American Journal of Anal yt ical Chemistry, 2010, 1, 135-143
doi:10.4236/ajac.2010.13017 Published Online November 2010 (
Copyright © 2010 SciRes. AJAC
Rapid and Sensitive Carvedilol Assay in Human Plasma
Using a High-Performance Liquid Chromatography with
Mass/Mass Spectrometer Detection Employed for a
Bioequivalence Study
Soo-Hwan Kim, Sang Hun Lee, Hye Jung Lee*
Hopkins Bio Research Center, Inc., Seoul, South Korea
Received July 15, 2010; revised October 25, 2010; accepted November 5, 2010
A method for the determination of carvedilol in human plasma was developed using a high-performance liq-
uid chromatography with tandem mass spectrometer (HPLC-MS/MS). Plasma samples were deproteinized
using acetonitrile and the supernatant was directly injected onto the HPLC column without any preparative
steps. Chromatography was performed on a reversed-phase (C18) column with isocratic mobile phase for 2
min. The calibration curve was linear over the range of 2 to100 ng/ml (R2 > 0.9998) and the lower limit of
quantitation (LLOQ) was 2 ng/ml. This method showed acceptable precision and accuracy, good recovery
from the plasma matrix, and stability during the analytical procedures. When its application to the bio-
equivalence test of two carvedilol 25 mg tablet formulations in male healthy 28 volunteers, this validated
analysis method was appropriate resulting in the bioequivalence of two formulations: no statistically signifi-
cant difference was observed between the logarithmic transformed area under curve (AUC) and maximum
plasma concentration (Cmax) values of the two formulations. The 90% confidence interval for the ratio of the
above mentioned two parameters were within the bioequivalence limit of 0.80-1.25. These results suggested
that the HPLC-MS/MS analysis method developed was suitable for the carvedilol analysis in human plasma.
Keywords: Carvedilol, Bioequivalence, HPLC-MS/MS, Validation
1. Introduction
Carvedilol, (±)-1-carbazol-4-yloxy-3-{[2-(O-methoxy-phe-
noxy)ethyl]amino}-2-propanol (Scheme 1(a)), is a non-
selective lipophilic β1- and β2- adrenoreceptor antagonist
with antioxidant and antiproliferative effects. It has
vasodilating properties that are attributed mainly to its
blocking activity at α1- receptors [1-4]. Carvedilol is
used in the treatment of mild to moderate hypertension
and angina pectoris [5] and is often used in combination
with other drugs [6].
Carvedilol has been quantified in biological fluids us-
ing high-performance liquid chromatography (HPLC)
with fluorescence [1-2,6-11] or electrochemical detector
[12], mass spectrometry [13-15], hydrophilic interaction
liquid chromatography (HILIC) with tandem mass spec-
trometry [16], capillary electrophoresis-ultra violet de-
tector [17] and gas chromatography (GC)-MS detector
[18]. The clean-up procedures for the extraction of
carvedilol from biological matrix has been carried out by
protein precipitation [8-9,13,15], solid-phase extraction
(SPE) [2,6,12], liquid-liquid extraction (LLE) [10,14,18],
combinations of protein precipitation with SPE [7] or
combinations of LLE with back-extraction [1,11]. How-
ever, these methods used a large volume of biological
fluids (0.15-1.0 ml plasma or 2-5 ml urine), time-con-
suming pre-treat procedures for LLE, SPE or derivatiza-
tion steps and required a long run-time (20 min) to obtain
high sensitivity. Besides, these methods employed a
large injection volume (20-100 μl) or low pH buffers (pH
2-2.5), which affect system stability. In addition, low pH
causes degradation of carvedilol [15].
We developed a rapid, simple and specific method for
quantitative analysis of carvedilol in human plasma em-
ploying HPLC-MS/MS with protein precipitation proce-
dure. This validated analysis method was applied to as-
136 S.-H. KIM ET AL.
sess the bioequivalence of two carvedilol 25 mg tablet
formulations in 28 healthy Korean human volunteers.
2. Experimental
2.1. Chemicals
Carvedilol was provided by Korea United Pharm. Co.,
Ltd. (Yeongi-Gun, South Korea). Toremifene (internal
standard (IS); Scheme 1(b)) and acetonitrile were pur-
chased from Ningbo Team Pharmaceutical. Co., Ltd.
(Ningbo, China) and Burdick & Jackson Inc. (Muskegon,
MI, USA), respectively. Other chemicals were of HPLC
grade or the highest quality available.
