lc–esi–ms for rapid and sensitive determination of aripiprazole in human plasma

LC–ESI–MS for Rapid and SensitiveDetermination of Aripiprazolein Human Plasma

Xiao-cong Zuo1,2, Feng Wang2, Ping Xu2, Rong-hua Zhu2, Huan-de Li2,&

1 Pharmacy Department, Third Xiangya Hospital, Central South University, Changsha 410013, People’s Republic of China2 Clinical Pharmaceutical Research Institute, Second Xiangya Hospital, Central South University, Changsha 410011, People’s Republic of China;

E-Mail: yzy1128@126.com

Received: 29 May 2006 / Revised: 28 June 2006 / Accepted: 4 July 2006Online publication: 29 August 2006

Abstract

A rapid, sensitive, and accurate high-performance liquid-chromatographic–mass spectrometric(HPLC–MS) method, with estazolam as internal standard, has been developed and validated fordetermination of aripiprazole in human plasma. After liquid–liquid extraction the compoundwas analyzed by HPLC on a C18 column, with acetonitrile—30 mM ammonium acetate con-taining 0.1% formic acid, 58:42 (v/v), as mobile phase, coupled with electrospray ioni-zation mass spectrometry (ESI-MS). The protonated analyte was quantified by selected-ionrecording (SIR) with a quadrupole mass spectrometer in positive-ion mode. Calibrationplots were linear over the concentration range 19.9–1119.6 ng mL)1. Intra-day and inter-day precision (CV%) and accuracy (RE%) for quality-control samples (37.3, 124.4, and622.0 ng mL)1) ranged between 2.5 and 9.0% and between 1.3 and 3.5%, respectively.Extraction recovery of aripiprazole from plasma was in the range 75.8–84.1%. The methodenables rapid, sensitive, precise, and accurate measurement of the concentration of aripip-razole in human plasma.

Keywords

Column liquid chromatographyLC–ESI–MSPharmacokineticsAripiprazole in human plasma

Introduction

Aripiprazole, 7-(4-[4-(2,3-dichlorophenyl)

-1-piperazinyl]butoxy)-3,4-dihydro-2(1H)

-quinolinone, is a novel, atypical anti-

psychotic drug for treatment of schizo-

phrenia and schizoaffective disorders. It

has potent partial agonist activity at

dopamine2 (D2) receptors, partial ago-

nist activity at serotonin1A (5-HT1A)

receptors, and antagonist activity at

5-HT2A receptors. As a result, aripip-

razole can improve both negative and

positive symptoms of schizophrenia with

lower propensity for extrapyramidal

symptoms (EPS). In addition to these

therapeutic advances, aripiprazole seems

to have a good side-effect pro-

file—research indicates that weight gain

and sedation are minimal and there is no

QTc interval prolongation or hyperp-

rolactinemia compared with placebo

[1–3].

Although all this evidence seems clear,

our knowledge of the pharmacokinetics

of aripiprazole in patients suffering from

schizophrenia is limited. Analytical

methods used to determine aripiprazole

include high-performance liquid chro-

matography (HPLC) with ultraviolet

detection [4, 5]. These methods are not

usually sufficiently sensitive and specific

to enable determination of aripiprazole in

schizophrenics’ plasma, because of the

effect of other drugs used in combination

with aripiprazole. One sensitive and

selective analytical method for determi-

nation of aripiprazole and its main

metabolite, OPC-14857, in biological

fluids is based on liquid chromatogra-

phy–tandem mass spectrometry (LC–

MS–MS) [6]. A large proportion of lab-

oratories have no access to such equip-

ment, however, because of its relatively

high price.

In this paper a rapid, specific, sensi-

tive and inexpensive high-performance

liquid-chromatographic–electrospray mass

spectrometric (HPLC–ESI–MS) method

is proposed for analysis of aripiprazole

in plasma samples from patients suffer-

ing from schizophrenia. This method

eliminates interference from other drugs

combined with aripiprazole and has been

successfully applied to pharmacokinetic

studies of aripiprazole after administra-

tion of multiple oral doses to patients

suffering from schizophrenia.

