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Journal of Chromatography B, 1038 (2016) 43–48

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom ep age: www.elsev ier .com/ locate /chromb

etermination of clozapine, and five antidepressants in humanlasma, serum and whole blood by gas chromatography–masspectrometry: A simple tool for clinical and postmortem toxicologicalnalysis

assiliki A. Boumba (Ph.D.) (Assistant Professor)a,∗, George Rallisa, Petros Petrikisb,heodore Vougiouklakisa, Venetsanos Mavreasb

Laboratory of Forensic Medicine and Toxicology, Faculty of Medicine, School of Health Sciences, University of Ioannina, 45110 Ioannina, GreecePsychiatric Clinic, University Hospital of Ioannina, 45110 Ioannina, Greece

r t i c l e i n f o

rticle history:eceived 7 July 2016eceived in revised form 17 October 2016ccepted 20 October 2016vailable online 21 October 2016

eywords:lozapinentidepressants

a b s t r a c t

In this study, we describe a simple and rapid method for the determination of the antipsychotic drugclozapine and five commonly co-administered antidepressants – bupropion, mirtazapine, sertraline,clomipramine and citalopram – in serum, plasma and whole blood. Sample preparation includes solidphase extraction of analytes and determination of drug concentrations by gas chromatography–massspectrometry without any derivatization steps. The method was fully validated according to internationalcriteria and can be successfully applied for routine analyses. Correlation coefficients of calibration curvesfor the tested drugs in the three specimens were in the range 0.9977–0.9999. Intra-day and inter-dayprecisions ranged from 0.81–7.85% and 3.60–12.91% respectively for the studied analytes and matrices.

onitoringC–MSutopsy

Recoveries were satisfactory for different concentrations of each drug in each specimen allowing accuratedeterminations in the range from sub-therapeutic to toxic levels. The presented method shows acceptablesensitivity, linearity in wide concentration ranges (sub-therapeutic, therapeutic, supra-therapeutic/toxiclevels), it is simple and rapid and it is applicable for qualitative and quantitative routine toxicologicalanalyses of clinical and postmortem cases.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

The concentrations of psychoactive drugs in human blood,lasma or serum are usually measured during either the ther-peutic drug monitoring (TDM) of psychiatric patients [1], orhe toxicological investigation of clinical poisoning cases (suicidettempts or accidental ingestions) [2], while antipsychotic andntidepressant drug levels should be determined in postmortemlood samples in order to assess their possible contribution to theanner of death [3].Clozapine is an atypical antipsychotic drug used to treat

chizophrenia which is included in the World Health Organization’sWHO) List of Essential Medicines, and it is considered as one of the

ost important medication needed in a basic health system [4].

∗ Corresponding author.E-mail addresses: vboumba@uoi.gr, vassiliki.boumba@gmail.com

V.A. Boumba).

ttp://dx.doi.org/10.1016/j.jchromb.2016.10.023570-0232/© 2016 Elsevier B.V. All rights reserved.

Clozapine is usually used only in patients that have not respondedto other anti-psychotic treatment because it can cause numeroussevere side effects. Antidepressants are frequently used for treatingdepressive symptoms associated with schizophrenia. The choice ofthe proper antidepressant is crucially important for the patientswho are treated with clozapine due to the possible complicationsby pharmacokinetic drug–drug interactions and by additive phar-macodynamic effects [5]. Therefore, it is highly recommended thatpatients under clozapine treatment should be subjected to TDM,weekly for the first six months, every two weeks for an additionalsix months and afterwards, every four weeks.

A significant number of publications report methods for thedetermination of antipsychotics and antidepressants, either aloneor in combination, in biological fluids, based on gas chromatog-raphy [6–16], or liquid chromatography with various detectors

[17–19]. These methods are often laborious, use tedious extractionprocedures or include derivatization of analytes.

The aim of this study was the development and validationof a simple, rapid, sensitive and specific method, based on gas

4 omatogr. B 1038 (2016) 43–48

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Table 1Retention times and characteristic ions (m/z) for the GC–MS analysis of the targetanalytes.

