe dried completely with on-line desorption, this may cause a decrease in therecovery of the analytes. The decrease in recovery due to the ethyl acetate thatremained in the disk was found to be 5%. Thus, due to mixing of the desorptionsolvent inside the pipette tip the loss caused by the “first in, last out principle” is


93

only small. In order to be able to quantify reliably the analytes present in theextracts an internal standard can be added to the ethyl acetate to correct for theloss of analytes that remain in the disk during on-line desorption. Usinguni-directional flow during desorption, that is only dispensing with desorptionsolvent, might prevent the possible loss of analytes with the “first in, last outprinciple”. However, this has the disadvantages that the pipettor and pipette hasto be disconnected and that desorption solvent can be present in the upper partof the pipette tip which has to be reconnected prior to injection and, so, cancontaminate the pipettor.

Chromatograms of in-vial and on-line desorption are presented in Fig. 2.No significant differences in peak heights of lidocaine and diazepam wereobserved between an analysis performed with in-vial desorption (referencepurposes) and one in which on-line desorption took place. With in-vialdesorption somewhat more impurities were present than with on-linedesorption. Optimisation of the extraction procedure with regard toconditioning, activation and washing solvents and volumes may producecleaner extracts. Besides solvent impurities the presence of impurities in theextracts might also be due to interferences that are being leached from thesorbent material or the sorbent holder. During some extractions white spotsappeared inside the pipette tip during the drying step and disappeared whenethyl acetate was drawn into the pipette tip. The white spots, which probablyoriginate from the stationary phase, seem to produce clusters of peaks in thechromatograms.

Fig. 2. Chromatograms of extracts of 200 µl phosphate buffer containing 200 nglidocaine (L) and diazepam (D) after desorption with 100 µl ethyl acetate:(A) in-vial desorption, (B) on-line desorption.

Recoveries of lidocaine and diazepam were about 75%. It should benoticed that the SPE procedure has still to be optimised, which may result inhigher recoveries. After elution of a disk with ethyl acetate, the carry-over from


94

the coupling device was checked by injection of 100 µl ethyl acetate via apipette tip without SPE disk and was found to be negligible.

3.2.4 Conclusions and perspectives

It is possible to perform an extraction off-line with SPEPTs and desorbthe analytes with on-line GC analysis with a laboratory-made coupling device toconnect the pipette tip with the PTV injector. This allows the injection of thecomplete eluate (100 µl). Since no significant difference between in-vial andon-line desorption and negligible carry-over from the coupling device isobserved, it is advantageous to desorb the analytes on-line, because thisincreases the rapidity and reliability of the extraction procedure.

The coupling device needs further optimisation and evaluation withregard to robustness of the system. The dimensions of the PTFE piece can bechanged so that a more optimal connection between the coupling device and apipette tip can be obtained. The inner diameter of the needle can be increasedwhich will result in a lower back-pressure when the eluent is pushed backthrough the coupling device into the PTV injector. However, increase of theinner diameter of the needle will also give an enhanced flow through the needle,which might result in leakage of eluate at the connection of the coupling deviceand the pipette tip.

Preliminary experiments have shown that the total set-up can also be usedfor plasma samples. The SPE procedure has still to be optimised with regard tosolvent and sample volumes and, if available, other SPE stationary phases,which might produce cleaner extracts and higher recoveries. Another possibilityto reduce the interference of impurities in the solvents and plasma extracts is theuse of more selective detectors, such as mass spectrometry [20] or anitrogen-phosphorous detection.

Acknowledgements

SPEC•PLUS•PT pipette tips were kindly provided by Ansys Diagnostics,Inc. This research was supported by the Technology Foundation STW, appliedscience division of NWO and the technology programme of the Ministry ofEconomic Affairs.


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3.2.5 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, H.G. Barth, C.H. Lochmüller. Anal Chem. 56 (1984) 300R.[4] X.H. Chen, J.P. Franke, R.A. de Zeeuw. Forensic Sci. Review 4 (1992) 147.[5] Z.E. Penton. Advances in Chromatogr. 37 (1997) 205.[6] J.P. Franke, R.A. de Zeeuw. J. Chromatogr. B 713 (1998) 51.[7] R.E. Majors. LC-GC Int. May (1998) 8.[8] R.W. Frei, K.Zech (Eds.). Selective Sample Handling and Detection in

High-Performance Liquid Chromatography, Part A, Elsevier, Amsterdam 1988.[9] E.R. Brouwer, H. Lingeman, U.A.Th. Brinkman. Chromatographia 29 (1990)

415.[10] E.R. Brouwer, D.J. van Iperen, I. Liska, H. Lingeman, U.A.Th. Brinkman. Intern.

J. Environ. Anal. Chem. 47 (1992) 257.[11] E.H.R. van der Wal, E.R. Brouwer, H. Lingeman, U.A.Th. Brinkman.

Chromatographia 39 (1994) 239.[12] E.C. Goosens, D. de Jong, G.J. de Jong, U.A.Th. Brinkman. Chromatographia 47

(1998) 313.[13] A.J.H. Louter, E. Bosma, J.C.A. Schipperen, J.J. Vreuls, U.A.Th Brinkman.

J. Chromatogr. B, 689 (1997) 35.[14] A. Namera, M. Yashiki, Y. Iwasaki, M. Ohtani, T. Kojima. J. Chromatogr. B 716

(1998) 171.[15] K.K. Verma, A.J.H. Louter, A. Jain, E. Pocurull, J.J. Vreuls, U.A.Th. Brinkman.

Chromatographia 44 (1997) 372.[16] D. Jahr. Chromatographia 47 (1998) 49.[17] A. Namera, M. Yashiki, K. Okada, Y. Iwasaki, M. Ohtani, T. Kojima.

J. Chromatogr. B 706 (1998) 253.[18] P. Enoch, A. Putzler, D. Rinne, J. Schlüter. J. Chromatogr. A 822 (1998) 75.[19] A.J.H. Louter, R.A.C.A. van der Wagt, U.A.Th. Brinkman. Chromatographia 40

(1995) 400.[20] M.W.J. van Hout, R.A. de Zeeuw, J.P. Franke, G.J. de Jong. J. Chromatogr. B

729 (1999) 199.[21] A. Koole, A.C. Jetten, Y. Luo, J.P. Franke, R.A. de Zeeuw. J. Anal. Toxicol. 23

(1999) 632.[22] H. Lingeman, S.J.F. Hoekstra-Oussoren. J. Chromatogr. B 689 (1997) 221.[23] D.A. Wells, G.L. Lensmeyer, D.A. Wiebe. J. Chromatogr. Sci. 33 (1995) 386.[24] S. Rudaz, W. Haerdi, J.L. Veuthey. Chromatographia 44 (1997) 283.[25] K. Hartonen, M.L. Riekkola. J. Chromatogr. B 676 (1996) 45.[26] K. Ensing, J.P. Franke, A. Temmink, X.H. Chen, R.A. de Zeeuw. J. Forensic Sci.

