Journal of Chromatography B, 974 (2015) 138146

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Simultaneous quantication of carvedilol and its metabolites in rat

plasma by ultra performance liquid chromatography tandem mass
spectrometry and pharmacokinetic application
Junwei Li a , Li Wang a , Shuanghu Wang c , Mengchun Chen a , Ermin Gu a ,
Guoxin Hu a , Renshan Ge b,
a

Institute of Molecular Pharmacology and Toxicology, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325000, China
The 2nd Afliated Hospital of Wenzhou Medical University, Wenzhou Medical University, Wenzhou, Zhejiang 325000, China
c
The Laboratory of Clinical Pharmacy, The Peoples Hospital of Lishui, Lishui, Zhejiang 323000, China
b

a r t i c l e

i n f o

Article history:
Received 10 July 2014
Received in revised form 19 October 2014
Accepted 26 October 2014
Available online 4 November 2014
Keywords:
Carvedilol
Metabolite
UPLCMS/MS
Assay

a b s t r a c t
A rapid-resolution ultra high-performance liquid chromatographytandem mass spectrometry separation method (UPLCMS/MS) was developed for the simultaneous determination of carvedilol, and its
three metabolites: 4 -hydroxyphenyl-carvedilol, 5 -hydroxyphenyl-carvedilol, o-desmethyl-carvedilol.
The effective UPLCMS/MS separation of the examined compounds was applied on an Acquity BEH C18
column (2.1 mm 50 mm, 1.7 m particle size) column with a gradient mobile phase system. The analysis
was performed in less than 6 min with a ow rate of 0.4 mL/min. The assay was validated over concentration ranges of 0.500100 ng/mL for carvedilol and 0.050010.0 ng/mL for its three metabolites. Intraand inter-assay precision values for replicate quality control samples were within 11.4% for all analytes
during the assay validation. Mean quality control accuracy values were within 11.5% of nominal values
for all analytes. Assay recoveries were high (>91%) and internal standard normalized matrix effects were
minimal. The four analytes were stable in rat plasma for at least 24 h at room temperature, 89 days at
20 C and 80 C, and following at least ve freezethaw cycles. The validated assay was successfully
applied to the quantication of carvedilol and its pharmacologically active metabolites in rat pharmacokinetic study. The accurate and simple method we developed could be applied to human pharmacokinetic
study in the near future.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Carvedilol, ()-1-(Carbazol-4yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propranolol, is a 1-, 2-, and 1-adrenoreceptor blocker drug with antioxidant and antiproliferative
effects that is indicated for treatment of hypertension, stable
angina pectoris, and congestive heart failure [1]. The compound
contains an oxyisopropanolamine moiety with aromatic substituents linked to both the oxy and amine ends of the molecule,
which provide its combined activities [2]. Carvedilol (CAR) is a
racemic mixture of (R+) and (S) enantiomers, and this drug
is used clinically as a racemic mixture of both enantiomers. Its
metabolism and pharmacokinetics have been described in Ref.
[38]. CAR is extensively metabolized to polar and mostly water

Corresponding author. Tel.:+86 13758702382.

E-mail addresses: raygee0828@163.com, gravity172@gmail.com,
sophyleo0803@126.com (R. Ge).
http://dx.doi.org/10.1016/j.jchromb.2014.10.037
1570-0232/ 2014 Elsevier B.V. All rights reserved.

soluble metabolites in vivo, such as o-desmethyl carvedilol (oDMC), 4 -hydroxyphenyl carvedilol (4 -HPC) and 5 -hydroxyphenyl
carvedilol (5 -HPC), which also are main metabolites in humans
[2,911]. 4 -HPC and 5 -HPC have been proven to be equipotent to
carvedilol with respect to -blockade [12], and o-DMC is also an
active metabolite of carvedilol. Preliminary data suggest that it is
a strong -adrenergic antagonist but a weak vasodilator [13].
The currently available methods for the simultaneous determination of carvedilol and its one or two active pharmacological
metabolites were mainly about the separation of the enantiomers,
and always using solid-phase extraction with derivatization or isotope labeling [1316]. However, there is no literature focused on
the ultra performance liquid chromatography tandem mass spectrometry (UPLCMS/MS) method without derivatization or isotope
labeling for the simultaneous determination of carvedilol, o-DMC,
4 -HPC and 5 -HPC. In order to routinely investigate this possibility in a clinical setting, a reliable assay capable of quantifying the
carvedilol, o-DMC, 4 -HPC and 5 -HPC in rat plasma is essential.
Therefore, in this study, a new method detecting the analytes in