2.2. Calibration Standards and Quality Control
Stock solutions of each carvedilol and toremifene (IS)
were prepared in acetonitrile at concentrations of 1 mg/ml.
The working standard solutions of each carvedilol and the
IS (1,000 ng/ml) were prepared by serial dilution of
stock solutions with acetonitrile. Each carvedilol and IS
solutions were stored at –20°C in polypropylene tubes in
the dark when not in use.
Quality control (QC) samples at 2, 5, 20 and 100 ng/ml
were prepared by adding 100 μl of the appropriate work-
ing standard solutions (40, 100, 400 and 2,000 ng/ml) to
1.9 ml of drug-free human plasma (Red Cross of Korea,
Seoul, South Korea). The QC samples were dispensed 50
μl into polypropylene tubes and stored at 20°C until
Samples for calibration curve were prepared by spik-
ing 100 μl of carvedilol working standard solutions (40,
100, 200, 400, 1,000 and 2,000 ng/ml) to 1.9 ml of
drug-free human plasma.
2.3. Sample Preparation
All frozen (20°C) human plasma samples were previ-
ously thawed at ambient temperature and mixed with a
vortex-mixer for 10 s. Five-microliter of IS solution was
spiked to 50 μl of human plasma samples or QC samples,
and then introduced into polypropylene tube which con-
taining 150 μl of acetonitrile and vortex-mixed for ap-
proximately 20 s. More than 75% of acetonitrile solution
was sufficient for the protein precipitation. After centri-
fuge at 20,000 g for 10 min, 150 μl of the supernatant
was transferred to 2 ml glass vial with insert for injec-
2.4. Apparatus
The HPLC system consisted of an Agilent 1200 series
Scheme 1. Chemical structures of (a) carvedilol and (b)
toremifene, IS.
LC (Agilent, Waldbronn, Germany) system consisting of
following modules; degasser (G1379B), binary pump
(G1321B), auto-sampler (G1367C), thermostat (G1330B)
and column oven (G1316B). System and data acquisition
were controlled by Agilent MassHunter software with a
security package built in.
2.5 Chromatographic Conditions
The separation was performed on Zorbax® XDB-C18
column (2.1 mm, i.d. × 50 mm, l; particle size, 3.5 μm;
Agilent, Littelfall, CA, USA) using the mobile phase [a
mixture of acetonitrile and 0.1% formic acid (70:30, v/v)]
at a flow-rate of 0.25 ml/min. The column and
auto-sampler tray were maintained at 25 and 4°C, re-
spectively. The analytical run-time was 2 min and injec-
tion volume was 2 μl. The eluent was introduced directly
into the turbo ionspray source of a tandem quadrupole
mass spectrometer (6410A, Agilent) with a positive
ionization mode. Multiple reaction monitoring (MRM)
mode was employed for the quantification; m/z 407.2
100.2 for carvedilol (Scheme 2), and m/z 406.2 205.2
for the IS. Fragmentor and collision energy parameters
for carvedilol were 145 and 29, respectively, and for IS
150 and 28, respectively. Gas temperature was 350°C
and gas flow-rate was 10 l/min. Nebulizer gas pressure
was 35 psi and capillary voltage was 4,000 V. Typical
standard retention times were 1.10 ± 0.1 min for
carvedilol and 1.20 ± 0.1 min for the IS and a back-
pressure value of approximately 30 bar was observed.
Although chemical structure of the IS, toremeifene, is
Copyright © 2010 SciRes. AJAC
Copyright © 2010 SciRes. AJAC
Scheme 2. Mass transition of carvedilol.
different from carvedilol, but it has a very similar mass
analysis parameters, such as fragmentor and collision
energy, and extraction recovery. In addition, separation
of carvedilol from toremifene was good in this analytical
condition of the mobile phase and the column used.
2.6. Method Validation
2.6.1. Selectivity
The selectivity was evaluated by analyzing blank plasma
samples obtained from 6 different subjects.
2.6.2. Linearity and Calibration Curve
Linearity was determined to assess the performance of
the method. The calibration curve was constructed in the
range of 2-100 ng/ml to encompass the expected plasma
concentrations after the oral administration of two
carvedilol 25 mg tablets to human subjects, and obtained
with no weighted function. The suitability of the curve
was confirmed by back-calculating the concentrations of
calibration standards.