37 (1992) 460.[27] D.F. Hagen, C.G. Markel, G.A. Schmitt, D.D. Blevins. Anal. Chim. Acta 236

(1990) 157.[28] P.J.M. Kwakman, J.J. Vreuls, U.A.Th. Brinkman, R.T. Ghijsen.

Chromatographia 34 (1992) 41.[29] D.D. Blevins, D.O. Hall. SPEC NEWS 3-1 (1998) 1.

Feasibility of the direct coupling of SPEPTs with PTV/GC

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3.3

Feasibility of the direct coupling ofsolid-phase extraction – pipette tips with aprogrammed temperature vaporiser forgas chromatographic analysis of drugs in plasma*

Summary

Solid-phase extraction – pipette tips (SPEPTs) were used for micro solid-phaseextraction of lidocaine and diazepam from plasma. Off-line extraction wasfollowed by on-line desorption. On-line desorption was carried out by directcoupling of the SPEPTs with the liner of the programmed temperaturevaporiser. This coupling only required shortening of the liner by maximally 16mm, cutting the SPEPT, and equipping the remaining part with two O-rings.Due to the heating of the injector the SPEPTs were heated as well, whichresulted in a significant amount of impurities. Pre-heating and pre-washing wasperformed prior to the extraction to reduce the impurity level. The internalcoupling device was applied successfully for the analysis of plasma sampleswith gas chromatography (GC) and mass selective detection. Detection limits of0.75 ng/ml and 2.5 ng/ml were obtained for lidocaine and diazepam,respectively, using 200 µl plasma. Recoveries for both compounds were about80%. Although it is possible, the internal coupling device was not developed tobe used as such. The main goal of this coupling was to show the feasibility ofthe integration of SPEPTs with GC and to realise an important step to newautomated SPE-GC systems.

*: M.W.J. van Hout, W.M.A. van Egmond, J.P. Franke, R.A. de Zeeuw, G.J. de Jong. J. Chromatogr. B 766(2002) 37-45.


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3.3.1 Introduction

Increasing knowledge of working mechanisms of drugs leads to thecoming on the market of more potent drugs. As a result, administered dosagesare decreasing. In order to be able to determine these drugs in lowconcentrations in biological matrices, more sensitive techniques are required. Apossibility to increase the sensitivity in a gas chromatographic (GC) system is toincrease the injected sample volume. Several techniques are available toperform large-volume injection (LVI) in GC [1]. On-column injection withretention gaps is a common technique [1]. A second option to allow LVI is theloop-type interface [2], which was originally designed for the coupling of liquidchromatography and GC. A third possibility for LVI is to use a programmedtemperature vaporiser (PTV). The PTV has been mainly applied forenvironmental analysis [1,3-6], although its potential for the analysis ofbiofluids has been explored as well [7]. With LVI, special attention must begiven to solvent purity and selectivity of the extraction procedure. Largevolumes imply the injection of an equivalent amount of impurities into theanalysing equipment [7].

Biological samples cannot be introduced directly into the GC.Furthermore, the decreasing concentrations of drugs in biological samplesrequire pre-concentration. For these purposes solid-phase extraction (SPE) isvery suitable. Originally, SPE was developed as an off-line sample clean-up andpre-concentration procedure [8-12]. In order to obtain high sample throughput,SPE can be coupled at-line and on-line to GC [13-18]. With on-lineSPE-LVI/GC, the aim is to introduce the total eluate of the SPE system into theGC system, thus increasing the sensitivity of the system. Furthermore, greaterprecision is obtained than with off-line SPE, since an error-prone step in theextraction procedure is eliminated. However, the critical aspect remains theamount of eluate that the GC system is able to accept. Moreover, the mainlimitation of the present on-line systems is the long drying step, which typicallytakes 10-30 min.

Miniaturisation of SPE has led to the development of SPE disks.Generally, SPE disks contain a small bed with small particles and have ahomogeneous particle size distribution [12,19-21]. An advantage of SPE disksis the possibility to use smaller solvent volumes during the several steps of theSPE procedure [19,22]. The use of smaller desorption volumes in combinationwith LVI/GC implies that no evaporation and reconstitution of the extracts isrequired, which eliminates critical and time-consuming steps in the extractionprocedure. Further miniaturisation of SPE has led to the development ofsolid-phase extraction – pipette tips (SPEPTs). An interesting aspect of SPEPTsis that bi-directional flow and cycling, i.e., aspirating and dispensing, can beapplied [22-24]. A disadvantage of micro-SPE may be that smaller sample


99

volumes have to be used, which leads to higher concentration detection limits(expressed in plasma concentrations). However, the injection of relatively largevolumes into a GC system can considerably increase the sensitivity. A firstattempt to perform on-line GC-analysis with SPEPTs was carried out using anexterior coupling device [22]. Extractions were performed off-line, andconsecutive desorption and GC analysis were performed on-line. The systemwas not applied to the analysis of biological samples.

The purpose of the present work was to investigate the further integrationof SPEPT and the PTV injector. The SPEPT was inserted into the injector ontop of the liner of the PTV to perform on-line desorption, i.e. an internalcoupling device. Special attention was paid to the impurity levels introduced bythe coupling of SPEPT and the liner. Also, several aspects regarding theextraction properties of the stationary phase of the SPEPT were investigated.The system was applied to the analysis of lidocaine and diazepam in plasma. Acomparison between the exterior coupling device [22] and the internal couplingdevice will be made. Both coupling devices, and in particular the internalcoupling device, can be considered as an intermediate step to the developmentof new, miniaturised, and automated SPE-GC systems. Therefore, a completeoptimisation and validation of the SPE procedure was not performed.

3.3.2 Experimental

3.3.2.1 Equipment and chromatographic conditions

SPEC•PLUS•PT pipette tips were obtained from ANSYS Diagnostics(Lake Forest, CA, USA). The extraction disk (4 mg) consisted of C18-ARstationary phase. The pipette tip had an inner diameter of 4.0 mm and an outerdiameter of 5.0 mm.

The PTV injection system was an OPTIC 2 (ATAS International,Veldhoven, The Netherlands), equipped with a shortened liner (64 mm×3.4 mmi.d.×5.0 mm o.d.). The liner was packed with ATAS “A” packing (a modifiedChromosorb-based material). The behaviour of this packing was previouslyinvestigated for the analysis of drugs in plasma, as well as for the settings of thePTV [7]. The injector was set at 50°C and evaporation of the solvent occurredusing the “AUTO vent mode” with a vent flow of 150 ml/min. After theevaporation of the solvent the valve was switched to the splitless mode and after10 s the temperature was increased at 10°C/s to 250°C. This final temperaturewas maintained during the analysis. The splitless mode was applied for1.75 min and, subsequently, the valve was switched to the split mode. The usedsplit flow was about 57 ml/min, whereas the septum purge flow and pressurewere 1.2 ml/min and 6.0 p.s.i. (1 p.s.i.=6894.76 Pa), respectively. A transfer


100

pressure of 12.0 p.s.i. was applied for 1.90 min. During the analysis the initialand final pressure were maintained at 8.0 p.s.i.

