American Journal of Anal yt ical Chemistry, 2011, 2, 447-455
doi:10.4236/ajac.2011.24054 Published Online August 2011 (http://www.SciRP.org/journal/ajac)
Determination of Clopidogrel Carboxylic Acid in Human
Plasma by LC-MS/MS
Mohamed El-Husseiny El-Sadek1, Samia Mahmoud Moustafa2, Hussien Omar Kadi3,
Abdul Moneim Ali Al-Hakami2
1Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
2Faculty of Pharmacy, Suez Canal University, Suez, Egypt
3Faculty of Medicine, Sana’a University, Sana’a, Yemen
Received April 2, 2011; revised May 12, 2011; accepted May 19, 2011
Abstract
Background: Clopidogrel, is a thienopyridine derivative labeled for use to prevent thrombosis after coro-
nary artery stenting. Pharmacokinetics of clopidogrel is studied indirectly by quantification of carboxylic
acid which is a major metabolite of Clopidogrel. Objective: The aim of this work is to develop and validate
a rapid, simple and sensitive LC/MS/MS assay method for the determination of Clopidogrel carboxylic acid
in human plasma using Clopidogrel-D4-carboxylic acid as internal standard. Methods: Analytes was ex-
tracted from 200 μl of plasma by a simple liquid-liquid extraction using diethyl ether – n-hexane (80:20, v/v).
The chromatographic separations were achieved on a C18 column using Methanol, de-ionized water and
formic acid as a mobile phase at flow rate of 0.5 ml/minute. Analysis was monitored by multiple reactions
monitoring mode based on m/z transition of 308.10→113 for Clopidogrel carboxylic acid and 312.10→129
for internal standard. Result: The method had a total run time of about 4 minutes. The lower limit of quanti-
fication (LLOQ) was 25 ng/ml showing good linearity over the working range of 25 – 3000 ng/ml (r ≥ 0.999).
The intra- and inter day accuracies were 90% - 98% and 92.138% - 96.889% respectively (deviation within
acceptable range ≤ 10%).Conclusion: It was shown that this method is suitable for pharmacokinetic study
following oral administration of Clopidogrel and can be successfully applied to the therapeutic drug moni-
toring of Clopidogrel in Clopidogrel-treated patients.
Keywords: Clopidogrel, Clopidogrel Carboxylic Acid, Liquid Chromatography, Tandem Mass
Spectrometry
1. Introduction
Clopidogrel hydrogen sulfate (Figure 1), methyl (+)-(S)-
α-(o-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridin-5(4
H)-acetate hydrogen sulfate, is a novel thienopyridine
derivative that irreversibly blocks adenosine diphosphate
(ADP) and is important in platelet aggregation, the
cross-linking of platelets by fibrin. Clopidogrel is chem-
ically related to Ticlopidine with superior side effects
profile and dosing requirements [1-3].
The empirical formula of Clopidogrel bisulfate is
C16H16ClNO2S.H2SO4 and its molecular weight is 419.9
[4].
The molecule of clopidogrel contains an asymmetrical
carbon at C-7 leading to the existence of two enanti-
omers (R and S). Studies showed that the active com-
pound clopidogrel is the S-enantiomer [5,6]. Clopidogrel
free base was unstable due to a labile proton in the chiral
center and was susceptible to racemization and hydroly-
sis of methyl ester group [6,7].
The drug is rapidly but incompletely absorbed after
oral administration and extensively metabolized to an
active metabolite. The parent drug or its active metabo-
lite remains undetectable in plasma. The major circulat-
ing compound, however, is an inactive carboxylic de-
rivative, which is used to document the pharmacokinetic
profile of clopidogrel [8].
The pharmacokinetic parameters of clopidogrel have
been characterized by this inactive carboxylic acid me-
tabolite in human plasma. An inactive carboxylic deriva-
tive represents 85% of circulating metabolites in human
plasma [9]. The elimination half life of this compound is

M. E.-H. EL-SADEK ET AL. 448
H3COOC H
N
SCl
H2SO4
Figure 1. Chemical structure of clopidogrel bisulfate.