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

short time and without derivatization and isotope labeling was

presented using UPLCMS/MS.
2. Experimental procedure
2.1. Chemicals
Carvedilol (CAR) (Cat: C184625), o-desmethyl carvedilol (oDMC) (Cat: D291475), 4 -hydroxyphenyl carvedilol (4 -HPC)
(Cat: H949120) and 5 -hydroxyphenyl-carvedilol (5 -HPC) (Cat:
H949125) were obtained from Toronto Research Chemicals, Inc.
(Ont., Canada), and the purity of them are all HPLC grade. Metoprolol tartrate (internal standard) (Cat: 15176062, HPLC grade) was
purchased from J&K Scientic Ltd. (Beijing, China). Methanol and
acetonitrile were obtained in HPLC grade from Merck KGaA (Darmstadt, Germany) and all other reagents were of analytical or HPLC
grade.
2.2. Instrumentation
CAR, o-DMC, 4 -HPC and 5 -HPC were analyzed by UPLCMS/MS
with ACQUITY UPLC H-Class and XEVO TQD triple quadrupole mass
spectrometer (Waters Corp., Milford, MA, USA) equipped with an
electrospray ionization (ESI) interface. Masslynx 4.1 software was
used to control the instrument and process all data (Waters Corp.,
Milford, MA, USA).
2.3. UPLCMS/MS conditions
Liquid chromatography was performed on an UPLC BEH C18
column (2.1 mm 100 mm, 1.7 m) kept at 40 C. The mobile phase
consisted of acetonitrile and water (containing 1% formic acid) with
gradient elution at a ow rate of 0.4 mL/min by injection volume
of 2 L. Elution was in a linear gradient, with acetonitrile content
changing from 25% to 95% between 0.3 and 3.5 min. Acetonitrile
content was maintained at 95% for 1 min and then decreased to 25%
over a time of 6.0 s. The overall run time was 5.5 min. A XEVO TQD
triple quadrupole mass spectrometer (Waters Corp., Milford, MA,
USA) equipped with an electrospray ionization (ESI) interface was
used for mass spectrometric detection. The main working parameters of the mass spectrometer are summarized in Table 1. The
dwell time was set to 63 ms for each MRM transition. Nitrogen
was used as the desolvation gas (1000 L/h) and cone gas (50 L/h).
The other parameters were as follows: capillary voltage, 4.0 kV;
source temperature, 150 C; desolvation temperature, 400 C. The
mass spectrums of the analytes are displayed in Fig. 1.
2.4. Standard solutions, calibration standards
The stock solutions of CAR, 4 -HPC, o-DMC and 5 -HPC used to
make the calibration standards and quality control (QC) samples
were prepared by dissolving 10 mg of each compound in 10 mL
methanol to obtain a concentration of 1.00 mg/mL of each compound. The IS stock solution was made by dissolving 10 mg of IS in
10 mL methanol at an initial concentration of 1 mg/mL. The stock
solution of analytes were further diluted with methanol to obtain

139

working solutions at several concentration levels. Calibration standards and QC samples in rat plasma were prepared by diluting the
corresponding working solutions with blank rat plasma. Final concentrations of the calibration standards in rat plasma were 0.5, 1,
2.5, 5, 10, 25, 50 and 100 ng/mL for CAR, and 0.05, 0.1, 0.25, 0.5,
1, 2.5, 5 and 10 ng/mL for 4 -HPC, 5 -HPC and o-DMC, respectively.
The concentrations of QC samples in rat plasma were 1, 10, and
100 ng/mL for CAR. The concentrations of QC samples in rat plasma
were 0.1, 1, and 10 ng/mL for 4 -HPC, o-DMC and 5 -HPC, respectively. All stock solutions, working solutions, calibration standards
were immediately stored at 80 C.
2.5. Extraction procedure
Before analysis, the rat plasma samples were thawed to room
temperature. In a 1.5 mL centrifuge tube, an aliquot of 30 L of
the internal standard working solution (50 ng/mL) was added to
0.1 mL of collected rat plasma sample followed by the addition of
0.2 mL of acetonitrile. The total volume of supernatant collected
after protein precipitation was 0.26 mL. The tubes were vortex
mixed for 1.0 min. After centrifugation at 13,000 rpm for 10 min,
the supernatant (2 L) was injected into the UPLCMS/MS system
for analysis.
2.6. In vivo studies
Male Sprague-Dawley rats (250280 g) obtained from the Laboratory Animal Center of Wenzhou Medical University (Wenzhou,
China) were used to study the pharmacokinetics of carvedilol. All
rats were housed at the Wenzhou Medical University Laboratory
Animal Research Center. All experimental procedures and protocols
were reviewed and approved by the Animal Care and Use Committee of Wenzhou Medical University and were in accordance with
the Guide for the Care and Use of Laboratory Animals. Animals were
housed under controlled conditions (25 1 C, RH 55 10%) and
the air was adequately recycled with a natural lightdark cycle. All
animals were fed standard rodent diet with the following composition (w/w): 20% proteins, 3% fat, 2% ber, 6% minerals, and 69%
starch and vitamin supplements, containing the same amount of
calories. They were allowed to adapt to the housing environment
for at least 1 week before the study. Rats were fasted for at least 16 h
prior to carvedilol administration. Blood samples (0.3 mL) were collected from the tail vein into 1.5 mL heparinized polythene tubes
at the time points of 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10,
12, 24 h after oral administration of carvedilol (6 mg/kg) [17]. The
samples were immediately centrifuged at 2500 g for 5 min. The
rat plasma obtained (100 L) was stored at 80 C until analysis. Rat
plasma concentration of carvedilol and its three metabolites versus
time data for each rat was analyzed by DAS software (Version 3.0,
Wenzhou Medical University, China).
2.7. Validation of the method
2.7.1. Calibration curve
To evaluate the linearity, calibration standards of eight concentrations of CAR (0.5100 ng/mL), 4 -HPC (0.0510 ng/mL), 5 -HPC