2.6.3. Accuracy and Precision
Batches were analyzed on consecutive five days to com-
plete the method validation. In each batch, QC samples
at 2, 5, 20 and 100 ng/ml were assayed in sets of five
replicates to evaluate the intra- and inter-batch precision
and accuracy [19]. The precision was calculated as
138 S.-H. KIM ET AL.
S.D./average (or mean) x 100, while the accuracy was
estimated as average (or mean)/nominal concentration ×
2.6.4. Stability Freeze and Thaw Stability
Three sets of QC samples of low and high concentrations
were stored at 70°C and subjected to three thawing and
freezing cycles. The QC samples were analyzed using a
freshly prepared calibration curve. Post Preparation Stability
Three sets of QC samples of low and high concentrations
were analyzed on the first validation day and left in the
auto-sampler at 4°C. The QC samples were then ana-
lyzed 24 h later using a freshly prepared calibration
curve. Short and Long Term Stability
Three sets of QC samples of low and high concentrations
were stored at 25°C for 24 h (short term) or at 70°C for
60 days (long term) and analyzed using a freshly pre-
pared calibration curve.
2.6.4. Recoveries
The sample preparation method is based on protein pre-
cipitation by acetonitrile. Usually, such procedure allows
higher recoveries for the target compounds, as the solu-
bility in the supernatant is enhanced by the organic sol-
vent [11]. The recoveries of carvedilol and the IS were
assessed by analyzing three sets of standards at three
concentrations (5, 20 and 100 ng/ml). The recoveries for
carvedilol and the IS were assessed the ratio of the mean
peak area of an analyte spiked before extraction to the
mean peak area of an analyte spiked post-extraction.
2.7. Bioequivalence Study
The developed method was applied to evaluate the bio-
equivalence of two tablet formulations of carvedilol. The
standard reference formulation was Dilatrend® tablet
(Lot No. JA002; Chong Kun Dang Co., Ltd., Seoul,
South Korea) and the test formulation was Carvedol®
tablet (Lot No. 518702; Korea United Pharm. Co., Ltd.,
Ywongi-Gun, South Korea). The study consisted of an
open study of 28 healthy Korean male volunteers (age,
22-27 years; body weight, 64.3-80.5 kg) who were quali-
fied according to inclusion and exclusion criteria. Each
formulation was administered to 28 healthy volunteers
under an open, randomized and two-period crossover
design with a week of washout period. The volunteers
were fasted for 12 h prior to the dosing, received a single
oral dose of carvedilol (25 mg tablet) with 240 ml of
water. Blood samples (2 ml) were withdrawn from the
forearm vein into glass tubes at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4,
6, 8, 12, 24 and 36 h after the oral administration of
carvedilol, and centrifuged. The plasma was divided into
two 0.5 ml aliquots for analysis and stored at 70°C. The
maximum plasma concentration (Cmax), time to reach
Cmax (Tmax) and total area under the plasma concentra-
tiontime curve from time zero to infinity (AUC) were
calculated by BA Calc 2007 (Version 1.0.0., KFDA).
The bioequivalence of two carvedilol 25 mg tablet for-
mulations was assessed by K-BE Test 2007 (Version
1.1.0., KFDA, Korea). Institutional Review Board ap-
proved the bioequivalence study protocol. The informed
consent was obtained from the subjects after explaining
the nature and purpose of the study in accordance with
Korean Guidelines for Bioequivalence Test [19].
3. Results and Discussion
3.1. Selectivity
As shown in Figures 1(a) and (b), no endogenous peak
was observed in the mass chromatogram from blank
plasma. The chromatogram for the standard lower limit
of quantitation (LLOQ; 2 ng/ml; the signal-to-noise ratio
of greater than 10) sample is also shown in Figures 1(c)
and (d), in which the retention times for carvedilol and
the IS were 1.10 and 1.20 min, respectively.
3.2. Linearity of Calibration Curve
Five calibration curves were constructed over the con-
centration ranges from 2 to 100 ng/ml of carvedilol in
human plasma (Table 1). Based on a linear least-squares
regression without a weighted function, the mean equa-
tion (± S.D.) of the calibration curve obtained from six
concentrations (n = 5) was y = 0.0294 (± 0.0081) x -
0.0028 (± 0.0044) (R2 > 0.9998), where y represents the
carvedilol concentration and x represents the ratio of
carvedilol peak area to that of the IS. The LLOQ was set
at 2 ng/ml. The precision and accuracy were ranged from
0.4 to 7.9% and from 99.1 to 104.5%, respectively, sug-
gesting that the method developed was linear, precise
and accurate over the concentration ranges employed.