8 hours [10-12]. The platelet inhibition with clopidogrel
lasts the lifespan of the platelet (7-10 days) [12-14].
Clopidogrel is inactive in vitro and hepatic biotrans-
formation via the cytochrome P450 pathway, is essential
for its in vivo antiplatelet activity [15]. The active me-
tabolite, 5-thiol compound, is formed by the oxidation of
clopidogrel to 2-oxoclopidogrel and subsequent hydroly-
sis. The active metabolite is highly labile and remains
undetected in plasma [6,16].
Clopidogrel and its metabolite bind reversibly to hu-
man plasma proteins (94% - 98%) and are excreted in
both urine and feces with an elimination half- life of
eight hours [17].
Clopidogrel is a potent, non-competitive inhibitor of
adenosine diphosphate (ADP) induced platelet aggrega-
tion, irreversibly inhibiting the binding of ADP to its
platelet membrane receptors [18]. The inhibition is spe-
cific and does not significantly affect cyclooxygenase or
arachidonic acid metabolism [19,20]. Clopidogrel is the
drug of choice to prevent thrombosis after coronary ar-
tery stenting [21,22].
Quantification of carboxylic acid metabolite would be
an indirect approach for studying the pharmacokinetics
of clopidogrel [12,23-25].
The present study report a simple and sensitive liquid
chromatography coupled to tandem mass spectrometric
method (LC-MS/MS) for determination of Clopidogrel
carboxylic acid in human plasma using Clopidogrel-d4-
-carboxylic acid as the internal standard. The developed
method was successfully applied to a pharmacokinetic
study after oral administration of Clopidogrel (75 mg) to
healthy volunteers.
2. Materials
2.1. Reagents and Chemicals
Clopidogrel carboxylic acid and Clopidogrel-d4-carbo-
xylic acid were obtained from TRC. Methanol and For-
mic acid, HPLC grade and n-hexane were purchased from
Merck, Germany. Diethyl ether was purchased from
VWR, EC. De-ionized water was purchased from Milli.
2.2. Instrumentation and LC/MS Conditions
HPLC1100 Agilant from Agilant, Germany consisting of
agilant 1100 binary pump, agilant 1100 autosampler and
agilant1100 degasser. The chromatographic separations
were achieved using a symmetryC18 column (3.5µm
4.6x75 mm; Waters, USA). The mobile phase consisted of
Methanol, de-ionized water and formic acid (75%:25%:
0.1%). The flow rate of the mobile phase was set at 0.5
ml/ min. the column oven temperature was 30˚C.
The HPLC system was coupled to an API 4000 MS/
MS triple-quadrupole system detector equipped with a
turbo ion spray ionization (ESI) source ( Applied Bio-
systems MDS, SCIEX, Canada). The turbo ion spray
ionization source was operated in a positive mode. The
ion spray voltage was adjusted to 5500 V. the mass spec-
trometer was operated at a unit resolution for both Q1
and Q3 in multiple reaction monitoring (MRM) mode.
The transition of precursor to product ion was monitored
at 308.10→113 for Clopidogrel carboxylic acid and
312.10→129 for internal standard. The method had a
total run time 4 minutes.
3. Methodology
3.1. Standard and Stock Solutions Preparation
 Clopidogrel carboxylic acid stock solution 1 mg/ml
prepared in methanol;
 Clopidogrel carboxylic acid working standard solu-
tion 100 μg/ml prepared in methanol;
 Clopidogrel-d4-carboxylic acid Stock Standard Solu-
tion 100 µg/ml prepared in methanol;
 Clopidogrel-d4-carboxylic acid working solution
1000 ng/ml prepared in de-ionized water;
3.2. Calibration Curve and Quality Controls
Spiking Preparation In Human Plasma
The calibration standards of Clopidogrel carboxylic acid
25, 50, 100, 200, 500, 750, 1000, 2000 and 3000 ng/ml,
and 75, 1500 and 2250 ng/ml quality controls were pre-
pared by diluting the clopidogrel carboxylic acid solution
(100 µg/ml) with drug-free human plasma. 200 µl ali-
quots were transferred to 10 ml glass tubes and stored at
–20oC. Calibration standards of Clopidogrel carboxylic
acid were extracted from all 9 concentration levels and
assayed.