Analyte Retention time (min) Ionsa (m/z)

Bupropion 4.62 44, 100, 224Cyclobenzaprine (IS) 8.58 58, 202, 215Mirtazapine 8.72 195, 180, 208Sertraline 9.50 274, 159, 304Citalopram 9.67 58, 190, 324Clomipramine 9.75 58, 227, 269Clozapine 13.45 243, 192, 256

4 V.A. Boumba et al. / J. Chr

hromatography–mass spectrometry (GC/MS) identification anduantification of clozapine, bupropion, citalopram, clomipramine,irtazapine and sertraline in whole blood, plasma and serum; thisethod can be applied for toxicological investigations, in clinical

nd forensic routine casework, allowing the determination of theelected drugs concentrations in sub-therapeutic, therapeutic andupra-therapeutic levels.

. Materials and methods

.1. Chemicals and reagents

Mirtazapine, was purchased from LGC GmbH (Luckenwalde,ermany). Clomipramine was purchased from Cerilliant (Roundock, TX, USA). Bupropion, clozapine, citalopram and sertralineere acquired from Sigma-Aldrich GmbH (Steinheim, Germany).ll solvents (methanol, dichloromethane, 2-propanol and ethylcetate) were analytical grade and were purchased from MerckDarmstadt, Germany). Ultra-pure water was supplied by an Aqua-ron Water Still A4000D purification system from Bibby Sterilinimited (Staffordshire, United Kingdom). For solid-phase extrac-ion, (SPE) Chem Elut (5 mL aqueous capacity, 100 unit/package)artridges were used (Agilent Technologies, Lake Forest, CA, USA).

.2. Standard and working solutions

Primary stock solutions of each analyte at 1 mg/mL, were pre-ared by dissolving the appropriate amount of pure analyte inethanol; secondary stock solutions at 0.1 mg/mL were prepared

y further diluting with methanol, both were stored light pro-ected at −20 ◦C. Working solutions were prepared further at 1nd 10 �g/mL by proper dilution with methanol of secondary stockolutions. After preparation, working solutions were stored lightrotected at −20 ◦C. Calibration standards were prepared by spik-

ng 1 mL of blank serum, plasma or blood with the respectiveolume of a working solution. Validation standards (at the respec-ive low, medium and high concentration for each drug) wererepared by adding the appropriate volume of a working solutionrom each drug to a propylene tube, drying the solvent under a gen-le stream of nitrogen and reconstituting in blank serum, plasma orlood. Primary stock solution of the internal standard was prepared

n methanol at a concentration 1 mg/mL and stored light protectedt −20 ◦C, the working solution was prepared by further dilutingith methanol at 10 �g/mL.

.3. Instrumentation

Chromatographic analysis of target compounds was carried outsing a Shimadzu GC-17A equipped with a Shimadzu QP 5050Aass spectrometer (Shimadzu Corporation, Kyoto, Japan). Analytesere separated with an Equity 5 capillary column 30 m × 0.25 mm,

ontaining 5% diphenyl/95% dimethylsiloxane with a phase thick-ess of 0.25 �m, supplied by Sigma-Aldrich Supelco (Steinheim,ermany). Oven temperature was programmed as follows: ini-

ial temperature 120 ◦C held for 2 min, ramped at 20 ◦C per mino 290 ◦C with a final hold time 5 min (total acquisition programime: 15.50 min). The injector port was set at 260 ◦C in the split-ess mode. Helium was used as the carrier gas with constant flowate at 1.5 mL/min. The interface temperature was set at 300 ◦C. Theass spectrometer was operated at electronic ionization mode (EI,

0 eV). In the full-scan mode, preliminary mass spectra of the tar-et analytes were obtained from m/z 40 to 450 AMU with a solvent

elay time of 3 min. In the selected-ion monitoring (SIM) modecquisition, four groups of target ions were monitored at differentime windows defined by the corresponding retention times. Threeons of each analyte were chosen for identification, according to the

a The ion used for quantitation (quantifier) is written in bold number.

mass spectra characteristic features obtained in the full-scan mode,as well as, by comparison with the mass spectra library. The basicion of each drug was used for the quantitation of target compoundsas it is shown in Table 1. The quality criteria were the following:retention times did not differ more than ±2% and the relative inten-sities of the selected ions did not differ more than 10% from therelative intensities of the same ions which were acquired from aspiked sample.