Table 1
Tandem mass-spectrometer main working parameters.
Parameter

[M + H]+ (m/z)
Cone voltage (V)
Mass transition (m/z to m/z)
Collision energy (eV)

Analyte
CAR

o-DMC

4 -HPC

5 -HPC

IS

407.1
50
407.3 99.2
30

393.05
50
393.05 209.88
23

423.03
50
423.03 99.73
23

423.1
50
423.1 99.73
23

268.09
40
268.09 115.92
18

140

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

Fig. 1. Mass spectrums of carvedilol, metoprolol and its three metabolites.

(0.0510 ng/mL) and o-DMC (0.0510 ng/mL) were separately

extracted and assayed on three separate days. The linearity for
carvedilol and its three metabolites were investigated by weighted
(1/x2 ) least-squares linear regression of peak area ratios against

concentrations. The lower limit of quantitation (LLOQ) was dened

as the lowest concentration of the analyte that can be quantied
with an acceptable level of precision and accuracy and for which
ion ratios fall within 20% of the established range and S/N ratios

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

are at least >10. The limit of detection (LOD) by this procedure is

the minimum concentration at which the analyte can be identied
(signal-to-noise [S/N] ratio > 3). The sensitivity of the method was
determined by quantifying the limit of detection (LOD).
2.7.2. Precision, recovery and matrix effect
The precision and accuracy of the method were assessed by
determination of QC samples in rat plasma at different concentrations (1, 10, and 100 ng/mL for CAR in rat plasma; 0.1, 1, and
10 ng/mL for 4 -HPC, 5 -HPC and o-DMC in rat plasma, respectively)
on three separate days. Precision was expressed as the percentage
relative standard deviations (RSD, %) and accuracy was expressed
as the relative error (RE = measured value/true value 1).
Extraction recovery experiments which showed an ability to
extract the analyte from the test biological samples were evaluated by comparing the peak areas obtained from extracted QC
samples with non-processed standard solutions at three concentrations with those at the same concentration. Recovery of IS was
determined similarly.
The matrix effect was evaluated by comparing the ratio
(A/B 100%) of peak response of the analytes containing an equivalent amount of blank rat plasma (A) dissolved in pure standard
solution (B). To determine the matrix effect, six different blank rat
plasma samples were utilized to prepare QC samples and used for
assessing the lot-to-lot matrix effect. Matrix effect was evaluated
at three QC levels. The matrix effect of IS was evaluated in the same
manner.
2.7.3. Stock solution stability
Stock solution stability assays were performed including room
temperature stability and freezer stability. Room temperature stability was evaluated by comparing the instrument response of a
portion of the stock solution stored at room temperature for 24 h
against the remainder of the solution stored in a 20 C freezer. Six
replicate injections at a single concentration (10 ng/mL) for CAR, 4 HPC, 5 -HPC, o-DMC and IS were made for each solution. For freezer
stability, frozen stock solutions were stored in a 20 C freezer for
three months. A solution of CAR, 4 -HPC, 5 -HPC, o-DMC and IS at
10 ng/mL each was prepared from the stock solutions. Six replicate
injections of the solution were made and the peak intensities were
compared against those obtained from a freshly prepared stock
solution from a new weighing. Solutions were considered to be
stable if assay values were within the acceptable limits of accuracy
(RE % 5%) and precision (RSD % 10%).
2.7.4. QC sample stability
QC sample stability assays comprising freezethaw stability,
short-term, and long-term stability were carried out to ensure the
reliability of the results. In the protocol, sample stability was tested
by analyzing QC samples after short-term (6 and 12 h) storage at
room temperature and on storage at 80 C for 30 days. At the same
time, the effect of three freeze (80 C)thaw (room temperature)
cycles was also investigated. Samples were considered to be stable
if assay values were within the acceptable limits of accuracy (RE
% 15%) and precision (RSD % 15%).

