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Journal of Chromatography B, 942– 943 (2013) 158– 164

Contents lists available at ScienceDirect

Journal of Chromatography B

j ourna l h o mepa ge: www.elsev ier .com/ locate /chromb

ydrophilic interaction liquid chromatography/positive ionlectrospray ionization mass spectrometry method for theuantification of alprazolam and �-hydroxy-alprazolam inuman plasma

leni Kalogriaa, Constantinos Pistosb, Irene Panderia,∗

University of Athens, School of Pharmacy, Division of Pharmaceutical Chemistry, Panepistimopolis – Zografou, 157 71 Athens, GreeceUniversity of Athens, School of Medicine, Department of Forensic Medicine and Toxicology, Micras Asias 75, Goudi, Athens, Greece

r t i c l e i n f o

rticle history:eceived 22 July 2013ccepted 26 October 2013vailable online 2 November 2013

eywords:ydrophilic interaction liquidhromatography (HILIC)iquid chromatography/mass spectrometrylprazolam

a b s t r a c t

A hydrophilic interaction liquid chromatography/positive ion electrospray-mass spectrometry (HILIC-ESI/MS) has been developed and fully validated for the quantification of alprazolam and its mainmetabolite, �-hydroxy-alprazolam, in human plasma. The assay is based on 50 �L plasma samples,following liquid-liquid extraction. All analytes and the internal standard (tiamulin) were sepa-rated by hydrophilic interaction liquid chromatography using an X-Bridge-HILIC analytical column(150.0 mm × 2.1 mm i.d., particle size 3.5 �m) under isoscratic elution. The mobile phase was com-posed of a 7% 10 mM ammonium formate water solution in acetonitrile and pumped at a flow rate of0.20 mL min−1. Running in positive electrospray ionization and selected ion monitoring (SIM) the massspectrometer was set to analyze the protonated molecules [M + H]+ at m/z 309, 325 and 494 for alprazo-

-Hydroxy-alprazolamiamulin

lam, �-hydroxy-alprazolam and tiamulin (ISTD) respectively. The assay was linear over the concentrationrange of 2.5–250 ng mL−1 for alprazolam and 2.5–50 ng mL−1 for �-hydroxy alprazolam. Intermediateprecision was less than 4.1% over the tested concentration ranges. The method is the first reported appli-cation of HILIC in the analysis benzodiazepines in human plasma. With a small sample size (50 �L humanplasma) and a run time less than 10.0 min for each sample the method can be used to support a wide

oncer

range of clinical studies c

. Introduction

Alprazolam, 8-chloro-1-methyl-6-phenyl-4H-[1,2,4]triazolo-4,3-a][1,4] benzodiazepine, is a triazolobenzodiazepine com-ound, with anti-anxiety and sedative-hypnotic actions. It bindso distinct benzodiazepine binding sites situated at the inter-ace between the �- and �-subunits of GABAA receptors and toeripheral benzodiazepine receptors inside various cells at several

ocations, mainly in the mitochondria [1]. Alprazolam is efficaciousn the treatment of panic disorders, with or without agoraphobia,nd in generalized anxiety disorders. Recent studies explored theotential efficacy of alprazolam extended release treatment tosers of stimulant drugs such as cocaine [2] and methamphetamine3]. In other studies it was found that alprazolam could induce

onformational changes in hemoglobin [4] and it also intercalatesnto the DNA [5], while in combination with caffeine augments celleath through necrosis [6].

∗ Corresponding author. Tel.: +30 2107274820; fax: +30 210 7274747.E-mail addresses: ipanderi@pharm.uoa.gr, irenepanderi@gmail.com (I. Panderi).

570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jchromb.2013.10.040

ning alprazolam quantification.© 2013 Elsevier B.V. All rights reserved.

