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SHORT COMMUNICATION

Analysis of carbamazepine and its active metabolite,


carbamazepine-10,11-epoxide, in human plasma
using high-performance liquid chromatography
Eun kyung Oh & Eunmi Ban & Jong Soo Woo &
Chong-Kook Kim
Received: 6 April 2006 / Revised: 25 June 2006 / Accepted: 1 August 2006 / Published online: 21 September 2006
# Springer-Verlag 2006
Abstract A sensitive method based on high-performance
liquid chromatography (HPLC) with ultraviolet (UV)
detection was developed for the determination of carbama-
zepine (CBZ) and one of its active metabolites, carbama-
zepine-10,11-epoxide (CBZ-E) in human plasma. CBZ,
CBZ-E and the internal standard (IS) 10,11-dihydrocarba-
mazepine were extracted from human plasma into methyl
tert-butyl ether. CBZ, CBZ-E and the IS were successfully
separated on an RP C18 column with a mobile phase of
acetonitrile:methanol:water (18:19:63, v/v/v) and monitored
via UV detection at 210 nm. The calibration curves were
linear over the concentration ranges of 0.0110 g/mL for
CBZ and 0.0055 g/mL for CBZ-E in human plasma,
respectively. The method displayed excellent sensitivity,
precision and accuracy, and was successfully applied to the
quantification of CBZ and CBZ-E in human plasma after
oral administration of a single 200 mg CBZ CR tablet. This
method is suitable for bioequivalence studies following
single doses given to healthy volunteers.
Keywords Carbamazepine
.
Carbamazepine-10,11-epoxide
.
High-performance liquid chromatography (HPLC)
.
Bioequivalence study
Introduction
Carbamazepine (CBZ), 5-H-dibenze[b,f]azepine-5-carboxa-
mide, is a tricyclic lipophilic compound that is widely used
as an antiepileptic drug in the treatment of partial and
generalized tonic-clonic seizures [1]. It is metabolized to
carbamazepine-10,11-epoxide (CBZ-E) and other metabo-
lites in liver by the CYP 3A4 and CYP 2C8 subtypes of the
cytochrome P450 system. From a clinical standpoint, CBZ-
E is the most important of the 33 metabolites of CBZ that
have been isolated, because CBZ-E appears to show
pharmacological activity, as does its parent compound
(CBZ).
Simultaneous determinations of CBZ and its metabolites,
including CBZ-E, in biological fluids and drug products [2]
have been published, including those based on the use of
HPLC methods with UV detection [36] after pretreatment
steps such as liquidliquid extraction [7], solidliquid
extraction [8], column-switching [9], and deproteinization
[10]. Also, LCmass spectrometry (MS) methods such as
LC/MS, LC/MS/MS and LC-Q-TOF MS [1115] have
been reported for the detection of CBZ and its metabolites
in aquatic environments and in plasma. However, MS
procedures are more sophisticated and more expensive than
HPLCUV, although they do provide improved sensitivity
and specificity compared with other analytical methods.
Thus, HPLC with UV detection is commonly used after
pretreatment.
The aim of this study was to establish a method based on
HPLCUV that is capable of analyzing CBZ and CBZ-E
simultaneously in order to evaluate their bioequivalence.
Previously published HPLC methods displayed inadequate
sensitivity for use in bioequivalence studies of CBZ and
CBZ-E because these methods were mainly focused on the
simultaneous analysis of CBZ and its metabolites. A more
Anal Bioanal Chem (2006) 386:19311936
DOI 10.1007/s00216-006-0724-7
E. k. Oh
:
E. Ban
:
C.-K. Kim (*)
Laboratory of Excellency for Drug and Gene Delivery,
College of Pharmacy, Seoul National University,
San 56-1, Shillim-dong, Kwanak-gu,
Seoul 151-742, South Korea
e-mail: ckkim@plaza.snu.ac.kr
J. S. Woo
Hanmi Pharm Co., Ltd.,
893-5, Hajeo-ri, Paltan-myeon, Hwasung-si,
Gyeonggi-do, South Korea
sensitive method is therefore required, based on HPLCUV
detection for the determination of trace CBZ and CBZ-E in
plasma.
In the present study, we describe the development and
validation of a sensitive and reproducible HPLC method
that uses enhanced liquidliquid extraction efficiency in the
detection of CBZ and CBZ-E in human plasma in order to
facilitate bioequivalence studies of CBZ.
Experimental
Materials and reagents
CBZ was provided by Whanin Pharm. Co. (Songpa-ku,
Seoul, Republic of Korea). CBZ-E and 10,11-dihydrocar-
bamazepine (internal standard, IS) were purchased from
Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade
acetonitrile, methanol, and methyl tert-butyl ether were
purchased from J.T. Baker (Phillipsburg, NJ, USA). All
other chemicals were of analytical grade and were used
without further purification.
Stock and working solutions of CBZ, CBZ-E (1 mg/mL)
was prepared by dissolving it in methanol. All solutions were
stable when stored at 20 C for several months. Standard
solutions of CBZ and CBZ-E in human plasma were
prepared by spiking the diluted stock solutions, yielding
final concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and
0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 g/mL (CBZ, CBZ-E,
respectively). An internal standard solution with a final
concentration of 400 ng/mL was also prepared.
HPLC condition and sample preparation
All experiments were performed using an HPLC system
consisting of a model LC-10AS solvent delivery pump, a
variable-wavelength UV detector from Shimadzu (Model
SPD-10A, Burkard Instrumente, Zurich, Switzerland) and a