2.4. Sample collection and storage

Serum, plasma (dipottasium EDTA) and whole blood (hep-arinized) were collected from drug-free donors. Heparinized wholebloods from different donors were mixed thoroughly, decanted in aglass vessel and hemolyzed by freezing [18]. Clinical samples wereobtained from psychiatric patients at the Psychiatric Clinic of theUniversity Hospital of Ioannina and autopsy samples from the Lab-oratory of Forensic Medicine and Toxicology, Faculty of Medicine,University of Ioannina. The patients received detailed informationabout the aim of the study and those included gave informed con-sent for their participation. Serum or plasma was retained aftersamples were centrifuged at 2415 × g (RCF) for 10 min, dividedinto aliquots of 1.0 mL, frozen immediately and stored at −20 ◦Cuntil analysis. Autopsy blood samples were collected and kept untiltheir analysis in test tubes containing anti-coagulant (EDTA K3) andpreservative (KF).

2.5. Sample preparation

Blood calibration and validation standards as well as real sam-ples were treated by a SPE procedure. The extraction conditions arereported below: 100 ng of cyclobenzaprine was added to 1.0 mLof sample and then it was diluted with 3.0 mL of 0.1 M sodiumphosphate buffer pH 9.5, vortexed, left to stand for 3 min and cen-trifuged at 945 × g, 4 ◦C, for 10 min. The sample was loaded ontothe SPE Chem Elut cartridge and allowed to stand for 10 min. Theelution was performed by 5 mL (2 × 2.5 mL) of a dichloromethane:2-propanol extraction mixture (90:10, v/v) by gravity flow. Theextract was evaporated to dryness under a gentle stream of nitro-gen at 40 ◦C, then reconstituted in 50 �L ethyl acetate, vortexedand, 1.0 �L of the extract was injected into the GC–MS system.

For samples with drug concentrations higher than the highestcalibration standard the following dilution procedures were fol-lowed for clinical or post-mortem samples: (i) 1:1 dilution: 1 mLinitial sample was diluted with 1.0 mL phosphate buffer saline(PBS), pH 7.4; (ii) 1:50 dilution: 1 mL initial sample was diluted with49 mL phosphate buffer saline (PBS), pH 7.4; finally, 1.0 mL of the

final mixture was undertaken the extraction procedure describedabove. These modifications were tested on five replicates to quan-tify plasma or blood samples overloaded with the respective drug.

V.A. Boumba et al. / J. Chromatogr. B 1038 (2016) 43–48 45

Fig. 1. Upper icons: [A] Bupropion (m/z 44) at 200 ng/mL spiked plasma concentration; [B] Cyclobenzaprine 100 ng/mL (m/z 58) and mirtazapine (m/z 195) at 200 ng/mLs aminea a chrof

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piked plasma concentration; [C] Sertraline (m/z 274), Citalopram (m/z 58), Clomiprt 200 ng/mL spiked plasma concentration. Lower icons: the respective blank plasmor each drug at the respective LOQ.

.6. Method validation

The analytical method was validated according to FDA guide-ines [20] for selectivity linearity, accuracy, precision, limits ofetection and quantification.