Gas chromatographic analyses were performed with a Hewlett-PackardHP 5890 series II instrument with a flame-ionisation detection (FID) or aGC-mass selective detection (MSD) system (HP 5972 series). The capillarycolumn was a HP-5 30 m×0.32 mm with 0.25 µm film thickness for analysiswith FID, whereas analyses with MSD were performed using a HP-5 MS30 m×0.25 mm with 0.25 µm film thickness. Helium was used as carrier gas.The column flow-rates were 1.1 and 0.5 ml/min for analysis with FID andMSD, respectively. The following temperature program was used for the GCsystem. The starting temperature was 40°C and after 3 min the temperature wasraised with 20°C/min at 215°C, followed by an increase of 5°C/min to 230°Cand a final rate of 25°C/min to 290°C. This final temperature was maintainedfor 3 min. The temperatures of the FID and mass selective detection systemswere set at 300°C and 280°C, respectively.

During analysis performed with GC-MSD in the total ion current (TIC)mode an m/z range of 50-350 was monitored. Using the selected ion monitoring(SIM) mode, an m/z value of 86, being the most intense fragment of lidocaine,was monitored from the start of the run to 16 min. From 16 min to the end ofthe run the m/z values 256 and 283 were monitored, corresponding to the mostintense fragment and the parent ion, respectively, of diazepam.

3.3.2.2 Chemicals

Methanol (Lab Scan, Dublin, Ireland) was of HPLC quality. K2HPO4 wasof analytical-reagent grade quality (Merck, Darmstadt, Germany). Ethyl acetateUltra resi-analysed (for organic residue analysis) was purchased fromMallinckrodt Baker (Deventer, The Netherlands). Water used during SPE wasultra pure (Elgastat maxima, Salm en Kipp, Breukelen, The Netherlands).Lidocaine (Eur. Ph., Holland Pharmaceutical Supply, Alphen A/D Rijn, TheNetherlands) and diazepam (Centrafarm, Etten-Leur, The Netherlands) wereused as test compounds and dissolved in ethyl acetate (for organic residueanalysis, Mallinckrodt Baker) or in phosphate buffer pH 8.0. Stock solutions of1 mg/ml were stored in the dark at 4°C.

3.3.2.3 SPE procedure and coupling to PTV

The SPE procedure was carried out by connecting a 10-ml gas tightplastic syringe (Omnifix syringe, Melsungen, Germany) to the upper end of thepipette tip. Liquid transport through the disk was done by applyingbi-directional flow with the exception of the desorption solvent. Air was only


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applied in the downward direction. Drawing air into the pipette tip through thedisk implies the possibility of dislodging the disk.

The SPEPTs were pre-heated in an oven at 145°C for 2 h. The SPE diskin the pipette tip was pre-washed with five times 300 µl ethyl acetate. The diskwas then activated with ca. 200 µl methanol followed by conditioning with twotimes ca. 100 µl of 0.1 M K2HPO4 buffer (pH 8.0). Subsequently, 200 µl spikedphosphate buffer or 200 µl spiked plasma diluted with 200 µl blank phosphatebuffer were extracted on the disk. The sample was drawn into and pushed out ofthe tip twice. Then the disk was washed twice with ca. 100 µl water and,subsequently, dried by pushing air through the disk (10×10 ml).

Fig. 1: Internal coupling device: integration of SPEPT and the liner of the PTV.Parts of a conventional PTV injector: (1) septum, (2) carrier gas, (3) split flow,(4) septum purge, (5) cooling pipe, (6) power supply, (7) thermocouple,(8) heating unit, (9) capillary column, (10) oven wall.


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The SPEPT was cut at the tip (4 mm) and the barrel (28 mm). Two O-ringswere placed around the remaining part of the pipette tip, one around the upperpart and a smaller one around the remainder of the tip. The silicone O-rings haddimensions of 6.0 mm and 1.3 mm, respectively. After opening the injector, theSPEPT system was inserted into the liner house on top of the shortened(16 mm) liner, and subsequently the injector was closed. For the desorption ofthe analytes from the disk 250 µl ethyl acetate was used. The ethyl acetate wasinjected through the septum of the GC injection port with a conventional GCsyringe with a shortened needle, so the ethyl acetate was injected into thepipette tip. The desorption occurs inside the injector. Due to the pressure andgas flow in the injector the ethyl acetate is transferred through the disk to theliner packing. The rest of the analysis is performed like conventional LVI/GCwith a PTV injector. The total system of the shortened liner and the cut pipettetip with the O-rings will be further mentioned as internal coupling device. Theset-up of the internal coupling device is depicted in Fig. 1. For each extraction anew SPEPT was used; the O-rings were used multiple times.

After the pipette tip was inserted into the injector, ethyl acetate as eluentcould be injected on top of the disk. To ensure that the injection needle did notperforate the disk, the needle of a conventional GC syringe was shortened to23 mm, positioning the tip of the needle about 1 mm above the disk of thepipette tip. If standard solutions were analysed, the fluids should not be injectedon top of a SPE disk. Therefore, a cut pipette tip without the disk assembly wasinserted into the injector, which mimicked the system with a disk inside thepipette tip.


3.3.3.1 Development of internal coupling device

To be able to perform on-line SPEPT-GC, solid-phase extraction− pipettetips should be directly attached to the injection system of the PTV. In a previouspaper [22], we described an exterior coupling device in which the SPEPT unitremained outside the injector. Though this device worked well, it had somedisadvantages, such as a high back-pressure during desorption and a loss ofabout 5% of analyte due to bi-directional flow during desorption. Furthermore,the system was not very robust.

Therefore, a further integration of SPEPT and GC was developed byinserting the pipette tip into the liner house (see Fig. 1). Since the outerdiameter of the pipette tip and the liner were similar, no changes had to be madeto the injector. Yet, to be able to accommodate the pipette tip into the linerhouse and install it on top of the liner, the 80-mm liner had to be shortened. If


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the injector was set at 250°C, a temperature distribution was observed as shownin Fig. 2. This implicates that the liner packing is at 250°C, and that there is atemperature drop to both ends of the liner. This allows insertion of the SPEPTinto the injection system. The plastic holder of the SPEPT was found to melt atabout 160°C. Using 150°C as maximum temperature (TMAX) to which theplastic of the SPEPT should be exposed, it can be concluded from Fig. 2 that theSPEPT can be inserted for maximally 18 mm. Shortening the liner bymaximally 16 mm and cutting off 4-5 mm from the tip of the SPEPT ensuresthat TMAX will never be exceeded. Cutting off about 28 mm (± 1 mm) from thebarrel of the SPEPT allows accommodation of the pipette tip into the linerhouse, thus replacing the removed part of the liner. The cutting should ensurethat maximum lengths are not exceeded. The shortened liner and the cut PTshould have a combined length of 80-85 mm. Slightly shorter or not very evencutting of the PT does not affect the performance of the system.

