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Simultaneous estimation of amlodipine and

atenolol in human plasma: a sensitive
LC–MS/MS method validation and its
application to a clinical PK study

Background: A highly sensitive, specific and rapid LC–ESI-MS/MS method has been developed and validated for
simultaneous quantification of amlodipine (AMD) and atenolol (ATL) in human plasma (200 µl) using AMD-d4 and
ATL-d7, respectively, as an internal standard (IS) as per the regulatory guidelines. Results: The SPE method was
used to extract the analytes and IS from human plasma. The chromatographic resolution of AMD, ATL and
corresponding IS was achieved using an isocratic flow on a C18 column. The total chromatographic run time was
3 min. A linear response function was established for the range of concentrations 50–8000 pg/ml and 10–800 ng/ml
for AMD and ATL, respectively, in human plasma. Conclusion: The intra- and inter-day accuracy and precision
values for AMD and ATL met the acceptance as per regulatory guidelines. The validated assay was applied to a
fixed-dose combination of AMD and ATL (Adopin-AT®) PK study in humans.

Across the world hypertension is a common
disorder and is associated with cardiovascular
risk. Hypertension remains the most common
cardiovascular disease in the USA affecting
nearly 60 million adults [1] . It is estimated that
50% of patients started on drug therapy fail
their initial treatment attempt. Antihypertensive
therapy with b1-blockers or calcium channel
blockers prevents the occurrence of further
complications, such as stroke, angina, ischemia
and myocardial infarction [2] . However, many
patients, after first-line treatment with classical
antihypertensive drugs, still remain at increased
cardiovascular risk due to insufficient blood
pressure reduction. As a result, most patients
need a combination of two or more hyperten-
sive drugs for adequate blood-pressure control.
Fixed-dose combination or a combination
of drugs with complementary mechanisms of
action can provide improved blood pressure
control, along with lesser side effects and bet-
ter patient tolerability. In combination therapy,
lower doses of individual drugs are adminis-
tered to produce the similar antihypertensive
effect (because of additive or synergistic effect)
than the higher doses used in monotherapy [3,4] .
This provides the pharmacological rationale of
combining a calcium channel blocker (amlo-
dipine [AMD]) and b1-adrenergic blocker or
b1-blocker (atenolol [ATL]).
AMD ( Figure 1, CAS no: 88150-42-9) is a
Raja Reddy Kallem1,
dihydropyridine calcium channel blocker, which Ramesh Mullangi2 ,
lowers blood pressure by direct peripheral arte- Kishore Kumar Hotha3,4,
rial vasodilation and decreasing the periph- LK Ravindranath3,
eral vascular resistance. Such as other calcium YN Spoorthy3 &
channel drugs, AMD inhibits the transmem- JVLN Seshagirirao*5
brane influx of extracellular calcium. Following 1
College of Pharmaceutical
Sciences, Andhra University,
oral administration of therapeutic doses (5 or Visakhapatnam 530 003, AP, India
10 mg) to humans, maximum plasma concen- 2
Jubilant Biosys, 2nd Stage,
trations (Cmax) attained between 6–12 h. AMD Industrial Suburb, Yeshwanthpur,
Bangalore 560 022, India
is extensively metabolized (90%) into its inac- 3
Sri Krishnadevaraya University,
tive pyridine metabolites via hepatic metabo- Anantapur 515 003, AP, India
4
Novel Laboratories Inc, Somerset,
lism, with 10% unchanged parent (AMD) NJ 08873, USA
and 60% of the metabolites excreted primarily 5
Yalamarty College of Pharmacy,
Tarluvada, Visakhapatnam 530 052,
through urine. The plasma protein binding is AP, India
approximately 93%. AMD eliminated from *Author for correspondence:
plasma in a biphasic manner with a half-life of Tel.: +91 949 176 6577
E-mail: jvlnsrao@rediffmail.com
30–50 h. The oral bioavailability was found to
be between 64–90% [5] . ATL ( Figure  1, CAS
no: 29122-68-7) is a selective b-blocker, which
acts preferentially on b1-adrenoceptors and pro-
duces negative chronotropic effect, by which it
reduces the cardiac output and systolic blood
pressure. Following oral administration, ATL is
rapidly and incompletely absorbed (50% of the
administered dose is absorbed) from the intestine
and attains Cmax between 2–4 h. Unabsorbed
drug eliminates unchanged through feces; how-
ever, the absorbed drug is eliminated primarily
through urine. ATL undergoes little (5%) or no

