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Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 467–471

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journa l homepage: www.e lsev ier .com/ locate / jpba

fast and feasible microextraction by packed sorbent (MEPS)rocedure for HPLC analysis of the atypical antipsychotic ziprasidone

n human plasma

aura Mercolinia,∗, Michele Protti a, Giulia Fulgenzia, Roberto Mandriolib, Nadia Ghedinia,ndreas Concac, Maria Augusta Raggia

Laboratory of Pharmaco-Toxicological Analysis, Department of Pharmacy and Biotechnology (FaBiT), Alma Mater Studiorum – University of Bologna, Viaelmeloro 6, 40126 Bologna, ItalyDepartment for Life Quality Studies (QuVi), Alma Mater Studiorum – University of Bologna, Corso d’Augusto 237, 47921 Rimini, ItalyMental Health Centre (C.S.M.), Local Health Authority of South Tyrol, Bolzano, Italy

r t i c l e i n f o

rticle history:eceived 23 July 2013eceived in revised form 4 September 2013ccepted 7 September 2013vailable online 5 October 2013

eywords:iprasidone

a b s t r a c t

An original high-performance liquid chromatographic method coupled to microextraction by packedsorbent (MEPS) was developed for the therapeutic drug monitoring (TDM) of psychiatric patients treatedwith the recent atypical antipsychotic ziprasidone.

The chromatographic separation was achieved on a RP C18 column, using an isocratic mobile phaseand setting the wavelength at 320 nm. The analyte was extracted from human plasma by means of a fastand feasible innovative MEPS procedure, optimised on C2 sorbent and requiring only 100 �L of biologicalsample. A second pre-treatment procedure, based on solid phase extraction (SPE), has been also developed

icroextraction by packed sorbent (MEPS)PLClasmaherapeutic drug monitoring (TDM)

for comparison. The availability of different pre-treatment procedures allows the choice of the one bestsuiting the specific clinical, economic and scientific needs. The extraction yield values were always higherthan 90% and sensitivity was also good, with a limit of quantitation (LOQ) of 1 ng/mL.

The method was successfully applied to plasma samples from ten subjects undergoing therapy withziprasidone, thus confirming its suitability for the TDM of psychiatric patients, in order to personalisetheir pharmacological treatments.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Ziprasidone (5-[2-[4-(1,2-benzisothiazol-3-yl)-1-iperazinyl]ethyl]-6-chloro-1,3-dihydro-2H-indol-2-one, ZPR,ig. 1a) is one of the most recent atypical antipsychotics introducednto the market. It is used for the treatment of schizophrenia, acute

ania and mixed state episodes associated with bipolar disorder1]. ZPR is also used off-label for depression, bipolar disorder

aintenance therapy, anxiety, aggression, dementia, attentioneficit hyperactivity, autism and stress disorders [2]. ZPR efficacy

n treating the positive symptoms of schizophrenia is primarilyediated by antagonism of the dopamine D2 receptors. Blockade

f 5-HT2A and 5-HT2C receptors and activation of 5-HT1A receptors,

s well as serotonin and norepinephrine reuptake inhibition, mayll contribute to its ability to alleviate also negative symptoms.nlike many other antipsychotics, ZPR has no significant affinity

∗ Corresponding author. Tel.: +39 051 2099726; fax: +39 051 2099726.E-mail address: laura.mercolini@unibo.it (L. Mercolini).

731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jpba.2013.09.019

for muscarinic cholinergic receptors (mAChRs), for this reason itdoes not show any anticholinergic side effects [3]. Moreover ZPRis not generally associated with weight gain or hyperlipidemia[4]. Clinically effective doses are 80–160 mg/day [5], correspond-ing to plasma levels ranging from 20 to 160 ng/mL [6]. Orallyadministered ZPR is easily absorbed and its absorption increasestwo-fold in the presence of food. ZPR is extensively metabolised byphase I (primarily cytochrome P450 3A4) and phase II metabolicpathways, resulting in several metabolites which do not seem tocontribute to the overall antipsychotic effect [7]. ZPR most frequentside-effects are mild or moderate headache, first-dose posturalhypotension and mild histaminergic sedative effect [8]. The mostimportant severe toxic effect of the drug is the QT syndrome: ZPRhas been shown to prolong the cardiac corrected QT (QTc) interval,which is linked to fatal ventricular tachyarrhythmias and torsadede pointes [9]. Thus, ZPR should be avoided in patients with some

types of cardiac diseases, uncontrolled electrolyte disturbance orin polypharmacy with other drugs that prolong the QT interval.