141

for 4 -HPC and Y = 0.0264093*X + 0.00144095 (r = 0.9996) for 5 HPC. A linear least-squares regression with a weighting index of 1/x
was carried out on the peak area ratios versus carvedilol and the
concentrations of its three metabolites in the 9 rat-plasma standards (in duplicate) to generate a calibration curve.
The current assay offered an LLOQ of 0.5 ng/mL for CAR and
0.05 ng/mL for o-DMC, 4 -HPC and 5 -HPC in rat plasma and an LLOD
of 0.1 ng/mL for CAR and 0.025 ng/mL for o-DMC, 4 -HPC and 5 HPC in rat plasma. The limits were sufcient for pharmacokinetics
studies following a single oral administration (6 mg/kg) of CAR.
The UPLCMS/MS chromatogram of the rat plasma standards
and blank rat plasma are shown in Fig. 2. The retention times
of CAR, o-DMC, 4 -HPC, 5 -HPC and IS are 3.73 min, 3.02 min,
2.24 min, 2.39 min, and 1.10 min, respectively. Compared with
the chromatogram of the blank blood sample, no interference of
endogenous peaks was observed.
The intra- and inter-day precision and accuracy of the method
were determined from the analysis of QC samples at three different
concentrations for each biological matrix. The results are summarized in Table 2. The method was reliable and reproducible since
RSD % was below 15% and RE % was between 5.29% and 4.85% for
all the investigated concentrations of CAR and its metabolites in rat
plasma.
The recovery of carvedilol was 92.2 (CV 7.4%), 91.5 (CV 7.2%) and
95.7 (CV 4.0%) for the 1.0, 10.0 and 100.0 ng/mL standard concentrations, respectively. The recovery of o-DMC was 91.2 (CV 6.5%),
103.2 (CV8.6%) and 105.8 (CV 4.5%) for the 0.1, 1.0 and 10.0 ng/mL
standard concentrations, respectively. The recovery of 4 -HPC was
92.2 (CV 7.4%), 91.5 (CV7.7%) and 95.7 (CV 4.0%) for the 0.1, 1.0 and
10.0 ng/mL standard concentrations, respectively. The recovery of
5 -HPC was 97.06 (CV 8.4%), 101.4 (CV7.2%) and 95.14 (CV 7.0%) for
the 0.1, 1.0 and 10.0 ng/mL standard concentrations, respectively.
The matrix effect in rat plasma was all from 92.23% to 105.89%
for CAR, o-DMC, 4 -HPC and 5 -HPC at different QC levels (Table 3).
The matrix effect for IS (50 ng/mL) was 96.57%. No apparent matrix
effect was found to affect the determination of CAR and its metabolites and IS in rat plasma. As a result, the matrix effect from rat
plasma was negligible in this method.
In the stock solutions, CAR, o-DMC, 4 -HPC and 5 -HPC did not
present degradation up to 24 h of storage at room temperature.
The REs of the mean test responses did not exceed the 5% limits of
variation during the 24 h. Stock solutions of CAR, o-DMC, 4 -HPC, 5 HPC and IS (1 mg/mL) were stored in a 20 C refrigerator without
problems of degradation or precipitation for 90 days (Table 4).
QC samples stability tests were performed at the low, medium
and high QC samples with ve determinations for each under different storage conditions. The RSDs of the mean test responses
were within 10% in all stability tests. There was no effect on
the quantitation for rat plasma samples kept at room temperature for 6 and 12 h. No signicant degradation was observed
when samples of carvedilol, metoprolol, 4 -hydroxycarvedilol, 5 hydroxycarvedilol and o-desmethyl carvedilol were taken through
three freeze (80 C)thaw (room temperature) cycles. As a result,
carvedilol and its three metabolites in samples were stable at
80 C for 30 days.
3.2. Method application

3. Results
3.1. UPLCMS/MS method validation
The simplest regression method for the calibration curves of
the carvedilol and its three metabolites was y = a + bx from 0.5
100 ng/mL to 0.0510 ng/mL (calibration curve Y = 0.0605694X +
0.0569335 (r = 0.9998) for carvedilol, Y = 0.127395*X + 0.00157365
(r = 0.9992) for o-DMC, Y = 0.0292282*X + 0.00399012 (r = 0.9991)

The validated UPLCMS/MS assay was successfully applied to

the quantication of both carvedilol and its pharmacologically
active metabolites in a pharmacokinetic study. Representative
MRM chromatograms for carvedilol and its metabolites are shown
in Fig. 3. As shown in Fig. 3, the 4 - and 5 -hydroxyphenyl metabolites and the o-desmethyl metabolite were all specically detected
in study sample chromatograms. The deployment of the gradient
elution in the validated assay was applied to separate the 4 - and

142

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

Fig. 2. Representative UPLCMS/MS chromatograms for CAR, o-DMC, 4 -HPC, 5 -HPC, IS and blank plasma.

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

143

Table 2
Precision and accuracy of method for the determination of CAR, 4 -HPC, 5 HPC and o-DMC in rat plasma (n = 6).
Analytes

Concentration added (ng/mL)

Intra-day precision (n = 6)
Mean SD

Car

1
10
100

Inter-day precision (n = 6)
Mean SD

RSD (%)

RE (%)

1.03 0.07
9.52 0.22
100.48 0.92

7.20
2.29
0.91

2.90
4.76
5.15

1.08 0.02
8.84 0.69
101.47 3.24

1.15
5.70
3.19

8.28
11.64
1.47

RSD (%)

RE (%)

o-DMC

0.1
1
10

0.09 0.01
1.05 0.12
10.91 0.21

7.10
11.35
1.89

12.6
5.10
9.10

0.11 0.01
0.97 0.06
10.30 0.39

1.06
7.84
3.87

9.33
2.76
2.98

4 -HPC

0.1
1
10

0.11 0.01
1.05 0.07
9.27 0.35

6.78
6.87
3.75

6.67
4.86
7.29

0.10 0.01
0.99 0.05
8.74 0.34

6.21
2.42
1.75

1.14
1.52
1.37

5 -HPC

0.1
1
10

0.01 0.01
1.00 0.11
10.10 0.13

10.86
10.71
3.87

3.03
0.17
0.95

0.10 0.01
1.06 0.05
9.62 0.57

1.17
4.86
5.91

1.57
6.08
3.85

Table 3
Recovery and matrix effect of CAR and its metabolites and internal standard (n = 6).
Analytes