Selectivity, towards endogenous interferences was tested bynalysing blank matrices (plasma, serum and whole blood) fromen different sources. Each blank sample was also tested for selec-ivity after spiked at the lower limit of quantification (LLOQ).otential interference with possible concomitant medication andther exogenous xenobiotics was evaluated by injecting into theC–MS system standard solutions of drugs such as antipsychotics

e.g. olanzapine, quetiapine, haloperidol, resperidone, paliperi-one, amisulpride), antidepressants (e.g. venlafaxine, fluoxetine,aroxetine), sedative hypnotics (e.g. diazepam, bromazepam, nor-iazepam, oxazepam, fluorazepam) and drugs of abuse (e.g.orphine, codeine, 6-monoacetyl-morphine, cocaine, benzoyl-

cgonine) in the concentration level of 1000 ng/mL.Analyte stability determinations comprised freeze-and-thaw

ycle stability (three cycles), short-term stability (room temper-ture) and long term stability in plasma, serum or blood and freezetability of the extracts. An acceptance interval of 80–120% waspplied for the mean stability sample concentration as compareso control samples mean (freshly prepared). The stability experi-

ents were performed for low, medium and high concentrationsith six repetitions.

The extraction efficiency of each drug was determined at low,edium and high concentration (n = 6). Therefore, blank plasma

amples were spiked with working solutions of drugs and internaltandard before or after the extraction procedure. The extrac-ion efficiency was expressed by its average and relative standardeviation (RSD). The internal standard was used to check theffectiveness of the extraction procedure and recovery of the realamples.

The method’s linearity was evaluated using five or six concen-ration levels, depending on the range of the therapeutic and toxicindow for each analyte (n = 5 or 6). The spiked concentration

evels were 20.83, 62.5, 125, 250, 500 and 1000 ng/mL for bupro-ion, 8.33, 25, 50, 100, 200 and 400 ng/mL for mirtazapine, 25, 50,00, 200 and 400 ng/mL for sertraline, 15.625, 31.25, 62.5, 250 and00 ng/mL for citalopram, 12.5, 25, 50, 100, 200 and 400 ng/mL for

(m/z 58), at 200 ng/mL spiked plasma concentration, and; [D] Clozapine (m/z 243),matograms for each ion set showed in the upper icons; the inserts show the peaks

clomipramine and 25, 100, 200, 400 and 800 ng/mL for clozapinein each specimen, respectively. Three spiked samples were pre-pared for each concentration of each analyte and for each specimen,respectively. Each extract was injected three times on the GC–MSsystem. Calibrator samples were fortified blank serum, plasma orblood samples and were treated similarly to the unknowns. Thesensitivity of the method was evaluated by determining the limitsof detection (LODs) and limits of quantification (LOQs) which weredefined as the lowest concentration giving a signal-to-noise ratio ofthree (S/N = 3) and ten (S/N = 10), respectively. Both, LODs and LOQs,were determined by analysing six spiked plasma/serum/wholeblood prepared at their respective concentrations.

Precision (RSD%) was calculated in terms of intra-day repeata-bility and inter-day reproducibility at three different concentrationlevels for each analyte (low, medium and high). Intra and inter-dayprecisions were assessed by six determinations per concentrationin the same day or on seven different days (five repeated determi-nations in each day), respectively.

Accuracy was evaluated by recovery experiments calculatedaccording to the following equation: Absolute analytical recov-ery = {Cfound/Cspiked} × 100% where Cfound is the determined drugconcentration in the extracts of spiked samples and Cspiked isthe drug concentration determined in the extraction eluents of ablank sample after they were spiked with the drug (same drugconcentrations are compared). Accuracy was calculated at threeconcentration levels (low, medium and high) for each drug andspecimen.

3. Results and discussion

In this study, we report the development of a simple, rapid selec-tive and sensitive method for the determination of clozapine andfive commonly co-prescribed antidepressants in human plasma,serum and whole blood by GC-MS. The developed method is suit-able for routine clinical and postmortem toxicological analysis.

Chromatographic separation of drugs is satisfactory and theproposed method is characterized by high selectivity due to the

lack of interfering peaks from endogenous compounds in the blanksamples (Fig. 1). The separation of the six drugs was achieved in13.6 min. The instrument was used in pulsed-split less injection andselected ion monitoring (SIM). The retention times and the respec-

46 V.A. Boumba et al. / J. Chromatogr. B 1038 (2016) 43–48

Table 2Validation values for linearity, LODs, LOQs and precision for each analyte and specimen.

