A novel microextraction by packed sorbent–gas chromatography procedure for the simultaneous...

J. Sep. Sci. 2012, 00, 1–8 1

Susheela RaniAshok Kumar Malik

Department of Chemistry,Punjabi University, Patiala, India

Received May 3, 2012Revised July 6, 2012Accepted July 7, 2012

Research Article

A novel microextraction by packedsorbent–gas chromatography procedurefor the simultaneous analysis of antiepilepticdrugs in human plasma and urine

A simple, accurate, and sensitive microextraction by packed sorbent–gas chromatography-mass spectrometry method has been developed for the simultaneous quantification of fourantiepileptic drugs; oxcarbazepine, carbamazepine, phenytoin, and alprazolam in humanplasma and urine as a tool for drug monitoring. Caffeine was used as internal standardsfor the electron ionization mode. An original pretreatment procedure on biological sam-ples, based on microextraction in packed syringe using C18 as packing material gave highextraction yields (69.92–99.38%), satisfactory precision (RSD < 4.7%) and good selectivity.Linearity was found in the 0.1–500 ng/mL range for these drugs with limits of detection(LODs) between 0.0018 and 0.0036 ng/mL. Therefore, the method has been found to besuitable for the therapeutic drug monitoring of patients treated with oxcarbazepine, carba-mazepine, phenytoin, and alprazolam. After validation, the method was successfully appliedto some plasma samples from patients undergoing therapy with one or more of these drugs.A comparison of the detection limit with similar methods indicates high sensitivity of thepresent method over the earlier reported methods. The present method is applied for theanalysis of these drugs in the real urine and plasma samples of the epileptic patients.

Keywords: Antiepileptics drugs / Gas chromatography / Micro extraction bypacked sorbentDOI 10.1002/jssc.201200439

1 Introduction

Therapeutic drug monitoring (TDM) of antiepileptic drugs isnecessary to optimize patients’ clinical outcome by manag-ing their medication regimen with the assistance of measureddrug concentrations [1]. Plasma concentration monitoring iswidely used for the clinical management of patients withepilepsy receiving phenytoin (PTN), alprazolam (ALP), oxcar-bazepine (OXC), and carbamazepine (CBZ). TDM is a well-established procedure that helps to maximize the effective-ness of antiepileptic therapy, increasing clinical efficacy whileminimizing adverse effects [2, 3]. Among the AEDs recentlyintroduced in therapeutic practice, oxcarbazepine (OXC) isone of the most widely used drug. Thus, the determination ofOXC as well as that of the other concomitantly administeredAEDs become relevant.

PTN, CBZ, ALP, and OXC (Supporting Information Fig.S1) are widely used as antiepileptic drugs (AED), which clin-ically control different types of seizures. Several methodshave been reported for the determination of one or more

Correspondence: Dr. A. K. Malik, Department of Chemistry,Punjabi University, Patiala, IndiaE-mail: [email protected]: 0091-175-2283073

antiepileptic drugs in biological fluids for therapeutic drugmonitoring (TDM). Various analytical tools have been devel-oped for therapeutic drug monitoring of AEDs in the biologi-cal and environmental samples including high-performanceliquid chromatography (HPLC) coupled with ultraviolet de-tection [4–8], diode array detector [9], fluorescent detector [10],mass spectrometry [11], mostly after sample pretreatmentby expensive solid-phase extraction [6, 7] or time-consumingsolid phase microextraction [9], and stir bar sorptive extrac-tion [12]. The availability of simple, accurate, and inexpensiveanalytical assays is still a crucial need for the successful useof TDM in clinical practice.

At present, analysis of the clinical samples demands sim-ple and fast sample clean up procedures and, therefore, find-ing an alternative sample preparation method or techniqueis crucial. The common sample preparation methods in bio-analytical laboratories are protein precipitation (PPT) [4, 11],liquid–liquid extraction (LLE), and solid phase extraction(SPE) [6, 7]. Protein precipitation as sample preparation canresult in high matrix background in the extracted samplesand significant signal suppression is often observed. LLEmethods require solvent or solvent mixtures of quite highpolarity and these often yield emulsions. For even more polaranalytes, such as drug conjugates and metabolites, recovery,and sample purity is further compromised. SPE is mostly

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2 S. Rani and A. K. Malik J. Sep. Sci. 2012, 00, 1–8

attractive in recovering drug measurement because of theability to efficiently retain highly functionalized compoundsfrom aqueous samples and then to release them into organicsolvents on elution.