Alprazolam is extensively metabolized by oxidation and con-jugation and the principal metabolites are �-hydroxy-alprazolamat 17% of a dose, 4-hydroxy-alprazolam at 0.3% of a dose and a,4-dihydroxyalprazolam at 0.2% of a dose [7]. Both metabolites havelower intrinsic benzodiazepine receptor affinity than alprazolam. Inparticular, �-hydroxy-alprazolam and 4-hydroxy-alprazolam haveapproximately 66 and 19% of the parent drug’s potency, respec-tively [8,9]. The clinical activity during treatment with alprazolamis essentially attributable to intact alprazolam. Plasma concentra-tions of alprazolam appear to be dose-proportional, patients given3 mg/day reached an averaged steady state-plasma concentrationof 29 ng mL−1, those on 6 mg/day averaged 61 ng mL−1 and thoseon 9 mg/day, 102 ng mL−1 [10].

Up to now, several high performance liquid chromatographicprocedures have been published in literature for the determinationof alprazolam alone or in combination with other benzodiazepinesin biofluids [11–14]. Other separation techniques, such as capillary

electrophoresis [15], and micellar electrokinetic chromatogra-phy [16,17] have also been reported. Dominant hyphenatedtechniques for the determination of alprazolam alone or in combi-nation with other benzodiazepines are gas chromatography-mass

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pectrometry (GC-MS) [18] and liquid chromatography–mass spec-rometry (LC–MS) [19–23].

To the best of our knowledge, no method has been reportedor the determination of alprazolam and �-hydroxy alprazolamy hydrophilic interaction liquid chromatography (HILIC). HILIC

n combination with mass spectrometry has been steadily gain-ng acceptance in the analysis of polar compounds in complexiological matrices [24,25]. This hyphenated technique offers thedvantages of improved sensitivity by employing high organicodifier fraction in the mobile phase, shortened sample prepara-

ion time and the potential shorter analytical run time [26]. The aimf the current study was to develop a sensitive, fast and convenientethod for the simultaneous identification and quantification of

lprazolam and its main metabolite, �-hydroxy alprazolam, usingiamulin as internal standard. The experiment was performedsing hydrophilic interaction liquid chromatography in combina-ion with electrospray ionization mass spectrometry. The methodas been successfully validated and applied to real samples and it

s suitable for monitoring these drugs in pharmacokinetic studies.

. Experimental

.1. Chemical and reagents

Alprazolam and �-hydroxy-alprazolam were obtained fromigma Aldrich, Germany, tiamulin (internal standard) was kindlyrovided from ILS AE (Athens, Greece). All solvents were of HPLCrade and were purchased from Merck, Darmstadt, Germany.mmonium formate was obtained from Acros Organics (New

ersey, USA). Acrodisk® GHP membrane syringe filters (13 mm, poreize 0.45 �m) were obtained from Pall Life Sciences (Ann Arbor, MI,SA). Pooled drug-free human plasma was obtained from Sotiriaeneral Hospital, Athens, Greece.

.2. Equipment

Experiments were performed using a Finnigan AQA sin-le quadrupole mass spectrometer (Thermo-Quest, Manchester,K) equipped with an electrospray ionization interface and apectraSeries P100 LC pump (SP ThermoSeparation, UK). A Nitrox-2, model UPLC-MS12E, nitrogen generator, Domnick hunter

Gateshead, England) was used to provide highly pure nitrogen.ata acquisition and analysis were performed using Xcalibur soft-are (v.1.2, ThermoQuest, Manchester, UK).

.3. Chromatographic conditions

Hydrophilic Interaction Liquid Chromatography was performedsing an Xbridge HILIC analytical column (150.0 mm × 2.1 mm i.d.,article size 3.5 �m, 135 A) under isocratic elution, (Merck-Seuant, Darmstadt, Germany). An XBridge HILIC guard cartridge

20 mm × 2.1 mm, 3.5 �m) was used to prolong column lifetime.obile phase was consisted of a mixture of acetonitrile/10 mM

mmonium formate (93/7, v/v) at a flow rate of 0.20 mL min−1.hromatography was performed at 25 ± 2 ◦C with a chromato-raphic run time of 10 min. Samples were injected into a 10-�Loop.