Parameter Bupropion Mirtazapine Sertraline Citalopram Clomipramine Clozapine

P S WB P S WB P S WB P S WB P S WB P S WB

Linearity, ng/mL 20.8–1000 8.33–400 25.00–400 15.625–500 12.5–400 25.0–800R2 0.9981–0.9991 0.9992–0.9999 0.9977–0.9985 0.9979–0.9999 0.9982–0.9998 0.9990–0.9999LOD, ng/mL 6.25 2.50 7.50 4.69 3.75 7.50LOQ, ng/mL 20.83 8.33 25.00 15.63 12.50 25.00

4.4 4.8 1.6 1.0 3.6 0.8 1.3 5.8 7.3 4.3 7.86.2 6.6 3.4 2.6 5.2 5.2 3.2 8.2 11.6 8.8 10.5

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Table 3Therapeutic and toxic drug concentrations reported for clozapine and the selectedantidepressant drugs.

Drug pKa [21] LOQ Therapeuticlevels, ng/mL

Toxic levels,ng/mL

[1] [22] [1] [22]

Bupropion 8.22 20.83 225–1500a 25–100 2000a 170Mirtazapine 6.67 8.33 30–80 20–100 160 –Sertraline 9.85 25.00 10–150 50–250 300 290b

Citalopram 9.78 15.63 50–110 20–200 220 500c

Clomipramine 9.20 12.50 230–450a 90–250 450a 400a

Clozapine 7.35 25.00 350–600 100–600 1000 800–1300

a Drug plus active metabolite.b Reported for one case.c Lethal concentration.

Table 4Determined values for recoveries of each drug from each specimen under the appliedexperimental conditions. In parentheses are reported the respective low, mediumand high concentrations of each drug that were spiked in each specimen.

Specimen Drug (CL/M/H ng/mL) Recovery% (mean ± SD)

Low Medium High

PlasmaBupropion (100/200/400) 83.01 ± 0.5 90.98 ± 1.0 91.02 ± 0.5Mirtazapine (30/90/200) 97.77 ± 1.0 74.74 ± 1.0 83.04 ± 0.5Sertraline (50/150/300) 18.02 ± 2.0 27.28 ± 2.0 9.33 ± 2.0Citalopram (80/160/500) 82.61 ± 1.0 52.05 ± 1.0 50.47 ± 0.5Clomipramine (50/200/400) 25.77 ± 1.0 38.62 ± 1.0 29.93 ± 1.0Clozapine (100/250/500) 41.13 ± 2.0 47.38 ± 3.0 45.42 ± 0.5

SerumBupropion (100/200/400) 78.65 ± 2.0 89.77 ± 2.0 95.15 ± 1.0Mirtazapine (30/90/200) 91.01 ± 1.0 79.00 ± 2.0 90.18 ± 2.0Sertraline (50/150/300) 20.15 ± 1.0 30.44 ± 1.0 10.88 ± 1.0Citalopram (80/160/500) 85.23 ± 2.0 65.00 ± 1.0 68.55 ± 0.5Clomipramine (50/200/400) 30.56 ± 2.0 35.22 ± 2.0 39.40 ± 0.5Clozapine (100/250/500) 55.67 ± 1.0 46.21 ± 1.0 60.00 ± 1.0

Whole BloodBupropion (100/200/400) 75.33 ± 2.0 82.15 ± 0.5 85.25 ± 2.0Mirtazapine (30/90/200) 89.56 ± 1.0 80.00 ± 2.0 82.87 ± 0.5Sertraline (50/150/300) 15.54 ± 1.0 29.11 ± 2.0 10.87 ± 1.0Citalopram (80/160/500) 90.150 ± 1.0 58.69 ± 1.0 59.82 ± 2.0