10.4155/BIO.13.39 © 2013 Future Science Ltd Bioanalysis (2013) 5(7), 827–837 ISSN 1757-6180 827
R esearch A rticle |
H estimation method would help the researchers, as
H3C N NH2
O H3C
H3COOC COOCH2CH3
Cl
Amlodipine (AMD)
OH
O
H
N CH3
O
H2N
Atenolol (ATL)
Figure 1. Structural representation of amlodipine, atenolol,
amlodipine-d4 and atenolol-d7.
Key Terms
Hypertension: Also called
high blood pressure.
Hypertension is a chronic
condition in which arteries
(pumps out the blood from the
heart to the whole body) have
persistent elevated blood
pressure.
b1-blockers: Also known as
b1-adrenergic blockers, which
block the binding of epinephrine
and norepinephrine in the heart
and reduce the heart rate and
blood pressure by dilating the
blood vessels.
Calcium channel blockers:
Calcium channel slows down the
entry of calcium ions into heart
cells and blood vessel walls,
which make it easier for heart
to pump blood and blood
vessels to dilate. As a result,
there is no strain on heart and
blood pressure lowers.
Fixed-dose combination:
Formulation of two or more
drugs in a single dosage form,
available in fixed-dose
combination. Fixed-dose
combination improves the
medication compliance, and
lower doses of individual drugs
are administered to produce the
beneficial effect than the higher
doses of individual drugs.
828
Kallem, Mullangi, Hotha, Ravindranath, Spoorthy & Seshagirirao
H D D
N NH2
O in the market as a fixed-dose combination, and
D D
H3COOC COOCH2CH3
Cl
Amlodipine-d4 (AMD-d4)
OH
H
O
O N CD3
CH3 D ing fixed-dose combination of AMD and ATL
CD3
H2N
Atenolol-d7 (ATL-d7)
metabolism in liver. The plasma protein bind-
ing is 6–16%. ATL mean elimination half-life is
6–7 h. The oral bioavailability was found to be
between 40–50% [6] . In clinical trials, in patients
with hyper­tension, AMD plus ATL fixed-dose
combination was significantly more effective
than a placebo and the respective monothera-
pies administered at the same dosages [7,8] . A few
commercially available fixed-dose combinations
of AMD and ATL (5 mg AMD and 50 mg ATL)
are Acard®, Acord-A®, Adipin Plus®, Adorant®,
Adipine-AT®, Alodip-AT®, Aglodepin-AT®,
Adopin-AT® and so on.
A thorough literature review revealed that sev-
eral LC–MS/MS methods have been published
for the determination of AMD and ATL individ-
ually (for AMD [9–16] and for ATL [17]), or along
with other drug(s) (ATL with other drugs [18–22]
and AMD with other drugs [23–27]) in various bio-
logical matrices. It is important to develop a sensi-
tive, rapid and reliable bioanalytical method for
simultaneous quantitation of two or more drugs
in combination therapy in human plasma. To
date there is no sensitive method published for
the simultaneous estimation of AMD and ATL
in any biological matrix. It is very challenging to
develop a simultaneous method for the estimation
of two drugs, whose LLOQ difference was 200-
fold (LLOQ for AMD and ATL was 50 pg/ml
and 10 ng/ml, respectively) and have logP (logP
for AMD and ATL is 4.16 and 0.5, respectively)
values, which pose challenges during the sample
clean-up process for optimal recovery. Accounting
for these challenges, we took up this method
Bioanalysis (2013) 5(7)
validation work as we felt that this simultaneous
both the drugs used in this method are available
has applications in PK and bioequivalence stud-
ies. The present work describes a simple, selec-
tive, rapid and sensitive method, which employs
the SPE technique for sample preparation and
LC–ESI-MS/MS for simultaneous quantitation
of AMD and ATL, using deuterated correspond-
ing internal standards (IS) in human plasma. The
application of this assay method to an oral PK
study in male human healthy volunteers follow-
(Adopin-AT) is described.
Experimental
„„Chemicals & materials
AMD besylate (purity 99.8%) was procured
from USP (Hyderabad, India) and ATL (purity
99.7%), AMD-d4 (purity 98.4%) and ATL-d7
(purity 98.8%) from Sigma-Aldrich Corporation
(Bangalore, India). HLB 1 cc 30-mg cartridges
were procured from Waters India (Bangalore,
India). HPLC grade acetonitrile and methanol
were purchased from Rankem, Ranbaxy Fine
Chemicals Limited (New Delhi, India). Ana-
lytical grade formic acid was purchased from
SD Fine Chemicals (Mumbai, India). All other
chemicals and reagents were of analytical grade
and used without further purification. The
control human K 2EDTA plasma sample was
procured from Cauvery Diagnostics and Blood
Bank (Secunderabad, India).
„„Instrumentation & chromatographic
conditions
A Shimadzu HT (Shimadzu, Japan) LC system
equipped with degasser (DGU-20A5), binary
pump (LC-20AD) along with autosampler
(SIL-HTC) was used to inject 20-µl aliquots
of the processed samples on a Discovery C18
column (50 × 4.6 mm, 5 µm), Supelco (MO,
USA), which was maintained at 40 ± 2°C in
column oven (CTO-10ASvp). The isocratic
mobile phase, a mixture of 0.01% formic acid
in water and methanol:acetonitrile (6:4, v/v) at
a ratio of 30:70 (v/v) was delivered at a flow-rate
of 0.5 ml/min into the MS-ESI chamber. The
mobile phase was filtered through a 0.45 µm
membrane filter (XI5522050) (Millipore, MA,
USA) and then degassed ultrasonically for 5 min
before being used for ana­lysis.
Quantitation was achieved by MS/MS
detection in positive ion mode for analytes and IS
future science group
 Simultaneous quantitation of amlodipine & atenolol in human plasma
using a MDS Sciex (CA, USA) API-4000 mass