3.3. Sample Extraction
The internal standard solution (100 µl of Clopidogrel-
-d4-carboxylic acid 1000 ng/ml in de-ionized water) was
added to each human plasma sample in glass tube, vortex
for 30 seconds. Then 30 µl of concentrated formic acid
were added, vortex for 30 seconds. Six ml of (20% n-

M. E.-H. EL-SADEK ET AL.449
-hexane:80% diethyl ether) were added, vortex for about
60 seconds followed by centrifugation for 5 minutes at
4000 r.p.m. The upper organic layer were decanted to a
clean 10 ml glass tubes and evaporated in water bath at
400C under gentle stream of nitrogen gas. The residue
was reconstituted with 200 µl of mobile phase and 25 µl
was injected into LC/MS/MS
4. Standardization and Calculation
The method's linearity for Clopidogrel carboxylic acid
determination in human plasma was confirmed for a
range of concentrations between (25 – 3000) ng/ml for
Clopidogrel carboxylic acid respectively. Calibration
curves were determined by a weighted quadratic regres-
sion equation (Y = a2X2 + a1X + a0) were used for Clo-
pidogrel carboxylic acid. The best-fit peak area ratios vs.
concentrations were used to back-calculate the concen-
trations, where y represents the (peak area of Clopidogrel
carboxylic acid /peak area of internal standard) and a
weighting of 1/x (a0, a1, a2, are the polynomial coeffi-
cients of quadratic equation). Chromatograms processing,
data generation and concentrations back calculations
were all performed by the Analyst software (Applied
Biosystems, MDS, SCIEX, Canada). Summary of de-
scriptive statistics was done by Excel spreadsheets.
4.1. Method Validation
Method validation was conducted in accordance with the
ICH and USFDA guidelines and satisfies the require-
ments of applicable statutes and regulations[26,27]. Ac-
cordingly, the method validation was evaluated in terms
of:
4.1.1. Specificity, Selectivity and Matrix Effect
The specificity of the method was determined by screen-
ing six different batches of human blank plasma and the
injection of six LLOQ samples prepared using six dif-
ferent batches of human plasma. Specificity of the me-
thod was verified by the absence of any co-eluted peaks
of endogenous plasma components at the retention times
of the drug or the internal standard. Five out of six sam-
ples of LLOQ should meet the LLOQ acceptance criteria
(not more than 20% deviation from the theoretical val-
ues). Specificity was conducted by analyzing 6 blank,
zero and 6 LLOQ samples from six different plasma
batches.
4.1.2. Linearity
For the determination of linearity, standard calibration
curves of 9 points (non-zero standards) in addition to the
blank and standard zero samples were prepared. On each
day of the validation course, a calibration curve was
prepared and its goodness of fit was calculated by least
square linear regression equation, excluding the results
of the blank and standard zero sample from the regres-
sion function. The concentrations of calibration standards
were back calculated applying the obtained regression
equation. At least 75% of non-zero standards, should
meet the following acceptance criteria.
Not more than 20% deviation at LLOQ. Not more than
15% deviation for standards above the LLOQ.
4.1.3. Accuracy and Precision
Accuracy and precision evaluation was conducted over
the first three days of the validation time course. At each
day, QC samples were calculated employing the regres-
sion of the calibration curve prepared at the same day.
The accuracy and precision deviation values should be
within 20% for the LLOQ and 15% for the QC's above
the LLOQ.
4.1.3.1. Intra-day Accuracy and Precision
The intra-day accuracy and precision of the assay was
measured by analyzing four QC samples at each concen-
tration level LLOQ, low QC, medium QC and high QC
samples (25, 75, 1500 and 2250 ng/ml) Clopidogrel car-
boxylic acid on three consecutive days with six replicate
sample each day, where their concentrations were back
calculated. The deviation of the mean from the nominal
value serves as the measure of accuracy.