3.3. Intra- and Inter-Batch Variability of QC Samples
The results of validating the precision and accuracy of
QC samples with intra- and inter-batch variability are
summarized in Table 2. At the LLOQ of 2 ng/ml, the
precision and accuracy for intra- and inter-batch were 5.1
and 114.3%, and 8.3 and 116.0%, respectively, satisfying
ith the acceptance criteria of less than ± 20% (from w
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Figure 1. Multiple reaction monitoring (MRM) chromatograms of blank human plasma of (a) carvedilol and (b) IS, MRM
chromatogram of lower limit of quantitation (LLOQ, 2 ng/ml) of (c) carvedilol and (d) IS.
140 S.-H. KIM ET AL.
Table 1. Linearity assessment of calibration curves of carvedilol in human plasma.
Concentration (ng/ml)
2.0 5.0 10.0 20.0 50.0 100.0
R2 Slope Intercept
1 2.0 5.3 10.0 19.4 50.2 100.0
0.99993 0.0363 -0.0023
2 2.2 5.0 9.8 20.9 48.4 100.6
0.99944 0.0362 -0.0025
3 2.0 4.7 10.1 20.4 49.8 100.0
0.99996 0.0165 -0.0023
4 1.9 4.9 9.7 20.3 50.5 99.8
0.99993 0.0278 0.0026
5 2.3 5.3 10.2 19.7 48.9 100.6
0.99975 0.0302 -0.0096
Mean 2.1 5.1 10.0 20.1 49.6 100.2 0.99980 0.0294 -0.0028
Precision (%) 7.9 5.1 2.3 3.0 1.8 0.4
Accuracy (%) 104.5 101.1 99.7 100.7 99.1 100.2
Table 2. Summary of precision and accuracy of quality control samples with inter- and intra-batch variability.
Batch No. 2 ng/ml 5 ng/ml 20 ng/ml 100 ng/ml
1 2.3 5.1 19.6 104.2
2 2.3 5.1 20.7 105.8
3 2.2 4.5 20.5 105.7
4 2.5 5.2 19.7 103.1
5 2.2 4.9 20.9 99.2
Mean 2.3 5.0 20.3 103.6
Precision (%) 5.1 5.9 2.8 2.6
Accuracy (%) 114.3 99.2 101.4 103.6
1 2.3 5.1 19.6 104.2
2 2.0 5.7 20.6 107.2
3 2.4 5.4 24.2 113.6
4 2.4 5.2 20.1 101.7
5 2.5 5.3 19.5 87.5
Mean 2.3 5.3 20.8 102.9
Precision (%) 8.3 4.1 9.3 9.4
Accuracy (%) 116.0 106.6 104.0 102.9
100% for accuracy and 0% for precision). For other
concentrations, those also meet the criteria of less than
15% for both intra- and inter-batch QC samples, also
suggesting the method developed was precise and accu-
rate for the intra- and inter-batch assays.
3.4. Stability
Stability studies for carvedilol made on QC samples at
concentrations of 2 and 100 ng/ml were considered to be
stable if the difference of concentrations for each proc-
essing are less than ±15%. The stability results after
three freeze-thawing, post preparation, and short and
long term studies are summarized in Table 3. For all
stability studies, % changes ranged from 94.7% (–5.3%,
long term, low concentration) to 106.4% (+6.4%, short
term, low concentration) satisfying the acceptance crite-
ria for the less than ±15% differences. These results in-
dicates that carvedilol in human plasma is stable during
sample preparation procedures, short and long term
Copyright © 2010 SciRes. AJAC
Table 3. Stability of quality control samples (carvedilol concentration, ng/ml).
Freeze and thaw Post preparation Short term Long term
Low* High** Low High Low High Low High
1 1.99 99.7 2.01 103.5 2.25 93.44 1.87 97.50
2 2.04 104.4 1.88 94.5 2.08 104.59 1.85 92.40
3 1.87 97.7 1.97 99.6 2.05 91.29 1.96 94.40
Average 1.97 100.6 1.95 99.2 2.13 96.44 1.89 94.77
RSD (%)*** 4.44 3.42 3.41 4.55 4.96 7.40 3.09 2.71
Stability (%) 98.3 100.6 97.7 99.2 106.4 96.4 94.7 94.8
*Low: 2 ng/ml; **High: 100 ng/ml; ***RSD (%): Relative Standard Deviation (percent) = S.D./average × 100.
