MS assay for olanzapine in human plasma and its application to a bioequivalence study

Acta Pharmaceutica Sinica B 2012;2(5):481–494 Institute of Materia Medica, Chinese Academy of Medical Sciences Chinese Pharmaceutical Association

Acta Pharmaceutica Sinica B www.elsevier.com/locate/apsb www.sciencedirect.com

ORIGINAL ARTICLE

LC–MS/MS assay for olanzapine in human plasma and its application to a bioequivalence study Dinesh S. Patela,b, Naveen Sharmab, Mukesh C. Patela, Bhavin N. Patelb,c,n, Pranav S. Shrivastavc,nn, Mallika Sanyald a

Chemistry Department, Pramukh Swami Science and H.D. Patel Arts College, Sarva Vidyalaya Campus, Kadi 382715, Gujarat, India b Bio-Analytical Laboratory, BA Research India Ltd., Bodakdev, Ahmedabad 380054, Gujarat, India c Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380009, Gujarat, India d Department of Chemistry, St. Xavier’s College, Navrangpura, Ahmedabad 380009, Gujarat, India Received 16 October 2011; revised 7 December 2011; accepted 29 December 2011

KEY WORDS Olanzapine; LC–MS/MS; Solid phase extraction; Bioequivalence; Incurred sample reanalysis

Abstract This paper describes a selective and sensitive assay for the determination of olanzapine (OLZ) in human plasma based on liquid chromatography–tandem mass spectrometry (LC–MS/ MS). The analyte and quetiapine as internal standard (IS) were extracted from 200 mL plasma via solid phase extraction on Waters Oasis HLB cartridges. Chromatographic separation was achieved on an ACE 5C18-300 column (100 mm  4.6 mm, 5 mm) under isocratic conditions in a run time of 3.5 min. Mass spectrometric detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring (MRM) of the transitions at m/z 313/256 for OLZ and m/z 384/253 for the IS. The assay was linear in the range 0.10–40.0 ng/mL with a lower limit of quantitation and limit of detection of 0.10 and 0.012 ng/mL, respectively. Intra- and inter-day precision (as coefficient of variation) and relative recovery were o5.0% and 490%, respectively.

n Corresponding author at: Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380009, Gujarat, India. Tel.: þ91 079 26300969; fax: þ91 079 26308545. nn Corresponding author. Tel.: þ91 079 26853088; fax: þ91 079 26853093. E-mail addresses: [email protected] (Bhavin N Patel), [email protected] (Pranav S Shrivastav). Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association.

2211-3835 & 2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsb.2012.02.009

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Dinesh Somabhai Patel et al. The method was successfully applied to a bioequivalence study of 5 and 10 mg OLZ disintegrating tablets in 40 healthy Indian males with reproducibility by incurred sample reanalysis in the range 7.43 to 8.07%. & 2012 Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. Production and hosting by Elsevier B.V. All rights reserved.

1.

Introduction

Olanzapine (OLZ), a thienobenzodiazepine derivative, is an antipsychotic drug which is highly effective in the treatment of schizophrenia and bipolar disorders1,2. It is an atypical antipsycotic with less extrapyrimidal side effects and greater positive effects on cognitive deficits than typical antipsychotics3. This is because of its affinity for multiple receptors including dopamine D2, 5-hydroxytryptamine (5-HT) 2A and 2C, histamine H1, a-adrenergic and muscarinic receptors4. OLZ has been approved by the US FDA for use as monotherapy or in combination with mood stabilizers for the treatment of acute mania in bipolar disorders1,5. It is available as coated tablets in dose strengths ranging from 2 to 20 mg under the brand name Zyprexa and as an orally disintegrating tablet known as Zyprexa Zydis. Following oral administration, OLZ is about 93% plasma protein bound, mainly to albumin and a-acid glycoprotein. It has an oral bioavailability of about 60% mainly due to hepatic first pass metabolism to the 10-N-glucuronide, 40 -N-desmethylolanzapine and olanzapine-N-oxide via uridine diphosphate glucuronyltransferase (UDPGT), cytochrome P450 (CYP) 1A2 and flavin monoxygenase (FMO), respectively. After absorption, OLZ reaches its maximum plasma concentration within 6 h and has a mean half life of about 33 h6. Numerous methods are described in the literature to determine OLZ in different biological fluids including GC with nitrogen phosphorous7 and mass spectrometric detection8, and HPLC with electrochemical9–16, ultraviolet17–22, diode array23 and mass spectrometric detection24–37. These methods either determine OLZ alone9,11,12,14,17,18,20,21,24,25,29,33 or OLZ and its metabolites10,13,15,16,19,28,37 or OLZ and other drugs particularly antipsychotics7,8,19,22,23,26,27,30–32,34–36. Of the methods applied to analysis of human plasma, either the chromatographic run time was long (45.0 min)9,12,13,15,16,18,20,23,27,35 and the plasma volume high (Z0.5 mL)9,15,18,20,23,27,29,35 or the method was too insensitive for routine application. However, the method reported by Nirogi et al.29 based on liquid chromatography– tandem mass spectrometry (LC–MS/MS) is highly sensitive with a lower limit of quantitation (LLOQ) of 100 pg/mL in human plasma and a demonstrated suitability for application to bioequivalence studies29. Recently, another LC–MS/MS method37 has been applied to the determination of OLZ and N-desmethylolanzapine in human serum and cerebrospinal fluid (CSF) from patients taking OLZ. The salient features of chromatographic methods developed for OLZ in human plasma are summarized in Table 1. The objective of the present study was to develop and validate a selective and sensitive method for the estimation of OLZ in human plasma based on LC–MS/MS. The method uses quetiapine as internal standard (IS) and has a sensitivity

equal to that of Nirogi et al.29 using a smaller plasma volume. The method is shown to be free of interference from other antipsychotic drugs commonly taken concomitantly and was successfully applied to a bioequivalence study of 5 and 10 mg OLZ tablets in healthy volunteers under fasting and fed conditions.

2. 2.1.

Materials amd methods Chemicals and materials

Chemicals and materials (suppliers) were as follows: Reference standard OLZ (99.5%) (Cadila Healthcare Ltd., Ahmedabad, India); quetiapine fumarate (IS, 99.2%) (Varda Biotech (P) Ltd., Mumbai, India); HPLC grade methanol and acetonitrile, analytical grade ortho-phosphoric acid, ammonia and ammonium formate (S.D. Fine Chemicals Ltd., Mumbai, India); Oasis HLB extraction cartridges (1 cc, 30 mg) (Waters Corporation, Milford, MA, USA); Control buffered (K2-EDTA) human plasma stored at 20 1C (Clinical Department, BA Research India Ltd., Ahmedabad, India); Mettler Toledo AG XP26DR micro balance (Greifensee, Switzerland); Eppendorf 5810 centrifuge (Hamburg, Germany); deionized water for LC–MS/MS prepared using a Milli Q water purification system (Millipore, Bangalore, India). 2.2.