.4. Mass spectrometric conditions

The electrospray interface was operated in the positive ion-

zation mode. The parameters of the source were used with theollowing settings: capillary voltage 3.7 kV, temperature 230 ◦C,one voltage (AQAmax) was set at 20.0 V. The MS system was oper-ted in SIM mode for the quantitation of the analytes.

42– 943 (2013) 158– 164 159

2.5. Stock and working solutions

Individual 500 �g mL−1 stock standard solutions of alpra-zolam, �-hydroxy-alprazolam and tiamulin, were prepared inmethanol and stored at −20◦C. Intermediate 5 �g mL−1 solu-tions were prepared from the stock solutions by dilution withwater. Mixed working standard solutions, containing alprazolamand �-hydroxy alprazolam, were prepared in the concentrationrange 2.5–250 ng mL−1 by appropriate dilutions of the interme-diate standard solutions in water. A 250 ng mL−1 solution of theinternal standard (tiamulin) was also prepared in water. Interme-diate and mixed working standard solutions were stored at 4 ◦C andremained stable for several weeks. Calibration spiked plasma sam-ples and quality control samples were prepared by spiking humanplasma with appropriated aliquots of the mixed working standardsolutions. Each calibration sample contained 250 ng mL−1 tiamulin(ISTD).

2.6. Sample preparation

A 50 �L aliquot of each plasma sample was transferred to a 2 mLeppendorf tube, followed by addition of 100 �L of ISTD solution(250 ng mL−1) and 100 �L of 10 mM ammonium acetate in water.The contents were mixed briefly by vortex and were subjected toliquid–liquid extraction by the addition of 1200 �L of tert butylmethyl ether. The mixture was vortexed for 2.5 min and centrifugedat 12,000 × g at 4 ◦C for 12 min. A 1100-�L aliquot of the super-natant was evaporated to dryness under a gentle steam of nitrogen.The residual was reconstituted in 400 �L of the mobile phase andfiltered through a 13 mm GHP Acrodisk® membrane syringe filter(pore size 0.45 �m) prior to the HILIC-ESI/MS analysis.

2.7. Validation procedure

Spiked plasma calibration standards were prepared and ana-lyzed in duplicate on three different analytical runs to evaluatethe linearity of the analytical method. The calibration curves wereconstructed in the concentration range 2.5–250 ng mL−1 for alpra-zolam and 2.5–50 ng mL−1 for �-hydroxy-alprazolam. Quantitationwas performed using the peak area ratio of each analyte to thatof the internal standard. Precision and accuracy were establishedby analyzing five replicates of QC samples at three concentrationlevels for alprazolam and two concentration levels for �-hydroxy-alprazolam. Recovery of the cleanup procedure was determined bycomparing the analytical response obtained from the analysis ofQC samples with the analytical response of blank plasma samplesspiked with the equivalent concentration of the analytes and the ISafter the sample preparation procedure.

The suppression or enhancement of ionization of the analytesand the IS by the presence of matrix components in plasma sam-ples (matrix effect) was also evaluated according to the methoddescribed by Matuszewski et al. [27]. For this purpose three individ-ual lots of drug-free human plasma were processed according to thesample preparation procedure and then spiked with alprazolam at50 ng mL−1 and 150 ng mL−1, �-hydroxy alprazolam at 50 ng mL−1

and with tiamulin (ISTD) at 250 ng mL−1. Subsequently, standardsolution of the analytes and the ISTD in equivalent concentrationswere prepared in mobile phase and were analyzed according tothe proposed method. The percentage matrix factor was used as aquantitatively measure of the matrix effect and it is defined as thepercentage of the ratio of the analyte peak response in the presenceof matrix ions to the analyte peak response in the absence of matrix

ions.