The micro extraction in packed syringe (MEPS) is a newtechnique for miniaturized SPE that can be connected on-line to GC or LC without any modification of the chromato-graph [13, 14]. MEPS differs from commercial SPE cartridgein that the solid sorbent is inserted into a syringe (100–250 �L) as a plug and it greatly reduces the sample volumeused for one analysis from the milliliter to the microliterrange. MEPS is fully automated, and takes only a few min-utes for one analysis, thus, it is suitable for the rapid analysisof biological samples. Further, the packed syringe can beused several times, more than 100 times with plasma [13]whereas a conventional SPE column is used only once. Com-pared to SPE or LLE, MEPS reduces sample preparation timeand organic solvent consumption. Unlike in solid-phase mi-croextraction, MEPS reduces sample preparation time (ap-proximately 1 min), sample volume (10–100 �L) and a muchhigher recovery (>70%) can be obtained. The MEPS tech-nique has been used to extract a wide range of analytes. Thus,several drugs such as local anesthetics and their metabo-lites [13–16]; UV filter and polycyclic musk [17], antipsychoticdrug risperidone [18], nonpolar heterocyclic amines [19], es-trogenic compounds [20], anticancer drugs roscovitine, olo-moucine, cyclophosphamide and busulfan [21, 22], atorvas-tatin and its metabolites [23], antidepressant drugs dopamineand serotonine [24], opoid analgesic drug remifentanil [25]piperazine-type stimulants [26, 27], methadone [28], variouspesticides [29], long acting opioids [30], clozapine and itsmetabolites [31], fluoroquinolone [32], nonpolar heterocyclicamines [33], parabens, triclosan, and methyl triclosan [34],and pravastatin and pravastatin lactone [35] have been ex-tracted successfully from biological samples such as plasma,urine or blood.

2 Experimental

2.1 Analytical reference standards and reagents

CBZ, OXC, PTN, ALP, and caffeine (internal standard)were supplied by Sigma-Aldrich (Steinheim, Germany).LC-grade methanol and acetonitrile were obtained fromRankem (New Delhi, India). Water was deionized (Riv-iera, SCHOTT DURAN, Mainz, Germany) and filtered us-ing 0.45-�m Nylon 66 membranes (Rankem, New Delhi,India).

Separate stock solutions were prepared in methanol toyield a 1 mg/mL concentration and stored in a refrigera-tor at −4�C. Standard solutions were obtained by dilutingstock solutions with methanol. Stock solutions were stablefor at least 6 months when stored at −4�C. Internal standardsolution of caffeine (100 ng/mL) was prepared in distilledwater.

2.2 GC-MS instrumentation and conditions

Gas chromatographic mass spectrometric (GC-MS) systemwith model GC-MS-QP2010 Plus (Shimadzu Corporation,Kyoto, Japan) was used for the analysis. The capillary columnused in the GC was Rtx-1MS (30 m × 0.25 mm id × 0.25 �m)supplied by Restek U.S. (Bellefonte, PA, USA). Chromato-graphic data were collected and recorded by GC-MS real-timeanalysis software. Sample injection was done in split mode(split ratio 10:1). Helium was used as the carrier gas at a flowrate of 1 mL/min. The GC injector temperature was set at270�C. The column oven temperature was optimized to holdat 100�C for 1 min and then to increase by 10�C/min up to200�C, then increase by 15�C/min up to 260�C and then by30�C/min up to 300�C. Mass spectrometry conditions wereas follows: electron ionization source set at 70 eV, MS sourcetemperature 200�C and solvent cut time was 3.5 min. Themass spectrometer was run in full scan mode (m/z 50–500)and in selected ion monitoring (SIM) mode. The quantitationof samples was done by using the SIM mode. The analysis ofthese drugs was completed in less than 15 min.

2.3 Preparation of biological samples

Blood (3 mL) and urine (10 mL) specimens were obtainedfrom the healthy volunteers (6 persons). Blood samples werestored in separate glass tubes containing ethylenediaminete-traacetic acid (EDTA) as the anticoagulant and then cen-trifuged (within 2 h from collection) at 4000 rpm for 10 minat 5�C. The supernatant (plasma) was then transferred topolypropylene tubes and stored at −4�C. These plasma sam-ples were stable over a period of 6 months. Before use, theplasma was thawed at room temperature and centrifuged at4000 rpm for 5 min. Prior to MEPS, 50 �L of the internalstandard working solution spiked plasma samples were pre-pared by adding a few micro liter of the analytes to 1 mLof centrifuged plasma. The samples were then extracted andanalyzed. The concentration range of the standard calibrationcurve was between 0.1 and 500 ng/mL.