Instrumentation and conditions

The LC system (Shimadzu, Kyoto, Japan) consisted of an LC10ADvp pump, an autosampler (SIL-HTc) and an on-line degasser (DGU-14A). Separation was performed by isocratic elution on an ACE 5C18-300 (100 mm  4.6 mm, 5.0 mm) column (Chromatopak Analytical Instrumentation (India) Pvt. Ltd., Mumbai, India) with a mobile phase consisting of acetonitrile:0.01% ammonia in 2 mM ammonium formate (85:15, v/v, pH 6.6) at a flow rate of 0.9 mL/min. The autosampler was maintained at 4 1C and the injection volume was 5.0 mL. Ionization and detection of analyte and IS was performed on an API-4000 triple quadrupole mass spectrometer equipped with Turbo Ion sprays (MDS SCIEX, Toronto, Canada) operating in the positive ion mode. Quantitation was performed using multiple reaction monitoring (MRM) of the protonated precursor to product ion transitions at m/z 313.2-256.2 for OLZ and 384.2-253.2 for IS (Figs. 1a and b). All LC and MS parameters were controlled by Analyst software version 1.4.2. Optimized source dependant MS parameters were as follows: Gas 1 (Nebulizer), 55 psi; Gas 2 (heater), 50 psi; ion spray voltage (ISV), 5500 V; turbo heater temperature (TEM), 550 1C; entrance potential (EP), 10 V; collision activation dissociation (CAD), 6 psi; curtain gas (CUR), 30 psi. Compound dependent

LC–MS/MS assay for olanzapine in human plasma

Table 1

Salient features of LC-MS methods developed for olanzapine in human plasma.

No. Extraction procedure (plasma volume); mean recovery; internal standard 1a

2

3b

4

483

Column; elution type; mobile phase; flow rate; injection volume

Liquid–liquid extraction with 2  5 mL of ether in presence of 0.1 mL, 0.1 M NaOH; (0.5 mL); 85.8%; diazepam

Macherry-Nagel C18 (125 mm  2.0 mm, 3 mm); isocratic; water containing 2.7 mM formic acid and 10 mM ammonium acetateacetonitrile (53:47, v/v); 0.16 mL/min, 5 mL Liquid–liquid extraction with 4 mL of Inertsil ODS C18 (100 mm  3.0 mm, diethyl ether–dichloromethane in 3 mm); isocratic; 10 mM ammonium presence of 50 mL, 0.1 M NaOH; acetate buffer-acetonitrile (10:90, v/v); (0.5 mL); 85.5%; loratadine 0.8 mL/min, 10 mL Solid phase extraction on 96-well Waters Xbridge C18 (100 mm  2.1 mm, plate Oasis MCX support; (0.5 mL); 3.5 mm); gradient; 10 mM ammonium 111%; remoxipride acetate, pH 8.1 adjusted with 25% ammonium hydroxide and acetonitrile; 0.3 mL/min, 5 mL Solid phase extraction on Oasis HLB ACE 5C18-300 (100 mm  4.6 mm, cartridge; (0.2 mL); 94%; quetiapine 5 mm); isocratic; acetonitrile-0.01% ammonia in 2 mM ammonium formate (85:15, v/v), pH 7.98; 0.9 mL/min; 5 mL

Detection; maximum on-column loading Ref. at ULOQ per injection volume; analytical run time; retention time; linear dynamic range; application LC–MS/MS; 2.5 ng; 10 min; 5.84 min; 1–50 ng/mL; analysis of plasma samples from 11 schizophrenia patients administered 10 mg/day olanzapine

27

LC–MS/MS; 0.6 ng; 2.0 min; 1.0 min; 0.1–30 ng/mL; bioequivalence study with 10 mg olanzapine in 18 healthy volunteers LC–MS; 2 ng; 14 min; 8.1 min; 2–200 ng/mL; analysis of plasma samples from 177 psychiatric patients

29

35

LC–MS/MS; 0.05 ng; 3.5 min; 2.36 min; PM 0.1–40 ng/mL; bioequivalence study with 5/10 mg olanzapine orally disintegrating tablets in 36 healthy Indian males (18–54 years) having body mass index between 18.5 and 30.0 kg/height2

a

Along with clozapine, risperidone and quetiapine. Along with aripiprazole, atomoxetine, duloxetine, clozapine, sertindole, venlafaxine and their active metabolites dehydroaripiprazole, norclozapine, dehydrosertindole and O-desmethylvenlafaxine; PM, present method. b

Figure 1 Product ion mass spectra of (a) olanzapine (m/z 313.2-256.2) and (b) quetiapine (IS, m/z 384.2-253.2) in positive ionization mode.

MS parameters viz declustering potential (DP), collision energy (CE) and cell exit potential (CXP) were, respectively 70, 35 and 15 V for OLZ and 85, 23 and 12 V for IS. Quadrupole 1 and quadrupole 3 were maintained at unit resolution and a dwell time of 600 ms was set for both OLZ and IS. 2.3.

Calibration standards and quality control (QC) samples

An OLZ stock solution (100 mg/mL) in methanol was diluted with methanol:water (80:20, v/v) to produce an intermediate

stock solution (800 ng/mL). This was further diluted to prepare standard and (independently) QC solutions. An IS stock solution (100 mg/mL) was also prepared in methanol and diluted with water to give an IS working solution (40.0 ng/mL). All solutions were stored at 4 1C when not in use. OLZ calibration standards (CS-1 to CS-10, 0.10, 0.20, 0.50, 1.00, 2.00, 5.00, 10.0, 20.0, 32.0 and 40.0 ng/mL), low (LQC, 0.30 ng/mL), medium (MQC-1, 16.0 ng/mL; MQC-2, 3.00 ng/mL) and high (HQC, 30.0 ng/mL) QC samples and QC samples at the lower limit of quantitation (LLOQ QC, 0.10 ng/mL) and upper limit of quantitation (ULOQ

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QC, 40.0 ng/mL) were prepared by spiking blank plasma with respective standard solutions (at 5% of the total volume of plasma). All CS, QC and study samples were stored at 20 1C pending analysis.

2.4.

Sample preparation

Plasma samples were thawed, allowed to equilibrate at room temperature and thoroughly vortexed prior to analysis. Aliquots of study samples (190 plus 10 mL methanol:water 80:20, v/v) or CS and QC samples (200 mL) and 190 mL blank plasma were transferred into vials to which 15 mL methanol:water (80:20, v/v) and 200 mL IS working solution were added. After vortexing, 100 mL 25% orthophosphoric acid solution was added and the mixture vortexed again. Samples were then loaded onto SPE cartridges pre-conditioned with 1.0 mL methanol followed by 1.0 mL water. After centrifugation for 2 min at 1811  g, cartridges were washed with 2  1.0 mL water and again centrifuged for 1 min at 1811  g. Analyte and IS were then eluted with 2  0.4 mL acetonitrile:water (90:10, v/v), centrifuged for 1 min at 1811  g and analyzed by LC–MS/MS.

2.5.