spectrometer, equipped with a TurboionsprayTM
interface at 550°C temperature and 5500 V ion
spray voltage. The source parameters (i.e., cur-
tain gas, GS1 and GS2) were set at 25, 45 and
50 psi. The compound parameters – decluster-
ing potential, entrance potential, collision energy
and collision cell exit potential – for AMD and
AMD-d4 were similar and were 38, 10, 17 and
7 V. For ATL and ATL-d7, the declustering
potential, entrance potential, collision energy
and collision cell exit potential were 52, 10, 25,
and 12 V, respectively. Detection of the ions was
performed in the SRM mode, monitoring the
transition of the m/z 409.2 precursor ion to the
m/z 238 product ion for AMD, and m/z 413/238
for AMD-d4 . ATL was monitored with m/z 267
precursor ion to the m/z 190.1 product ion and
m/z 274/190.1 for ATL- d7. Quadrupole Q1 and
Q3 were set on unit resolution. The dwell time
was 200 msec. The analytical data were processed
by Analyst software (version 1.5.2).
„„Standard solutions
AMD, ATL and their corresponding IS were
weighed accurately into volumetric flasks using
an analytical micro balance. They were then
diluted with methanol. The primary stock solu-
tions of AMD and ATL, and both IS solutions
were prepared at 0.5 mg/ml. The stock solutions
of AMD, ATL and ISs were stored at 4°C, which
were found to be stable for 28 days (data not
shown) and successively diluted with methanol
to prepare secondary stocks and working solutions
to prepare calibration curve (CC) for AMD and
ATL. Another set of secondary and working stock
solutions of AMD and ATL were made in metha-
nol (from primary stock) for preparation of QC
samples. Working stock solutions were stored at
approximately 4°C for a week (data not shown).
Working stocks were used to prepare plasma
calibration standards. A working IS solution of
AMD-d4 (3000 pg/ml) and ATL-d7 (400 ng/ml)
was prepared in methanol. Calibration samples
were prepared by spiking 5 µl of appropriate
working solution (pooled for both analytes) into
495 µl of control human plasma on the day of
ana­lysis. Samples for the determination of pre-
cision and accuracy were prepared by spiking
control human plasma in bulk with AMD and
ATL at appropriate concentrations – for AMD:
50 pg/ml (LLOQ), 150 pg/ml (low quality con-
trol [LQC]), 3000 pg/ml (medium QC [MQC])
and 6500 pg/ml (high QC [HQC]); for ATL:
10 ng/ml (LLOQ), 30 ng/ml (LQC), 300 ng/ml
future science group
| R esearch A rticle
(MQC) and 650 ng/ml (HQC)]; and 200 µl
plasma aliquots were distributed into different
tubes. All the samples were stored at -65 ± 15°C.
„„Sample preparation
EZYPRESS™ 48 position positive pressure
processor (Orochem Technologies Inc., IL, USA)
was employed in the sample preparation. A simple
generic SPE procedure was adopted for the extrac-
tion of AMD and ATL from human plasma. To
an aliquot of 200 µl plasma, 5 µl of IS solution was
added, vortex mixed for 5 s on a vortex mixer and
added to the preconditioned HLB 1 cc 30 mg neu-
tral cartridge. The samples were washed with 1 ml
of water followed by 1 ml of 5% methanol. The
analytes were slowly eluted using 0.5 ml of mobile
phase and 5 µl was injected onto LC–MS/MS
system for ana­lysis.
„„Method validation
A full validation according to the US FDA and
European Medicines Agency guidelines was
performed for the assay in human plasma [28,101] .
„„Specificity & selectivity
The specificity of the method was evaluated by
analyzing human plasma samples from at least six
different lots to investigate the potential interfer-
ences at the LC peak region for analytes and IS.
The acceptance criterion for experiment was that
at least four out of six lots should have <20% area
response to that of the LLOQ level response in
the same matrix.
„„Recovery
The efficiency of AMD, ATL and IS extraction
from human plasma was determined by com-
paring the responses of the analytes extracted
from replicate QC samples (n = 6) with those of
neat standard solutions spiked in post-extracted
plasma blank sample at equivalent concentra-
tions by SPE. Recoveries of AMD and ATL were
determined at LQC, MQC and HQC concen-
trations; that is, for AMD: 150 pg/ml (LQC),
3000 pg/ml (MQC) and 6500 pg/ml (HQC);
for ATL: 30 ng/ml (LQC), 300 ng/ml (MQC)
and 650 ng/ml (HQC); whereas the recovery
of AMD-d4 and ATL-d7 (IS) were determined
at a single concentration of 3000 pg/ml and
400 ng/ml, respectively.
„„Matrix effect
The matrix effect has been investigated to ensure
that precision and accuracy of the method is not
compromised with the co-eluting interferences
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R esearch A rticle | Kallem, Mullangi, Hotha, Ravindranath, Spoorthy & Seshagirirao
present in the biological matrix. Stable-labeled
isotopes (AMD-d 4 and ATL-d7 ) employed in
the method are expected to nullify the matrix
effects. However, a quantitative matrix effect
experiment was performed at the LQC, MQC
and HQC levels. Matrix effect was determined
by comparing the response of the neat analytes
spiked into post-extracted plasma blanks (n = 4)
with that of neat solutions prepared in mobile
phase at equivalent concentrations. Chro-
matographic method and sample preparation
techniques were fine tuned in such a way that
the amount of matrix effect should be ±15%
deviation from the nominal concentration
values.
„„Calibration curve
The seven-point CC for AMD (50, 100, 200,
500, 2000, 5000 and 8000 pg/ml) and ATL