The importance of having at disposal feasible but reliable analyt-ical methods for the therapeutic drug monitoring (TDM) of patients

468 L. Mercolini et al. / Journal of Pharmaceutical an

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ig. 1. Chemical structures of (a) ziprasidone (ZPR) and (b) mirtazapine, used as thenternal standard (IS).

reated with ZPR is thus evident, in order to personalise and opti-ise pharmacological therapies, minimising side and toxic effects.The method presented herein allows ZPR plasma determina-

ion by means of a fast and feasible microextraction by packedorbent (MEPS) pre-treatment and HPLC-UV analysis. The method-logy does not require expensive instrumentations and exploits aample pre-treatment procedure, based on a miniaturised extrac-ion, that reduces sample volumes and analysis time, while grantingound results.

To the best of our knowledge, only a few papers can be found inhe literature, which specifically deal with the analysis of ZPR in bio-ogical fluids: those take advantage of chromatographic separationsHPLC) coupled to spectrophotometric (UV) [10,11], spectroflu-rometric (F) [12], mass spectrometry (MS) [13–19] detectionnd describe pre-treatment techniques based on liquid–liquidxtractions with organic solvents [11–13,15–18] or on protein pre-ipitation [14,19].

This original MEPS-HPLC method is therefore proposed as aromising analytical improvement compared to already publishedapers.

. Materials and methods

.1. Chemicals

ZPR and mirtazapine ((±)-2-methyl-1,2,3,4,10,14b-exahydropyrazino[2,1-a]pyrido[2,3-c][2]benzazepine, Fig. 1b),sed as the Internal Standard (IS), reference pure compounds,ere purchased from Sigma–Aldrich (St. Louis, USA). HPLC-grade

cetonitrile and methanol, monobasic sodium phosphate, sodiumydroxide, 85% (w/w) phosphoric acid and diethylamine, all pure

or analysis, were bought from Sigma–Aldrich. Ultrapure water

d Biomedical Analysis 88 (2014) 467–471

(18.2 M� cm) was obtained by means of a MilliQ apparatus byMillipore (Milford, USA).

2.2. Instrumentation and chromatographic conditions

The chromatographic system was composed of a Jasco (Tokyo,Japan) PU-980 isocratic pump, equipped with a Jasco UV-975 spec-trophotometric detector, set at 320 nm. Separations were obtainedon a Phenomenex (Torrance, CA, USA) Gemini® reversed-phase col-umn (50 mm × 3.0 mm I.D., 5 �m), kept at room temperature. Themobile phase was a mixture of acetonitrile (30%, v/v) and a pH 2.5,50 mM phosphate buffer containing 0.2% (v/v) diethylamine (70%,v/v), flowing at 0.5 mL/min. The injections were carried out througha 10-�L loop. Data processing was handled by means of a JascoChromNAV 1.16 software.

2.3. Stock and standard solution preparation

Stock solutions of ZPR and IS (1 mg/mL) were prepared by dis-solving suitable amounts of each pure substance in methanol.Standard solutions were obtained by diluting stock solutions withthe mobile phase and injected into the HPLC-UV system. Stock solu-tions were stable for at least one month when stored at −20 ◦C (asassessed by HPLC assays), while standard solutions were preparedfresh every day.

2.4. Plasma collection and pre-treatment

Blood samples were centrifuged (within 1 h from collection) at1400 × g for 10 min, the supernatant (plasma) was transferred intoglass vials and stored at −20 ◦C until analysis.