717 plus autosampler (Waters, Milford, MA, USA), as well
as a column temperature controller. The signals were
processed by dsChrom2000 (Donam, Seoul, Korea).
The separation of CBZ, CBZ-E, and IS in plasma samples
was accomplished using a mobile phase consisting of a
mixture of acetonitrile:methanol:water (18:19:63, v/v/v) and
a CAPCELL PAK C18 column (250 mm 4.6 mm i.d.,
5 m, Shiseido, Tokyo, Japan) at a flowrate of 1.2 mL/min at
30 C. The analytes were monitored with a UV detector at
210 nm.
Five microliters of internal standard solution (400 ng/mL)
were added to 0.5 mL of the plasma sample in a tube. The
tube was vortex-mixed for 10 s and then 4 mL of methyl tert-
butyl ether was added as extraction solvent. After 15 min of
shaking, the samples were centrifuged at 1000g for
15 min. The organic phase was then evaporated under a
steam of nitrogen gas at 35 C. The dried analytes were
reconstituted using 100 l of mobile phase and 40 l were
injected into the HPLC system.
Validation of the method
The HPLC method was validated based on linearity,
precision, accuracy, specificity, and sensitivity.
Specificity was determined by examining peak interfer-
ence from endogenous substances. Linearity was determined
froma calibration curve of the ratio of the area under the drug
peak to that under the internal standard peak over the
concentration ranges of 0.0110 g/mL and 0.0055 g/mL
for CBZ and CBZ-E, respectively.
The lower limit of quantitation (LLOQ) is defined as the
lowest concentration on the calibration graph for which
acceptable accuracy (80120, bias %) and precision (<20%)
of the CV was obtained and the signal-to-noise ratio was
better than 10. The intra- and interday precisions (coeffi-
cients of variation, CV %) and the interday accuracy (bias %)
of the assay procedure were determined by analyzing four
samples at concentrations of 0.01, 0.1, 1 and 10 g/mL for
CBZ and 0.005, 0.05, 0.5 and 5 g/mL for CBZ-E
throughout the same day and one sample at each concentra-
tion on four different days, respectively.
The robustness of a method is its ability to remain
unaffected by small and deliberate variations of parameters
of the method [16]. A study of robustness was carried out to
evaluate the influence of the column temperature and the
mobile phase. The robustness of this HPLC method was
determined by analyzing samples with various acetonitrile
contents between 16 and 22% and at various temperatures.
The methanol ratio was maintained at 18%.
Analyte recovery was determined in triplicate at high,
medium and low concentrations in plasma by extracting
drug-free plasma samples spiked with CBZ and CBZ-E.
The freezethaw stability of the samples was obtained over
three freezethaw cycles, by thawing at room temperature
and refreezing at 70 C for 24 h. The short-term stability
was examined by maintaining the plasma samples at room
temperature for 24 h. The long-term stability was tested
after storage at 70 C for nine weeks. The autosampler
stabilities of CBZ and CBZ-E were tested by analyzing
processed samples stored in the autosampler tray for 24 h.
Application of the method to three volunteers plasma
Informed consent was obtained from healthy male volun-
teers. After an overnight fast, a catheter was introduced in a
forearm vein and a predosing blood sample was collected. A
volunteer was then orally administered one tablet of 200 mg
CBZ CR (Controlled Release) Tegretol CR 200 mg
1932 Anal Bioanal Chem (2006) 386:19311936
(Norvartis, Seoul, Korea) with 240 mL of water. Heparinized
venous blood samples (8 mL) were withdrawn from the
forearm vein before administration and at 0, 2, 4, 6, 8, 10, 12,
14, 24, 30, 36, 48, 72, 120 and 144 h postdosing, transferred
to vacutainer tubes and centrifuged at 2000g for 20 min.
After centrifugation, plasma samples were stored at 70 C
prior to analysis. The pharmacokinetic parameters were
calculated by an analytical bioavailability program, BA
Calc 2002, provided by the College of Pharmacy at Seoul
National University [17].
Results and discussion
Optimization of the extraction method
Several parameters, such as types of organic solvents used,
the volumes of organic solvents used, the shaking time and
the alkalinization, were evaluated in order to improve the
sensitivity of the method to CBZ and CBZ-E in plasma.
First, methyl tert-butyl ether [18], acetonitrile [4], and ethyl
acetate [12] were tested as organic extraction solvents as
described in previous papers. We found that acetonitrile had
a high extraction yield but poor specificity. Other organic
solvents showed similar extraction yields for CBZ and
CBZ-E, but methyl tert-butyl ether presented higher
extraction yields than other organic solvents. Therefore,
methyl tert-butyl ether was used in all subsequent extrac-
tion procedures (data not shown). To obtain an effective
extraction yield, the volume of methyl tert-butyl ether used
and the shaking time were varied. As shown in Fig. 1, the
extraction yields of CBZ and CBZ-E from plasma increased
as the volume of methyl tert-butyl ether used was
increased, but did not increase at a higher volume than
4 mL. In spite of the properties of the drugs, increasing the
shaking time allowed the amount of extraction solvent to be
(a)
(b)
Added volume (mL)
1 2 3 4 5 6 7
R
e
c
o
v
e
r
y