(10, 20, 50, 100, 200, 500 and 800 ng/ml)
was constructed by plotting the peak area ratio
of each analyte:IS against the nominal con-
centration of calibration standards in human
plasma. Following the evaluation of different
weighing factors, the results were fitted to lin-
ear regression ana­lysis with the use of 1/X 2
weighing factor. The CC had to have a cor-
relation coefficient (r) of 0.99 or better. The
acceptance criteria for each back-calculated
standard concentration were ±15% deviation
from the nominal value except at LLOQ, which
was set at ±20% [28] .
„„Precision & accuracy
The intra-assay precision and accuracy were
estimated by analyzing six replicates contain-
ing AMD and ATL at four different QC levels;
that is, for AMD: 50 (LLOQ ), 150 (LQC),
3000 (MQC) and 6500 pg/ml (HQC); for
ATL: 10 (LLOQ), 30 (LQC), 300 (MQC) and
650 ng/ml (HQC) in plasma. The inter-assay
precision was determined by analyzing the four
level QC samples on five different runs. The
acceptance criteria for each back-calculated
standard concentration were 85–115% accu-
racy from the nominal value except at LLOQ,
which was set at 80–120%. A precision of
within ±15% RSD, except for the LLOQ,
where it should not exceed ±20% of RSD [28] .
„„Stability experiments
Stability tests were conducted to evaluate the
AMD and ATL stability in plasma samples
under different conditions. Bench-top stability
(8 h), processed samples stability (auto­sampler
830 Bioanalysis (2013) 5(7)
stability for 24 h at 10°C), freeze–thaw sta-
bility (three cycles) and long-term stability
(30 days at -65 ± 15°C) were performed at
LQC and HQC levels using six replicates at
each level. Samples were considered stable if
assay values were within the acceptable limits
of accuracy (i.e., 85–115% from fresh samples)
and precision (i.e., ±15% RSD) [28] .
„„Dilution integrity
Dilution integrity was investigated to ensure
that samples could be diluted with blank
matrix without affecting the final concentra-
tion. Dilution integrity experiment will be
performed for study sample concentrations
crossing the ULOQ. AMD and ATL spiked
human plasma samples prepared at twofold
above the ULOQ concentration for each of
the analyte; that is, 16000 pg/ml for AMD;
1600 ng/ml for ATL, and diluted fivefold with
human blank plasma to obtain the final test
concentrations of 3200 pg/ml and 320 ng/ml
(n = 4) for AMD and ATL, respectively. The
back-calculated standard concentrations had
to comply to have both precision of <15% and
accuracy of 100 ± 15% similar to other QC
samples [28] .
PK
„„ study
A PK study was performed in healthy male
human volunteers (n = 6). The study proto-
col was approved by the Wellquest Clinical
Research Laboratory’s Institutional ethics com-
mittee and the volunteers provided informed
written consent. Following oral co-administra-
tion of Adopin-AT tablet to human volunteers,
blood samples were collected at predose and 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 24, 36, 48, 72
and 96 h post-dose samples for both AMD and
ATL in K 2EDTA vacutainer collection tubes
(BD, NJ, USA). The tubes were centrifuged
at 4000 rpm for 10 min and the plasma was
collected. The collected samples were stored at
-65 ± 15°C until ana­lysis. An aliquot of 200 µl
of thawed plasma samples were spiked with
both IS and processed as mentioned in the
sample preparation section. Along with clini-
cal samples, QC samples at LQC, MQC and
HQC concentrations were assayed in triplicate,
and were distributed among unknown samples
in the analytical run.
The criteria for acceptance of the analytical
runs encompassed the following: 67% of the
QC samples accuracy must be within 85–115%
of the nominal concentration and not less than
future science group
 Simultaneous quantitation of amlodipine & atenolol in human plasma | R esearch A rticle
50% at each QC concentration level must meet ISR should be less than ±20% of their means
the acceptance criteria. Plasma concentration- for at least 67% of the repeats. Large differences
time data of AMD and ATL were analyzed by between results may indicate analytical issues
noncompartmental method using WinNonlin and should be investigated.
Version 5.1 (Pharsight Corporation, CA, USA).
Results
„„Incurred samples reana­lysis „„LC
The recent European Medicines Agency and The final chromatographic retention and
FDA guidelines have emphasized the necessity appropriate resolution among analytes was
of ensuring incurred sample reproducibility [29] . achieved with the combination of acetoni-
European Medicines Agency 2011 guidelines trile and methanol at 4:6 v/v ratio as mobile
on bioanalytical method validation provided phase A, and 0.01% formic acid in water as
the rationale and procedure for conductance of mobile phase B. An isocratic flow (70:30% v/v)
incurred sample reana­lysis (ISR) [101] . As per of mobile phase A:B was used at 0.5 ml/min
the guidance, 10% of the samples should be flow rate for 3 min. The chromatographic reso-
reanalyzed in case the number of samples is lution of peaks was achieved between AMD
<1000 [101] . Furthermore, it is advised to obtain and ATL, along with their IS, by combining
samples around Cmax and in the elimination appropriate volumes of methanol and acetoni-
phase. As per the guidance, the difference in trile into mobile phase A, rather than any single
concentrations between the initial value and the component. Discovery C18 column was found