MEPS procedure was performed on a SGE Analytical Science(Ringwood, Australia) apparatus, consisting of a 100-�L HPLCsyringe with a removable needle, fitted with a BIN (Barrel Insertand Needle) containing a C2 sorbent. The sorbent was conditionedwith 200 �L of methanol and equilibrated with 200 �L of ultrapurewater. The loading solution was a mixture of 100 �L of plasma,100 �L of ultrapure water and 5 �L of IS solution prepared in mobilephase; the loading mixture was drawn into the syringe and dis-charged back 10 times. The cartridge was then washed with 100 �Lof water, 100 �L of a water/methanol mixture (90/10, v/v) andeluted by drawing and discharging 500 �L of methanol. The elu-ate was dried under vacuum, re-dissolved with 100 �L of mobilephase and injected into the HPLC-UV system.

The SPE procedure, developed as a comparison, was carried outon a Macherey-Nagel (Düren, Germany) Chromabond® vacuumapparatus, using Biotage (Uppsala, Sweden) Isolute C2 cartridges(50 mg, 1 mL) that were activated by passing 2 mL of methanol andthen conditioned by passing 2 mL of ultrapure water. To 250 �L ofplasma, 500 �L of ultrapure water and 10 �L of IS solution preparedin mobile phase were added and the resulting mixture was loadedonto a previously conditioned cartridge. The cartridge was thenwashed with 1 mL of ultrapure water and 1 mL of a water/methanolmixture (90/10, v/v), finally eluted with 1 mL of methanol. The elu-ate was dried under vacuum, re-dissolved with 125 �L of mobilephase and injected into the HPLC-UV system.

2.5. Method validation on blank plasma samples

Method validation was carried out on blank plasma samplesfrom ten healthy volunteers, not treated with ZPR, according toUSP [20] and Crystal City [21] guidelines.

2.5.1. LinearityAliquots of 10 �L of ZPR standard solutions at seven different

concentrations, containing IS at a constant concentration, were

L. Mercolini et al. / Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 467–471 469

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ig. 2. Chromatograms of a standard mixture containing 100 ng/mL of ZPR and 50 nample spiked with a mixture of ZPR and IS both at a concentration of 50 ng/mL (c)

dded to blank plasma samples. The resulting “spiked” plasmaamples were subjected to the previously described MEPS pre-reatment and for comparison to SPE procedure.

The procedure was carried out in triplicate for each concentra-ion. The analyte/IS peak area ratios (pure numbers) were plottedgainst the corresponding concentrations of the analyte (expresseds ng/mL) and the calibration curves were set up by means of theeast-square method. The values of limit of quantification (LOQ) andimit of detection (LOD) were calculated as the analyte concentra-ions, which give rise to peaks whose heights are 10 and 3 timeshe baseline noise, respectively.

.5.2. Extraction yield and precisionExtraction yield and precision were evaluated by adding known

mounts of ZPR (at three different concentrations, correspondingo the lower limit, a middle point and a high value of the cali-ration curve) to blank plasma samples, subjecting them to MEPSre-treatment and for comparison to SPE procedure. The analyteeak areas were compared to those obtained by injecting standardolutions at the same theoretical concentrations and the extractionield was calculated. The assays described above were repeatedix times within the same day to obtain intraday precision andix times over six different days to obtain interday precision, bothxpressed as percentage relative standard deviation (RSD%).

.5.3. SelectivityBlank biological samples from ten different volunteers were

ubjected to the pre-treatment procedures and injected into thePLC system; the resulting chromatograms were checked forossible interference from endogenous compounds. Furthermore,tandard solutions of several different drugs active on the Cen-ral Nervous System were injected at concentrations higher than

he respective therapeutic levels. If the resulting chromatogramsontained any interference peak, the potentially interfering com-ounds were subjected to MEPS procedure to ascertain if they coulde extracted. The acceptance criterion was that no interference

f IS (a), a blank plasma sample from a healthy volunteer (b), the same blank plasmaplasma sample from a psychiatric patient taking 140 mg/day of oral ZPR (d).

peak has to be higher than the analyte peak corresponding to itsLOD.