The stability of the analytes in spiked human plasma sampleswas investigated under various storage conditions. The stabilitysamples were stored at ambient temperature for 4 hours and at

160 E. Kalogria et al. / J. Chromatogr. B 942– 943 (2013) 158– 164

Fig. 1. ESI mass spectra of 25.0 �g mL−1 alprazolam standard solution, 25.0 �g mL−1 �-hydroxy alprazolam standard solution and 10.0 �g mL−1 tiamulin standard solutionprepared in a mixture of 7% 10.0 mM ammonium formate water solution pH = 5.0 in acetonitrile under the optimal cone voltage conditions of 20 V. MS conditions: positiveE in−1.

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SI mode; ESI probe temperature 230 ◦C; capillary voltage 3.7 kV; flow rate 50 �L m

20 ◦C for 4 weeks. Freeze-thaw stability was also evaluated by suc-essive cycles of freezing and thawing; four complete freeze-thawycles were performed with samples frozen at −20 ◦C for 7 days perycle and thawed (without warming) at room temperature. Resultsbtained from the analysis of the stored samples were compared tohose were obtained from the analysis of freshly prepared spikedlasma samples.

. Results and discussion

.1. Method development

The acquisition parameters were determined by direct infusionnto the mass spectrometer of 25 �g mL−1 solution in mobile phasef each one of the analytes and 10 �g mL−1 solution of the ISTDt a flow rate of 50 �L min−1. The electrospray ionization inter-ace parameters were optimized for maximum abundance of the

olecular ions of the compounds. ESI probe temperature was set athe minimum acceptable value (230 ◦C) regarding the mobile phaseow rate (0.20 mL min−1), capillary voltage was kept at 3.7 kV andhe source block voltage was set at 20 V. Mass spectra of the ana-ytes and the IS obtained in ESI positive ion mode and under theptimum mass spectrometric conditions are presented in Fig. 1.lprazolam and �-hydroxy- alprazolam were evidenced in ESI posi-

ive ion mode using a cone voltage of 20 V to obtained the maximumbundance of the protonated molecules [M + H]+ at m/z 309 and 325espectively, while tiamulin (ISTD) was quantitated by measuringhe protonated molecule at m/z 494.

The BEH XBridge®-HILIC column that was used in this workonsists of bis-triethoxysilyl ethane (BEH) particles. In BEH par-icle synthesis the bis (triethoxysilyl) ethane monomer contains a

reformed ethylene bridge which is responsible for the high pHissolution resistance of polymers into the silica backbone. Highlyrganic HILIC mobile phases usually enhance ionization in thelectrospray ion source of a mass spectrometer, in comparison to

mobile phases with higher concentrations of water generally usedin reversed-phase (RP) LC separations. The separation mechanismin HILIC is complex and under varying experimental conditions hasbeen shown to involve hydrophilic partitioning into an absorbedwater layer, adsorption onto the water layer or stationary phase, ionexchange onto a charged stationary phase and hydrophobic inter-actions with the stationary phase [25]. When developing robustmethods in HILIC it is important to understand the dominant mech-anism that governs retention.

To optimize the chromatographic conditions for alprazolam,�-hydroxy alprazolam and tiamulin (ISTD) on the BEH XBridge®-HILIC column, the percentages of water and the concentration ofammonium formate in the mobile phase were selected for exami-nation using acetonitrile as organic modifier.

The percentage of water, ϕwater, which is the strongest eluent onHILIC, was varied from 5 to 25%, while ammonium formate concen-tration was maintained constant at 1.0 mM in whole mobile phase.The dependencies of log k′ values of the analytes and the ISTD onthe percentage of water are presented in Fig. 2a. Alprazolam and�-hydroxy alprazolam are basic compounds and they are 100% pos-itively charged at pH = 4.5 of the mobile phase. It was found that thelog k values of the analytes and the ISTD decrease linearly (r ≥ 0.91)as the percentage of water, ϕwater, in the mobile phase increasespointing to a partition mechanism for the separation. The effect ofthe water content of the mobile phase in the MS signal of the ana-lytes, is presented in Fig. 2b. As the aqueous content of the mobilephase is decreased the MS signal is increased probably due to theimprovement of the desolvation process. The analytes and the ISTDare adequately retained and exhibit the best MS signal using a 7%of water in the mobile phase.