Assay validation

Assay validation was carried out according to US FDA guidelines38. Validation included evaluation of selectivity, interference, carryover, linearity, precision and accuracy, reinjection reproducibility, recovery, ion suppression/enhancement, matrix effects, stability and dilution integrity. Selectivity was assessed by assay of 12 different lots of blank human plasma (including haemolysed and lipemic samples) collected with K2-EDTA as anticoagulant. For each lot, two replicates (190 mL) were spiked with 10 mL methanol:water (80:20, v/v) in one case containing IS. (total 24 samples). In addition, a system suitability sample (SSS) with the same concentration as CS-2 and two replicates of CS-1 were prepared. The blank human plasma used for spiking these samples was chosen from one of the 12 lots. The acceptance criterion was that at least 90% of samples should be free from any interference at the retention times of analyte and IS. Potential interference from acetaminophen, aspirin, caffeine, cetirizine, chlorpheniramine maleate, ibuprofen and pseudoephedrine were evaluated. Additionally, the antipsychotics, clozapine, risperidone and aripiprazole, were studied for ion suppression/enhancement, analytical recovery (precision and accuracy) and chromatographic interference at the MRM transitions of analyte and IS. Their stock solutions (100 mg/mL) were prepared in methanol and diluted in methanol:water (80:20, v/v) to 20.0 mg/mL working solutions. They were then analyzed in triplicate under the same conditions as the LQC and HQC samples along with freshly prepared CS and two sets (8 samples) of HQC, MQC-1, MQC-2 and LQC. The acceptance criteria was that the accuracy should be in the range 85–115%. MRM transitions (positive ionization mode) for clozapine (327.1/270.2), risperidone (411.3/191.2) and aripiprazole (448.0/285.2) were studied. Carryover was assessed by injecting the following sequence of samples; double blank plasma, two LLOQ samples, double

blank plasma, an ULOQ sample, double blank plasma, an ULOQ sample and finally double blank plasma. Linearity was determined by construction of six calibration curves based on peak area ratios at ten non-zero concentrations. Each calibration curve was analyzed individually by least squares weighted (1/x2) linear regression. A correlation coefficient (r2)40.99 was desirable for all calibration curves. The lowest standard on the calibration curve was accepted as the LLOQ if the analyte response was at least 10 times more than that of blank plasma. To determine intra-day accuracy and precision, a calibration curve and six replicates of LLOQ QC, LQC, MQC-2, MQC-1, HQC and ULOQ QC were analysed on the same day. Inter-day accuracy and precision were assessed by analyzing three batches of samples on three consecutive days. The deviation from the nominal concentration is required to be o715% except at the LLOQ where 720% is allowed. Similarly, accuracy should be within 715% except at the LLOQ where it can be within 720% of nominal concentrations. Reinjection reproducibility was performed by reinjecting one complete validation batch. Relative recovery (RE), matrix effects (ME) and process efficiency were assessed as recommended by Matuszewski et al.39. All three parameters were evaluated by assay of six replicates of HQC, MQC-1, MQC-2 and LQC samples. RE was calculated by comparing the mean area responses of samples spiked before extraction with those of samples spiked after extraction at each QC level. The recovery of IS was estimated in the same way. Absolute ME were assessed by comparing the mean area responses of samples spiked after extraction with those of standard solutions in mobile phase. Overall ‘‘process efficiency’’ (PE, %) was calculated as (ME  RE)/100. In addition, the effect of the plasma matrix was checked using eight different lots of K2-EDTA anticoagulated plasma including haemolysed and lipemic samples. For each lot, four samples at LQC and HQC levels were spiked after extraction and checked for accuracy and precision. The effect of the matrix on ion suppression was evaluated by post-column infusion40 at 5.0 mL/min of a standard solution containing 16.0 ng/mL OLZ and 40.0 ng/ mL IS in mobile phase via a ‘T’ connector employing a Harvard infusion pump (Harvard Bioscience, USA). Aliquots (5.0 mL) of extracted blank plasma (without OLZ and IS) were then injected and chromatograms acquired. Any reduction in the baseline on injection of the blank plasma was taken to indicate ion suppression whereas a peak at the retention time of either OLZ or IS ws taken to indicate ion enhancement. Stability was evaluated by comparing concentrations of LQC and HQC samples subjected to various conditions with those of freshly prepared samples. Samples were considered stable if deviation from nominal values was o710.0%. Stock solutions of OLZ and IS were checked for short term stability at room temperature and long term stability at 4 1C. Bench top stability, processed sample stability at room temperature and at 4 1C, freeze–thaw stability and long term stability at 20 1C were performed by assay of six replicates. To meet acceptance criteria, concentrations in at least 2/3 of QC samples should remain within 715% with accuracy in the range 85–115%. Dilution integrity was assessed by assay of five samples with analyte concentrations above the ULOQ i.e., at 200 ng/mL and at the HQC level. Six replicates of samples after ten-fold dilution (20.0 and 3.0 ng/mL) were prepared and their OLZ

LC–MS/MS assay for olanzapine in human plasma

485

concentrations calculated by applying the dilution factor of 10 to concentrations obtained from the calibration curve. 2.6.

Bioequivalence study design

The design of the study comprised an open label, randomized, two period, two treatment, two sequence, crossover, balanced, single dose, evaluation of the relative oral bioavailability of a 5 mg OLZ disintegrating tablet from a sponsor company (test) and a 5 mg OLZ disintegrating tablet (ZYPREXAs ZYDISs) from Eli Lilly and Co., Indianapolis, Indiana 46285, USA (reference) in 40 healthy Indian males under fasting and fed conditions. The study also included evaluation of a 10 mg OLZ disintegrating tablet from a Sponsor company (test) and a 10 mg OLZ disintegrating tablet (ZYPREXAs VELOTABTM) from Eli Lilly and Co., Netherlands (reference) in 40 healthy Indian males under fasting conditions. All participants were informed of the aims and risks of the study and gave written consent. Inclusion criteria were; age 18–45 years, body mass index 18.5–30.0 kg/height2, and no abnormalities on general physical examination, electrocardiogram and laboratory tests (hematology, blood and urine chemistry and immunological tests). Exclusion criteria were; allergy to OLZ, alcoholism, smoking, diabetes, psychosis, and any disease which could compromise the haemopoietic, gastrointestinal, renal, hepatic, cardiovascular, respiratory or central nervous systems. The protocol was approved and subject to review by the relevant Institutional Ethics Committee. All procedures in dealing with human subjects were based on the International Conference on Harmonization, E6 Good Clinical Practice (ICH, E6 GCP) guidelines41. Subjects in fasting studies were required to fast for 10 h before administration of drug. Subjects in fed studies were given a high fat, high (969) calorie breakfast (consisting of 200 mL milk with 16 g sugar, 80 g black gram, two slices bread and butter and two cheese cutlets) 30 min prior to drug adminstration. Blood samples were collected in vacutainers containing K2-EDTA anticoagulant before and at 1–10, 12, 16, 24, 36, 48, 72, 96, 120, 144 and 168 h after administration of drug. Plasma obtained by centrifugation at 1811  g and 4 1C for 15 min was stored at 20 1C until assayed. An incurred sample reanalysis (ISR) was also conducted for all three studies by computerized random selection of 442 samples (10% of the total study sample) with concentrations near the Cmax or during the elimination phase. The results were compared with corresponding values obtained earlier for the same sample using the same procedure. The percent change should not be more than 720%42 where Change ð%Þ ¼ ½ðRepeat valueInitial valueÞ= Mean of repeat and initial values  100% 2.7.

