lc–esi–ms for rapid and sensitive determination of aripiprazole in human plasma
TRANSCRIPT
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: [email protected]
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.
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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