DOI: 10.1365/s10337-006-0037-10009-5893/06/10 � 2006 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

2006, 64, 387–391

Original Chromatographia 2006, 64, October (No. 7/8) 387

Experimental

Chemicals and Reagents

An aripiprazole standard (Fig. 1a) was

kindly provided by Chongqing pharma-

ceutical research institute, China, and the

internal standard, estazolam (Fig. 1b),

was from the National Dope Laboratory,

China. HPLC-grade methanol, acetoni-

trile, and methyl t-butyl ether were ob-

tained from Tedia (Fairfield, USA).

Other chemicals and reagents were of

analytical grade from the Chemical Re-

agent Factory of Hunan (Changsha,

China). Control human plasma was ob-

tained from the Blood Center of Shang-

hai (Shanghai, China). Whole-blood

samples were obtained from patients

suffering from schizophrenia.

Preparation of Stock Solutions,Calibration Standards, andQuality-Control Samples

Stock solutions of aripiprazole

(186.6 lg mL)1) and estazolam

(1.32 mg mL)1) were prepared in metha-

nol. Working solutions were obtained by

diluting the stock solutionswithmethanol.

All the stock and working solutions were

stored at )10 �C. Calibration standards

and quality-control (QC) samples were

prepared on the same day by spiking blank

plasma, obtained from patients, with

appropriate amounts of stock solutions.

Calibration plots were constructed using

seven concentrations, 19.9, 37.3, 99.5,

124.4, 248.8, 622.0, and 1119.6 ng mL)1.

This range covers the concentrations ex-

pected in plasma in our experimental

studies. QC samples were prepared at

concentrations of 37.3 ng mL)1 (low),

124.4 ng mL)1 (medium), and

622.0 ng mL)1 (high). All standards and

QC samples were stored at )80 �C.

Pharmacokinetic Studies

The experimental procedure was ap-

proved by the Ethics Committee of the

Xiangya Second Hospital, Central South

University. Twelve schizophrenics, aged

18–60 years, were treated with aripipraz-

ole 10 mg day)1 for the first 2 days, then

with 20 mg day)1 (given as 10 mg twice a

day) for the remaining 19 days. The

pharmacokinetic study was performed on

day 21. Blood samples were taken pre-

dose and 1, 3, 4, 5, 12, 24, 48, 72, 96, 144,

and 192 h thereafter, into EDTA tubes,

mixed gently, then centrifuged at 3,000g

(TGL-16G, China) for 5 min to furnish

500-lL plasma. The plasma was stored

frozen at )80 �C until analysis.

Sample Preparation

Plasma samples were vortex-mixed briefly

and portions (150 lL) were transferred to

glass centrifuge tubes. After addition of

internal standard working solution

(1.3 lg mL)1, 10 lL) to the tubes, the

samples were made alkaline by addition

of 30 lL aqueous ammonia. The samples

were vortex-mixed for 10 s then methyl t-

butyl ether (4 mL) was added and the

mixture was vortex-mixed for 2 min then

centrifuged for 5 min at 3,500g. The

supernatant was transferred to a clean

tube and evaporated under nitrogen at

50�C on a water bath. The residue was

dissolved in 150 lL mobile phase con-

taining 0.1% trifluoroacetic acid and 5-

lL samples were the injected for analysis

by HPLC–MS.

HPLC–ESI–MS Analysis

HPLC–MS was performed with a Waters

2690 pump, a refrigerated Waters 2690

autosampler with 20-lL loop, a Waters

2690 separation module (HPLC), and a

Waters Micromass (Wythenshawe, Man-

chester, UK) ZQ mass spectrometer

equipped with ESI. HPLC was performed

on a Thermo Hypersil Gold C18 column

(2.1 mm · 150 mm, 5-lm particles); the

column temperature was 40 �C. The

mobile phase was a 58:42 (%, v/v) mix-

ture of acetonitrile and distilled water

containing 0.1% formic acid and 30 mM

ammonium acetate. The mobile phase

was filtered and degassed before use.