Urine samples were collected from the healthy people andfrozen at −4�C in glass tubes until the time of sample pre-treatment. After centrifugation at 4000 rpm for 10 min at 5�C,the assays were carried out on the clear supernatant. Spikedurine samples were prepared using the same procedure asdescribed above for plasma samples.

2.4 MEPS conditions

MEPS was carried out on a BIN (Barrel Insert and NeedleAssembly) containing 4 mg of solid-phase silica-C18 material,inserted into a 250 �L gas-tight syringe from SGE AnalyticalScience (Melbourne, Australia). This sorbent has irregularparticles with an average size of 45 �m and nominal porosity60 A. Before being used for the first time, the sorbent hadbeen manually conditioned with 100 �L methanol followed


J. Sep. Sci. 2012, 00, 1–8 Sample Preparation 3

Table 1. Linearity parameters derived from assays on spiked urine and plasma samples

Analyte Linearity Retention Monitoring Equation coefficients, R2 LOD LOQrange (ng/mL) time (min) ions (y = mx + c) (ng/mL) (ng/mL)

m c

PTN 0.1–500 12.1 182a), 209, 252 0.0201 (0.0292 )b) 0.5788 (0.9876 ) 0.9944 (0.9886 ) 0.0027 (0.0029 ) 0.0080 (0.0087 )ALP 0.1–500 12.7 205, 281a), 309 0.0115 (0.0042 )b) 2.3154 (4.8994 ) 0.9925 (0.9886 ) 0.0021 (0.0023 ) 0.0062 (0.0069)OXC 0.1–500 11.05 208, 236a), 253 0.0389 (0.0238 )b) 2.5712 (1.0904 ) 0.9933 (0.9922 ) 0.0020 (0.0018 ) 0.0060 (0.0056 )CBZ 0.1–500 13.3 165, 193a), 253 0.0466 (0.0433 )b) 1.3141 (0.6820 ) 0.9912 (0.9925 ) 0.0029 (0.0036 ) 0.0088 (0.0108 )

a) Most abundant ion; (underlined) molecular ion.b) The values in italics and in the brackets are referred to linearity parameters on plasma samples.

by 100 �L water. The volumes of methanol and water weredrawn up and then discarded every time at an approximateflow rate of 20 �L/s (±5 �L/s).

The plasma and urine samples (50 �L each) were drawnthrough the syringe ten times manually. It is important thatsamples are drawn slowly (approximately 20 ± 5 �L/s) andwith caution to obtain good percolation between sample andsolid support. The sorbent was then washed once with 50 �Lof water to remove proteins and other interferences. The ana-lytes were then eluted with 30 �L of methanol into a vial andthen 1 �L of it was injected into GC injector. The sorbent wasused for multiple extractions. The multiple pulling/pushingof the sample by the syringe increases the extraction recov-ery. To increase the sorbent lifetime, the C18 adsorbent in thebarrel insert and needle assembly was washed with methanol(4 × 50 �L) and water (4 × 50 �L). This step not only decreasedmemory effects, but also functioned as a conditioning stepbefore the next extraction. The same packing bed was usedfor about 100 extractions before it was discarded.

2.5 Method validation

Calibration curves of urine and plasma samples spiked withantiepileptics standards were performed in the range 0.1–500 ng/mL on GC-MS with seven concentration levels sep-arately. The calibration curves were described by the linearregression equation:

y = mx + c

where y is peak area, x is the concentration, m is the slope,and c is the intercept. The limit of detection (LOD) was setat the concentration when the signal/noise ratio was equal to3:1. The quality control (QC) samples were prepared with theconcentration of 0.1, 1, 10, and 100 ng/mL for antiepileptics.The accuracy and precision were calculated for the QC sam-ples, both within and between days. The experiments weredone six times during six different days. The extraction re-coveries of the drugs were calculated by comparing the peakareas of extracted QC samples from plasma and urine to thepeak areas of analyte standard solutions.

Figure 1. MEPS/GC chromatogram of a standard solution con-taining 0.1 ng/mL of PTN, OXC, CBZ, and ALP.