Fig. 2: Temperature distribution (A) over the range of the entire liner of PTV. Themaximum temperature of SPEPTs defines the allowable position of the tip ofSPEPTs (B, dashed line) inside the injector and the shortened liner (C, dottedline).

An O-ring was placed around the barrel of the PT. This ring wasnecessary to prevent gas to go from the carrier gas line to the split flow linewithout going through the disk of the pipette tip and the liner packing. A secondO-ring was placed around the tip of the PT. This ring was essential to preventleakage of elution solvent and carrier gas. Without the latter O-ring carrier gas


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could flow from the carrier gas line through the disk to the split flow line. TheO-rings and the combination of SPEPT and liner, which is slightly longer than aconventional unshortened liner of 80 mm, ensured a gas-tight connectionbetween PT and liner. No fluids came into contact with the O-ring. The totaltime required for the cutting and installation of the PT is less than two min,whereas the extraction time is less than 4 min.

3.3.3.2 Effect of pre-heating and pre-washing

Upon heating the liner packing, the temperature at the position of thepipette tip also increased. This heating of the internal coupling device resultedin a significant amount of compounds in the front of the chromatogram (up to16 min). Thus, (semi-) volatile compounds were released by this heatingprocess. The impurities probably originate from both the disk and thePT-housing. After 2 h pre-heating at 145°C in an oven, less than 1% of theinitial amount of impurities was still present. No visible changes of the diskwere observed. An important drawback of pre-heating is that the extractionproperties changed. Extracting lidocaine and diazepam from buffer afterpre-heating the pipette tip resulted in lower recoveries (from about 80% to 40%)with more variation.

Cutting of the pipette tip and insertion of the remaining part of the pipettetip into the injector and subsequent injection of ethyl acetate on top of apre-heated pipette tip resulted in a significant amount of medium- andless-volatile impurities which were observed in the chromatograms as clustersof peaks (Fig. 3A). These impurities interfered with the determination oflidocaine and diazepam and, therefore, had to be removed. Removal of theimpurities was done by applying a pre-wash step prior to the actual SPEprocedure. After pre-washing the disk with 1.5 ml ethyl acetate usingbi-directional flow, over 99% of the impurities were removed (Fig. 3B). Therecoveries of lidocaine and diazepam were not effected by this pre-wash step.This means that the properties of the stationary phase are not changed by thewashing.

Since the (semi-) volatile impurities did not interfere with thedetermination of lidocaine and diazepam, no pre-heating was applied prior touse of the SPEPTs for further experiments. Furthermore, since the pre-washingremoved interfering impurities and did not affect the extraction process, thisstep was applied during further experiments. Each SPEPT was used for onlyone extraction, since the heating of the injector during analysis could also affectthe properties of the stationary phase.


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Fig. 3: Effect of pre-washing SPEPTs on the impurities in the chromatogram:(A) without pre-washing (offset signal: 2000); (B) 1.5 ml ethyl acetate.Note: for both chromatograms pre-heating is applied.

3.3.3.3 Application of internal coupling device

Practical aspectsInjection of ethyl acetate on the disk and consecutive evaporation resulted

in some tailing of the peak of the organic solvent. This was caused by theincomplete transfer of ethyl acetate from the disk to the liner packing. Uponheating the injector, the remaining ethyl acetate was evaporated from the diskand transferred to the GC column. The tailing of the solvent peak did notinterfere with the analysis of lidocaine and diazepam.

With slow injection of ethyl acetate (100 µl in 7-10 s), only a part of thedisk was moistened with ethyl acetate, which resulted in low recoveries.Injecting the organic solvent rapidly, i.e. injection of 100 µl in less than asecond, produced higher recoveries of lidocaine and diazepam and smallervariations in recovery. Upon rapid injection of ethyl acetate, a reservoir ofsolvent was formed on top of the disk and this ensured that the entire disk wasmoistened by the solvent. Desorption of lidocaine and diazepam with 250 µlethyl acetate resulted in still higher recoveries (increase from about 60 to 80%)than desorption with 100 µl ethyl acetate. However, the liquid capacity (Vmax)of the ATAS “A” liner is only 150 µl [7]. If Vmax is exceeded solvent will enterthe column. Therefore, the injection of 250 µl ethyl acetate was performed in athree-step injection. First, 100 µl ethyl acetate was rapidly injected on top of thedisk. After 2.25 min, a second portion of 75 µl ethyl acetate was injectedrapidly, and after 4.25 min the remaining 75 µl ethyl acetate was injected


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rapidly. The total evaporation time was about 6.25 min. This injectionprocedure ensured that the next amount of ethyl acetate was never injected afterthe previous amount was completely evaporated. Too late injection of the nextamount of solvent implies injection while the actual GC analysis has alreadybeen started. The three-step injection procedure also ensured that Vmax of theliner was not exceeded at any time.

Injection of 100 µl ethyl acetate at a pressure of 2.5 p.s.i. instead of6.0 p.s.i. resulted in a small loss in recovery. The evaporation time was alsolonger (about 0.3 min) than the three-step injection of 250 µl ethyl acetate at6.0 p.s.i. Therefore, in the present work desorption was performed using 250 µlethyl acetate while the pressure was maintained at 6.0 p.s.i.

Analytical dataIn previous experiments the use of MSD proved to be necessary to

determine analytes in low concentrations after extraction from buffer andespecially from biological samples if LVI/GC was applied [7,22]. With thepresent system, i.e. the internal coupling device, extraction of lidocaine anddiazepam from buffer already showed the necessity of MSD, since aninterfering peak was observed for lidocaine.

The injection of 100 µl of standard solutions using the internal couplingdevice without SPE disk resulted in good linearity (R>0.998, range fromdetection limit to 250 ng/ml), demonstrating the reliability of the device. Thedetection limit (LOD), which was determined as S/N 3 or three times the blankpeak, was 10 ng/ml for both lidocaine and diazepam using the TIC mode. TheLOD was decreased to 0.5 ng/ml for both compounds if the SIM mode wasused. For the determination of lidocaine and diazepam the m/z values 86 and283 were used, respectively. Many silica-based compounds have a fragmentwith m/z 86. The m/z value of 283 corresponds with compounds fromsilicone-based materials [25]. These compounds probably originate from thesepta used on top of the vials in which the samples were stored and/or from thesilicone O-ring of the internal coupling that was used for the connection of thepipette tip and the liner.