Car

Concentration added (ng/mL)

1
10
100

Recovery (%)

Matrix effect (%)

Mean SD

RSD (%)

Mean SD

RSD (%)

92.21 5.99
91.53 6.60
95.73 3.85

6.57
7.21
4.02

95.65 5.95
94.31 3.11
98.12 2.52

6.22
3.29
2.57

o-DMC

0.1
1
10

91.26 3.49
97.36 5.55
97.96 11.11

3.87
5.70
1.13

92.23 6.81
103.2 8.95
105.89 4.86

2.45
8.67
4.59

4 -HPC

0.1
1
10

91.67 3.05
97.84 4.81
91.13 5.78

3.33
4.92
6.34

104.5 7.38
101.7 7.42
94.92 3.48

7.06
7.30
3.67

5 -HPC

0.1
1
10
50

97.06
96.76
95.14
93.42

IS

8.21
5.77
6.71
4.63

8.26
5.97
7.05
4.95

101.77
101.4
99.46
96.57

4.00
7.89
3.80
3.78

3.92
7.78
3.82
3.91

Table 4
Stock solution stability tests for the determination of CAR, 4 -HPC, 5 HPC and o-DMC in rat plasma (n = 6).
Stock solution stability

Nominal concentration
(ng/mL)

Mean area ratio of

comparison samples

Mean area ratio of

stability samples

RSD (%)

RE (%)

Room temperature
24 h

CAR
4 HPC
5 HPC
DMC
IS

10
10
10
10
10

0.9805
0.9876
0.9533
1.0670
0.9486

0.9813
1.0275
1.0333
0.9837
1.0254

2.41
2.01
2.67
1.96
2.78

1.87
2.75
3.3
1.6
2.54

Freezer stability 91
day

CAR
4 -HPC
5 HPC
DMC
IS

10
10
10
10
10

1.0072
1.0342
1.1351
0.9901
0.9741

0.9743
0.9660
0.9657
0.9585
0.9851

3.15
3.61
4.89
2.52
3.09

2.5
3.3
3.4
4.1
1.5

Table 5
Pharmacokinetic parameters of CAR and its metabolites after oral administration of carvedilol (n = 6, 6 mg/kg).
Parameter

CAR

4 -HPC

5 -HPC

o-DMC

t1/2 (h)
Cmax (g/L)
AUC0 (g/L h)

3.18 0.23
27.02 6.94
138.91 22.76

19.78 4.83
4.44 1.23
86.97 21.23

1.42 0.55
7.91 2.46
41.72 12.56

2.07 1.07
4.73 1.63
37.28 10.09

5 -hydroxyphenyl metabolites which were quite similar in their

chemical structure in the pharmacokinetic samples. Fig. 4 shows
mean (SD) rat plasma concentrationtime proles for carvedilol
and its three metabolites, along with their corresponding single
dose pharmacokinetic parameters in the rats in Table 5. The disposition of carvedilol and its three metabolites were adequately
described by a non-compartment model using DAS version 3.0.

4. Discussion
CAR and its metabolites 4 -HPC, 5 -HPC and o-DMC are basic
compounds containing secondary amino groups that can be easily protonated under acidic chromatographic conditions. Therefore,
electrospray ionization in the positive ion mode was used for MRM
analyses. The most stable and abundant product ions at m/z 99.2

144

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

Fig. 3. Representative MRM chromatograms for CAR, o-DMC, 4 -HPC, 5 -HPC and IS in rat plasma samples.

for CAR can be attributed to the elimination of the 9H-carbazol4-yloxy and 2-methoxy-phenoxy groups from the precursor ion
(Fig. 1a). For o-DMC, the most stable and abundant product ions at
m/z 209.88 can be attributed to the elimination of the 9H-carbazol4-yloxy fragment from the precursor ion (Fig. 1b). For 4 -HPC, the
most consistent and abundant ion at m/z 99.73 corresponded to 2hydroxypropyl ethylamine substructure (Fig. 1c). For 5 -HPC (m/z
423.1), the same fragment with 4 -HPC (m/z 99.73) corresponded
to almost the same substructure (Fig. 1d). The quantication of
the analytes was performed using the MRM mode due to the high
selectivity and sensitivity of MRM data acquisitions.
Plasma extraction procedures reported for the simultaneous
determination of CAR and its metabolites have either used
liquidliquid extraction [12,18] or SPE [12,13,15,16]. Chandiran
et al. [19] demonstrated the use of robotic on-line extraction for
the extraction of racemic CAR and 4 -HPC from plasma. However,
this on-line process requires signicant modications when compared with off-line extraction and may not be available in every
laboratory. In the present work, liquidliquid extraction was tried
initially using diethylether, ethyl acetate, methylene chloride and
methyl tert-butyl ether under alkaline conditions (NaOH/Na2 CO3 )
as reported previously [12,18]. However, the recovery for 4 -HPC,
5 -HPC and o-DMC were less than 65% at LQC level. Thus, extraction was carried out by the addition of 0.2 mL of acetonitrile. With
acetonitrile addition, not only was the protein precipitated, but the
drug was extracted from the rat plasma. The recovery of this onestep precipitation as sample preparation procedure was easy and
sufciently consistent.
Carvedilol and its metabolites have been determined in plasma
and other biological uids such as high performance liquid chromatography coupled to uorometric detection [13,17,2027], high
performance liquid chromatography coupled to ultraviolet detection [27], capillary electrophoresis coupled to ultra-violet detection
[27,28], capillary electrophoresis coupled with laser-induced uorescence [29], high performance liquid chromatography coupled
to electrochemical detection [30], liquid chromatography coupled