Precision, RSD%Intraday 5.1 4.0 6.1 3.6 5.5 5.6 6.4

Interday 12.9 10.5 10.5 5.6 5.9 8.8 7.7

ive ions selected for monitoring each analyte are shown in Table 1.electivity towards other xenobiotics was evaluated by injectingnto the GC–MS system standard solutions of drugs such as antipsy-hotics (e.g. olanzapine, quetiapine,haloperidol, resperidone,aliperidone, amisulpride) antidepressants (e.g. amitriptyline, ven-

afaxine, fluoxetine, paroxetine), sedative hypnotics (e.g. diazepam,romazepam, nordiazepam, oxazepam, fluorazepam) and drugs ofbuse (e.g. morphine, codeine, 6-monoacetyl-morphine, cocaine,enzoyl-ecgonine). None of the tested compounds interfered withhe chromatographic peaks of the determined analytes within theun time of 13.6 min, in the concentration level of 1000 ng/mL, usinghe respective retention times and the respective mass ions as cri-eria of selectivity. In this assay, cyclobenzaprine was used to testhe performance of the extraction procedure because it is chemi-ally similar compound to the tested analytes, it can be extractedrom the three specimens with the developed SPE procedure withery good extraction efficiency (mean ± SD = 96.3% ± 2.2) and it iseparated from all the analytes using the optimized GC/MS param-ters.

.1. Method validation

Clozapine and the five ADs were stable after 4 h at room tem-erature or after 48 h at 4

◦C. The freeze thaw stability was within

cceptance range except for sertraline. Long term stability of drugsn serum, plasma and blood was acceptable for three months stor-ge at −20

◦C.

The method was evaluated by characterizing its analyticalerformance in terms of linearity, precision, recovery, limit ofetection (LOD) and limit of quantification (LOQ) for each ana-

yte and specimen as described under materials and methodsTable 2). Coefficients of correlation between 0.9977 and 0.9999ere obtained for calibration curves of all analytes and matrices.

he LODs and LOQs of each analyte were the same for plasma,erum and whole blood. The intra-day repeatability, ranged from.81% to 7.33%, while the inter-day reproducibility, ranged from.40% to 12.91% for all analytes and specimens, respectively, andet validation requirements.The linearity range covers sub-therapeutic, therapeutic and

upra-therapeutic (toxic) levels for clozapine, mirtazapine,lomipramine and citalopram and therapeutic and toxic levels forupropion and sertraline as it is shown in Table 3 [1,21–22]. TheOQs were lower than the lower reported therapeutic level for allrugs with the exception of sertraline. For sertraline the lowerherapeutic level was lying between the respective LOD and LOQLOD < 10 ng/mL < LOQ). The proposed assay was efficient enoughor the quantification of clozapine and the selected antidepres-ants through a wide detection window and for the three testedatrices. More specifically, the method enables the determination

f sub-therapeutic, therapeutic and supra-therapeutic (toxic)evels for clozapine, mirtazapine, clomipramine and citalopramnd therapeutic and toxic levels for bupropion and sertraline beingherefore more sensitive than previous methods [16].

Clomipramine (50/200/400) 23.48 ± 2.0 30.25 ± 2.0 32.28 ± 0.5Clozapine (100/250/500) 40.69 ± 2.0 44.40 ± 2.0 50.42 ± 1.0

The extraction recoveries of each drug from plasma, serum andwhole blood, were defined as percentage of the extracted amountsof each analyte into the final phase (presented in Table 4). Analy-ses of quality control samples in three concentration levels (low,medium and high with declared values for each drug) confirmedthe validity of the method. Furthermore, peak shapes and reso-lution were satisfactory and similar to those obtained by injectingstandard solutions. The extraction procedure was reproducible and

leaded to satisfactory extraction efficiencies with the exception ofsertraline. The recoveries were more satisfactory for most drugs atthe low concentration level. The higher recovery values in the three

V.A. Boumba et al. / J. Chromato

Table 5Analysis results of some representative cases: clinical samples were plasma frompatients with schizophrenia treated with clozapine; postmortem (PM) case wasautopsy whole blood (WB).