2.6. Analysis of patient plasma samples and accuracy

ZPR standard solutions at three different concentrations wereadded to plasma samples from psychiatric patients treated withZPR, whose concentrations were previously analysed. Thesepatients were treated at the Mental Health Centres (C.S.M.) inBolzano or in Bologna (Italy).

The samples were subjected to MEPS procedure and for com-parison to SPE pre-treatment. The method accuracy was tested andexpressed as percentage recovery.

3. Results and discussion

3.1. Choice of the chromatographic conditions

Our previous experience with the analysis of some classical andatypical antipsychotics in human plasma prompted us to study ZPRstarting from similar experimental conditions [22,23]. The use of aC18 column with smaller diameter and length (50 mm × 3.0 mmI.D.), and a lower flow rate (0.5 mL/min) allowed the use of smallervolumes of solvents, while the composition of the mobile phasewas adjusted to obtain shorter retention times while retainingsatisfactory resolution and selectivity. Regarding the IS, differ-ent compounds were tested, such as citalopram, venlafaxine andmirtazapine; mirtazapine resulted to have chemical–physical andretention properties sufficiently similar to those of the analyte,while being well resolved from it. For this reason, it was cho-sen as a prospective IS. Under these conditions the analyte and ISprovide neat, symmetric peaks with retention times shorter than

9 min. Fig. 2a shows the chromatogram of the HPLC-UV analysis ofa standard mixture containing ZPR at a concentration of 100 ng/mLand IS at a concentration of 50 ng/mL. As one can note, separationis fast and resolution is satisfactory.

4 cal and Biomedical Analysis 88 (2014) 467–471

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Table 1Linearity data on blank spiked plasma samples.

Pre-treatment ZPR linearityrange (ng/mL)

r2 LOQ (ng/mL) LOD (ng/mL)

MEPS 1–500 0.9993 1 0.3

TE

70 L. Mercolini et al. / Journal of Pharmaceuti

.2. Development of the microextraction by packed sorbentMEPS) procedure

In the last few years, miniaturisation, short extraction times,ower analysis cost and consumption of smaller volumes of samplend of organic solvents are increasingly investigated, especially forhose analyses with TDM purposes [23]. MEPS is a recent technique,ased on the general principle of solid phase extraction, but withhe sorbent contained within a very small barrel (BIN), which con-titutes the needle assembly of an HPLC syringe. MEPS grants highelectivity, good sample purification and extraction yields, whileeing faster, more feasible and using smaller amounts of biolog-

cal samples and solvents, when compared to SPE, liquid–liquidxtraction [11] or protein precipitation [19].

The first MEPS sorbents tested were C18 and C8 ones, but theyere not satisfactory in terms of either ZPR extraction yield or

ample clean-up. The weakly lipophilic C1 and C2 sorbents gaveromising results also in terms of selectivity and for this reasonhey were chosen for extraction parameter optimisation. All the

ain steps of the procedure (loading, washing and elution) wereptimised. An aliquot of the sample can be drawn up and downhrough the MEPS syringe, at a suitable speed, once or severalimes (cycles). Thus, in the loading step, the number of cycles andhe speed performed are two of the parameters which affect thenalyte retention. Different cycles and speeds were tested: theetention was satisfactory after 10 draw/discharge cycles of theoading mixture at a speed of 5 �L/s (extraction yields: <40% withne cycle, <70% with 5 cycles). Good purification was obtained byashing the sorbent with 100 �L of water followed by 100 �L ofater/methanol 90/10 (v/v) mixture; an elution step consisting in

wo cycles with 250 �L of pure methanol (for a total of 500 �L)roved sufficient for the complete elution (>92%) of ZPR (extrac-ion yields: <50% with 3 × 100 �L, <80% with 3 × 200 �L, <93% witholumes higher than 3 × 250 �L). After each extraction, a sorbentleaning step was carried out with 200 �L of methanol, followedy 200 �L of water. This step decreased carryover effects, but alsocted as the conditioning step for the next extraction. The eluateas dried under vacuum and re-dissolved with 100 �L of mobilehase. Using this MEPS procedure, good extraction yields of thenalyte and IS were reached, while eliminating all endogenousnterference.