A m/z 409.2/238 m/z 413/238 2.19

2.0 1.6
Intensity (cps)

Intensity (cps)

1.5 1.2 2.03

0.29 0.45 1.06 1.74
1.0 0.8
0.5 0.4

0.0 0.0
0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5
Time (min) Time (min)
B
800 0.96 0.97
700 3.5e4
Intensity (cps)

Intensity (cps)
Intensity (cps)

600 3.0e4
500 2.5e4
400 2.0e4
300 1.5e4
200 1.0e4
100 5000
0 0
0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5
Time (min) Time (min)
C
0.97 0.97
3.5e4 3.5e4
Intensity (cps)
Intensity (cps)

Intensity (cps)

3.0e4 3.0e4
2.5e4 2.5e4
2.0e4 2.0e4
1.5e4 1.5e4
1.0e4 1.0e4
5000 5000
0 0
0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5
Time (min) Time (min)

Figure 2. Typical SRM chromatograms. Amlodipine (left panels) and internal standard (right
panels) in (A) human blank plasma; (B) human blank plasma spiked with amlodipine at LLOQ
(50 pg/ml) and internal standard; and (C) a 5 h in vivo plasma sample showing amlodipine peak
obtained following oral dosing of Adopin-AT to humans along with internal standard.

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R esearch A rticle | Kallem, Mullangi, Hotha, Ravindranath, Spoorthy & Seshagirirao

A m/z 267/190.1 m/z 274/190.1

2.5 5

Intensity (cps)

Intensity (cps)
2.0 4
1.5 3
1.0 2
0.5 1
0.0 0
0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5
Time (min) Time (min)
B
800
0.90 0.89
700
Intensity (cps)

Intensity (cps)
600 1.5e 4

500
400 1.0e4
300
200 5000
100
0 0
0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5
Time (min) Time (min)
C
3.5e4 0.88 0.89
3.0e4
Intensity (cps)

Intensity (cps)
2.5e4 1.5e 4

2.0e4
1.0e4
1.5e4
1.0e4 5000
5000
0 0
0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5 2.0 2.5
Time (min) Time (min)

Figure 3. Typical SRM chromatograms. Atenolol (left panels) and internal standard (right panels)
in (A) human blank plasma; (B) human blank plasma spiked with atenolol at LLOQ (10 ng/ml) and
internal standard; and (C) a 5 h in vivo plasma sample showing atenolol peak obtained following
oral dosing of Adopin-AT to humans along with internal standard.

to be suitable with sharp and symmetric peak
shapes among few other columns tested in the
method optimization process (data not shown).
The observed retention times of analytes were
0.97, 0.97, 0.89 and 0.89 min for AMD, AMD-
d 4 , ATL and ATL-d7, respectively. The mea-
sured retention factors (k’) for AMD and ATL
are 4.4 and 3.9, respectively.
„„MS
In order to optimize ESI conditions for AMD,
ATL and IS, quadrupole full scans were carried
out both in positive and negative ion detec-
tion mode and found that good response was
achieved in positive ionization mode. During a
direct infusion experiment, the mass spectra for
AMD and AMD-d4 revealed spectral peaks at
m/z 409.2 and 413, respectively. The parent mass
spectra for ATL and ATL-d7 were found to be
m/z  267 and 274, respectively, as protonated
molecular ions, [M+H]+. Following detailed
optimization of MS conditions (provided in the
‘MS operating conditions’ section) m/z 409.2
precursor ion to the m/z 238 was used for quan-
tification of AMD and m/z 413 precursor ion
to the m/z 238 was used for quantification of
AMD-d4 . Similarly, for ATL m/z 267 precursor
ion to the m/z 190.1 and m/z 274 precursor ion
to the m/z 190.1 was used for quantification of
ATL-d7. Massaroti et al. [12] and Johnson and
Lewis [18] have discussed in detail on fragmen-
tation pattern of AMD and ATL, respectively;
hence, we are not presenting the data pertaining
to this.
„„Recovery
SPE process proved to be robust and provided
cleanest samples. The results of the comparison