Acceptance Criteria: ±20% for QCs at LLOQ and
±15% for QCs at low, mid and high concentrations. Ac-
cepted ration is 67% of each level and 67% for the total.
4.1.3.2. Inter-day Accuracy and Precision
The inter day accuracy and precision was investigated at
each concentration level (25, 75, 1500 and 2250) ng/ml
for Clopdogrel carboxylic acid over three days. Analysis
was carried out using eighteen QC samples at each con-
centration level.
Acceptance criteria: ±20% for QCs at LLOQ and
±15% for QCs at low, mid and high concentrations. Ac-
cepted ratio is 67% of each level and 67% for the total.
4.1.3.3. Absolute Analytical Recovery
The detector response obtained from the injection of the
extracted QC samples was compared to the detector re-
sponse of an equivalent pure authentic standard solution
that was prepared to contain a drug and internal standard
concentration assuming 100% recovery and reconstituted
in dry extracted blank samples from six different batches.
The absolute recoveries were calculated for Clopidogrel
carboxylic acid and internal standards by comparing the
relevant peak areas of the extracted QC samples with the

M. E.-H. EL-SADEK ET AL. 450
peak areas of an equivalent un-extracted pure authentic
standard solution reconstitute in the dry extracted blank
samples. The absolute analytical recovery was calculated
for Clopidogrel carboxylic acid at three concentration
levels (75, 1500 and 2250) ng/ml, while for the internal
standard, absolute analytical recovery was calculated at
the nominal concentration of 500 ng/ml for Clopido-
grel-d4-carboxylic acid
Acceptance criteria: Recovery close to 100% are de-
sirable, the extent of absolute analytical recovery of an
analyte and/or the internal standard may be as low as
50% to 60% but not more than 110%; if the recovery is
precise, accurate, and reproducible.
4.1.4. Sensiti vi ty
The lowest standard concentration in the calibration
curve is considered as the lower limit of quantification
(LLOQ) that meets the following acceptance criteria:
LLOQ response is five times the response of the blank
sample.
The sensitivity was tested by preparing and analyzing
six QC samples at the LLOQ with a complete set of cali-
bration curve on each day of the three days of validation.
4.1.5. Stability
4.1.5.1. Stability in Biological Plasma Samples
The stability of Clopidogrel carboxylic acid and the in-
ternal standards (Clopidogrel-d4-carboxylic acid) was
studied using six QC samples for each time interval ses-
sion, at both the low and the high concentration levels
(75 and 2250) ng/ml Clopidogrel carboxylic acid.
1) Short-term stability
Sufficient number of QC samples at each concentra-
tion level were allocated to carry out the short-term sta-
bility. Six low and the high concentration levels were
prepared and frozen at –20oC for more than 12 hrs then
thawed at room temperature (RT) and left on the bench
top at room temperature for 6 hours and then analyzed.
Stability was calculated by comparing the tested QC
samples with theoretical nominal concentrations.
Acceptance criteria: % CV is within ±15% for QCs
at low and high concentrations. Accepted ratio is 67% of
each level and 67% for the total, stability ±15%.
2) Maximum number of injections per run
The maximum number of injections per run is vali-
dated by injecting a standard calibration curve, and QC's
at three levels (low, mid, high) in duplicate or more with
blank samples spaced between the QC's. QC samples are
distributed as the unknown samples in routine analysis.
The maximum number of injections per run was stud-
ied for 104 and it was passed.
4.1.5.2. Stock Solution Stability
Clopidogrel carboxylic acid and Clopidogrel-d4-carbox-
ylic acid stock solution stability (prepared in methanol)
were evaluated for 6 hours at room temperature, and for
23 days at –20oC.
Clopidogrel carboxylic acid and Colpidogre-d4-carb-
oxylic acid working solution stability (prepared in de-io-
nized water) were evaluated for 6 hours at room tem-
perature, and 10 days at –20oC.
Stability was calculated by comparing the pertinent
responses obtained from the test stock solutions(s) with
the responses of freshly prepared ones.