The flow-rate was constant at

0.35 mL min)1. The single quadrupole

MS was operated in selected-ion-moni-

(A)

N

N

O

NH O

Cl

Cl

(B)

N

N N

N

Cl

Fig. 1. The chemical structures of aripiprazole(a) and estazolam (b)

Fig. 2. Positive-ion electrospray mass spectra obtained in full-scan mode from authentic samples ofaripiprazole and estazolam

388 Chromatographia 2006, 64, October (No. 7/8) Original

toring (SIR) mode with compounds being

ionized in the positive electrospray ioni-

zation ion source (ESI+) of the mass

spectrometer. To achieve the best sensi-

tivity the MS was adjusted to facilitate

the ionization process. The detection

conditions were: capillary potential 3 kV,

cone potential 34 V, extractor potential

7.5 V, source temperature 100 �C, desol-vation temperature 280 �C, cone gas flow73 L h)1, and desolvation gas flow

413 L h)1. These conditions resulted in

minimum fragmentation of the parent

compound and the optimum yield of its

quasi-molecular ion, and minimized

interference from the matrix. Target ions

were monitored at m/z 448 and 450 for

aripiprazole and at m/z 295 (Fig. 2) for

estazolam, in SIR mode.

Assay Validation

Specificity

The specificity of the assay for the ana-

lytes in the presence of endogenous sub-

stances in the matrix was assessed by

comparing the response obtained from

the lowest concentration used to produce

the calibration plots with that for recon-

stitutions prepared in blank plasma from

six different patients.

Linearity and LLOQ

Calibration standards containing aripip-

razole at seven concentrations from 19.9

to 1119.6 ng mL)1 and the LLOQ plas-

ma sample of aripiprazole were extracted

and assayed. A calibration plot was

constructed by plotting aripiprazole-to-

estazolam peak-area ratios against the

concentration of aripiprazole in the

plasma. LLOQ for aripiprazole was

established on the basis of a signal-to-

noise ratio (S/N) of 10 with accuracy and

precision better than 20%.

Precision and Accuracy

The precision and accuracy of the assay

were determined using QC samples. Pre-

cision was assessed as the coefficient of

variation (CV, %) of measured concen-

trations in a set of replicate analyses.

Intra-day precision was determined by

replicate analysis of QC samples (n = 5

for each concentration) on the same day.

Inter-day precision was determined by

replicate analysis of QC samples (n = 5

for each concentration) on three consec-

utive days. Accuracy was determined

from the mean relative error for a set of

replicate analyses (i.e. the difference be-

tween measured and nominal concentra-

tions for spiked samples).

Extraction Recovery

Extraction recovery from human plasma

was determined by comparison of MS

responses from extracted samples con-

Fig. 3. Typical mass chromatograms (TIC and SIR) obtained from aripiprazole (channel 2) andthe internal standard (channel 1). a Drug-free plasma. b Blank plasma spiked with 19.9 ng mL)1

aripiprazole and 1.3 lg mL)1 estazolam. c Plasma sample from a patient 1 h after oraladministration of 10 mg aripiprazole, under steady-state conditions

Original Chromatographia 2006, 64, October (No. 7/8) 389

taining known amounts of each drug

(aripiprazole QC samples; internal stan-

dard 1.3 lg mL)1) with those from un-

extracted and directly injected standards

spiked with the same amounts.

Stability

The effect of different storage conditions

on sample stability was determined for

each analyte using QC samples. In one set

of experiments, QC samples (n = 5 for

each concentration) were kept at room

temperature for 36 h or at )80 �C for

6 months before sample preparation. In a

different set of experiments the stability

of processed samples in the autosampler

was determined using extracts of QC

samples (n = 5 for each concentration)

that were stored in capped 120-well plates

at 4 �C (the temperature of the auto-

sampler) for 24 h. Stability was assessed

by comparing the mean concentration of

the stored QC samples with the mean

measured concentration of freshly pre-

pared QC samples.