832 Bioanalysis (2013) 5(7) future science group

 Simultaneous quantitation of amlodipine & atenolol in human plasma | R esearch A rticle
Table 1. Intra- and inter-day precision and accuracy determination of amlodipine
and atenolol QCs in human plasma.
Theoretical Mean ± SD RSD† Accuracy
concentration (%)
Intra-day variation (six replicates at each concentration)
AMD (50 pg/ml) 48.9 ± 2.06 4.21 97.8
ATL (10 ng/ml) 10.1 ± 1.21 12.00 101.0
AMD (150 pg/ml) 163 ± 6.48 3.98 109.0
ATL (30 ng/ml) 30.9 ± 1.73 5.61 103.0
AMD (3000 pg/ml) 3020 ± 189 6.26 101.0
ATL (300 ng/ml) 305 ± 27.7 9.23 102.0
AMD (6500 pg/ml) 6402 ± 438 6.84 98.5
ATL (650 ng/ml) 633 ± 23.8 3.76 97.4
Inter-day variation (24 replicates at each concentration)
AMD (50 pg/ml) 49.8 ± 4.96 9.96 99.6
ATL (10 ng/ml) 9.67 ± 1.01 10.40 96.7
AMD (150 pg/ml) 160 ± 2.94 1.84 107.0
ATL (30 ng/ml) 30.1 ± 1.11 3.69 100.0
AMD (3000 pg/ml) 2996 ± 312 10.40 100.0
ATL (300 ng/ml) 319 ± 17.8 5.58 106.0
AMD (6500 pg/ml) 6978 ± 263 3.77 107.0
ATL (650 ng/ml) 641 ± 22.0 3.43 98.6
RSD = SD × 100/mean.
†

AMD: Amlodipine; ATL: Atenolol.

of plasma-extracted standards versus the neat
solution spiked into post-extracted blank sam-
ple at equivalent concentration were estimated
for AMD and ATL. The recovery at LQC and
HQC for AMD was 93 ± 13.0 and 92 ± 12.6%,
whereas for ATL it was found to be 95 ± 7.43
and 96 ± 11.21%, respectively. The recovery
of the stable-labeled IS were similar to their
analytes at the tested single concentration level.
„„Specificity & selectivity
F igure   2A–2C show chromatogramsfor the
human blank plasma (free of analytes and
IS), human blank plasma spiked with AMD at
LLOQ, and an in vivo plasma sample obtained
at 5 h after oral administration of Adopin-AT,
respectively. Similarly, F igure  3A–3C show
chromatograms for the human blank plasma
(free of analytes and IS), human blank plasma
spiked with ATL at LLOQ, and an in vivo
plasma sample showing the peak of ATL at
5 h following oral administration of Adopin-
AT, respectively. No interfering peaks from
endo­genous compounds were observed at the
retention times of AMD, ATL and IS in the
matrix. The retention time of AMD, AMD-d4 ,

Table 2. Stability data of amlodipine and atenolol QCs in human plasma.

Nominal Stability AMD ATL
concentration
Mean ± SD †
Accuracy Precision Mean ± SD †
Accuracy Precision
(n = 6) (%) ‡ (% CV) (n = 6) (%) ‡ (% CV)
AMD 0 h (for all) 154 ± 6.51 103 4.23 30.6 ± 2.92 102.0 9.54
(150 pg/ml) 8 h (bench-top) 160 ± 2.94 107 1.84 30.1 ± 1.11 100.0 3.69
ATL 24 h (in-injector) 157 ± 8.48 105 5.40 31.2 ± 2.99 104.0 9.58
(30 ng/ml) 30 days (-65°C) 163 ± 4.90 109 3.01 30.4 ± 2.00 101.0 6.58
AMD 0 h (for all) 7008 ± 295 108 4.21 633 ± 32.00 97.5 5.06
(6500 pg/ml) 8 h (bench-top) 6978 ± 263 107 3.77 601 ± 22.00 92.5 3.66
ATL 24 h (in-injector) 6531± 370 100 5.67 653 ± 43.20 100.0 6.62
(650 ng/ml) 30 days (-65°C) 7039 ± 228 108 3.24 661 ± 42.50 102.0 6.43
Back-calculated plasma concentrations.
†
‡
(Mean assayed concentration/mean assayed concentration at 0 h, [i.e., fresh samples]) × 100.
AMD: Amlodipine; ATL: Atenolol.