4.1.6. Matrix Dilution Integrity
Samples of concentration higher than ULOQ should be
diluted with a complementary volume of the same bio-
logical matrix. Thus, the resultant dilute sample would
be processed and the obtained area ratio can be easily
fitted to the regression equation of the calibration curve.
The dilution should bring down the concentration to fall
within the linear range.
Acceptance criteria: % CV and accuracy should be
within ±15% for QC dilution and the accepted ratio is
67% per level.
5. Result
5.1. Mass Spectrometry and Chromatography
The MRM chromatograms of Clopidogrel carboxylic
acid and Clopidogrel-d4-carboxylic acid obtained by ex-
traction of human plasma are shown in (Figures 2-5).
The retention times for Clopidogrel and internal standard
was 1.82. No endogenous or extraneous peaks interfered
with the assy. The lower limit of quantification of Clo-
pidogrel carboxylic acid was 25 ng/ml. the calibration
curve was linear over a concentration from 25 – 3000
ng/ml. In this study, matrix matched standards were used
in constructing the calibration curve, therefore, any po-
ssible indirect interference caused by the endogenous
components was minimized.
5.2. Method Validation
Specificity of the method was verified by the absence of
any co-eluted peaks of endogenous plasma component at
the retention times of the drug or the internal standard.
All samples passed the acceptance criteria. The chroma-
togram for a blank plasma sample indicating that the
method is specific, and the results of the six LLOQ
shows that there is no matrix effect on LLOQ (Figure 2).
The method is linear over the range (25 – 3000) ng/ml
for Clopidogrel carboxylic acid, (r ≥ 0.999), where at the
LLOQ accuracy obtained was 94% (less than 20% devia-
tion) and 96.76% - 103% for standard points higher than

M. E. SADEK ET AL.451
Clopidogrel d4 carboxylic acid(IS) (QC) 312.1/129.0 amu –
230509D019.wiff
Area: 2469344 counts Height: 2.66e+005 cps RT: 1.82 min
0.025/25 ng/ml –Clopidogrel carboxylic acid (QC) 308.1/113.0
amu – 230509D009.wiff
Area: 109142 counts Height: 1.164e+004 cps RT: 1.82 min
Figure 2. LC/MS/MS chromatogram showing human plasma containing 25 ng/ml Clopidogrel carboxylic acid (LLOQ) and
500 ng/ml internal standard (Clopidogrel-d4-carboxylic acid).
0.075/75 ng/ml –Clopidogrel carboxylic acid (QC) 308.1/113.0 amu
– 230509D025.wiff
Area: 289967 counts Height: 3.176e
+
004cpsRT:1.82min
0.075/75 ng/ml –Clopidogrel d4 carboxylic acid(IS) (QC) 312.1/129.0 amu –
230509D025.wiff
Ar
ea:
22
05
211
cou
nt
s
H
e
i
g
ht
:
2
.36e
+
005
cps
RT
:
1
.8
2 min
Figure 3. LC/MS/MS chromatogram showing human plasma containing 75 ng/ml Clopidogrel carboxylic acid (QC LOW)
and 500 ng/ml internal standard (Clopidogrel-d4-carboxylic acid).
LLOQ (less than 15% deviation), and 75% ratio of total
standard points were accepted. LLOQ response is identi-
fiable, discrete and reproducible with precision and ac-
curacy of ±20%. The peak is identifiable, precise and
accurate at this concentration. The intra- and inter-day
accuracies were 90% - 98% for LLOQ and 92.138% -
96.889% for QC's above LLOQ respectively, and the
intra- and inter-day precisions were 2.43% - 5% for
LLOQ and 1.31-2.64 for QC's above LLOQ and 5.47%
for LLOQ and 1.40-2.64 for QC's above LLOQ respec-
tively (Table 1). This Results support the fact that the
method is accurate and precise, where deviations ob-
tained were less than 20% for LLOQ and less than 15%
for the QC's above the LLOQ. The average extraction
recoveries of Clopidogrel carboxylic acid determined at
75, 1500 and 2250 ng/ml were 67.57, 73.51 and 69.03
respectively (Table 2), while that of internal standard
determined at 500 ng/ml was 70.91, 74.46 and 68.66 res-

M. E. SADEK ET AL. 452
Clopidogrel d4 carboxylic acid(IS) (QC) 312.1/129.0 amu –
230509D031.wiff
Area: 2329774 counts Height: 2.48e+005 cps
RT: 1.82 min
3.000/1500 ng/ml –Clopidogrel carboxylic acid (QC) 308.1/113.0 amu –
230509D031.wiff
Area: 5234674 counts Height: 5.397e+005 cps RT: 1.82 min
Figure 4. LC/MS/MS chromatogram showing human plasma sample containing 1500 ng/ml Clopidogrel carboxylic acid (QC
Medium) and 500 ng/ml internal standard (Clopidogrel-d4-carboxylic acid) .