The analysis of 200-µl plasma samples was performed using both the TICand SIM mode. Correlation coefficients (R) and LODs are presented in Table 1.Both scan modes showed good linearity over the entire concentration range.Also for plasma recoveries of about 80% were observed. Use of the SIM moderesulted in a lower LOD as compared with the TIC mode. A similar differencein LOD between TIC and SIM was observed for plasma extracts as comparedwith standard injections. If the SIM mode was used the background peaks werereduced, but not completely eliminated. This is partly due to the fact that nopre-heating was applied and due to the low m/z value of lidocaine. Moreover, as


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with standard injections m/z 86 and 283 were present due to the silica-basedcompounds and the silicone-based compounds, respectively.

Table 1: Detection limits (LODs) and correlation coefficients (R) for lidocaine anddiazepam in 200 µl plasma with GC-MS analysis using the internal couplingdevice for the coupling with SPE (desorption with 250 µl ethyl acetate).

TIC SIMLOD

(ng/ml)R* LOD

(ng/ml)R*

Lidocaine 15 0.9990(n=4)

0.75 0.9999(n=7)

Diazepam 60 0.9998(n=3)

2.5 0.9995(n=6)

*: Ranges from LOD to 250.0 ng/ml.

In Fig. 4, representative chromatograms are depicted for the TIC mode(Figs. 4A and B) and the SIM mode (Figs. 4C and D) of blank and spikedplasma extracts, respectively. In Fig. 4D, the peaks of silicone-basedcompounds are lower than in Fig. 4C. This can be explained by the fact that thesilicone O-ring between SPEPT and liner was used before. Furthermore, whileclosing the injector the SPEPT and liner are pressed together. The tighter theseparts are pressed together, the greater the risk of tearing the ring, thus the O-ringcan release more compounds.

3.3.4 Conclusions and perspectives

The direct coupling of SPEPTs to the liner of the PTV injector couldeasily be established. The SPEPTs, and thus the extracts, can be purified bysimple pre-heating and pre-washing. However, the extraction properties of thestationary phase of the SPEPTs can be altered during the pre-heating, which isonly needed for the analysis of relatively volatile compounds. Other compoundspresent in the extracts originate from the biological matrix and solventimpurities. With LVI the use of selective detection, such as MSD, is essential todetermine drugs at low concentrations in biological samples.

The system is better applicable than the exterior coupling device [22],since the pipette tips and the liner are directly connected, which makes thesystem more robust. Furthermore, no carry-over is observed, and no leakage ofdesorption solvent can occur. The exterior coupling device has the advantage ofbi-directional flow during desorption, which allows in principle use of lessdesorption solvent. However, with this device 5% loss of analyte was observedbecause of incomplete desorption caused by the high back-pressure.


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Fig. 4: GC-MS chromatograms of extracts of 200 µl plasma: (A) blank (TIC mode),(B) 62.5 ng/ml (TIC mode), (C) blank (SIM mode), (D) 5.5 ng/ml (SIMmode). L = lidocaine, D = diazepam, C = caffeine, * = silicone-basedcompounds.


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The necessity of high sample-throughput requires automation. On-linesystems for SPE-GC already exist, but the main limitation of these systems isthe relatively long drying of the SPE cartridge, which typically takes 10-30 min.The use of SPEPTs enables decrease of the extraction time and especially thedrying step, which now only takes about 30 s. Automation of a system based onSPEPT and GC can be obtained more easily with the internal coupling devicethan with the exterior coupling device. A pipette robot based on a 96-well platedesign can simultaneously perform the extractions at-line in pre-cut SPEPTswith O-rings and a metal cap at the top. A recently developed liner exchanger[26] can be used to combine the SPEPT with the PTV liner, after which theinjector is closed automatically via a pneumatic system. Subsequently, thedesorption solvent can be injected on top of the disk. For automated injectionthe injection height should to be adjusted, or a shorter needle must be used inorder to prevent perforation of the disk. In such an approach, high-throughputanalysis is performed by means of a combination of miniaturised SPE and GCusing at-line extraction and on-line desorption. In the future, an even furtherintegration of SPE and GC might be obtained by performing the extraction inthe liner of the GC system.

Acknowledgements

SPEC•PLUS•PT pipette tips were kindly provided by ANSYSDiagnostics (Lake Forest, CA, USA). Jan Henk Marsman and Ronald Veenhuis(Department of Chemical Engineering, University of Groningen, Groningen,The Netherlands) are gratefully acknowledged for the use of their GC-MSDsystem and their assistance. This research was supported by the TechnologyFoundation STW, applied science division of NWO and the technologyprogramme of the Ministry of Economic Affairs.

3.3.5 References

[1] H.G.J. Mol, H.-G.M. Janssen, C.A. Cramers, J.J. Vreuls, U.A.Th. Brinkman.J. Chromatogr. A 703 (1995) 277.

[2] K. Grob, J.-M. Stoll. J. High Resolut. Chromatogr. Commun. 9 (1986) 518.[3] J.J. Vreuls, U.A.Th. Brinkman, G.J. de Jong, K. Grob, A. Artho. J. High Resolut.

Chromatogr. 14 (1991) 455.[4] J.J. Vreuls, G.J. de Jong, R.T. Ghijsen, U.A.Th. Brinkman. J. Microcol. Sep. 5

(1993) 317.[5] A.J.H. Louter, J. van Doornmalen, J.J. Vreuls, U.A.Th. Brinkman. J. High

Resolut. Chromatogr. 19 (1996) 679.


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[6] R.J.C.A. Steen, I.L. Freriks, W.P. Cofino, U.A.Th. Brinkman. Anal. Chim. Acta353 (1997) 153.

[7] M.W.J. van Hout, R.A. de Zeeuw, J.P. Franke, G.J. de Jong. J. Chromatogr. B729 (1999) 199.

[8] M. Dressler. J. Chromatogr. 165 (1979) 167.[9] A. Lagana, B.M. Petronio, M. Rotatori. J. Chromatogr. 198 (1980) 143.[10] R.E. Majors, H.G. Barth, C.H. Lochmüller. Anal Chem. 56 (1984) 300R.[11] X.H. Chen, J.P. Franke, R.A. de Zeeuw. Forensic Sci. Rev. 4 (1992) 147.[12] Z.E. Penton. Adv. Chromatogr. 37 (1997) 205.[13] E.C. Goosens, D. de Jong, G.J. de Jong, U.A.Th. Brinkman. Chromatographia 47

(1998) 313.[14] A.J.H. Louter, E. Bosma, J.C.A. Schipperen, J.J. Vreuls, U.A.Th. Brinkman.

J. Chromatogr. B 689 (1997) 35.[15] A. Namera, M. Yashiki, Y. Iwasaki, M. Ohtani, T. Kojima. J. Chromatogr. B 716

(1998) 171.[16] P. Enoch, A. Putzler, D. Rinne, J. Schüter. J. Chromatogr. A 822 (1998) 75.[17] A.J.H. Louter, C.A. van Beekvelt, P.Cid Montanes, J. Slobodník, J.J. Vreuls,

U.A.Th. Brinkman. J. Chromatogr. A 725 (1996) 67.[18] R. Sasano, T. Hamada, M. Kurano, M. Furuno. J. Chromatogr. A 896 (2000) 41.[19] D.A. Wells, G.L. Lensmeyer, D.A. Wiebe. J. Chromatogr. Sci. 33 (1995) 386.[20] E.H.R. van der Wal, E.R. Brouwer, H. Lingeman, U.A.Th. Brinkman.