3 Results and discussion

3.1 GC-MS analysis

A GC-MS method has been developed and optimized in orderto determine the extraction recovery of antiepileptics drugsin spiked urine and blood samples. The developed GC-MSmethod was optimized for: column temperature program,flow rate of carrier gas and temperatures of injector, ionsource, and interface. The final optimized GC separation ofanalytes of interest was achieved within 14 min and the totalchromatographic run time was of 30.33 min. Due to higherspecificity of GC-MS, a compound can be easily recognized byits molecular ion and any other interference from coexistingcompounds can be identified, even if they coelute.

For detection in SIM mode, the ions selected from theirmain fragmentation pattern according to their abundance areshown in Table 1. The procedure yielded excellent separationand symmetrical peaks for each antiepileptic. Figure 1 showsthe GC/MS chromatogram of these antiepileptics at a con-centration of 0.1 ng mL−1.

3.2 MEPS method development

The method using microextraction in packed syringe wasused with C18 (4 mg) as sorbent material. The recoveries fromspiked samples were compared to that of calibration curve of



Figure 2. (A) Effect of number of extraction cycles on extraction efficiency. (B) Comparison of the antiepileptics response using differentwashing solutions for MEPS (C18). (C) Evaluation of the elution efficiency of different organic solvents using the MEPS (C18) syringe. (D)Effect of the sample pH on the response of the antiepileptic drugs. Extracted conditions: C18-BIN, sample concentration is 10 ng/mL, andsample volume is 500 �L.

pure standard solutions by calculating the peak areas fromchromatograms. The factors affecting the absolute recoverywere investigated such as the draw/eject speed, sample load-ing amount, sort of eluents, elution volume, and carry overeffects.

In MEPS the retention of the analytes to the sorbent phaseis affected by the number of extraction cycles performed andthe speed applied. Practically, an aliquot of the volume of thesample can be drawn up and down through the syringe, onceor several times (cycles) without discarding it. The multipleextraction cycles can be carried out from the same aliquot(draw eject in the same vial) or by draw up from aliquot anddiscard in waste (extract–discard). The last option was selectedin this study. The influence of the number of extraction cycleson extraction efficiency of antiepileptics studied is illustratedin Fig. 2A. Sample recovery was increased as we increasedthe number of extraction cycles up to ten but after that recov-ery was almost same. In this way, the extraction cycles wereoptimized for the sample preparation step.

The influence of different washing solutions on the ab-solute recovery of the analytes was investigated. The employ-ment of methanol and its different aqueous washing mix-tures were tried for the better removal of the interferences ofthe matrices, but it increased the leakage and decreased the

extraction yields (Fig. 2B). However, clean extracts were ob-tained using only 50 �L of water, drawn once at a flow rate ofabout 20 �L/s to remove the interference from the biologicalmatrix.

The influence of both desorbing solvent and its volumewere investigated, to ensure effective elution of the trappedanalytes from the sorbent. The elution solvent should be ableto displace the target analytes from the sorbent at the low-est possible volume. Different organic solvents (methanol,acetonitrile, toluene, hexane, and acetone) with various func-tionality and polarity were used to investigate the optimumdesorption condition. The extraction was performed using500 �L of aqueous sample spiked with the target analytes ata concentration level of 10 ng/mL, and 30 �L of desorbingsolvent was used. Methanol exhibited the highest desorptionefficiency (Fig. 2C) and was, therefore, chosen as desorptionsolvent for further experiments. In this study, the effect ofdesorbing solvent volume was also investigated. The analytesresponses were enhanced as eluting solvent volume was in-creased up to 30 �L and remained almost constant after thispoint. Therefore, a solvent volume of 30 �L was chosen forthe elution of analytes.

The sample pH is a significant factor, which could af-fect the analytes extraction recovery from water samples. The



Figure 3. MEPS/GC chromatograms obtained from (A) blankplasma (B) the same blank plasma spiked with 0.1 ng/mL of PTN,OXC, CBZ, and ALP. (C) A plasma sample (diluted 10 times) froma patient taking 300 mg of PTN.

effects of pH on the extraction efficiency of the selected an-alytes from water samples were evaluated at the pH intervalof 2–8. Initial pH of the 10 ng/mL solution was 5.5. ForMEPS experiments, pure water spiked with these drugs wasanalyzed with the C18 BIN at different pH-values: pH 2 and4 (both adjusted with 0.1 M hydrochloride acid) and pH 6and 8 (both adjusted with 0.1 M sodium hydroxide solution)(Fig. 2D). The extraction at pH 5.5 was found to be the mostsuitable condition. At pH 2 and 4, the signal intensity ofdrugs decreased. Perhaps, hydrolysis can reduce the amountof drugs at pH 2 and 4 but this factor was not investigatedfurther. In this way, pH values of the sample solutions wereoptimized. Thus, analogous to SPE and LLE, the sensitivityof MEPS protocols can be controlled by the pH conditions ofthe sample solution.