4.500/2250 ng/ml –Clopidogrel carboxylic acid (QC) 308.1/113.0 amu –
230509D037.wiff
Area: 6294723 counts Height: 6.374e+005 cps RT: 1.82 min
4.500/2250 ng/ml –Clopidogrel carboxylic acid (QC) 308.1/113.0 amu –
230509D037.wiff
Area: 6294723 counts Height: 6.374e+005 cps RT: 1.82 min
Figure 5. LC/MS/MS chromatogram showing human plasma containing 2250 ng/ml Clopidogrel carboxylic acid (QC High)
and 500 ng/ml internal standard (Clopidogrel-d4-carboxylic acid).
pectively with high degree of precision, accuracy and
reproducibility. Clopidogrel carboxylic acid is stable up
to 6 hours at room temperature as supported by the % CV
obtained (2.12%) for the low concentration (75 ng/ml)
and (1.35%) for the high concentration (2250 ng/ml) for
all the QC samples. The method has shown that the stock
solution for Clopidogrel carboxylic acid and Clopidogrel
d4-carboxylic acid in methanol is stable for 6 hours at
room temperature and up to 23 days at –20oC, while that
for the serial solution in de-ionized water is 6 hours at
room temperature and up to 10 days at –20oC. Clopido-
grel carboxylic acid was stable with absolute percentages

M. E. SADEK ET AL.453
Table 1. Accuracy and precision for the analysis of Clopidogrel carboxylic acid in human plasma obtained after analysis of
three days with six replicates per day.
QC
Intra-day mean (±SD) concentrations
(intra-day CV %) ng/ml
ng/ml Day 1 Day2 Day 3
Inter-day mean
concentration
ng/ml ± SD
Inter-day
CV % Accuracy %
QC LLOQ 25
ng/ml 24.5 ± 1.2 (5)
22.8 ± 1
(4.31)
22.5 ± 0.5
(2.43) 23.3 ± 1.3 5.47 93.111
QC Low 75
ng/ml 72.8 ± 1.3 (1.82)
72.8 ± 1.2
(1.61)
72.3 ± 1.4
(1.89) 72.7 ± 1.2 1.70 96.889
QC Mid 1500
ng/ml 1404.2 ± 20.6 (1.46)
1403.5 ± 19.7
(1.40)
1418.3 ± 18.6
(1.31) 1408.7 ± 19.8 1.40 93.911
QC High 2250
ng/ml 2033.2 ± 53.8 (2.64)
2088.2 ± 55.8
(2.67)
2098 ± 34.9
(1.66) 2073.1 ± 54.6 2.64 92.138
Table 2. Extraction recovery data of Clopidogrel carboxylic acid in human plasma (n = 6).
Spiked
Concentration ng/ml Extraction recovery% Mean recovery% SD CV%
75 67.57
1500 73.51
2250 69.03
70.037 3.059 4.42
nnumber of replicates
of deviation of calculated vs theoretical concentration
being less than 15% for stock solution, auto sampler,
freeze-thaw, short-term and long-term stabilities deter-
mined at three concentrations of 75, 1500 and 2250
ng/ml. The stability was within the limit of 85.00% -
115.00% & the CV% less than 15.00%.The method has
shown that the dilution factor of (3, 4) for Clopidogrel
carboxylic acid concentration (6000, 9000) ng/ml is pre-
cise since the obtained CV% was 1.18 & 1.01.