(
%
)
50
60
70
80
90
100
0.01 g/mL
10 g/mL
Added volume (mL)
1 2 3 4 5 6 7
R
e
c
o
v
e
r
y

(
%
)
50
60
70
80
90
100
0.005 g/mL
1 g/mL
Fig. 1 Extraction recovery efficiency versus volume of methyl tert-
butyl ether added: (a) 0.01, 10 g /mL for CBZ; (b) 0.005, 1 g /mL
for CBZ-E
Fig. 2 Chromatograms of (a) blank plasma, (b) a plasma standard
spiked with CBZ, CBZ-E (1 g /mL) and IS (400 ng/mL), (c) a
plasma sample obtained from a human subject 36 h after oral
administration of a 200 mg CBZ CR tablet. Peaks: 1 CBZ-E, 2
CBZ, 3 IS
Anal Bioanal Chem (2006) 386:19311936 1933
reduced and made it possible to achieve lower LODs (limit
of detections) and LOQs (limits of quantification) for CBZ
and CBZ-E compared to the previous method [18]. In our
test, increasing the shaking time was shown to improve the
extraction efficiency but the extraction yield did not
improve at longer shaking times than 15 minutes.
Besides the type of organic solvent, the effect of
alkalinization on the extraction efficiency was tested.
Alkalinization of the sample solution did not affect the
extraction yields of either CBZ or CBZ-E in our study,
although Pienimaki et al. [18] proposed that the recoveries
of CBZ and CBZ-E were increased upon the alkalinization
of the sample solution. Considering our results, we found
that the optimal extraction of CBZ and CBZ-E in plasma
was conducted by using using 4 mL of methyl tert-butyl
ether and by shaking for 15 min without any alkalinization
of the solution.
Method validation
As shown Fig. 2a,b, CBZ-E, CBZ and IS showed complete
baseline separation, and no interfering peaks from the
endogenous plasma components were observed at the
retention times of CBZ-E, CBZ and IS. Figure 2c shows a
chromatogram for a plasma sample obtained from a human
subject 36 h after administration of CBZ.
The calibration curves were linear over the concentration
ranges of 0.0110 g/mL for CBZ and 0.0055 g/mL for
CBZ-E in human plasma. The mean calibration equations
were y=2.1084x+0.0847 with a correlation coefficient of
r
2
=0.9998 for CBZ and y=1.6880x0.0139 with a correla-
tion coefficient of r
2
=0.9997 for CBZ-E.
The current assay has a LOQ of 10 ng/mL for CBZ and
5 ng/mL for CBZ-E in human plasma. LOD was less than
2 ng /mL for CBZ and 3 ng /mL for CBZ-E. These results
show that the sensitivity was fourfold better for CBZ and
twofold better for CBZ-E than the previous HPLC method
[18], respectively. The method gave a quantification limit
that was lower than the minimum concentration recom-
mended for plasma samples obtained after the administra-
tion of a 200 mg CBZ CR tablet.
Table 1 summarizes the within- and between-day
precisions and accuracies for CBZ and CBZ-E, as evaluated
by assaying the samples. The values obtained were lower
than the limits required for biological samples; the
precisions and accuracies of the LOQs (10 ng/ mL for
CBZ, 5 ng/mL for CBZ-E) were less than 20%, and those
of the other concentrations were less than 15%.
In order to evaluate the robustness of the method, the
influence of small variations in column temperature and
mobile phase composition on the retention times of CBZ,
CBZ-E and IS was studied. The results showed that signi-
ficant changes in the retention times of CBZ, CBZ-E and IS
occurred when the concentration of acetonitrile was varied
by 2% (13 min for CBZ and IS and 3 min for CBZ-E). Also,
we found that the retention times of CBZ, CBZ-E and IS
were significantly influenced by the temperature. The
retention times of the molecules were shorter at 30 C
compared to those observed at 20 C (31 min for CBZ and
IS and 21 min for CBZ-E).
The mean extraction recoveries for 10, 1 and 0.01 g/mL
CBZ were 107.1%, 92.4% and 90.7%, respectively (n=5),