Results

Method Validation

Specificity

Potential interference from endogenous

substances was investigated. Representa-

tive chromatograms obtained from blank

plasma, blank plasma spiked with ari-

piprazole and estazolam, and a plasma

sample from a patient after administra-

tion of aripiprazole are shown in Fig. 3.

No interferences of endogenous sub-

stances with the analyte or with estazo-

lam were detected. The HPLC–MS

method has high specificity compared

with UV measurement, because only the

objective ions derived from the analytes

of interest are monitored. Compounds

with different ions are, therefore, not

detected under these MS conditions.

Complete separation of the analytes was

achieved within 4.20 min. Retention

times were 2.80 min for aripiprazole and

2.48 min for estazolam.

Calibration Plots and Sensitivity

The calibration plot was linear over the

concentration range 19.9–

1119.6 ng mL)1 and the peak-area ratio

of aripiprazole to estazolam was closely

related to aripiprazole concentration

(correlation coefficient 0.9996). The lin-

ear regression equation for the calibra-

tion plot was y = 0.0199x + 0.0395 (i.e.

the slope and intercept were 0.0199 and

0.0395, respectively). The LLOQ for ari-

piprazole was 19.9 ng mL)1.

Precision and Accuracy

The accuracy and intra-day and inter-day

precision data are shown in Table 1.

Intra-day precision was 2.5–7.5% and

inter-day CV was 4.8–9.0%. Accuracy

was well within 5%.

Extraction Recovery

Methyl t-butyl ether was chosen as the

extraction solvent because it enabled

efficient extraction of the target com-

pound. Recovery of aripiprazole and the

internal standard from patient plasma

with methyl t-butyl ether was 75.8–84.1

and 76.7%, respectively. Extraction

recovery data are listed in Table 2.

Stability

The effects of different storage condi-

tions on sample stability are listed in

Tables 3 and 4. The experimental pro-

cedures were selected to allow enough

time for sample preparation and over-

night injection. The results revealed that

reconstituted samples stored in the

refrigerated autosampler and samples

kept at room temperature for 36 h or at

)80 �C for 6 months did not deteriorate

substantially. For all samples the differ-

ence (%) between the concentration

after storage under different conditions

and the initial concentration of the QC

Table 1. Intra-day and inter-day precision and accuracy for analysis of aripiprazole in patient plasma

QC sampleconcn (ng mL)1)

Intra-day (n = 5) Inter-day (n = 15)

Measured concentration(ng mL)1; mean ± SD)

CV (%) RE (%) Measured concentration(ng mL)1; mean ± SD)

CV (%) RE (%)

37.3 38.6 ± 2.9 7.5 3.5 37.8 ± 3.4 9.0 1.3124.4 121.3 ± 7.9 6.5 )2.5 120.9 ± 9.1 7.5 )2.8622.0 607.7 ± 15.2 2.5 )2.3 613.9 ± 29.5 4.8 )1.3

Fig. 4. Mean plasma concentration–time curves (mean ± SD) for aripiprazole after multiple oraladministration to patients of 10 mg twice a day for 19 days (n = 11)

390 Chromatographia 2006, 64, October (No. 7/8) Original

standards was within 5%, indicating that

aripiprazole was stable under these

conditions.

Pharmacokinetic Studies

The method has been successfully ap-

plied to pharmacokinetic studies of ari-

piprazole in schizophrenic patients.

Mean plasma concentration–time curves

for aripiprazole after oral administra-

tion of 10 mg twice a day for 19 days

to 11 patients are shown in Fig. 4.

Maximum concentration (Cmax) and

time to maximum concentration (Tmax)

were experimentally observed values.