6. Discussion
Several analytical methods have been reported for de-
termination of Clopidogrel carboxylic acid in biological
fluids. A sensitive GC/MS method with LOQ of 5 ng/ml
has been published [23]. In this method, a complex two
steps extraction method using both liquid–liquid and
solid phase extraction procedures as well as derivatiza-
tion of the analyte are required. LC/UV method for de-
termination of clopidogerel carboxylic acid in Wistar rat
plasma has been reported using HPLC and UV detection
[24]. In this method however, LOQ of 125 ng/mL has
been reported using 50 μL injection. Furthermore, this
method involves long run time of analysis (16 min) and
gradient elution of the mobile phase. In HPLC–UV tech-
nique described by Souri et al. [25] ticlopidine has been
used as internal standard. To overcome close retention
times of clopidogrel carboxylic acid and ticlopidine, they
used a mobile phase with low flow rate (0.9 mL/min) and
high percent of aqueous phase which leads to long ana-
lytical run time (12 min) and reduction of sensitivity
(LOQ 0.2 μg/mL). Two LC–MS methods [10,28] have
been reported for the determination of Clopidogrel car-
boxylic acid in plasma. In the method described by Mi-
takos and Panderi [28], extraction of the analyte from the
serum has been achieved using single step solid phase
extraction however, their method is not sensitive enough
(LOQ 100 ng/mL) to be used in single-dose pharma-
cokinetic studies of the drug. In the other published
LC–MS method by Ksycinska et al. [10] two steps
time-consuming extraction procedure using both liq-
uid–liquid and solid phase extraction techniques have
been used however, the method is sensitive enough
(LLOQ 20 ng/mL) for measuring of the analyte up to at
least three half lives post-dose. To improve run time and
sensitivity of the analysis, the flow rate should be in-
creased and it is preferred to select a mobile phase with
higher proportion of organic solvent. LC/MS/MS meth-
ods [16,29-31] have been reported for the determination
of clopidogrel in plasma . Taubert et al. [32] reported an
LC/MS/MS method for the simultaneous quantification
of clopidogrel and its carboxylic acid in human plasma,
in which no internal standards were used. Recently, Mani
et al. [33] and Abib E et al. [34] developed a LC/MS/MS
method for the simultaneous determination of clopido-
grel carboxylic acid in human plasma, with the LLOQ of
25 and 10 ng/mL respectively, but the employed proce-
dure of solid–phase extraction (SPE) was complicated. In
order to monitor the therapeutic drug in clopido-
grel-treated patients, a more sensitive and simple method
was required. In the present study, a sensitive
LC/MS/MS method is reported for the simultaneous de-
termination of clopidogrel carboxylic acid in human
plasma. The internal standards were Clopidogrel-d4-car-
boxylic acid, and the LLOQ values of 25 ng/mL were
found to be reproducible. The major advantages of the

M. E.-H. EL-SADEK ET AL. 454
method are selectivity and sensitivity (LLOQ 25 ng/ml)
reduction in run time of analysis (4.0 min) which allows
determination of the analyte more precisely. Representa-
tive chromatograms are shown in Figures 2-5. All sam-
ples were found to show no interference at the retention
times of the analytes.
7. Conclusions
The developed method showed a linear dynamic range of
25-3000 ng/ml, with good intra-and inter-day accuracy
and precision. It was shown that this method is suitable
for pharmacokinetic study following oral administration
of Clopidogrel to male volunteers under fasting condi-
tions. The method has been successfully applied to the
therapeutic drug monitoring of clopidogrel in clopido-
grel-treated patients. The LLOQ of the assay was suffi-
cient to characterize the absorption kinetics of Clopido-
grel in human.
8. References
[1] D. C. Mills, R. Puri, C. J. Hu, C. Minniti, G. Grana, M. D.
Freedman, R. F. Colman and R. W. Colman, “Clopido-
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