Cases Samples Drug Concentration (ng/mL)

Patient 1a 1a 447.71b 852.3

Patient 2a 2a 30.3Patient 3a 3a 106.4

3b 568.83c 601.8

PM caseb WB 12500

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a Drug concentration is for clozapine.b Drug concentration is for mirtazapine.

oncentration levels were observed for mirtazapine, citalopramnd bupropion. Sertraline had the lowest recovery at its high con-entration level (9.33% from plasma). However, the recovery valuesre considered less important as long as the data for LOQ, accuracynd precision are acceptable [23]. Recovery values, although quietifferent between analytes, were comparable or enhanced (partic-larly for mirtazapine) compared to previously reported [7,8,12].hese values indicate the method’s ability for accurate and preciseetermination of each drug in complicated matrices such as humanlasma, serum and whole blood, after pre-treatment according tohe stated protocol.

It is worth mentioning that this assay does not include anyerivatization of analytes being therefore simpler and faster thanreviously reported methods which include laborious derivatiza-ion steps [8–9,11,14].

Sample dilutions beyond the calibration range were essential inpplying the extraction process, given the wide range of concentra-ions expected for clozapine and the other drugs (from therapeutico toxic levels) and depending on the matrix. Dilutions of samplesere performed with PBS which is isotonic to human blood. Theilution process was effective for clozapine in plasma, with accu-acy 42.4% ± 2.2 and precision less than 10.9%, and for mirtazapinen whole blood with accuracy 101.5% ± 3.0 and precision less than.7%.

The current method relies on simple and basic analytical instru-entation and can be applied for routine toxicological analyses, in

hree widely analyzed human specimens, serum, plasma and wholelood, with comparable analytical effectiveness between differentatrices.

.2. Method application in clinical and postmortem cases

The proposed method was used for monitoring psychiatricatients being under clozapine therapy and also for post-mortemoxicological analysis. The results of some representative casesre shown in Table 5. Clinical samples were from patients withchizophrenia treated with clozapine. Patient 1 appeared seriouslyll on the day of the first blood sampling (sample 1a), althoughhe was receiving 450 mg clozapine daily. Clozapine levels wereound within therapeutic range (sample 1a) and a month laterhe had moderate symptoms although with alert clozapine lev-ls (sample 1b). Patient 2 was under chronic clozapine treatment300 mg daily), and the drug analysis proved that probably he hadiscontinued his medication for some days. Patient 3 had startedlozapine monotherapy. A week after the titration of clozapine to50 mg per day, clozapine levels were sub-therapeutic (sample 3a).

month later and after the dose was adjusted to 600 mg clozap-ne per day, clozapine levels were close to the upper therapeuticimit (sample 3b), remained so even after six months of treatmentut with improved clinical symptoms. The post-mortem blood (PM

[

gr. B 1038 (2016) 43–48 47

case) was from a suicide victim who ingested unknown number ofmirtazapine pills.

4. Conclusions

A simple, rapid, sensitive and specific method has been devel-oped and validated for the determination of the antipsychoticclozapine and the antidepressants, bupropion, mirtazapine, ser-traline, clomipramine and citalopram in three widely analyzedhuman specimens, serum, plasma and whole blood. Sample prepa-ration includes SPE and no derivatization of analytes. The presentedmethod is selective, sensitive, accurate and precise, it shows lin-earity in wide concentration ranges, it relies on simple and basicanalytical instrumentation and it can be applied for routine toxico-logical analyses, with comparable analytical effectiveness betweendifferent matrices. The current validated GC–MS represents asignificant tool which enables measurement and monitoring of fre-quently taken psychoactive drugs and also is suitable for routinepostmortem toxicological analysis. The clinical significance of thecurrent assay was verified during the investigation of clinical andpostmortem toxicology cases from our routine casework.

Acknowledgments

This study has been funded by the European Regional Develop-ment Fund—ERDF (MIS: 380379) through the Operational Program“THESSALY, MAINLAND GREECE AND EPIRUS 2007–2013” of theNational Strategic Reference Framework (NSRF 2007–2013).

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