In Fig. 2b the chromatogram of a blank plasma sample iseported to prove MEPS high performance in terms of matrix purifi-ation. The chromatogram of the same blank plasma sample, spikedith ZPR and IS at the concentration of 50 ng/mL, is shown in Fig. 2c.s can be seen, peak shape and resolution are good, with asymme-

ry factors always lower than 1.3 and resolution values higher than.

.3. Comparison with a solid-phase extraction (SPE) procedure

SPE was tested as a comparative extraction technique on C2artridges and being the preliminary results rather promising,ssays were carried out on this sorbent for extraction parameters

able 2xtraction yield and precision data on blank spiked plasma samples.

Compound Concentration (ng/mL) Mean extraction yield (%

MEPS SPE

ZPR 10 92 9050 92 90

250 95 91IS 50b 98 92

a n = 6.b In the injected solutions.

SPE 0.5–250 0.9989 0.5 0.2

optimisation. The washing steps were initially carried out withwater and water/methanol mixtures. The methanol percentage wasvaried in the 5–30% range and it was observed that more than 10%methanol caused a sharp drop in extraction yields. Finally, 1 mLof pure methanol was used to elute ZPR and IS. The eluate wasthen dried under vacuum and re-dissolved in 125 �L of mobilephase; ZPR concentration in the final, injected solution was thustwice that of the original sample. SPE was used under these condi-tions to compare the results obtained by MEPS on blank and realsamples.

3.4. Method validation

The analytical method was validated according to internationalregulatory guidelines [20,21] in terms of linearity, extraction yield,precision and accuracy. A very good linearity (r2 > 0.9992) wasfound in the 1–500 ng/mL range. The limit of quantitation (LOQ)and the limit of detection (LOD) were 1 and 0.3 ng/mL, respectively.The complete linearity data are reported in Table 1, where one canfind also a comparison with SPE data.

Extraction yield and precision assays were carried out at threedifferent concentrations of ZPR, corresponding to the lowest level,a middle point and a high level of the calibration curve. The resultswere satisfactory, being ZPR extraction yields higher than 91% andthose of IS always higher than 97%. The precision was also good,with relative standard deviations values always lower than 4.2%both for intraday and interday data. The complete results of theseassays are reported in Table 2, compared to those obtained fromSPE assays.

Method selectivity was evaluated injecting standard solutionsof several drugs usually co-administered to psychiatric patients,such as antidepressants, sedative-hypnotics and mood-stabilizers.Retention times of the tested compounds were compared to that ofthe analyte and none of them interfered with the chromatographicpeaks of ZPR or IS. Moreover, the analysis of blank plasma sam-ples from ten healthy volunteers showed no evidence of significantinterference from endogenous compounds.

3.5. Analysis of patient plasma samples

The MEPS-HPLC method was applied to the analysis of plasma

samples from some psychiatric patients treated with ZPR at MentalHealth Centres of Bolzano or of Bologna (Italy).

The chromatogram of a plasma sample from a patient taking140 mg/day of oral ZPR is reported in Fig. 2d. As one can see, no

)a Intraday precision (RSD%)a Interday precision (RSD%)a

MEPS SPE MEPS SPE

3.1 3.5 4.0 4.43.0 3.5 4.1 4.43.0 3.3 4.0 4.03.1 3.2 3.3 3.7

L. Mercolini et al. / Journal of Pharmaceutical an

Table 3Results from the analysis of patient plasma samples.

Sample No. ZPR oral daily dosage (mg/day) ZPR plasmaconcentration found(ng/mL)

MEPS SPE

1 40 22 212 40 19 213 80 73 704 80 77 755 120 96 996 120 90 927 140 109 106

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nterference from the biological matrix is apparent and the analytend the IS peaks are evident and well resolved. The analyte levelound in this sample was 134 ng/mL, which can be considered inhe therapeutic range (20–160 ng/mL) [6].