ð1Þ

Statistical analysis

Pharmacokinetic parameters for OLZ were estimated by noncompartmental analysis using WinNonlin software version 5.2.1 (Pharsight Corporation, Sunnyvale, CA, USA). The Cmax values and the time to reach maximum plasma concentration (Tmax) were determined directly from plasma concentration-time curves. Area under plasma concentration-time curves from time 0 to 168 h (AUC0–168) was calculated using the linear trapezoidal rule. AUC0–N was calculated as AUC0–168þCt/kel,

where Ct is the last plasma concentration measured and kel is the elimination rate constant determined by linear regression of the logarithm linear part of the plasma concentration-time curve. The T1/2 of OLZ was calculated as ln 2/kel. To determine whether test and reference formulations were pharmacokinetically bioequivalent, the mean and 90% confidence intervals (CI) of log transformed Cmax, AUC0–168, AUC0–N and their ratios (test/reference) were determined using SASs software version 9.1.3 (SAS Institute Inc., Cary, NC, USA). The formulations were considered bioequivalent if the differences between the compared parameters were not significantly different (PZ0.05) and the 90% CIs for these parameters fell within the range 0.8–1.25.

3. 3.1.

Results and discussion Method development

The objective of the present work was to develop and fully validate an LC-MS/MSmethod for the determination of OLZ in human plasma with sensitivity adequate to monitor the concentration of OLZ for least five half lives after a therapeutic dose. To realize this aim, the extraction procedure, mass spectrometry and chromatographic conditions were optimized. The ESI was operated in the positive ion mode as both OLZ and IS are basic in nature. OLZ and IS gave predominant, singly charged protonated precursor [MþH]þ ions at m/z 313.2 and 384.2, respectively in Q1 full scan spectra. Furthermore, the most abundant ion in the product ion mass spectra of OLZ was at m/z 256.2, resulting from cleavage of the piperazine ring to the neutral fragment, CH3NHCH¼ CH2. As previously reported43, other characteristic fragments were observed at m/z 282.4 and 213.0 attributed to formation of CH3NH2 and elimination of the piperazine ring, respectively. The proposed fragmentation pathway of OLZ is depicted in Fig. 2a. For quetiapine, the most stable and reproducible product ion at m/z 253.2 arises from cleavage of the piperazine ring44. The other product ion at m/z 279.4 is due to breaking of two C–N bonds from the precursor ion to eject the fragment, (HOCH2CH2OCH2CH2NH2) m/z 105, as shown in Fig. 2b. To attain an ideal Taylor cone and a better impact on spectral response, the nebulizer gas (gas 1) pressure was optimized at 55 psi. Fine tuning of the nebulizer and CAD gas was carried out to obtain a consistent and stable response. Ion spray voltage and temperature did not affect analyte response and were maintained at 5500 V and 550 1C, respectively. A dwell time of 600 ms was found adequate for OLZ and IS and no cross talk was observed between the MRMs of analyte and IS. Chromatographic conditions including mobile phase selection, flow rate, column type and injection volume were optimized. Mobile phases containing different ratios (5:95, 10:90, 15:85, 20:80 and 30:70, v/v) of water–methanol/acetonitrile together with either formic acid, ammonia (0.01–0.005%), ammonium trifluoroacetate, ammonium acetate or ammonium formate buffers in varying strengths (2–20 mM) at flow rates of 0.5–1.0 mL/min were evaluated. A number of columns including Hypurity C8 (50 mm  4.6 mm, 5 mm), Hypurity cyano (50 mm  4.6 mm, 5 mm), Beta basic cyano (100 mm  2.1 mm, 5 mm), BDS Hypersil C18 (50 mm  4.6 mm, 5 mm) and ACE 5C18-300 (100 mm  4.6 mm, 5 mm) were also evaluated. Finally it was found that the ACE

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Figure 2 Proposed fragmentation pathways for (a) olanzapine and (b) quetiapine.

5C18-300 column with a mobile phase consisting of acetonitrile : 0.01% ammonia in 2 mM ammonium formate solution (pH 6.6) (85:15, v/v) at a flow rate of 0.9 mL/min provided the best combination of efficiency, peak shape and resolution within 3.0 min. Under these conditions, the retention times for OLZ and IS were 2.36 and 2.05 min, respectively with a reproducibility for the OLZ retention time (as CV %) of r1.3% for 100 injections on the same column. Capacity factors for OLZ and IS based on a solvent front of 1.1 min were 0.86 and 1.15, respectively with a selectivity factor (a) of 0.74. The number of theoretical plates for OLZ and IS were 660 and 620, respectively with a resolution factor of 0.9.

In terms of the choice of IS, a deuterated analogue is preferable but was not available. Therefore an atypical antipsychotic belonging to the same class of dibenzothiazepines was selected in the present work. Quetiapine showed similar chromatographic behavior and did not affect analyte recovery, sensitivity or ion suppression. As part of the investigation into the extraction efficiency of OLZ from plasma, the effect of the anticoagulants K3EDTA, K2EDTA and Na-heparin was evaluated. Chin et al.28 extensively studied the potential matrix effects of anticoagulants and lipemia on an LC-MS/MS assay for OLZ and its metabolite. As a result, they suggested that K3EDTA and

LC–MS/MS assay for olanzapine in human plasma

487

Figure 3 MRM ion-chromatograms of olanzapine (m/z 313.2-256.2) and quetiapine (IS, m/z 384.2-253.2) in (a) blank plasma without analyte and IS, (b) blank plasma with IS, (c) blank plasma with olanzapine at the LLOQ with IS and (d) a study sample at Cmax after administration of a 5 mg dose of olanzapine.

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Na-heparin should be avoided, especially for lipemic samples. Consistent with their observation, the best result was obtained using K2EDTA as anticoagulant. As is clear from Table 1, both liquid–liquid extraction (LLE) and solid phase extraction (SPE) have been used to extract OLZ. In fact, SPE has been successfully carried out on Varian C8 BondElut12, StepBio C815, Oasis HLB13 and Oasis MCX35 columns. Similarly, LLE using heptane–isoamyl alcohol18, hexane–dichloromethane20, ethyl acetate-n-hexane–isopropanol23 and diethyl ether–dichloromethane29 has provided quantitative recoveries. Nevertheless, LLE sometimes required an additional back extraction step under acidic conditions to produce clean extracts20,23. In the present work, protein precipitation (PP) using methanol and acetonitrile was first evaluated but gave poor recovery and considerable ion suppression. LLE under alkaline conditions using different solvents (diethyl ether, n-hexane, dichloromethane, methyl tert-butyl ether and ethyl acetate) alone and in combination was then evaluated23,27,29 but, although diethyl ether–dichloromethane gave promising results, the recovery was not consistent at all QC levels. As regards the previous use of SPE, Oasis MCX columns under acidic conditions (citric acid) gave quantitative

Table 2 QC

LLOQ LQC MQC-2 MQC-1 HQC ULOQ

recovery of psychotropic drugs and their metabolites28,35 and Raggi et al.13 obtained a recovery of OLZ of 97.9% using Oasis HLB under neutral conditions. Accordingly SPE on Oasis columns with hydrophilic–lipophilic balance (HLB) were evaluated under both acidic and neutral conditions and shown to provide quantitative recovery with minimum matrix effects in both cases. However, given that a slightly higher recovery was obtained using an acidic media (100 mL 25% orthophosphoric acid), the latter conditions were used in the present work.

3.2.