Chromatographia 47 (1994) 239.[21] K. Hartonen, M.L. Riekkola. J. Chromatogr. B 676 (1996) 45.[22] M.W.J. van Hout, R.A. de Zeeuw, G.J. de Jong. J. Chromatogr. A 858 (1999)

117.[23] R.E. Majors. LC•GC Int. May (1998) 8.[24] D.D. Blevins. D.O. Hall. SPEC News 3-1 (1998) 1.[25] M. Spiteller, G. Spiteller. Massenspektrensammlung von Lösungsmitteln,

Verunreinigungen, Säulenbelegmaterialien und Einfachen AliphatischenVerbindungen, Springer-Verlag, Vienna, 1973, S70.

[26] ATAS International, Veldhoven, The Netherlands, http://www.ATAS-INT.com

SPETD-GC-MSD for determination of drugs in urine

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3.4

Solid-phase extraction – thermal desorption –gas chromatography with mass selective detectionfor the determination of drugs in urine*

Summary

Solid-phase extraction (SPE) was combined with thermal desorption (TD) andgas chromatographic (GC) analysis to determine drugs in urine. The extractionwas performed inside a fritted GC liner using about 5 mg Tenax that wasinserted into the liner on top of the frit. After the extraction the liner was placedinto the injector of the GC and the analytes were thermally desorbed by usingprogrammed temperature vaporiser. Several stationary phases were investigatedfor the applicability of SPETD-GC analysis. Tenax proved to be the mostsuitable extraction phase, since hardly any interferences were observed andacceptable absolute recoveries (73 and 74%) were obtained for lidocaine anddiazepam. A mass selective detector (MSD) in the selected ion monitoringmode allowed the detection of lidocaine and diazepam down to 0.5 ng/ml using50 µl urine. The use of 5 mg stationary phase allowed a rapid extractionprocedure, while a 10-m GC column provided a fast chromatographic system.As a result, the total analysis time was less than 20 min, including 5 min dryingof Tenax and 5 min thermal desorption. Thus, SPETD-GC-MSD appears to be apowerful tool for the rapid analysis of biological samples.

*: M.W.J. van Hout, R.A. de Zeeuw, J.P. Franke, G.J. de Jong. Accepted for publication in Chromatographia.


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3.4.1 Introduction

In bioanalysis, the numbers of samples are increasing, thus requiringrapid analytical systems. Not only the separation must be performed rapidly, butalso the extraction may not be time-limiting. Consequently, the extraction iscoupled more and more on-line with the separation step. For example,solid-phase extraction (SPE) is now coupled on-line with liquidchromatography (LC) on a routine basis [1-11], and on-line SPE-gaschromatography (GC) is also gaining interest [12-20]. Besides the approach ofon-line extraction and separation, another possibility is miniaturisation of theextraction [21,22]. Some time-limiting steps can then be shortened or evenomitted. On-line SPE-GC implies the desorption of the analytes with relativelylarge volumes of eluent, and thus the injection of large volumes of solvent intothe GC. This can be achieved by using a retention gap or a programmedtemperature vaporiser (PTV). The elution and injection of large solvent volumescan be critical with regard to flow-rate during elution and evaporation of thesolvent [22,23].

Another option of the PTV is thermal desorption, since the PTV allowsrapid heating of the injector. When applying solid-phase extraction – thermaldesorption (SPETD), the extraction procedure is basically the same as foron-line SPE up to and including the drying step. Subsequently, the analytes arethermally desorbed instead of using liquid desorption. Thus, no injection andevaporation of the eluate in the injector is required. In order to perform SPETD[17,18,20,24-31], the stationary phase of the extraction unit is critical. Thephase must have good extraction properties and must also be thermostable inorder to prevent interference of the phase components during analysis.Polydimethylsiloxane (PDMS) [29,30] and Tenax (a porous polymer basedupon 2,6-diphenyl-p-phenylene oxide) [24,27,28,31] have been claimed topossess these characteristics. Another important aspect is the drying of thephase prior to thermal desorption. In previous studies the drying step was oftenvery time-consuming (up to 30 min) and/or high gas flow-rates (>100 ml/min)were required [24,26,27,29,30,32]. Minimising the amount of extraction phasecan reduce the length of the drying step.

The goal of the present study was to explore the possibilities of SPETDfor the analysis of biological samples, setting up a system with good sensitivity,selectivity and speed. The amount of the extraction phase was kept to aminimum. In order to obtain an integrated design, the extraction was carried outoff-line in the liner of the GC, after which the liner was put into the GC andon-line thermal desorption took place.


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3.4.2 Experimental

3.4.2.1 Chemicals

Methanol (Lab Scan, Dublin, Ireland) was of HPLC quality. KH2PO4

(Merck, Darmstadt, Germany) was of analytical-reagent grade quality. Ethylacetate Ultra resi analysed (for organic residue analysis) was purchased fromMallinckrodt Baker (Deventer, The Netherlands). Water used during SPE wasultra pure (ElgaStat Maxima, Salm & Kipp, Breukelen, The Netherlands).Lidocaine (Eur. Ph., Holland Pharmaceuticals Supply, Alphen a/d Rijn, TheNetherlands) and diazepam (Centrafarm, Etten-Leur, The Netherlands) wereused as test compounds and dissolved in ethyl acetate or phosphate buffer(0.1 M, pH 8.0). Stock solutions of 1 mg/ml were stored in the dark at 4ºC.

Stationary phases that were investigated for thermal desorption wereResin GP, PLRP-S and Styrene-divinylbenzene (all from Spark Holland,Emmen, The Netherlands), Tenax, Hayesep Q, ATAS Focus Trap and PDMS(all from Varian-Chrompack, Middelburg, The Netherlands), Bond Elut LMSand Abselut Nexus from Varian Sample Preparation Products (Harbor City, CA,USA), and C18 SPEC disks (ANSYS Diagnostics, Lake Forest, CA, USA).

3.4.2.2 Equipment and chromatographic conditions

The PTV injection system was an OPTIC 2 (ATAS International,Veldhoven, The Netherlands), with a fritted liner (80 mm×3.4 mm i.d.×5.0 mmo.d., frit 15 mm from the bottom, ATAS International) to hold the stationaryphase. The extraction was also performed inside these liners. A Visiprep system(Supelco, Bellefonte, PA, USA) was used to apply vacuum under the linersduring the extraction. After extraction the liner was inserted into the injector,which was set at 50°C. Then the temperature of the injector was increased at15°C/s to 350°C. This final temperature was maintained during the analysis.The splitless mode was applied for 5.10 min and, subsequently, the valve wasswitched to the split mode. The split flow was about 57 ml/min, whereas theseptum purge flow and pressure were 1.1 ml/min and 1.5 p.s.i. (1 p.s.i. =6894.76 Pa), respectively. A transfer pressure of 1.5 p.s.i. was applied for5.25 min. During the analysis the initial and final pressure were maintained at1.0 p.s.i.