The concentration of ammonium formate in the aqueous con-

tent of the mobile phase was varied from 5.0 to 25.0 mM, while thewater content of the mobile phase was kept constant at the previ-ously determined optimum value of 7%. The retention of alprazolamand �-hydroxy alprazolam was almost unaffected upon increasing

E. Kalogria et al. / J. Chromatogr. B 9

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ig. 2. (A) Plots of the logarithm of capacity factor, log k′ , versus the percentage ofater content of the mobile phase. (B) Plots of the MS signal versus the percentage

f water content of the mobile phase.

he concentration of ammonium formate. On the contrary theecrease of retention factor of tiamulin (ISTD) with increasedmmonium formate concentration is probably due to ionic reten-ion associated with ionized silanols in the BEH HILIC column. Aecrease in MS signal is observed upon increasing the concen-ration of the buffering salt probably due to ion suppression. Anmmonium formate concentration of 10.0 mM in the aqueous con-ent of the mobile phase was chosen as the optimum to performhe HILIC-ESI/MS analysis.

A mobile phase consisting of 7% 10.0 mM ammonium formateater solution pH = 5.0, adjusted with formic acid, in acetonitrileas finally used and pumped at a flow rate of 0.20 mL min−1.

ach chromatographic run was completed within 10.0 min. A rep-esentative MS chromatogram obtained from the analysis of aalibration plasma sample spiked with 2.5 ng mL−1 alprazolam and-hydroxyalprazolam (LOQ level) and 250 ng mL−1 tiamulin (IS) isresented in Fig. 3 and illustrate the selectivity of the proposedhromatographic procedure. Under the current chromatographiconditions �-hydroxy-alprazolam, alprazolam and tiamulin wereluted at 3.49, 3.81 and 8.16 min, respectively.

.2. Sample extraction

Due to the high partition coefficient (log P) of alprazolam and-hydroxy alprazolam, liquid–liquid extraction was considered

able 1nalytical concentration parameters of the calibration equations for the determination o

Compound Concentration range Regression equationsa

(ng mL−1)

Mean of three calibration curves over a period of 1 monthAlprazolam 2.5–250 RAlp = 0.0915 × CAlp − 0.0171

�-Hydroxy-alprazolam 2.5–50 Ra-OH-Alp = 0.0429 × Ca-OH-Alp +

a Ratios of the peak areas signals of alprazolam, RAlp, and �-hydroxy-alprazolam, Ra-OH-Al

f alprazolam, CAlp and �-hydroxy-alprazolam, Ca-OH-Alp.b Correlation coefficient.c Standard error of the estimate.d Theoretical value of t for alprazolam at P = 0.05 and f = n − 2 = 5 df, 2.57 and for �-hydr

42– 943 (2013) 158– 164 161

the optimal extraction technique. Hexane, ethyl acetate, diethylether and tert-butyl methyl ether were all tested as solvents forextraction of the analytes from human plasma, both alone andin combination. Tert-butyl methyl ether produced a clean chro-matogram for blank human plasma samples, gave the best recoverywith negligible matrix effects and reduced the amount of interfer-ences that could cause ion suppression. Liquid–liquid extraction isalso advantageous as it has a low experimental cost compared withother analytical techniques.