Figure 2. Mean plasma concentrationtime curves of carvedilol obtained after the single oral administration of 25 mg of test
(--) and reference (--) carvedilol formulations to humans (n = 28). Vertical bars represent S.D.
storage, and on the auto-sampler before injection made
onto the column.
3.5. Recovery
From the five replicates of the recovery study at 5, 20
and 100 ng/ml of carvedilol concentrations, the % recov-
ered at each concentration were 85.8 ± 4.2% (coefficient
of variation), 85.4 ± 4.9% and 84.9 ± 2.0%, respectively.
The recovery of IS was 85.7 ± 3.2% (coefficient of
variation). Possible cause for the less than 100% of the
recovery could be due to the co-precipitation of
carvedilol and the IS with plasma proteins. However,
these results were satisfied with the reproducible and
reliable recovery for both carvedilol and the IS from hu-
man plasma matrix.
According to the results of selectivity, linearity, stabil-
ity and recovery studies, this analytical method was con-
sidered to meet the acceptance criteria in all areas of in-
dustrial guidance for the bioanalytical method validation
set by the regulatory agency in US and Korea [19,20].
3.6. Application to Bioequivalence Study
The analytical method developed above was applied to
assess the bioequivalence of two formulations of
carvedilol in human. After analyzing 700 plasma sam-
ples from 28 healthy male volunteers, the mean plasma
concen tr a t iontime curves of carvedilol are shown in
Figure 2. The plasma concentrations of carvedilol ranged
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142 S.-H. KIM ET AL.
Table 4. Mean pharmacokinetic parameters of two carvedilol
formulations after oral administration to 28 healthy male
Test Reference
Mean S.D. Mean S.D.
AUC (ng/ml·h) 364.0 217.9 359.4 202.1
Cmax (ng/ml) 111.2 75.5 107.7 64.3
T1/2 (h) 8.2 2.6 8.4 3.1
Tmax (h) 1.4 1.1 1.2 0.7
from 2.1 to 362.4 ng/ml for both test and reference for-
mulations. The Cmax and Tmax appeared to be around 100
ng/ml and 1 h, respectively. In Figure 2, the mean
plasma curves of two formulations were apparently
similar and shown typical pattern for carvedilol plasma
concentration curves after the oral administration to
healthy humans [21].
The relevant pharmacokinetic parameters of carvedilol
are listed in Table 4. The Cmax and AUC values were
111.2 ng/ml and 364.0 ng·h/ml, respectively, for the test
formulation while those for the reference formulation
107.7 ng/ml and 359.4 ng·h/ml, respectively. The AUC of the
test and reference formulations were 364.0 ± 217.9 ng·h/ml
and 359.4 ± 202.1 ng·h/ml, respectively showing less
than 1.5% difference in AUC. The values of the Tmax and
half-life (T1/2) of the two formulations were very similar
ranging from 1.2 to 1.4 h and from 8.2 and 8.4 h, respec-
tively (Table 4). The calculated 90% confidence interval
(CI) for mean Cmax and AUC of test/reference individual
ratios were 89.5-114.5% and 93.5-108.6%, respectively,
and within the 80–125% interval which is defined as
bioequivalent by the regulatory agency [19,20].
4. Conclusions
A rapid and simple HPLC–MS/MS method for the de-
termination of carvedilol in human plasma has been suc-
cessfully developed and validated using an one-step pro-
tein precipitation procedure. Although this method has
simple sample preparation procedure, it demonstrated
acceptable selectivity, sensitivity, precision and accuracy.
The validated method is suitable for high throughput
assays such as bioequivalence studies and was success-
fully applied to assay human plasma samples for bio-
equivalence study of carvedilol. Carvedilol is unstable at
low pH but there was no perceivable degradation of the
analyte under the described stability test conditions. The
mobile phase containing a small amount of formic acid
did not interfere with the bioanalysis and presence of the
acid was necessary in order to improve the detection of
the compounds in the positive mode.
The 90% CI for Cmax and AUC ratios were within the
80-125% interval and the Tmax and T1/2 for the two for-
mulations were quite similar. Therefore, the tested
carvedilol formulation can be considered as bioequiva-
lent to the reference formulation for both the rate and the
extent of absorption.
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