to tandem mass spectrometry [2,31,32], gas chromatography

coupled to mass spectrometry [9], ultra performance liquid chromatography coupled to tandem mass spectrometry [16]. There
are two existing methods in the literature analyzing CAR and its
three metabolites (4 -HPC, 5 -HPC and o-DMC) simultaneously,
which are a LC method reported by Gehr et al. [12], and a GC/MS
method reported by Myung et al. [9]. However, both of them used
derivatization with the chiral reagents like N-methyl-N-trimethylsilyltri-uoroacetamide (MSTFA), N-methyl-bis-triuoraceamide
(MBTFA) and 2,3,4,6-tetra-O-acetyl--d-glucopyranosyl isothiocyanate (GITC), demonstrated limits of quantitation of the
metabolites ranging from 0.75 to 3.0 ng/mL and their retention
times were from 12 to 20 min. The method reported in our work
has a LLOQ of 0.05 ng/mL for the three metabolites (4 -HPC, 5 -HPC
and o-DMC) and 0.5 ng/mL for CAR and run time of 5.5 min with
a ow rate of 0.4 mL/min, which showed that the developed
method is rapid and highly sensitive. It is very difcult to separate
4 -HPC and 5 -HPC in the normal mobile phase, because the
only dissimilarity between them is that the different hydroxyl
sites on the same benzene ring. With the deployment of the
gradient elution in this work, 4 -HPC and 5 -HPC were separated
without any derivatization. The results of the precision and
accuracy indicate that the assay performs as well as any of the
published methods on analysis of CAR and its three metabolites
from plasma matrices, and there was no evidence of matrix
effect.
Although this method was a non-stereoselective assay, it
showed a favorable recovery (97.891.3%) as well as good
reproducibility (C.V. 1.18.3%). The method also enabled the determination of the rat plasma concentration using a small volume of
rat plasma (100 L). In addition, the assay was rapid and easy to
perform.
The 4 -, 5 -hydroxyphenyl and o-desmethyl metabolites were
found in the rat plasma samples, same as the in vitro study previously conducted by our group with rat liver microsomes, and
their pharmacokinetic parameters showed that 4 -HPC, 5 -HPC may

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

145

Fig. 4. Plasma concentrationtime proles for carvedilol and its three metabolites. (A) Carvedilol, (B) 4 -hydroxylphenyl-carvedilol, (C) 5 -hydroxylphenyl-carvedilol and
(D) o-desmethyl-carvedilol.

have enterohepatic recycle as the other hydroxyl metabolites (8hydroxycarvedilol and 1-hydroxycarvedilol), similarly reported by
Schaefer. However, Schaefer did not nd 4 -, 5 -hydroxyphenyl and
o-desmethyl metabolites in rat plasma [2] because of the low sensitivity of the detecting method. Moreover, the 8-hydroxycarvedilol
and 1-hydroxycarvedilol were most abundant (18.5% and 37.8%),
while 4 -, 5 -hydroxyphenyl and o-desmethyl metabolites were
found in trace amounts in the rat plasma. In this study, the
amount of 8-hydroxycarvedilol and 1-hydroxycarvedilol were also
determined and were duplicates of the 4 -, 5 -hydroxyphenyl and
o-desmethyl metabolites; but they had not been quantied in rat
plasma because they were not pharmacologically active and have
no signicant clinical application. Additionally, the o-desmethyl
metabolite did not show a second peak in the concentrationtime
curve, possibly indicating that this metabolite may not be excreted
into the biliary system.
There are few reports on carvedilol pharmacokinetic study in
rats [17,3337]. When CAR was given orally, the elimination halflife (t1/2 ) usually varied between 3.1 and 18 h [17,3337]. In this
study, the t1/2 was 3.2 h after administration of a 1.51.8 mg doses
for each rat in non-compartment model analysis, which is in good
agreement with the reported value.