3.3. Statistical analysis of data

Spiked plasma calibration standards containing alprazolam and�-hydroxy alprazolam were analyzed in duplicate in three differentanalytical runs for the calibration procedure. Linear relationshipsbetween the ratios of the peak area signals of the analytes to that ofthe internal standard and the corresponding concentrations wereobserved as shown by the results presented in Table 1. In all cases, aweighting factor of 1/y2 was used due to data heteroscedascity andbecause of the consistently better results regarding to other weight-ing schemes (1/y, 1/x, 1/x2) and to unweighted linear regression thatwere also tested. Back-calculated concentrations in the calibrationcurves were less than 11.0% of the nominal, which are in agreementwith international guidelines [28]. The insignificance of interceptsthat was proven by Student’s t tests indicates that there is no matrixeffect. The limits of detection, LOD, and the limits of quantitation,LOQ, for each analyte were calculated taking under considerationdata obtained from the calibration equations. The LODs were foundto be at the level of 0.68 and 0.67 ng mL−1 for alprazolam and �-hydroxy alprazolam, respectively, while the LOQs were found to beat the level of 2.26 and 2.21 ng mL−1 for alprazolam and �-hydroxyalprazolam, respectively.

One-way analysis of variance (ANOVA) was used to evaluate theintra- and inter-assay precision. Results presented in Table 2 indi-cate that intra-assay coefficients of variations, %CV, were between2.4 and 6.8% for alprazolam and between 4.5 and 5.3% for �-hydroxyalprazolam. The inter-assay %CV was no more than 1.9% for alpra-zolam and between 1.0 and 3.0% for �-hydroxy alprazolam. Theoverall assay was assessed by the relative percentage error, abso-lute %Er that was ranged from −3.1 to 4.1 for alprazolam and from0.1 to 2.2 for �-hydroxy alprazolam.

The selectivity toward endogenous plasma compounds wasalso tested in five different lots of drug-free human plasma. Masschromatograms of five batches of drug-free plasma contained noco-eluting peaks greater than 20% of the area of the analytes at theLOQ level, and no co-eluting peaks greater than 5% of the area of theinternal standard tiamulin. The concentrations of alprazolam and�-hydroxy alprazolam obtained after the analysis of the six

different lots of human plasma were found to be2.54 ± 0.25 ng mL−1 and 2.53 ± 0.28 ng mL−1, respectively, indi-cating that the matrix effect does not appreciably affect theassay.

f alprazolam and �-hydroxy alprazolam by LC–ESI/MS.

rb Standard deviation Src ˛/S�

d

Slope Intercept

≥0.996 0.0063 0.0046 ≤0.0061 ≤1.56 0.00049 ≥0.993 0.0019 0.00068 ≤0.0049 ≤0.19

p, to that of the internal standard (tiamulin) versus the corresponding concentration

oxy-alprazolam at P = 0.05 and f = n − 2 = 3 df, 3.18.

162 E. Kalogria et al. / J. Chromatogr. B 942– 943 (2013) 158– 164

Fig. 3. Smoothed and normalized mass chromatogram of a calibration plasma sample spiked with 2.5 ng mL−1 alprazolam and �-hydroxy alprazolam (LOQ level) and2 m andh l columfl

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50 ng mL−1 of the ISTD; the retention times of �-hydroxy alprazolam, alprazolaydrophilic interaction liquid chromatography on a BEH XBridge®-HILIC analyticaow rate 0.20 mL min−1.

The suppression or enhancement of ionization of the analytesnd the ISTD by the presence of endogenous components in plasmaamples (matrix effect) was also evaluated by analyzing plasmaxtracts spiked with the analytes and the ISTD after the samplereparation procedure. The MS response (peak areas) of the ana-

ytes and the ISTD at any given concentration spiked after theample preparation procedure into a plasma extract was thenompared to the MS response of the analytes and the ISTD in

cetonitrile. To evaluate potential matrix effects, the percentageatrix factors were calculated by the ratio of the analyte peak

esponse in the presence of matrix ions to the analyte peak response

able 2ccuracy and precision evaluation of quality control samples for alprazolam and-hydroxy-alprazolam (n = 5 runs, five replicates per run).