Figure 4. MEPS/GC chromatograms obtained from (A) blankurine (B) the same blank urine spiked with 0.1 ng/mL of PTN,OXC, CBZ, and ALP. (C) A urine sample (diluted 10 times) from apatient taking 300 mg of PTN.

Using this MEPS procedure, no interference from thematrices was present as it is evident from the chromatogramsfrom blank plasma and urine samples (Figs. 3A and 4A, re-spectively). Figures 3B and 4B show blank plasma and urinesamples spiked with 0.1 ng/mL of PTN, CBZ, and OXC andALP. The peaks of all the analytes are symmetric and wellresolved without any interference.

When plasma and urine samples spiked with a mixtureof analytes were analyzed and compared to blank plasma, nointerfering compounds were detected at the same retentiontimes as the studied compounds. Figures 3B and 4B showgood selectivity for MEPS as a sample preparation method.



Table 2. Validation parameters

Analyte Amount Extraction yield (%) Intraday RSD (%)a) Interday precisionadded (ng/mL) (RSD%)a)

Urine Plasma Urine Plasma Urine Plasma

PTN 0.1 87.04 80.22 4.2 4.5 4.4 4.61 94.18 95.32 4.1 4.3 4.2 4.410 98.15 96.83 3.7 3.9 4.1 3.6100 98.86 98.31 3.1 3.5 3.5 3.5

ALP 0.1 97.84 89.89 4.1 3.8 4.1 4.41 98.14 95.58 3.9 3.7 3.8 4.110 98.30 96.38 3.5 3.1 3.6 3.8100 99.12 98.44 2.7 2.6 2.5 3.2

OXC 0.1 84.46 89.74 3.8 4.1 3.9 4.71 94.32 89.34 3.4 3.8 3.4 4.210 98.78 95.27 3.3 3.6 3.1 3.9100 99.38 96.87 2.9 3.1 2.4 3.5

CBZ 0.1 86.24 69.92 3.7 3.4 3.8 4.31 94.58 82.24 3.4 3.3 3.5 3.810 96.47 93.13 3.1 3.1 2.9 3.4100 98.36 95.68 2.9 2.6 2.3 2.9

a) Each value is the mean of six independent assays. The extraction yield was calculated from the analyte peak area from spiked urine andplasma samples compared to those obtained from the same analyte concentration in standard solutions.

3.3 Method validation

Good linearity was obtained for all analytes, with correlationcoefficients r > 0.981 over a range of 1–500 ng/mL. Limitsof detection (LOD) were calculated based on signal to noise(S/N = 3) from spiked samples at low concentrations. LODsof these antidepressants were ranged between 0.0020–0.0029ng/mL for the urine samples and 0.0018–0.0036 ng/mL forthe plasma samples. Limit of quantifications (LOQs) werecalculated based on signal to noise (S/N = 10) for the analytesstudied and in the range of 0.0060–0.0088 ng/mL and 0.0056–0.0108 ng/mL for the urine and plasma samples, respectively(Table 1).

Stability of all analytes in blank was studied after 4, 8, and24 h at room temperature. The stability of studied antiepilep-tic drugs in urine and plasma samples at room temperaturewas studied at 4 and 24 h. The stability studies were studiedusing middle and high QC samples in three replicates for eachpoint. Long-term storage stability was tested by storing the QCsamples at −4�C for 8 weeks. Stability during three cycles offreeze and thawing was also investigated. No obvious changesor degradation of any of the compounds was observed.

3.4 Recovery

Extraction yield and precision assays were carried out at fourdifferent concentration levels, corresponding to the lowestlevel, one higher level and two middle point of each calibrationcurve. Table 2 shows the average recoveries of antiepilepticdrugs from spiked urine and plasma samples at four different

levels (100, 10, 1, and 0.1 ng mL−1) were in the range of 69.92–99.38% in the urine and blood samples with relative standarddeviations (RSDs) lower than 4.7%. The carry over was foundto be less than 0.1%.