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R esearch A rticle | Kallem, Mullangi, Hotha, Ravindranath, Spoorthy & Seshagirirao

A „„Matrix effect
4000 The results of the comparison of analyte peak
responses from neat standard solution spiked
Mean ± SD concentration of AMD

3500
into plasma blank (post-extracted) samples to
in human plasma (pg/ml)

3000 that of neat standard solutions in mobile phase

at equivalent concentration. The results have
2500
shown that the precision and accuracy for ana-
2000 lyzed samples at LQC, MQC and HQC lev-
els were within the acceptance range of ±15%
1500
deviation from the nominal concentration (data
1000 not shown).

500 „„Calibration curve

0 The plasma CC was constructed in the linear
range using seven calibration standards (i.e., 50,
0 20 40 60 80 100
100, 200, 500, 2000, 5000 and 8000 pg/ml
Time (h)
B
for AMD and 10, 20, 50, 100, 200, 500 and
500 800 ng/ml for ATL). The calibration standard
450 curve had a reliable reproducibility over the
standard concentrations across the calibra-
Mean ± SD concentration of ATL

400
tion range. CC was prepared by determining
in human plasma (ng/ml)

350
the best fit of concentration versus peak-area
300 ratios (peak area analyte/peak area correspond-
250 ing deuterated IS), and fitted to the y = mx +
c using weighing factor (1/X 2 ). The average
200
regression (n = 5) was found to be >0.997 for
150 both AMD and ATL. The lowest concentration
100 with the RSD <20% was taken as LLOQ, and
50
was found to be 50 pg/ml and 10 ng/ml for
AMD and ATL, respectively. The percentage
0 accuracy observed for the mean of back-calcu-
-50 lated concentrations for five CCs for AMD and
0 5 10 15 20 25 30 35 40 ATL was within 88.9–107.4 and 92.7–112.1,
Time (h) respectively; while the precision (% CV) values
ranged from 2.46–12.72 and 1.64–13.49 for
Figure 4. Mean ± SD plasma concentration–time profiles. (A) AMD and AMD and ATL, respectively.
(B) ATL in human plasma following oral administration Adopin-AT tablet.
AMD: Amlodipine; ATL: Atenolol. „„Accuracy & precision
Accuracy and precision data for intra- and inter-
ATL and ATL-d7 were approximately 0.97, day plasma samples for AMD and ATL are pre-
0.97, 0.89, 0.89 min, respectively. The total sented in Table 1. The assay values on both the
chromatographic run time was 3 min. The occasions (intra- and inter-day) were found to
specificity of the method was evaluated by be within the accepted variable limits.
analyzing human plasma samples from six dif-
ferent lots to investigate the potential interfer- „„Stability
ences at the LC peak region for analytes and The predicted concentrations for AMD at
IS. No significant response was observed in the 150 and 6500 pg/ml, and for ATL at 30 and
LC region for any of the blank samples ana- 650 ng/ml samples deviated within ±15% of
lyzed; as compared with corresponding LLOQ the fresh sample concentrations in a battery
level response in the same matrix lot (data not of stability tests: in-injector (24 h), bench-
shown). We have also investigated and con- top (8 h), repeated three freeze–thaw cycles
firmed that, in the presence of AMD HQC, and freezer stability at -65 ± 15°C for at least
there was no interference at the retention time 30 days (Table 2) . The results were found to be
of ATL at LLOQ concentration and vice versa within the assay variability limits during the
(data not shown). entire process.

834 Bioanalysis (2013) 5(7) future science group

 Simultaneous quantitation of amlodipine & atenolol in human plasma | R esearch A rticle
„„Dilution effect A
The dilution integrity was confirmed for QC

Deviation from initial value (%)

20
samples that exceeded the upper limit of stan- n = 6 samples at each time point
15
dard CC. The results have shown that the pre-
10
cision and accuracy for five-times diluted test
5
samples were within the acceptance range (data
0
not shown).
-5
-10
„„PK study 4 5 6 24 36
-15
In order to verify the sensitivity and selectivity
of this method in a real-time situation, the pres- -20
ent method was used for the plasma ana­lysis of Time (h)
AMD and ATL obtained from healthy human B

Deviation from initial value (%)