System suitability and carryover

During assay validation, the precision (CV, %) of a system suitability test was found to be in the range 0.11–0.54% for retention time and 1.6–2.2% for the area response of OLZ and IS. The signal-to-noise ratio for system performance was Z25 for both analyte and IS. Carryover was shown to be negligible (r0.51%) in that no enhancement in response was observed in extracted blank plasma (without IS and analyte) after subsequent injection of the ULOQ standard at the retention times of OLZ or IS.

Intra-batch and inter-batch accuracy and precision for olanzapine. Conc. added (ng/mL)

0.10 0.30 3.00 16.0 30.0 40.0

Intra-batch

Inter-batch

n

Mean conc. found (mg/mL)a

Accuracy (%)

CV (%)

n

Mean conc. found (mg/mL)b

Accuracy (%)

CV (%)

6 6 6 6 6 6

0.10 0.28 2.89 15.9 29.3 40.8

100.0 93.3 96.3 99.4 97.7 102.3

4.3 2.3 3.2 1.1 0.6 1.9

18 18 18 18 18 18

0.10 0.29 3.00 15.7 29.8 41.1

100.0 96.7 100.0 98.1 99.3 102.8

4.6 2.1 3.2 2.1 1.9 2.6

n: total number of observations. CV: coefficient of variation. a Mean of six replicate observations at each concentration. b Mean of 18 replicate observations over three different analytical runs.

Table 3

Absolute matrix effect, relative recovery and process efficiency for olanzapine.

QC

Aa (CV, %)

Bb (CV, %)

Cc (CV, %)

Absolute matrix effect (ME, %)d

LQC MQC-2 MQC-1 HQC

0.0324 0.3389 1.9075 3.4498

0.0318 0.3317 1.8637 3.4185

0.0290 0.3081 1.6792 3.3059

98.2 97.9 97.7 99.1

(2.00) (0.65) (1.23) (1.24)

(0.44) (2.54) (3.09) (2.29)

(2.09) (1.47) (1.46) (1.71)

(101.3)g (101.7)g (101.8)g (100.4)g

Relative recovery (RE, %)e 91.1 92.9 90.1 96.7

(97.5)g (96.4)g (96.9)g (94.1)g

CV: coefficient of variation. a Mean area ratio response of six replicate samples prepared in mobile phase (neat samples). b Mean area ratio response of six replicate samples prepared by spiking in extracted blank plasma. c Mean area ratio response of six replicate samples prepared by spiking before extraction. d (B/A)  100%. e (C/B)  100%. f (C/A)  100%¼ (ME  RE)/100. g Values for internal standard, quetiapine.

Process efficiency (PE, %)f 89.5 90.9 88.0 95.8

(98.8)g (98.0)g (98.7)g (94.5)g

LC–MS/MS assay for olanzapine in human plasma

Table 4 Relative matrix effect in different lots of human plasma at LQC and HQC levels for olanzapine (n ¼ 4). Plasma lot

Lot-1 Lot-2 Lot-3 Lot-4 Lot-5 Lot-6 Lot-7 (haemolysed) Lot-8 (lipemic)

Mean calculated conc.a (CV, %) LQC (0.30 ng/mL)

HQC (30.0 ng/mL)

0.30 0.28 0.31 0.31 0.29 0.31 0.28 0.28

31.8 31.4 31.4 31.8 32.0 32.4 30.4 30.3

(2.9) (3.6) (2.3) (2.3) (2.8) (0.8) (0.5) (1.2)

(0.3) (0.4) (0.6) (1.0) (0.8) (0.8) (0.7) (1.0)

489 3.3.

All samples were found to be free of interference from endogenous substances in plasma as shown in Figs. 3a–c. Similarly, no interference was observed from commonly used medications such as acetaminophen, aspirin, caffeine, cetirizine, chlorpheniramine maleate, ibuprofen and pseudoephedrine as shown in Fig. 3d. In addition, the three antipsychotic drugs (clozapine, risperidone and aripiprazole) did not interfere in the determination of OLZ. This was because, although their retention times were 2.72, 2.60 and 3.21 min, respectively, their MRM transitions were different from that of OLZ. Accuracy (%) for OLZ at all QC levels was in the range 93.4–101.4%. 3.4.

CV: coefficient of variation. a Mean of four replicate observations at each concentration.

Table 5

Linearity, sensitivity, accuracy and precision

All six calibration curves were linear over the concentration range 0.10–40.0 ng/mL with correlation coefficients rZ0.9996. The equation of the calibration curve was y¼ (0.119670.0106)x (0.000770.0001) where y is analyte/IS peak area ratio and x is analyte concentration. Accuracy (%) and precision (CV, %) of calibration standards were 97.0–103.1% and 1.2–3.9%, respectively. The LLOQ was 0.10 ng/mL at a signal-to-noise ratio Z25 and the limit of detection (LOD) was 0.012 ng/mL. Intra-day precision and accuracy (Table 2) were 0.6–4.3% and 93.3–102.3%, respectively. Inter-day precision and accuracy were 1.9–4.6% and 96.7–102.8%, respectively. 3.5.

Figure 4 Representative post column analyte infusion chromatograms for olanzapine and quetiapine (IS). (a) Exact ion current (XIC) chromatogram of olanzapine (m/z 313.2-256.2) (b) XIC of quetiapine (m/z 384.2-253.2).

Selectivity and interference

Recovery and matrix effects

The relative recoveries, absolute matrix effects and process efficiencies for OLZ and IS are presented in Table 3. The relative recovery of analyte is the ‘‘true recovery’’ since it is calculated by comparing the response (analyte/IS) of samples spiked before extraction with the response of samples spiked after extraction. The process efficiency/absolute recovery obtained for OLZ and IS was Z88% at all QC levels. Furthermore, relative matrix effects which compares the precision (CV, %) between different lots of plasma samples spiked after extraction varied from 0.3 to 3.6% for OLZ at the LOQ and HQC levels (Table 4). Postcolumn analyte infusion chromatograms (Fig. 4) indicate there

Stability results for olanzapine under different conditions (n¼ 6).

Stability

Storage condition

Level

Mean stability sample (mg/mL)

CV (%)

Change (%)

Bench top stability

Room temperature (24 h)

Processed sample stability (extracted samples) Processed sample stability (extracted samples) Freeze and thaw stability

Auto sampler (4 1C, 97 h)

After 6th cycle at 20 1C

Long term stability

90 days at 20 1C

LQC HQC LQC HQC LQC HQC LQC HQC LQC HQC

0.28 29.1 0.29 29.7 0.30 29.5 0.29 29.5 0.28 28.4

1.9 0.5 2.7 0.9 7.6 0.6 1.4 0.6 3.7 2.7

6.7 3.0 3.3 1.0 0.0 1.7 3.3 1.7 6.7 5.3

Room temperature (45 h)

CV: coefficient of variation. n: number of replicates at each level. Meanstability samples Meancomparison samples  100% Change ð%Þ ¼ Meancomparison samples

490

was no ion suppression or enhancement at the retention times of OLZ and IS. The average matrix factor value calculated as the response of post spiked samples divided by the response of neat solutions in mobile phase at the LLOQ level was 0.98 indicating suppression was only 2%.

Stability, dilution integrity and ruggedness

Summary of mean pharmacokinetic parameters for bioequivalence studies with olanzapine in healthy volunteers.