The results of method application to this and nine other patientsre reported in Table 3. It is possible to observe how the plasmaevels found within the TDM of patients treated with ZPR wereonsistent with the administered dosages corresponding to initial,aintenance and high doses.The same plasma samples have been processed also by SPE,

btaining quantitative results very similar to those got by MEPS,hich possesses anyway the great advantage of being faster and

easible.Method accuracy was evaluated by means of recovery assays,

piking patient plasma samples with known amounts of thenalyte at three different concentrations; the obtained resultsere satisfactory, with mean recovery values always higher than

0%.

. Conclusion

An original HPLC method has been developed and fully vali-ated for the TDM of schizophrenic patients treated with the recenttypical antipsychotic ZPR. The development of a fast and feasi-le MEPS pre-treatment procedure granted satisfactory results inerms of extraction yield and of purification of the biological matrixnd represents an analytical improvement over already publishedethods. MEPS represents an innovative miniaturised extraction

echnique, cheaper and less time-consuming than liquid–liquidxtraction [11–13,15–18] and more efficient than protein precip-tation procedures [14,19], often adopted for the same purpose.

EPS, even when compared to SPE, allows reducing plasmand organic solvent volume requirements (only 100 �L of loadedlasma), thus making the method more feasible, but still reliable.uring method validation, MEPS performance has demonstrated toe advantageous with respect to SPE also in terms of linearity andrecision (r2 > 0.9992, RSD% < 4.2).

To the best of our knowledge, this is the first methodology thatddresses the issue of TDM of psychiatric patients treated with ZPRhrough the use of MEPS pre-treatment procedure. The obtainedesults give interesting insights on the perspectives for ZPR analysisn biological fluids. Furthermore, compared to the methods alreadyresent in the scientific literature exploiting the same detection

eans (UV), the method described here allows to obtain better sen-

itivity (1 ng/mL vs 10 ng/mL) [10], shorter analysis times (8 minor each chromatographic run vs 20 min) [10,11] and also betterccuracy [11].

[

d Biomedical Analysis 88 (2014) 467–471 471

After validation, the proposed method has been successfullyapplied to the analysis of plasma samples from several psychiatricpatients undergoing monotherapy or polypharmacy.

In conclusion, the method seems to be suitable for the TDM ofpatients subjected to therapy with ZPR and is a significant improve-ment over currently available procedures.

References

[1] W.M. Greenberg, L. Citrome, Ziprasidone for schizophrenia and bipolar disor-der: a review of the clinical trials, CNS Drug Rev. 13 (2007) 137–177.

[2] M. John, Eisenberg Center for Clinical Decisions and Communications Science,Off-label use of atypical antipsychotics: an update, in: Comparative Effective-ness Review Summary Guides for Clinicians [Internet], Agency for HealthcareResearch and Quality (US), Rockville, MD, 2012.

[3] S.M. Stahl, D.K. Shayegan, The psychopharmacology of ziprasidone: receptor-binding properties and real-world psychiatric practice, J. Clin. Psychiatry 64(2003) 6–12.

[4] J.W. Newcomer, Second-generation (atypical) antipsychotics and metaboliceffects: a comprehensive literature review, CNS Drugs 19 (2005) 1–93.

[5] A. Rossi, A. Vita, P. Tiradritti, F. Romeo, Assessment of clinical and metabolicstatus, and subjective well-being, in schizophrenic patients switched from typ-ical and atypical antipsychotics to ziprasidone, Int. Clin. Psychopharmacol. 23(2008) 216–222.

[6] M.C. Mauri, A. Colasanti, M. Rossattini, L.S. Volonteri, F. Dragogna, A. Fiorentini,A. Valli, P. Papa, Ziprasidone outcome and tolerability: a practical clinical trialwith plasma drug levels, Pharmacopsychiatry 40 (2007) 89–92.

[7] C. Prakash, A. Kamel, J. Gummerus, K. Wilner, Metabolism and excretion of anew antipsychotic drug, ziprasidone, in humans, Drug Metab. Dispos. 25 (1997)863–872.