Compound Concentration (ng mL−1)

AlprazolamAdded concentration 2.5 50 250Overall mean 2.42 52.1 249.5Intra-assay CV (%)a 6.8 6.5 2.4Inter-assay CV (%)a 1.9 N.V.c 1.7Overall accuracy % Er

b −3.1 4.1 −0.2

�-Hydroxy-alprazolamAdded concentration 2.5 50Overall mean 2.51 51.1Intra-assay CV (%)a 5.3 4.5Inter-assay CV (%)a 3.0 1.0Overall accuracy % Er

b 0.1 2.2

a Coefficient of variation; intra- and inter-assay CVs were calculated by ANOVA.b Relative percentage error = [(overall mean assayed concentration − added con-

entration)/(added concentration) × 100].c N.V. No significant additional variation was observed as a result of performing

he assay on different runs.

tiamulin are 3.49, 3.81 and 8.16 min, respectively. Chromatographic conditions:n; mobile phase: 7% 10.0 mM ammonium formate water solution in acetonitrile;

in the absence of matrix ions [27]. Results presented in Table 3indicate low ion suppression.

Recovery data presented in Table 3 indicate average recovery ofmore than 77.3 and 88.1% for alprazolam and �-hydroxy alprazo-lam, respectively, while the recovery for tiamulin (ISTD) was foundto be 84.5% at 250 ng mL−1.

The carry-over test met the pre-defined criteria as no interferingpeaks with peak areas greater than 10% of the peak areas at the LOQlevel of each analyte in blank plasma samples injected after a highconcentration calibration plasma sample.

Specificity was further evaluated by analyzing six QC sam-ples containing 50 ng mL−1 of alprazolam and 50 ng mL−1 of�-hydroxy-alprazolam and fortified with 50 ng mL−1 of twentyseven other drugs (11-hydrohy-tetrahydrocannabinol, cannabinol,

cannabidiol, �9-tetrahydrocannabinol, acetaminophen, aspirin,cetirizine, pseudoephedrine, methamphetamine, amphetamine,ephedrine, morphine, codeine, 6-monoacetylmorphine,methadone, ketamine, diazepam, flunitrazepam, bromazepam,

Table 3Recovery and ion suppression data for the determination of alprazolam, �-hydroxy-alprazolam and tiamulin (ISTD) by LC–ESI/MS.

Compound Concentration levels (ng mL−1)

Alprazolam 50 150% Recovery (mean ± s.d.)n=3 77.9 ± 0.4 79.5 ± 0.3% Matrix factor (mean ± s.d.)n=3 82.1 ± 1.2 83.3 ± 1.2

�-Hydroxy-alprazolam 50% Recovery (mean ± s.d.)n=3 87.7 ± 1.9% Matrix factor (mean ± s.d.)n=3 85.1 ± 1.2

Tiamulin (ISTD) 250% Recovery (mean ± s.d.)n=3 82.9 ± 2.8% Matrix factor (mean ± s.d.)n=3 91.6 ± 0.1

E. Kalogria et al. / J. Chromatogr. B 942– 943 (2013) 158– 164 163

Table 4Stability data for alprazolam and �-hydroxy-alprazolam in human plasma under various storage conditions.

Compound Storage conditions/time

Ambient temperature/4 h −20 ◦C/28 days −20 ◦C/4 Freeze-thaw cycles

Concentration levels (ng mL−1) Mean ± s.d. (n = 3) %Er Mean ± s.d. (n = 3) %Er Mean ± s.d. (n = 3) %Er

Alprazolam50 50.1 ± 1.3 1.7 49.1 ± 2.1 −1.9 48.3 ± 1.3 −3.4150 150.2 ± 2.1 −0.1 147.7 ± 1.1 −1.6 145.6 ± 2.3 −2.9

�-Hydroxy-alprazolam

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50 49.2 ± 1.4 −1.6

Er: Relative percentage error = (overall mean assayed concentration–added concen

idazolam, oxazepam, temazepam, phenobarbital, cocaine, ben-oylecgonine, ecgonine methylester and �-hydroxybutyriccid). All twenty-seven drugs did not yield any interfer-nce at the retention time of the detection for alprazolamnd �-hydroxy-alprazolam. The concentration of alprazolamnd �-hydroxy-alprazolam in the samples were found to be9.6 ± 1.3 ng mL−1 and 47.4 ± 1.9 ng mL−1, respectively.