Formulation and dose strength

a

s

Study condition; No. of subjects; wash out period; measurement time period

b

s

T -10 mg OLZ tablet, Integrol , R -10 mg OLZ tablet, Zyprexa

Tc-10 mg OLZ tablet, Rd-10 mg OLZ tablet, Zyprexas VelotabTM Tc-5 mg OLZ tablet, Re-5 mg OLZ tablet, Zyprexas Zydiss c

3.6.

The stock solution of OLZ was stable for 6 h at room temperature and for 41 days at 4 1C. The intermediate stock solution of OLZ was stable for 24 h at room temperature and for 15 days at 4 1C with percent changes of 0.9 and 2.7%, respectively. OLZ was found to be stable in plasma for up to 24 h at room temperature and during six freeze–thaw cycles.

Figure 5 Mean plasma concentration-time profiles of olanzapine after oral administration of test (5 mg olanzapine disintegrating tablet) and reference (ZYPREXAs ZYDISs, 5 mg olanzapine disintegrating tablet from Eli Lilly and Co., USA) formulations to 40 healthy Indian males under (a) fasting and (b) fed conditions. (c) shows the profile for 10 mg olanzapine tablet in 40 healthy males under fasting condition. Table 6

e

s

Zydis

Tmax7SD (h)

AUC0–t7SD (ng.h/mL)/ AUC0–N7SD (ng.h/mL)

t1/27SD (h)/ Kel7SD (1/h)

T

R

T

R

T

R

T

R

13.0774.47

11.6074.08

6.0472.77

6.4274.04

10.877 2.44

5.8172.27

5.5871.96

5.0271.59

4.9871.36

8.2371.40

7.9071.67

5.0571.14

4.9771.26

8.0071.82

7.4771.91

363.387129.28/ 466.877165.38 473.147134.57/ 494.787146.06 204.19746.33/ 213.55748.42 214.36754.50/ 223.49756.47

367.267119.22/ 477.987137.38 445.317117.10/ 474.747126.81 206.45747.56/ 215.57747.73 212.22758.14/ 221.81761.41

30.5079.15/ 0.0270.01 36.2676.38/ 0.0270.003 35.6376.94/ 0.0270.005 35.3477.05/ 0.0270.004

32.66711.34/ 0.0270.01 34.3875.42/ 0.0270.004 35.86714.29/ 0.0270.004 35.3176.14/ 0.0270.003

11.9273.35

Ref

45

PW PW PW

T, test formulation; R, reference formulation; OLZ, olanzapine; SD, standard deviation. Cmax, maximum plasma concentration; Tmax, time point of maximum plasma concentration; T1/2, half life of drug elimination during the terminal phase; AUC0t: area under the plasma concentration-time curve from zero hour to 168 h; AUC0N: area under the plasma concentration-time curve from zero hour to infinity; PW, present work. a Global Napi Pharmaceuticals, Cairo, Egypt. b Eli Lilly and Company, Basingstoke, Hampshire, United Kingdom. c Orally disintegrating tablets from a Sponsor company. d Orally disintegrating tablets from Eli Lilly and Company, Netherland. e Orally disintegrating tablets from Eli Lilly and Company, Indianapolis, USA. f In the age group of 18–45 years and having body mass index between 18.5 and 30.0 kg/height2.

Dinesh Somabhai Patel et al.

T -5 mg OLZ tablet, R -5 mg OLZ tablet, Zyprexa

s

Fasting; 24 male subjects; 14 days; 0–72 h Fasting; 40 Indian male subjectsf; 21 days; 0–168 h Fasting; 40 Indian male subjectsf; 21 days; 0–168 h Fed; 40 Indian male subjectsf; 21 days; 0–168 h

Cmax7SD (ng/mL)

LC–MS/MS assay for olanzapine in human plasma OLZ in extracted plasma samples was stable for 45 h at room temperature and for 97 h at 4 1C. OLZ was stable in plasma stored at 20 1C for a minimum of 90 days. The values of percent changes for all stability experiments are compiled in Table 5. The precisions for ten-fold dilutions of 5  ULOQ (20.0 ng/mL) and HQC (3.00 ng/mL) were 0.5 and 1.2%, respectively with accuracies of 101.6 and 103.2%, respectively. These values are within the acceptance limits of 15% for precision and 85–115% for accuracy. Method ruggedness was evaluated for QC samples by (a) reanalysis on a different column of the same make and (b) reanalysis by a different analyst. Precision (CV, %) and accuracy (%) for reanalysis on different columns were in the ranges 1.6–2.8% and 94.9–99.2%, respectively. Corresponding ranges for reanalysis by a different analyst were 1.9–3.2% and 95.8–101.6%, respectively.

3.7.

Bioequivalence study

All subjects completed the studies with no evidence of adverse events. Fig. 5 shows plasma concentration-time profiles of OLZ in healthy subjects under fasting and fed conditions. The method allowed plasma concentrations to be monitored for up to 168 h. In total, 6183 samples were successfully analyzed with precision and accuracy within acceptable limits. Table 6 compares pharmacokinetic parameters for the 10 mg tablet with values reported in a similar study45. Cmax and Tmax values were marginally lower than in the previous study while AUC0–t, AUC0–N and T1/2 values were somewhat higher. This may be due to differences in genetics, race, age and gender (body size and muscle mass) of the study subjects or to differences in the type of food consumed. The effect of food was negligible in the study of the 5 mg tablet. Comparison of the two dose strengths (5 and 10 mg) reveals dose-dependent pharmacokinetics. The bioequivalence statistics of the two formulations are summarized in Table 7. No statistically significant differences were found in any parameter between the two formulations. The mean log-transformed ratios of the parameters and their 90% CIs were all within the required ranges confirming the bioequivalence of the test and reference products. The percent change in the randomly selected samples for incurred sample (assay reproducibility) reanalysis (Fig. 6) was in the range

491 7.43 to 8.07%. This authenticates the reproducibility and ruggedness of the method. 3.8.

Comparison with previous methods

The method reported here has high sensitivity with the use of only 200 mL plasma samples. Moreover, the total analysis time (including extraction and chromatography) is the shortest of all reported methods except one29. Also, the on-column loading of OLZ at the ULOQ is only 50 pg/injection which is about 12 times lower than for the equally sensitive method reported by Nirogi et al.29. Additionally, the bioequivalence study was conducted in both fasting and fed conditions. Incurred sample reanalysis, which has now become mandatory for clinical and non-clinical studies proves the reproducibility of the proposed method in samples from healthy subjects. A detailed comparison of the salient features of the present method with those of reported asays for OLZ in human plasma is given in Table 8.

4.

Conclusions

The objective of this work was to develop a selective and sensitive method for the determination of olanzapine in human plasma suitable for pharmacokinetic studies. The method was shown to be free of interference from other antipsychotic drugs namely clozapine, risperidone and aripiprazole and from other commonly used medications. Moreover, the sensitivity was sufficient to monitor olanzapine plasma concentration for at least five half lives after a therapeutic dose with good intra- and inter-day accuracy and precision. In fact, the maximum on-column loading was

Figure 6 Graphical representation of the results for 160 incurred sample reanalyses of olanzapine.