The carvedilol rat-plasma concentrations under the clinical

doses in the literature are 2379 ng/ml as the Cmax [7]. The present
method is adequate for detecting concentrations of 2.3 ng/ml (one
tenth of the above Cmax ), because the detection limit in this method
was 0.5 ng/mL. The pharmacokinetic parameters of 4 -HPC, 5 -HPC
and o-DMC in rats was never reported in the literature, so, to conrm the results, further studies such as pharmacokinetic studies
in rats with higher doses (20 mg/kg), have been performed. And it
still arrived at the same conclusion with this study. The Cmax of 4 HPC, 5 -HPC and o-DMC in rats was 4.40, 7.9, 4.7 ng/mL respectively,
which are higher than values reported by Patel et al. [16] in healthy
volunteers; but are all lower than the Day 9 results from the Gehrs
results in patients with hypertension and renal insufciency [12].
Overall, the present method is suitable for the clinical detection of
the three pharmacologically active metabolites in patients.
5. Conclusion
We have described the development, validation and successful application of the rst assay capable of quantifying
carvedilol and its three pharmacologically active metabolites (4 ,
5 -hydroxyphenyl and o-desmethyl) in rat plasma. A combination

146

J. Li et al. / J. Chromatogr. B 974 (2015) 138146

of ultra liquid chromatographic and mass spectrometric techniques

was used to separate these two metabolites (4 , 5 -hydroxyphenyl)
known to be almost the same in their chemical structure, without radiolabeling and derivatization. The method is accurate,
reproducible, specic, requires relatively small volumes of serum
(100 L), and is applicable to the evaluation of pharmacokinetic
proles of carvedilol in rats. The developed UPLCMS/MS method
was found to be suitable for the analysis of carvedilol and its
metabolites in rat serum. The three metabolites in rats are the same
as in humans; thus, this method is also equally suitable for the
estimation of carvedilol and its metabolites in human biological
samples.
Acknowledgement
This research program was supported by the National Health
and Family Planning Commission, PRC (Project No. 201302008).

[17]

[18]

[19]

[20]

[21]
[22]

[23]

References
[24]
[1] K. Beattie, G. Phadke, J. Novakovic, Carvedilol, Proles Drug Subst. Excip. Relat.
Methodol. 38 (2013) 113157.
[2] W.H. Schaefer, J. Politowski, B. Hwang, et al., Metabolism of carvedilol in dogs,
rats, and mice, Drug Metab. Dispos. 26 (1998) 958969.
[3] H.H. Zhou, A.J. Wood, Stereoselective disposition of carvedilol is determined by
CYP2D6, Clin. Pharmacol. Ther. 57 (1995) 518524.
[4] H.G. Oldham, S.E. Clarke, In vitro identication of the human cytochrome P450
enzymes involved in the metabolism of R(+)- and S()-carvedilol, Drug Metab.
Dispos. 25 (1997) 970977.
[5] K. Fukumoto, T. Kobayashi, K. Komamura, et al., Stereoselective effect of
amiodarone on the pharmacokinetics of racemic carvedilol, Drug Metab. Pharmacokinet. 20 (2005) 423427.
[6] Y. Liu, C. Lu, Q. Chen, et al., Bioequivalence and pharmacokinetic evaluation
of two tablet formulations of carvedilol 25-mg: a single-dose, randomizedsequence, open-label, two-way crossover study in healthy Chinese male
volunteers, Drug Res. (Stuttg.) 63 (2013) 7478.
[7] M. Fujimaki, Y. Murakoshi, H. Hakusui, Assay and disposition of carvedilol
enantiomers in humans and monkeys: Evidence of stereoselective presystemic
metabolism, J. Pharm. Sci. 79 (1990) 568572.
[8] E. Yang, S. Wang, J. Kratz, et al., Stereoselective analysis of carvedilol in human
plasma using HPLC/MS/MS after chiral derivatization, J. Pharm. Biomed. Anal.
36 (2004) 609615.
[9] S.W. Myung, C.H. Jo, Gas chromatograph-mass spectrometric method for the
determination of carvedilol and its metabolites in human urine, J. Chromatogr.
B Analyt. Technol. Biomed. Life Sci. 822 (12) (2005) 7077.
[10] M. Fujimaki, H. Hakusui, Identication of two major biliary metabolites of
carvedilol in rats, Xenobiotica 20 (1990) 10251034.
[11] G. Neugebauer, P. Neubert, Metabolism of carvedilol in man, Eur. J. Drug Metab.
Pharmacokinet. 16 (1991) 257260.
[12] T.W. Gehr, D.M. Tenero, D.A. Boyle, et al., The pharmacokinetics of carvedilol
and its metabolites after single and multiple dose oral administration in
patients with hypertension and renal insufciency, Eur. J. Clin. Pharmacol. 55
(1999) 269277.
[13] E.J. Eisenberg, W.R. Patterson, G.C. Kahn, High-performance liquid chromatographic method for the simultaneous determination of the enantiomers of
carvedilol and its O-desmethyl metabolite in human plasma after chiral derivatization, J. Chromatogr. 493 (1989) 105115.
[14] F. Behn, S. Michels, S. Laer, et al., Separation of carvedilol enantiomers in
very small volumes of human plasma by capillary electrophoresis with laserinduced uorescence, J. Chromatogr. B Biomed. Sci. Appl. 755 (2001) 111117.
[15] M.T. Furlong, B. He, W. Mylott, et al., A validated enantioselective LCMS/MS
assay for the simultaneous determination of carvedilol and its pharmacologically active 4 -hydroxyphenyl metabolite in human plasma: application to a
clinical pharmacokinetic study, J. Pharm. Biomed. Anal. 70 (2012) 574579.
[16] D.P. Patel, P. Sharma, M. Sanyal, et al., UPLCMS/MS assay for the simultaneous quantication of carvedilol and its active metabolite 4 -hydroxyphenyl