Aripiprazole disposition was best de-

scribed by a two-compartment model

with first-order absorption. The area

under the plasma concentration–time

curve (AUC) was calculated using the

trapezoidal rule. AUC(0–12) was calcu-

lated from time zero to 12 h and

AUC(0–¥) from time zero to infinity.

Terminal elimination (b) was calculated

from the slope of the regression line of

the last four natural points on the log-

transformed plasma concentration–time

curve. The terminal elimination half-life

(T1/2) was calculated from 0.693/b. Theoral clearance at steady state (CL/F)

was calculated by use of the equation

CL/F = 10 mg/AUC(0–12). The appar-

ent volume of distribution (V/F) was

calculated by use of the equation

V/F = CL/b. The estimated pharmaco-

kinetic data are shown in Table 5.

Conclusion

We have developed a simple LC–ESI–

MS method for determination of ari-

piprazole in the plasma of schizophrenic

patients. The method is rapid, sensitive,

accurate, precise, and capable of han-

dling large batches of samples with

short analysis time. The HPLC–ESI–

MS technique was used to determine

pharmacokinetic data for aripiprazole in

schizophrenic patients for the first time.

Among these data, T1/2 was similar

to the results published for healthy

volunteers [7] but CL/F was somewhat

lower than the value reported in the

literature.

This method has the advantages of

being relatively simple, specific, and

practical and requiring small plasma

samples; it is suitable for pharmacoki-

netic studies under steady-state condi-

tions.

Acknowledgment

We thank Dr Tie-qiao Liu, Dr Zhe-ning

Liu, and Nurse Meng-xian Deng for their

clinical assistance.

References

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Table 2. Recovery of aripiprazole from patient plasma (n = 5)

Analyte QC sample concn(ng mL)1)

Measured concentration(ng mL)1; mean ± SD)

Recovery(%; mean ± SD)

CV (%)

Aripiprazole 37.3 28.3 ± 2.0 75.8 ± 5.4 7.1124.4 97.2 ± 6.4 78.1 ± 5.2 6.6622.0 523.1 ± 13.5 84.1 ± 1.9 2.6

Estazolam 1,300 997.3 ± 14.1 76.7 ± 1.1 1.4

Table 3. Stability of untreated QC samples of aripiprazole under different storage conditions(n = 5)

QC sampleconcn(ng mL)1)

Measured concentration(ng mL)1; mean ± SD)

Difference(%)

Measuredconcentration(ng mL)1;mean ± SD)

Difference(%)

Initial (t = 0) 25 �C, 36 h )80 �C, 6 months

37.3 37.9 ± 2.5 36.3 ± 3.6 4.2 36.0 ± 3.1 5.0124.4 122.5 ± 7.2 119.3 ± 7.5 2.6 120.2 ± 7.2 1.9622.0 610.5 ± 19.9 600.1 ± 20.2 1.7 597.6 ± 22.9 2.1

Table 4. Stability of extracts of QC samples of aripiprazole at 4 �C for 24 h in an autosampler(n = 5)

QC sample concn(ng mL)1)

Measured concentration(ng mL)1; mean ± SD)

Difference (%)

Initial (t = 0) t = 24 h

37.3 37.9 ± 2.5 37.0 ± 3.3 2.4124.4 122.5 ± 7.2 120.7 ± 8.3 1.5622.0 610.5 ± 19.9 617.3 ± 18.5 1.1

Table 5. Pharmacokinetic data (mean ± SD) for aripiprazole after oral administration of 10 mgtwice a day for 19 days (n = 11)

Property Mean ± SD Property Mean ± SD

Tmax (h) 2.6 ± 1.1 V/F (L) 173 ± 48Cmax (ng mL)1) 557.3 ± 135.5 CL/F (L h)1) 1.9 ± 0.5b (h)1) 0.011 ± 0.002 AUC(0–12) (ng h mL)1) 5,492 ± 1,390T1/2(b) (h) 62.2 ± 9.0 AUC(0–¥) (ng h mL)1) 38,678 ± 12,639

Original Chromatographia 2006, 64, October (No. 7/8) 391