[8] B. Green, Focus on ziprasidone, Curr. Med. Res. Opin. 17 (2001) 146–150.[9] Y.G. Yap, A.J. Camm, Drug induced QT prolongation and torsades de pointes,

Heart 89 (2003) 1363–1372.10] J. Sachse, S. Härtter, C. Hiemke, Automated determination of ziprasidone by

HPLC with column switching and spectrophotometric detection, Ther. DrugMonit. 27 (2005) 158–162.

11] G. Zhang, A.V. Terry Jr., M.G. Bartlett, Simultaneous determination of fiveantipsychotic drugs in rat plasma by high performance liquid chromatographywith ultraviolet detection, J. Chromatogr. B 856 (2007) 20–28.

12] R.F. Suckow, M. Fein, C.U. Correll, T.B. Cooper, Determination of plasma ziprasi-done using liquid chromatography with fluorescence detection, J. Chromatogr.B 799 (2004) 201–208.

13] O.Y. Al Dirbashi, H.Y. Aboul-Enein, A. Al-Odaib, M. Jacob, M.S. Rashed, Rapidliquid chromatography-tandem mass spectrometry method for quantificationof ziprasidone in human plasma, Biomed. Chromatogr. 20 (2006) 365–368.

14] H. Kirchherr, W.N. Kühn-Velten, Quantitative determination of forty-eightantidepressants and antipsychotics in human serum by HPLC tandem massspectrometry: a multi-level, single-sample approach, J. Chromatogr. B 843(2006) 100–113.

15] G. Zhang, A.V. Jr Terry, M.G. Bartlett, Sensitive liquid chromatography/tandemmass spectrometry method for the simultaneous determination of olanzapine,risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone inrat brain tissue, J. Chromatogr. B 858 (2007) 276–281.

16] M. Aravagiri, S.R. Marder, B. Pollock, Determination of ziprasidone in humanplasma by liquid chromatography-electrospray tandem mass spectrometryand its application to plasma level determination in schizophrenia patients,J. Chromatogr. B 847 (2007) 237–244.

17] G. Zhang, A.V. Terry Jr., M.G. Bartlett, Liquid chromatography/tandem massspectrometry method for the simultaneous determination of olanzapine,risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone inrat plasma, Rapid Commun. Mass Spectrom. 21 (2007) 920–928.

18] G. Zhang, A.V. Terry Jr., M.G. Bartlett, Determination of the lipophilicantipsychotic drug ziprasidone in rat plasma and brain tissue using liquidchromatography-tandem mass spectrometry, Biomed. Chromatogr. 22 (2008)770–778.

19] J. Hasselstrøm, Quantification of antidepressants and antipsychotics in humanserum by precipitation and ultra high pressure liquid chromatography-tandemmass spectrometry, J. Chromatogr. B 879 (2011) 123–128.

20] United States Pharmacopeial Convention, 32th ed., United States Pharma-copeia, Rockville, MD, 2009, pp. 734.

21] V.P. Shah, K.K. Midha, J.W.A. Findlay, H.M. Hill, J.D. Hulse, I.J. McGilveray, G.McKay, K.J. Miller, R.N. Patnaik, M.L. Powell, A. Tonelli, C.T. Viswanathan, A.Yacobi, Bioanalytical method validation – a revisit with a decade of progress,Pharm. Res. 17 (2000) 1551–1557.

22] L. Mercolini, M. Grillo, C. Bartoletti, G. Boncompagni, M.A. Raggi, Simultaneous

analysis of classical neuroleptics, atypical antipsychotics and their metabolitesin human plasma, Anal. Bioanal. Chem. 388 (2007) 235–243.

23] R. Mandrioli, L. Mercolini, D. Lateana, G. Boncompagni, M.A. Raggi, Analysis ofrisperidone and 9-hydroxyrisperidone in human plasma, urine and saliva byMEPS-LC-UV, J. Chromatogr. B 879 (2011) 167–173.