volunteers (n = 6), following oral administration
of Adopin-AT. The mean ± SD plasma concen-
tration versus time of AMD and ATL are shown
in Figure 4, which indicates the suitability of the
proposed method for PK studies of AMD and
ATL in humans. AMD and ATL were detect-
able in human plasma up to 96 and 36 h, respec-
tively. The maximum concentration (Cmax) in
plasma (2.88 ± 0.77 ng/ml) for AMD attained
at 8.17 ± 2.86 h (Tmax). The AUC 0–∞ (area
under the plasma concentration–time curve
from 0 h to infinity) and t1/2 (half-life) values
for AMD were found to be 140 ± 3.47 ng.h/ml
and 47.9 ± 17.4 h, respectively. Similarly for
ATL the Cmax (0.33 ± 0.12 µg/ml) attained at
3.67 ± 0.81 h (Tmax), whereas the AUC0–∞ and
t1/2 were found to be 3.31 ± 0.85 µg.h/ml and
7.77 ± 0.90 h, respectively. The PK param-
eters obtained in this study for AMD and
ATL following co-administration were in close
agreement with earlier reported values for AMD
and ATL individually [30,102] .
„„Incurred sample reana­lysis
For ISR we have selected plasma samples at five
different time points from each subject: three
samples at Cmax range (4, 5 and 6 h) and two sam-
ples from the elimination phase (24 and 36 h).
A total of 30 samples were re-assayed under a
separate batch. As shown in Figure 5A & 5B, the
obtained concentrations from ISR were found
within ±20% of their mean value, indicating
good reproducibility of the current validated
method.
20
n = 6 samples at each time point
15
10
5
0
-5
-10
-15 4 5 6 24 36
-20
Time (h)
Figure 5. Graphical representation of results from incurred sample
reana­lysis – percentage change of incurred sample reana­lysis value from
their initial mean value. (A) Amlodipine and (B) atenolol.
extraction efficiency was not optimum for one of
the analytes due to the differences in logP (logP
for AMD and ATL is 4.16 and 0.5, respectively)
values. Protein precipitation technique was also
tried where the recoveries were found to be less.
Finally, after few logical trials, the SPE tech-
nique gave us the required recoveries (>90%)
for analytes and IS. In order to avoid the matrix
effect and similar recovery efficiency for the IS,
we have utilized corresponding deuterated ISs
in the validation process. Validated methods
are essential for the determination of AMD and
ATL concentrations in human plasma for bio-
equivalence studies. To the best of our knowl-
edge, this is the first report on the simultaneous
ana­lysis of AMD and ATL in human plasma.
The validated method is sensitive, specific,
rugged and rapid, with a short run time of 3 min
for further application.

Discussion
So far there are no published methods available
for the simultaneous quantification of AMD and
ATL in any of the biological matrices. The major
limitation could be the simultaneous extrac-
tion of both analytes due to the differences in
their physicochemical properties. Liquid–liquid
Conclusion
In summary, we have developed and validated
a highly sensitive, specific, reproducible and
high-throughput LC–MS/MS assay to quan-
tify AMD and ATL simultaneously in human
plasma as per regulatory guidelines. The
method showed suitability for PK studies in

future science group www.future-science.com 835

R esearch A rticle | Kallem, Mullangi, Hotha, Ravindranath, Spoorthy & Seshagirirao

humans. The extraction method gave consistent Acknowledgements

and reproducible recoveries for AMD and ATL
from plasma. The simplicity and ruggedness
of the assay and sample turnover rate of 3 min
per sample, make it an attractive procedure in
high-throughput bioana­lysis of AMD and ATL.
From the results of all the validation parameters,
we can conclude that the developed method
can be useful for bioavailability/bioequivalence
studies and in preclinical PK studies, with the
desired precision and accuracy.
Future perspective
We trust that this simultaneous quantification
of AMD and ATL by LC–MS/MS method in
human plasma will have application in bioavail-
ability/bioequivalence studies, and also in pre-
clinical studies, because it is simple, sensitive
and reproducible. This method offers simulta-
neous quantification of two drugs in a single
run with a short run time; hence, it is cost
effective and time-saving. We can anticipate
that our method can be easily extended to other
preclinical species with little or no modification.
The authors gratefully acknowledge Wellquest Clinical
Research Laboratories, Hyderabad, for providing the
necessary facilities for carrying out this study.
Financial & competing interests disclosure
The authors have no relevant affiliations or financial
involvement with any organization or entity with a
financial interest in or financial conflict with the subject
matter or materials discussed in the manuscript. This
includes employment, consultancies, honoraria, stock
ownership or options, expert t­estimony, grants or patents
received or pending, or royalties.
No writing assistance was utilized in the production of
this manuscript.
Ethical conduct of research
The authors state that they have obtained appropriate
insti­tutional review board approval or have followed the
princi­ples outlined in the Declaration of Helsinki for all
human or animal experimental investigations. In
addition, for investi­gations involving human subjects,
informed consent has been obtained from the participants
involved.

Executive summary
Objective
„„ To develop and validate an LC–MS/MS method for simultaneous quantification of amlodipine (AMD) and atenolol (ATL) in human
plasma, and show its application in a clinical PK study.
Experimental
„„ The method utilizes low sample volume (200 µl), SPE extraction, isocratic mobile phase for chromatography, rapid ana­lysis time (3 min)
and good sensitivity.
Results
„„ The method was validated as per regulatory guidelines and the results met the acceptance criteria.
Conclusion
„„ This is the first report on simultaneous quantitation of AMD and ATL in human plasma using an LC–ESI-MS/MS method.
„„ The method was successfully applied in human PK studies to characterize the PK parameters for AMD and ATL postdosing Adopin-AT®
tablet.

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