The stability results presented in Table 4 indicate that thenalytes can be considered stable under the various condi-

ions investigated since the concentration of alprazolam and-hydroxy alprazolam deviate by no more than −3.4 and −2.9%,

espectively, relative to the reference for any of the tests ando degradation products were detected at the selected mass

ig. 4. Smoothed and normalized mass chromatogram of a blank plasma sample (dashed h after the administration of Xanax® at a dose of 2 mg/day (dashed line) containing 39.3f �-hydroxy alprazolam, alprazolam and tiamulin are 3.51, 3.86 and 8.11 min, respective

BEH XBridge®-HILIC analytical column; mobile phase: 7% 10.0 mM ammonium formate

51.9 ± 1.3 1.1 50.8 ± 1.9 1.6

n)/(added concentration) × 100.

spectrometric conditions. Plasma samples containing alprazolamand �-hydroxy alprazolam may therefore be kept for up to 4 h atambient temperature, 28 days at −20 ◦C, and after 4 freeze-thawcycles (7 days per cycle) at −20 ◦C without any significant degrada-tion.

3.4. Application of the method to the analysis of real samples

The method was applied to the analysis of plasma samples

obtained after oral administration of alprazolam (Xanax®, PfizerHellas AE). The local ethics committee approved the studies onhuman patients. The first sample was collected from a 40 yearsold female patient that was in continuous oral administration

line), along with a smoothed and normalized LC–ESI/MS patient’s plasma sample ng mL−1 alprazolam and 10.30 ng mL−1 �-hydroxy alprazolam; the retention timesly. Chromatographic conditions: hydrophilic interaction liquid chromatography on

water solution in acetonitrile; flow rate 0.20 mL min−1.

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64 E. Kalogria et al. / J. Chromatog

f 3 mg/day dose of alprazolam. The second sample was col-ected from a 60 years old male patient that received a singleral administration of a 2 mg dose of alprazolam. Blood sam-les were collected 2 h after dosing in Venoject® tubes containing

ithium heparin as anticoagulant. Immediately after drawing, theamples were shaken gently and centrifuged at 4000 rpm for0 min at 4◦C. All plasma samples were analyzed in triplicate oneeek after storage at −20◦C, according to the sample preparationrocedure Plasma sample obtained from the female patient exhib-

ted alprazolam and �-hydroxy alprazolam plasma concentrations9.3 ± 3.51 ng mL−1 and 10.30 ± 2.1 ng mL−1, respectively. Plasmaample of the male patient exhibited alprazolam plasma concentra-ion 14.3 ± 2.11 ng mL−1, while �-hydroxy alprazolam was belowhe limit of quantitation. Fig. 4 illustrates a MS chromatogram of aatient’s plasma sample along with a MS chromatogram obtainedrom the analysis of a blank plasma samples to demonstrate thathere is no interference at the retention times of the analytes andhe ISTD.

. Conclusions

The proposed LC/ESI-MS method enables an accurate and selec-ive assay for the determination of alprazolam and �-hydroxylprazolam in human plasma. The method is a part of a larger studyhat aims to investigate the chromatographic behavior of severalrugs on HILIC columns. Hydrophilic interaction liquid chromatog-aphy was performed on a BEH XBridge®-HILIC column, 3.5 �marticle size, with a run time less than 10.0 min. Mass spectrometryas conducted in ESI positive ion mode and enabled the sensitive

nd selective quantitation of the analytes. The method was suc-

essfully applied to the analysis of plasma samples obtained fromatients treated with alprazolam. The proposed method with a LOQf 2.5 ng mL−1 for alprazolam and �-hydroxy alprazolam is suitableo support a wide range of clinical studies.

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42– 943 (2013) 158– 164

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