3.5 Analysis of patient plasma and urine samples

The validated method was applied to the analysis of plasmaand urine samples from some patients. Figures 3C and 4Cshow the chromatograms corresponding to plasma and urinesamples (diluted 10 times) from a patient treated simultane-ously with PTN (300 mg per day). As can be seen, no interfer-ence from the matrices or coadministered drug is detected.The analyte levels found in the urine and plasma samples(taken 12 h after the last antiepileptic drug administration)were 142.34 and 134.86 ng/mL, respectively. Method accuracywas evaluated by means of recovery assays at four differentconcentration levels for all the analytes (n = 6 for each level),as reported in Section 3.4.

Results were satisfactory as recovery values were alwayshigher than 69.92% for all analytes in both urine and plasmasamples spiked with antiepileptic drugs at different concen-tration levels.

3.6 Carryover and matrix effects

The carryover effect was investigated on the column by in-jecting into the gas chromatographic system three successivealiquots of a standard mixture containing all the analytes ata high concentration followed by three successive aliquots of



Table 3. Comparison of LOQs and recoveries of antiepileptics between present study and earlier studies

Drugs used Method used Sample preparation technique LOQ (ng/mL) Recovery (%) RSD (%) Reference

CBZ, CBZE, PHB, LTG, PTN, ZNS, OXC LC-DAD SPE 14–65 92–103 1.2–1.6 [7]CBZ, CBZE, PTN, PHB LC-UV SBSE 80 62.9–86.9 5.0–9.8 [12]LTG, PHB, PTN, CBZ LC-UV Centrifugation 200 94.1–102 2.7–7.8 [36]OXC and metabolites LC-DAD MEPS 50–125 86.6–96.8 3.3–4.2 [37]CBZ and acidic compounds GC-MS ASE 0.5 75–118 5.0–8.0 [39]OXC, CBZ, PTN, PRM, PHB GC-MS MEPS 0.0056–0.0108 69.9–98.4 2.3–4.7 Present method

ASE, accelerated solvent extraction; LTG, lamotrigine; ZNS, zonisamide; LC, liquid chromatography; DAD, diode array detection; SBSE, Stirbar sorptive extraction.

extracted blank urine and plasma. A nonsignificant carryovereffect (less than 0.1%) was evident. Moreover, assays were alsocarried out to ensure that the small quantity of sorbent phase(4 mg) in the MEPS can be easily and effectively washed be-fore the subsequent pre-treatment, by reducing the possibilityof carryover. For this assay, the MEPS sorbent was washedfour times with the eluting solvent (4 × 50 �L of methanol)after the extraction of urine or plasma sample spiked with ahigh concentration of the analytes and then injected into theGC system.

The chromatograms of blank urine and plasma samplesshowed that matrix effect was minimal. Thus MEPS proce-dure coupled with suitable chromatographic conditions en-sured that there was no matrix effect among different samplesof urine and plasma.

4 Conclusions

A new MEPS sample preparation method for the deter-mination of antiepileptics was developed. The results ofthe present study were compared with the results fromother sample preparation techniques such as SPE and SBSE(Table 3) and are found to be better than the earlier publisheddata. This method (MEPS) showed comparable values of theaccuracy and precision with other methods for the analysisof antiepileptics. The major advantage is that MEPS reducesthe extraction time for the analytes studied by 30–100 timescompared to earlier studies [7, 12, 36] using SPE and SBSE.Furthermore, the present method required a very low samplevolume of approximately 1 mL compared to 10 and 30 mL us-ing SPE and SBSE, respectively. This study demonstrated thatthe present method gives better results than those obtainedwith LC coupled with SPE [7, 38] in terms of sensitivity androbustness. Moreover, the GC-MS method requires no deriva-tization and this method can be used in conjunction with avariety of extraction methods. This procedure therefore, pro-vides a useful and more efficient approach that is alternativeto previous reported methods.

MEPS extraction procedure is fast and simple samplepreparation method using small volume of sample, wash-ing, and elution solvent. Therefore, it is highly useful in theanalysis of patient’s samples and is found to be environment

friendly. Because MEPS is less time-consuming, simpler, andmore useful than SPE and SBSE methods, this technique isproposed as an alternative to SPE and SBSE for the routineanalyses of biological samples in clinical laboratories.

The authors are pleased to acknowledge the financial supportfrom UGC, New Delhi vide Major Research Project No. F-33–293(2007).

The authors have declared no conflict of interest.

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A novel microextraction by packed sorbent–gas chromatography procedure for the simultaneous...

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