while those for 5, 0.5 and 0.005 g/mL CBZ-E were
87.4%, 83.9% and 85.6%, respectively (n=5). Table 2 lists
Table 1 Precisions and accuracies found for the analysis of CBZ and CBZ-E in human plasma (n=4)
CBZ Concentration
(g/mL)
Precision (CV %) Accuracy (%) CBZ-E Concentration
(g/mL)
Precision (CV %) Accuracy (%)
Intraday Interday Intraday Interday
0.01 (LOQ) 2.6 3.5 96.9 0.005 (LOQ) 15.9 16.6 96
0.1 0.9 2.8 92.6 0.05 13.8 5.9 93.8
1 4.4 4.5 102.4 0.5 3.2 7.8 104.2
10 5.6 0.6 100.2 5 6.5 10.0 100.1
Table 2 Stabilities of CBZ and CBZ-E under various conditions (n=3)
CBZ (g/mL) CBZ-E (g/mL)
0.01 10 0.005 5
Freezethaw stability (%) 97.3 84.7 91.6 103.3
Short-term stability (%) 110.0 88.7 112.7 94.4
Long-term stability (%) 96.2 111.6 92.3 98.3
Autosampler stability (%) 97.5 106.9 100.8 103.1
1934 Anal Bioanal Chem (2006) 386:19311936
data for the autosampler, freezethaw, and storage stabili-
ties. The results indicate that the analytes are stable under
any of the storage conditions described above and that no
stability-related problems would be expected during routine
sample analysis for pharmacokinetics, bioequivalence and
bioavailability studies.
Application of the method to three volunteers plasma
The method was employed to analyze plasma samples
containing CBZ and CBZ-E collected after giving a single
oral dose of a 200 mg CBZ CR tablet to three healthy
volunteers. Figure 3a,b represent the mean concentration
time profiles of CBZ and CBZ-E obtained from the plasma
of human volunteers given a single oral dose of a 200 mg
CBZ CR tablet. Table 3 shows the mean values of the
pharmacokinetic parameters. The maximum plasma concen-
tration of CBZ was 2.40.5 g/mL 20.75.8 h after
administration. The half-life of CBZ and the area under the
curve (AUC) were 57.311.4 h and 195.634.6 g h/mL,
respectively. After 44.06.9 h, the plasma concentration of
CBZ-E peaked at 0.080.01 g/mL with a half-life of
61.716.8 h and an AUC of 7.71.1 g h/mL.
Conclusion
A method based on HPLC with a UV detector and
improved liquidliquid extraction efficiency was developed
for the determination of CBZ and CBZ-E in human plasma.
This method showed excellent sensitivity (10 and 5 ng/mL
for CBZ and CBZ-E, respectively), reproducibility and
specificity. In particular, the LOQs and LODs of the
method were close to those obtained by using LC/MS/MS
[12, 15]. In conclusion, this paper describes a simple,
reproducible and sensitive HPLC method for the determi-
nation of CBZ and CBZ-E in human plasma. Moreover, the
limits of quantification obtained permit its application to
pharmacokinetic studies and bioequivalence studies of CBZ
using human plasma.
Acknowledgements This study was supported by Whanin Pharm.
Co. (Seoul, Republic of Korea) through the Research Institute of
Pharmaceutical Sciences, Seoul National University.
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(a)
(b)
Time (h)
0 20 40 60 80 100 120 140 160
P
l
a
s
m
a

c
o
n
c
e
n
t
r
a
t
i
o
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(

g

/
m
L
)
0
1
2
3
Time (h)
0 20 40 60 80 100 120 140 160
P
l
a
s
m
a

c
o
n
c
e
n
t
r
a
t
i
o
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(

g

/
m
L
)
0.000
0.025
0.050
0.075
0.100
Fig. 3 The concentrationtime profiles for CBZ (a) and CBZ-E (b)
obtained from plasma from three human volunteers given an oral
administration of a 200 mg CBZ CR tablet
Table 3 CBZ and CBZ-E pharmacokinetic parameters in healthy
subjects (n=3)
Pharmacokinetic parameters CBZ CBZ-E
T
max
(h) 20.75.8 44.06.9
C
max
(g/mL) 2.40.5 0.080.01
AUC
0144 h
(gh/mL) 195.634.6 7.71.1
T
1/2
(h) 57.311.4 61.716.8
Anal Bioanal Chem (2006) 386:19311936 1935
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1936 Anal Bioanal Chem (2006) 386:19311936