Gas chromatographic analyses were performed with a Hewlett-PackardHP 5890 series II instrument with flame-ionisation detection (FID) or aHP 6890 series GC- mass selective detection (MSD) system (HP 5972 series).The capillary column was a HP-5 10 m×0.32 mm with 0.25 µm film thickness.Helium was used as carrier gas. The column flow was 2.2 ml/min. The startingtemperature was 50°C and after 5.25 min the temperature was raised with 30 or


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50°C/min for FID or MSD, respectively, to 325°C. This final temperature wasmaintained for 0.50 min. The temperature of the FID and MSD was set at300°C and 280°C, respectively.

During analysis performed with GC-MSD in the total ion current (TIC)mode an m/z range of 50-350 was monitored. Using the selected ion monitoring(SIM) mode, an m/z value of 86, the most intense fragment of lidocaine, wasmonitored from the start of the run to 8.50 min. From 8.50 min to the end of therun the m/z values 283 and 284 were monitored, corresponding to the parent ionand its protonated form, respectively, of diazepam.

3.4.2.3 SPETD procedure

About 5.0 mg stationary phase was brought into the fritted liner. Thelatter was tapped carefully to pack the stationary phase more tightly on top ofthe frit, resulting in about a 2-mm height of the phase. The C18 SPEC disks(4.0 mg) were inserted as such on top of the frit. The extraction unit, existing ofthe liner and the stationary phase, was then placed onto the Visiprep systemafter which 250 µl ethyl acetate was flushed through the unit as pre-wash step.Subsequently, 100 µl methanol was pipetted into the liner to remove theremaining ethyl acetate prior to conditioning the stationary phase with 100 µlphosphate buffer (0.1 M, pH 8.0). Then 100 µl sample was applied. The latterexisted of 50 µl untreated calf urine (blank or spiked), diluted with 50 µlphosphate buffer, unless stated otherwise. The sample was introduced on top ofthe stationary phase inside the liner and then passed through the phase by meansof gravity, which took about 15-20 s. This was followed by washing of theextraction unit with 150 µl water. Then vacuum (-15 mm Hg) was applied underthe extraction unit for 5 min at room temperature to remove the remainingwater. Finally, the extraction unit was inserted into the PTV injector, afterwhich thermal desorption took place at 350°C for 5 min. The liners werere-used but the stationary phase was replaced after each analysis.


3.4.3.1 Selection of the stationary phase

Several stationary phases were investigated. In order to determine theirsuitability for thermal desorption, about 5 mg was filled into the liner afterwhich the liner was inserted into the injector and subsequently, heating at 300°Ccombined with GC was performed. Various well-known LC phases wereselected because of their proven extraction properties. These phases could notwithstand high temperatures and, consequently, a high background was


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observed. In a previous study [22] it was shown that the (volatile) impuritiescould be removed by pre-heating the SPEC disk. However, the extractionproperties also changed. Therefore, the LC phases were not further consideredfor SPETD purposes.

Subsequently, some typical GC phases were treated in the same way.ATAS Focus Trap proved to be clean upon heating. Yet, its extractionproperties were very poor, i.e., no retention of lidocaine and diazepam wasobserved upon extraction of buffer solutions. The impurity level of PDMS wasalso considerable. The suitability of this phase for thermal desorption has beenclaimed [29], though it was also noticed that disturbing siloxane breakdowncould also occur [17,18]. Pre-washing of about 5 mg PDMS with 750 µl ethylacetate and subsequent pre-heating at 300°C for 24 hours decreased theimpurity level, but severe interferences remained visible, even with massspectrometric detection in the SIM mode. Therefore, PDMS was notinvestigated further. Another GC stationary phase, Tenax, also showed asignificant amount of interferences. In several reports, a pretreatment of Tenaxwas applied prior to analysis to clean the phase. Vreuls et al. performedwashing of Tenax with acetone [28], whereas pre-heating Tenax for a certainperiod of time [27] or repeatedly pre-heating the phase in the extraction unit(2-5 times) prior to using it for the actual analysis [24] was also applied. In thepresent set-up, pre-heating Tenax (at 300°C for 15 min) in the liner eliminatedmost of the interferences, but pre-washing was also suitable. The latter waspreferred as this could be integrated into the extraction procedure more easily.About 5 mg Tenax in the liner was pre-washed with 250 µl ethyl acetate prior tothe extraction, after which only a few interferences were observed. As thisphase had shown acceptable extraction properties with typical recoveries of80-110% [24,26,27], this phase was selected for further exploration using theliner as the extraction tube.

3.4.3.2 Optimisation of SPETD procedure

Initially, 5 mg Tenax was inserted into the liner and, after pre-washing thephase with 250 µl ethyl acetate, 100 µl methanol and subsequently 150 µlphosphate buffer were used to condition the extraction phase. Then 400 µlsample (100 µl urine diluted with 300 µl buffer pH 8.0) was extracted, and theextraction unit was washed with 500 µl water. Vacuum (-15 mm Hg) at roomtemperature was applied to dry the phase during 0.5 min, which was thenfollowed by insertion of the extraction unit into the injector and 0.5 min thermaldesorption at 300°C and analysis with GC-FID.

Increasing the desorption temperature proved favourable for bothlidocaine and diazepam. For both compounds a gain in signal was observed upto 350°C, and no higher signals were observed with higher desorption


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temperatures. Furthermore, the signals of both compounds also increased bylengthening the desorption time from 0.5 min to 5 min. No higher signals wereobserved with desorption times longer than 5 min. Therefore, the desorptiontemperature was set at 350°C with a desorption time of 5 min.

At pH 8.0 diazepam is in its neutral form, whereas lidocaine is partiallyprotonated. When using Tenax it might be favourable for the recovery to havethe compounds in their non-protonated form. Increasing the pH to 10 ensuresthat lidocaine is also non-protonated. However, no gain in recovery wasobserved.

Then, the focus was on the time required for the entire extraction/dryingprocess. It is essential to avoid water entering the GC, so, the drying should becomplete. In Fig. 1 the drying time was varied, and the amount of remainingwater in the stationary phase was calculated by weighing the extraction unit.After 1 or 2 min drying both the liner and the stationary phase were still visiblywet. After 3 min, the liner appeared to be dry, but as can be seen in Fig. 1, itstill retained a substantial amount of water. The drying was near complete atabout 5 min, thus shortening of the extraction time was not possible. Anyremaining water traces after 5 min drying did not interfere with the analysis. Itshould be noted that in many other studies [24,26,29,30,32] 10-30 min of dryingis recommended. In our study, the drying time can be kept relatively short dueto the limited amount of stationary phase in the extraction. Furthermore, in thepresent set-up, only vacuum is applied at room temperature. In other systemsheating of the SPE device and a high gas flow-rate during the drying step areneeded [24,26], which can be determinative in the final analysis.