Table 7 Comparison of treatment ratios and 90% CIs of natural log(Ln)-transformed parameters for 5 mg and 10 mg olanzapine test and reference formulations under fasting and fed condition. Dose

Parameter

Ratio (test/reference, %)

90% CI (Lower–Upper)

Power

Fast

Fed

Fast

Fed

Fast

Fed

Fast

Fed

97.2–107.7 97.4–105.7 97.1–105.8

1.00 1.00 1.00

1.00 1.00 1.00

9.7 8.4 8.6

12.9 10.3 10.7

– – –

1.00 1.00 1.00

– – –

5 mg

Ln Cmax (ng/mL) Ln AUC0–168 (h  ng/mL) Ln AUC0–N (h  ng/mL)

100.3 99.1 99.1

102.3 101.5 101.4

10 mg

Ln Cmax (ng/mL) Ln AUC0–168 (h  ng/mL) Ln AUC0–N (h  ng/mL)

108.5 103.4 103.6

– – –

CI: confidence interval; CV: coefficient of variation.

96.7–104.1 96.0–102.3 95.9–102.4 102.6–114.9 98.2–108.8 98.5–109.0

Intra subject variation (CV, %)

14.3 13.0 12.8

– – –

492

Dinesh Somabhai Patel et al.

Table 8

Comparison of salient features of the present method with reported procedures for olanzapine in human plasma.

No. Lower limit of Plasma quantitation volume for (ng/mL) processing (mL)

Retention time; run time; maximum on-column loading at ULOQ per injection volume

Organic solvent consumption (extraction and chromatography) per sample analysis (mL)

Post-column infusion study; relative matrix effect results (% CV)

Incurred sample reanalysis results

Ref.

1a 2a 3 4 5b 6c 7 8d 9

6.1 min; 15.0 min; 3.0 ng 4.2 min; 8.0 min; 0.8 ng 8.0 min; 15.0 min; 3.3 mg 5.4 min; 12.0 min; 60 ng 6.24 min; 20.0 min; – 5.84 min; 10.0 min; 2.5 ng 1.0 min; 2.0 min; 0.6 ng 8.1 min; 14.0 min; 2.0 ng 2.36 min; 3.5 min; 0.05 ng

6.0 7.0 10.5 12.0 12.4 6.0 5.5 5.0 4.5

NA NA NA NA NA Yes; NA Yes; NA NA Yes; 0.3% to 3.6% for 8 lots of plasma

NA NA NA NA NA NA NA NA Change (%) from 7.43 to 8.07%

15

5.0 0.4 1.0 2.0 0.5 1.0 0.1 2.0 0.1

500 250 1000 1000 1000 500 500 500 200

16 18 20 23 27 29 35

PM

ULOQ: upper limit of quantitation. NA: data not available. PM: present method. a Along with its metabolite desmethylolanzapine. b Along with clozapine, N-desmethylclozapine, quetiapine and perazine. c Along with clozapine, risperidone and quetiapine. d Along with aripiprazole, atomoxetine, duloxetine, clozapine, sertindole, venlafaxine and their active metabolites dehydroaripiprazole, norclozapine, dehydrosertindole and O-desmethylvenlafaxine.

considerably less than in other reported procedures which contributes to maintaining the efficiency and life of the column. Finally the simplicity of sample preparation and the short chromatographic run time of 3.5 min gives the method the capability for high sample throughput. The proposed method provides the means to carry out a wide range of pharmacokinetic/bioequivalence studies. Acknowledgment The authors are indebted to Mr. Vijay Patel, Executive Director, BA Research India Ltd., Ahmedabad for providing the necessary facilities to carry out this work. We gratefully acknowledge Mr. Anshul Dogra, Study Director and Mrs. Arpana Prasad, R&D Supervisor, BA Research India Ltd. for their continuous support, motivation and assistance during the course of this project. References 1. Scherk H, Pajonk FG, Leucht S. Second-generation antipsychotic agents in the treatment of acute mania: a systematic review and meta-analysis of randomized controlled trials. Arch Gen Psychiatry 2007;64:442–55. 2. van der Zwaal EM, Luijendijk MC, Adan RA, la Fleur SE. Olanzapine-induced weight gain: chronic infusion using osmotic minipumps does not result in stable plasma levels due to degradation of olanzapine in solution. Eur J Pharmacol 2008;585: 130–6. 3. Leucht S, Wahlbeck K, Hamann J, Kissling W. New generation antipsychotics versus low-potency conventional antipsychotics: a systematic review and meta-analysis. Lancet 2003;361:1581–9.

4. Bymaster FP, Calligaro DO, Falcone JF, March RD, Moore NA, Tye NC, et al. Radioreceptor binding profile of the atypical antipsychotic olanzapine. Neuropsychopharmacology 1996;14: 87–96. 5. Sweetman SC, editor. Martindale: The complete drug reference, 33rd edn., London: Pharmaceutical Press; 2002. 6. Callaghan JT, Bergstrom RF, Ptak LR, Beasley CM. Olanzapine. Pharmacokinetic and pharmacodynamic profile. Clin Pharmacokinet 1999;37:177–93. 7. Sa´nchez de la Torre C, Martinez MA, Almarza E. Determination of several psychiatric drugs in whole blood using capillary gas– liquid chromatography with nitrogen phosphorous detection: comparison of two solid phase extraction procedures. Forensic Sci Int 2005;155:193–204. 8. Adamowicz P, Kala M. Simultaneous screening for and determination of 128 date-rape drugs in urine by gas chromatography– electron ionization-mass spectrometry. Forensic Sci Int 2010;198: 39–45. 9. Catlow JT, Barton RD, Clemens M, Gillespie TA, Goodwin M, Swanson SP. Analysis of olanzapine in human plasma utilizing reversed-phase high-performance liquid chromatography with electrochemical detection. J Chromatogr B Biomed Sci Appl 1995;668: 85–90. 10. Chiu JA, Franklin RB. Analysis and pharmacokinetics of olanzapine (LY170053) and two metabolites in rat plasma using reversed-phase HPLC with electrochemical detection. J Pharm Biomed Anal 1996;14:609–15. 11. Aravagiri M, Ames D, Wirshing WC, Marder SR. Plasma level monitoring of olanzapine in patients with schizophrenia: determination by high-performance liquid chromatography with electrochemical detection. Ther Drug Monit 1997;19:307–13. 12. Raggi MA, Casamenti G, Mandrioli R, Fanali S, Ronchi DD, Volterra V. Determination of the novel antipsychotic drug olanzapine in human plasma using HPLC with amperometric detection. Chromatographia 2000;51:562–6.