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

carvedilol in human plasma to support a bioequivalence study in healthy volunteers, Biomed. Chromatogr. 27 (2013) 974986.
N. Hokama, N. Hobara, H. Kameya, et al., Rapid and simple micro-determination
of carvedilol in rat plasma by high-performance liquid chromatography, J. Chromatogr. B Biomed. Sci. Appl. 732 (1999) 233238.
G. Neugebauer, W. Akpan, E. von Mollendorff, et al., Pharmacokinetics and disposition of carvedilol in humans, J. Cardiovasc. Pharmacol. 10 (Suppl 11) (1987)
8588.
I. Sarath Chandiran, K.N. Jayaveera, S. Raghunadha Reddy, High-throughput
liquid chromatographytandem mass spectrometric method for simultaneous
quantication of carvedilol and its metabolite 4 -hydroxyphenyl carvedilol in
human plasma and its application to bioequivalence study, J. Chem. Pharm. Res.
2 (2011) 341353.
C. de Mey, K. Breithaupt, J. Schloos, et al., Dose-effect and
pharmacokineticpharmacodynamic relationships of the beta 1-adrenergic
receptor blocking properties of various doses of carvedilol in healthy humans,
Clin. Pharmacol. Ther. 55 (1994) 329337.
F. Varin, L.X. Cubeddu, J.R. Powell, Liquid chromatographic assay and disposition of carvedilol in healthy volunteers, J. Pharm. Sci. 75 (1986) 11951197.
K. Reiff, High-performance liquid chromatographic method for the determination of carvedilol and its desmethyl metabolite in body uids, J. Chromatogr.
413 (1987) 355362.
H. Spahn, W. Henke, P. Langguth, et al., Measurement of carvedilol enantiomers
in human plasma and urine using S-naproxen chloride for chiral derivatization,
Arch. Pharm. (Weinheim) 323 (1990) 465469.
G. Neugebauer, W. Akpan, B. Kaufmann, et al., Stereoselective disposition of
carvedilol in man after intravenous and oral administration of the racemic
compound, Eur. J. Clin. Pharmacol. 38 (1990) S108S111.
D. Tenero, S. Boike, D. Boyle, et al., Steady-state pharmacokinetics of carvedilol
and its enantiomers in patients with congestive heart failure, J. Clin. Pharmacol.
40 (2000) 844853.
P. Ptacek, J. Macek, J. Klima, Liquid chromatographic determination of carvedilol
in human plasma, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 789 (2003)
405410.
L. Clohs, K.M. McErlane, Development of a capillary electrophoresis assay for
the determination of carvedilol enantiomers in serum using cyclodextrins, J.
Pharm. Biomed. Anal. 24 (2001) 545554.
J. Oravcova, D. Sojkova, W. Lindner, Comparison of the HummelDreyer method
in high-performance liquid chromatography and capillary electrophoresis conditions for study of the interaction of (RS)-, (R)- and (S)-carvedilol with isolated
plasma proteins, J. Chromatogr. B Biomed. Appl. 682 (1996) 349357.
F. Behn, S. Laer, H. Scholz, Determination of carvedilol in human cardiac tissue by high-performance liquid chromatography, J. Chromatogr. Sci. 39 (2001)
121124.
M. Machida, M. Watanabe, S. Takechi, et al., Measurement of carvedilol in
plasma by high-performance liquid chromatography with electrochemical
detection, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 798 (2003)
187191.
M. Gergov, J.N. Robson, E. Duchoslav, et al., Automated liquid chromatographic/tandem mass spectrometric method for screening beta-blocking drugs
in urine, J. Mass Spectrom. 35 (2000) 912918.
N.C. do Carmo Borges, G.D. Mendes, D. de Oliveira Silva, et al., Quantication
of carvedilol in human plasma by high-performance liquid chromatography
coupled to electrospray tandem mass spectrometry: Application to bioequivalence study, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 822 (2005)
253262.
F.M. Bertera, et al., Enantioselective pharmacokinetic and pharmacodynamic
properties of carvedilol in spontaneously hypertensive rats: focus on blood
pressure variability, Naunyn Schmiedebergs Arch. Pharmacol. 385 (2012)
325335.
F. Bertera, C.A. Di Verniero, M.A. Mayer, et al., Pharmacokinetic and pharmacodynamic properties of carvedilol in fructose hypertensive rats, Xenobiotica 42
(2012) 206219.
E. Stahl, U. Baumgartner, D. Henke, et al., Rats with portacaval shunt as a
potential experimental pharmacokinetic model for liver cirrhosis: application
to carvedilol stereopharmacokinetics, Chirality 5 (1993) 17.
E. Stahl, D. Henke, E. Mutschler, et al., Saturable enantioselective rst-pass
effect for carvedilol after high oral racemate doses in rats, Arch. Pharm. (Weinheim) 326 (1993) 123125.
E. Stahl, E. Mutschler, U. Baumgartner, et al., Carvedilol stereopharmacokinetics
in rats: afnities to blood constituents and tissues, Arch. Pharm. (Weinheim)
326 (1993) 529533.