Fig. 1: Remaining amounts of water in the stationary phase at different drying times;calculated by weighing of the total extraction unit.

02468

10121416

0 2 4 6 8 10

Drying time (min)

Rem

ain

ing

wat

er in

st

atio

nar

y p

has

e (m

g)


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Using the optimised conditions as described above, the system wasapplied to the analysis of 400 µl spiked phosphate buffer prior to analysis ofurine. Both lidocaine and diazepam could be analysed with GC-FID withoutinterference of compounds originating from the stationary phases or solventsused during the extraction (Fig. 2A).

Fig. 2: (A) SPETD-GC-FID of blank buffer (lower line) and spiked buffer (200 ng/ml;upper line), (B) SPETD-GC-FID of blank urine/buffer (1:3; lower line) andspiked urine/buffer (1:3; 100 ng/ml lidocaine in urine; upper line),(C) SPETD-GC-FID of blank (lower line) and spiked urine/buffer (1:3;100 ng/ml diazepam in urine; upper line).


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When extracting urine, it was necessary to dilute with buffer to ensure properflowing of the sample through the stationary phase. However, if 100 µl spikedurine was diluted with 300 µl buffer and the sample was extracted and thenanalysed with GC-FID, severe interference of the urine matrix was observed.Only enlarging the time scale revealed the peaks of lidocaine and diazepam(Figs. 2B and C). Using 100 µl urine, the absolute recovery was in the order of56% for lidocaine and 65% for diazepam at a level of 200 ng/ml, and a limit ofdetection (LOD) of about 50 ng/ml could be obtained for both compounds. Therecoveries for urine were similar to those observed upon the extraction frombuffer.

The low recovery may be caused by occupation of sorption sites bymatrix components or by chromatographic breakthrough. Therefore, 50 µl urinewas diluted with 50 µl buffer, resulting in a 100-µl sample. The recoveries nowincreased to 74 and 73% for lidocaine and diazepam, respectively, at aconcentration of 200 ng/ml. To obtain even higher recoveries, the amount ofstationary phase was increased to 10 mg. The recoveries were now about 90%for both compounds, but the peaks became very broad. This is probably due tolarger amounts of water being retained by the stationary phase after drying.Consequently, the broader peaks led to higher LODs and the water can damagethe chromatographic system. The latter could be overcome by lengthening thedrying time. However, in order to keep the extraction time as short as possible,the amount of stationary phase was kept at 5 mg, accepting the slightly lowerrecoveries.

3.4.3.4 Application of SPETD-GC-MS

To improve the applicability of SPETD-GC for biological samples, anMSD was used. Now, 50 µl spiked urine diluted with 50 µl buffer was extractedwithin 8 min, including 5 min drying, and using 5 mg Tenax. Thermaldesorption was performed at 350°C for 5 min, after which the GC temperaturewas increased rapidly. The analysis time could also be kept very short, since acapillary GC column of only 10 m was used. The total GC-MSD time, including5 min thermal desorption, took about 11 min, thus a total analysis time of about19 min was obtained. In the TIC mode, significant interference of the samplematrix was observed. By extracting the desired m/z values, the LODs(signal-to-noise ratio 3), 10 ng/ml, were lower than with FID (50 ng/ml). Evenbetter results were obtained using the SIM mode due to the increased sensitivityand selectivity (LOD about 0.5 ng/ml), but as can be seen in Fig. 3, theinterference was not completely eliminated. The difference between the blank(Fig. 3A) and spiked urine (Fig. 3B) between 6.0 and 8.5 min may be due to themanual packing of the stationary phase. The latter may result in a difference infirmness of the packing and thus in a change in the filtration properties of the


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stationary phase. However, no adverse effect on the determination of lidocaineand diazepam was observed.

Fig. 3: SPETD-GC-MSD in the SIM mode: (A) Blank urine (50 µl; diluted withbuffer 1:1), (B) 0.5 ng/ml lidocaine and diazepam in urine (50 µl; diluted withbuffer 1:1).

The results of this SPETD-GC-MSD system are presented in Table 1.Even though only 50 µl urine was used, acceptable LODs (sub-ng/ml) wereobtained for both compounds with good linearity over a range of 0.5-200 ng/ml.A quantitation limit of about 1 ng/ml could be obtained. The absolute recoveriesas well as the reproducibilities (n=8) were also satisfactory. The use of asuitable internal standard may help to obtain even better reproducibilities.


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Table 1: Analytical data of urine analysis using SPETD-GC-MSD.

Lidocaine DiazepamAbsolute recovery ± RSDa (%) 74 ± 6.5 (n=8) 73 ± 5.9 (n=8)LOD (ng/ml) – SIM mode 0.5 0.5R 0.9999 0.9971Linear range (ng/ml) 0.5-200 0.5-200

a: at 10 ng/ml.

3.4.4 Conclusions

The applicability of solid-phase extraction combined with thermaldesorption strongly depends on a useful, clean stationary phase with goodextraction properties and thermostability. Most stationary phases seem to lackone or more of these characteristics, as the phases are usually developed foreither LC or GC purposes. In this study, Tenax was a suitable compromise interms of acceptable recoveries and impurity levels, but the ideal stationaryphase for SPETD purposes seems at yet unavailable.

In this study, only 5 mg of stationary phase was required, herebydecreasing the time-consuming drying step in the extraction procedure. As theextraction was carried out in a GC liner, the thermal desorption was very easy toperform. Despite the small sample volume (50 µl urine + 50 µl buffer), goodsensitivity was obtained by using MS detection. If a higher sensitivity isimportant, more Tenax and probably also more urine can be used. Heating ofthe SPE unit during the drying might help to minimise the time required fordrying of the stationary phase. However, this will also make the extraction unitmore complex. Moreover, it is important to investigate if the stationary phasecan be used more than once for thermal desorption.

So far, the extraction was performed off-line with on-line thermaldesorption. By using a suitable robotic system, the extraction can be performedat-line and the liner can be installed and removed by the liner exchanger. In thisway automation of the entire extraction procedure can be obtained and thenSPETD seems a powerful approach for combination of SPE and GC for theanalysis of biological samples.

Acknowledgements

Wil van Egmond (ATAS International) is gratefully acknowledged for theuse of the MSD. The authors thank René de Nijs (Varian-Chrompack), BertOoms (Spark Holland), Dennis Blevins (ANSYS Diagnostics) and NigelSimpson (Varian Sample Preparation Products) for their donation of the


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stationary phases. This research was supported by the Technology FoundationSTW, applied science division of NWO and the technology programme of theMinistry of Economic Affairs.

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university of groningen on-line coupling of sample ...plasma concentrations of 250 pg/ml can be...

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