LC–MS/MS assay for olanzapine in human plasma 13. Raggi MA, Mandrioli R, Sabbioni C, Ghedini N, Fanali S, Volterra V. Determination of olanzapine and desmethylolanzapine in the plasma sample of schizophrenic patients by means of an improved HPLC method with amperometric detection. Chromatographia 2001;54:203–7. 14. Bao J, Potts BD. Quantitative determination of olanzapine in rat brain tissue by high-performance liquid chromatography with electrochemical detection. J Chromatogr B Biomed Sci Appl 2001;752:61–7. 15. Raggi MA, Casamenti G, Mandrioli R, Volterra V. A sensitive high-performance liquid chromatographic method using electrochemical detection for the analysis of olanzapine and desmethylolanzapine in plasma of schizophrenic patients using a new solid-phase extraction procedure. J Chromatogr B Biomed Sci Appl 2001;750: 137–46. 16. Sabbioni C, Saracino MA, Mandrioli R, Albers L, Boncompagni G, Raggi MA. Rapid analysis of olanzapine and desmethylolanzapine in human plasma using high-performance liquid chromatography with coulometric detection. Anal Chim Acta 2004;516:111–7. 17. Olesen OV, Linnet K. Determination of olanzapine in serum by high-performance liquid chromatography using ultraviolet detection considering the easy oxidability of the compound and the presence of other psychotropic drugs. J Chromatogr B Biomed Sci Appl 1998;714:309–15. 18. Boulton DW, Markowitz JS, DeVane CL. A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine. J Chromatogr B Biomed Sci Appl 2001;759:319–23. 19. Weigmann H, Hartter S, Maehrlein S, Kiefer W, Kramer G, Dannhardt G, et al. Simultaneous determination of olanzapine, clozapine and demethylated metabolites in serum by on-line column-switching high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl 2001;759:63–71. 20. Dusci LJ, Peter Hackett L, Fellows LM, Ilett KF. Determination of olanzapine in plasma by high-performance liquid chromatography using ultraviolet absorbance detection. J Chromatogr B Biomed Sci Appl 2002;773:191–7. 21. Jasin´ska A, Starczewska B, Polkowska M, Puzanowska-Tarasiewicz H. Solid phase extraction of olanzapine with reverse phase sorbents prior to UV and HPLC analysis. Anal Lett 2005;38: 815–24. 22. Zhang G, Terry Jr AV, Bartlett MG. Simultaneous determination of five antipsychotic drugs in rat plasma by high performance liquid chromatography with ultraviolet detection. J Chromatogr B Analyt Technol Biomed Life Sci 2007;856:20–8. 23. Madej K, Biedron A, Garbacik A. Study of separation and extraction conditions for five neuroleptic drugs by an LLE– HPLC–DAD method in human plasma. J Liq Chromatogr Relat Technol 2009;32:3025–37. 24. Bogusz MJ, Kruger KD, Maier RD, Erkwoh R, Tuchtenhagen F. ¨ Monitoring of olanzapine in serum by liquid chromatography– atmospheric pressure chemical ionization mass spectrometry. J Chromatogr B Biomed Sci Appl 1999;732:257–69. 25. Berna M, Ackermann B, Ruterbories K, Glass S. Determination of olanzapine in human blood by liquid chromatography–tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2002;767:163–8. 26. Josefsson M, Kronstrand R, Andersson J, Roman M. Evaluation of electrospray ionization liquid chromatography–tandem mass spectrometry for rational determination of a number of neuroleptics and their major metabolites in human body fluids and tissues. J Chromatogr B Analyt Technol Biomed Life Sci 2003;789: 151–67. 27. Zhou Z, Li X, Li K, Xie Z, Cheng Z, Peng W, et al. Simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in plasma by high-performance liquid chromatography–electrospray ionization mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2004;802:257–62.

493 28. Chin C, Zhang ZP, Karnes HT. A study of matrix effects on an LC/MS/MS assay for olanzapine and desmethylolanzapine. J Pharm Biomed Anal 2004;35:1149–67. 29. Nirogi RV, Kandikere VN, Shukla M, Mudigonda K, Maurya S, Boosi R, et al. Development and validation of a sensitive liquid chromatography/electrospray tandem mass spectrometry assay for the quantification of olanzapine in human plasma. J Pharm Biomed Anal 2006;41:935–42. 30. Zhang G, Terry Jr AV, Bartlett MG. Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat brain tissue. J Chromatogr B Analyt Technol Biomed Life Sci 2007;858:276–81. 31. Zhang G, Terry Jr AV, Bartlett MG. Liquid chromatography/ tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat plasma. Rapid Commun Mass Spectrom 2007;21:920–8. 32. Williamson LN, Zhang G, Terry Jr AV, Bartlett MG. Comparison of time-of-flight mass spectrometry to triple quadrupole tandem mass spectrometry for quantitative bioanalysis: application to antipsychotics. J Liq Chromatogr Relat Technol 2008;31: 2737–51. 33. Nielsen MKK, Johansen SS. Determination of olanzapine in whole blood using simple protein precipitation and liquid chromatography–tandem mass spectrometry. J Anal Toxicol 2009;33:212–7. 34. Saar E, Gerostamoulos D, Drummer OH, Beyer J. Comparison of extraction efficiencies and LC–MS–MS matrix effects using LLE and SPE methods for 19 antipsychotics in human blood. Anal Bioanal Chem 2009;393:727–34. 35. Choong E, Rudaz S, Kottelat A, Guillarme D, Veuthey JL, Eap CB. Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC–MS. J Pharm Biomed Anal 2009;50:1000–8. 36. Sturm S, Hammann F, Drewe J, Maurer HH, Scholer A. An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography–tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:2726–32. 37. Josefsson M, Roman M, Skogh E, Dahl ML. Liquid chromatography/tandem mass spectrometry for determination of olanzapine and N-desmethylolanzapine in human serum and cerebrospinal fluid. J Pharm Biomed Anal 2010;53:576–82. 38. US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research (CDER), Centre for Veterinary Medicine (CVM). Guidance for Industry, Bionanlytical Method Validation. 2001 May. Available from: URL: /http://www.fda.gov/downloads/Drugs/GuidanceCom plianceRegulatoryInformation/Guidances/UCM070107.pdfS. 39. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal Chem 2003;75: 3019–30. 40. King R, Bonfiglio R, Fernandez-Metzler C, Miller-Stein C, Olah T. Mechanistic investigation of ionization suppression in electrospray ionization. J Am Soc Mass Spectrom 2000;11: 942–50. 41. US Department of Health and Human Services Food and Drug Administration, Centre for Drug Evaluation and Research (CDER), Centre for Biologics Evaluation and Research (CBER). Guidance for Industry: ICH E6 Good Clinical Practice. 1996 Apr. Available from: URL:/http://www.fda.gov/downloads/Drugs/Gui danceComplianceRegulatoryInformation/Guidances/UCM073122. pdfS. 42. Rocci Jr ML, Devanarayan V, Haughey DB, Jardieu P. Confirmatory reanalysis of incurred bioanalytical samples. AAPS J 2007;9:E336–43.

494 43. Smyth WF, Ramachandran VN, O’Kane E, Coulter D. Characterization of selected drugs with nitrogen-containing saturated ring structures by use of electrospray ionization with ion-trap mass spectrometry. Anal Bioanal Chem 2004;378:1305–12. 44. Barrett B, Holcapek M, Huclova´ J, Borek-Dohalsky´ V, Fejt P, Nemeca B, et al. Validated HPLC–MS/MS method for

Dinesh Somabhai Patel et al. determination of quetiapine in human plasma. J Pharm Biomed Anal 2007;44:498–505. 45. Elshafeey AH, Elsherbiny MA, Fathallah MM. A single-dose, randomized, two-way crossover study comparing two olanzapine tablet products in healthy adult male volunteers under fasting conditions. Clin Ther 2009;31:600–8.