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

validation work as we felt that this simultaneous


H estimation method would help the researchers, as
H3C N NH2 H D D
O H3C N NH2
both the drugs used in this method are available
O in the market as a fixed-dose combination, and
H3COOC COOCH2CH3 D D
H3COOC COOCH2CH3 has applications in PK and bioequivalence stud-
Cl
Cl ies. The present work describes a simple, selec-
tive, rapid and sensitive method, which employs
the SPE technique for sample preparation and
Amlodipine (AMD) Amlodipine-d4 (AMD-d4) LC–ESI-MS/MS for simultaneous quantitation
of AMD and ATL, using deuterated correspond-
ing internal standards (IS) in human plasma. The
OH
O
H
N CH3
OH application of this assay method to an oral PK
H
O
O
O N CD3 study in male human healthy volunteers follow-
CH3 D ing fixed-dose combination of AMD and ATL
H2N CD3
H2N (Adopin-AT) is described.
Atenolol (ATL) Atenolol-d7 (ATL-d7)
Experimental
Figure 1. Structural representation of amlodipine, atenolol, „„Chemicals & materials
amlodipine-d4 and atenolol-d7. AMD besylate (purity 99.8%) was procured
from USP (Hyderabad, India) and ATL (purity
99.7%), AMD-d4 (purity 98.4%) and ATL-d7
metabolism in liver. The plasma protein bind- (purity 98.8%) from Sigma-Aldrich Corporation
ing is 6–16%. ATL mean elimination half-life is (Bangalore, India). HLB 1 cc 30-mg cartridges
6–7 h. The oral bioavailability was found to be were procured from Waters India (Bangalore,
Key Terms between 40–50% [6] . In clinical trials, in patients India). HPLC grade acetonitrile and methanol
Hypertension: Also called with hyper­tension, AMD plus ATL fixed-dose were purchased from Rankem, Ranbaxy Fine
high blood pressure. combination was significantly more effective Chemicals Limited (New Delhi, India). Ana-
Hypertension is a chronic than a placebo and the respective monothera- lytical grade formic acid was purchased from
condition in which arteries
pies administered at the same dosages [7,8] . A few SD Fine Chemicals (Mumbai, India). All other
(pumps out the blood from the
heart to the whole body) have commercially available fixed-dose combinations chemicals and reagents were of analytical grade
persistent elevated blood of AMD and ATL (5 mg AMD and 50 mg ATL) and used without further purification. The
pressure. are Acard®, Acord-A®, Adipin Plus®, Adorant®, control human K 2EDTA plasma sample was
b1-blockers: Also known as Adipine-AT®, Alodip-AT®, Aglodepin-AT®, procured from Cauvery Diagnostics and Blood
b1-adrenergic blockers, which Adopin-AT® and so on. Bank (Secunderabad, India).
block the binding of epinephrine
A thorough literature review revealed that sev-
and norepinephrine in the heart
and reduce the heart rate and eral LC–MS/MS methods have been published „„Instrumentation & chromatographic
blood pressure by dilating the for the determination of AMD and ATL individ- conditions
blood vessels. ually (for AMD [9–16] and for ATL [17]), or along A Shimadzu HT (Shimadzu, Japan) LC system
Calcium channel blockers: with other drug(s) (ATL with other drugs [18–22] equipped with degasser (DGU-20A5), binary
Calcium channel slows down the and AMD with other drugs [23–27]) in various bio- pump (LC-20AD) along with autosampler
entry of calcium ions into heart
cells and blood vessel walls,
logical matrices. It is important to develop a sensi- (SIL-HTC) was used to inject 20-µl aliquots
which make it easier for heart tive, rapid and reliable bioanalytical method for of the processed samples on a Discovery C18
to pump blood and blood simultaneous quantitation of two or more drugs column (50 × 4.6 mm, 5 µm), Supelco (MO,
vessels to dilate. As a result, in combination therapy in human plasma. To USA), which was maintained at 40 ± 2°C in
there is no strain on heart and
blood pressure lowers.
date there is no sensitive method published for column oven (CTO-10ASvp). The isocratic
the simultaneous estimation of AMD and ATL mobile phase, a mixture of 0.01% formic acid
Fixed-dose combination:
Formulation of two or more
in any biological matrix. It is very challenging to in water and methanol:acetonitrile (6:4, v/v) at
drugs in a single dosage form, develop a simultaneous method for the estimation a ratio of 30:70 (v/v) was delivered at a flow-rate
available in fixed-dose of two drugs, whose LLOQ difference was 200- of 0.5 ml/min into the MS-ESI chamber. The
combination. Fixed-dose fold (LLOQ for AMD and ATL was 50 pg/ml mobile phase was filtered through a 0.45 µm
combination improves the
medication compliance, and
and 10 ng/ml, respectively) and have logP (logP membrane filter (XI5522050) (Millipore, MA,
lower doses of individual drugs for AMD and ATL is 4.16 and 0.5, respectively) USA) and then degassed ultrasonically for 5 min
are administered to produce the values, which pose challenges during the sample before being used for ana­lysis.
beneficial effect than the higher clean-up process for optimal recovery. Accounting Quantitation was achieved by MS/MS
doses of individual drugs.
for these challenges, we took up this method detection in positive ion mode for analytes and IS

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Simultaneous quantitation of amlodipine & atenolol in human plasma | R esearch A rticle
using a MDS Sciex (CA, USA) API-4000 mass (MQC) and 650 ng/ml (HQC)]; and 200 µl
spectrometer, equipped with a TurboionsprayTM plasma aliquots were distributed into different
interface at 550°C temperature and 5500 V ion tubes. All the samples were stored at -65 ± 15°C.
spray voltage. The source parameters (i.e., cur-
tain gas, GS1 and GS2) were set at 25, 45 and „„Sample preparation
50 psi. The compound parameters – decluster- EZYPRESS™ 48 position positive pressure
ing potential, entrance potential, collision energy processor (Orochem Technologies Inc., IL, USA)
and collision cell exit potential – for AMD and was employed in the sample preparation. A simple
AMD-d4 were similar and were 38, 10, 17 and generic SPE procedure was adopted for the extrac-
7 V. For ATL and ATL-d7, the declustering tion of AMD and ATL from human plasma. To
potential, entrance potential, collision energy an aliquot of 200 µl plasma, 5 µl of IS solution was
and collision cell exit potential were 52, 10, 25, added, vortex mixed for 5 s on a vortex mixer and
and 12 V, respectively. Detection of the ions was added to the preconditioned HLB 1 cc 30 mg neu-
performed in the SRM mode, monitoring the tral cartridge. The samples were washed with 1 ml
transition of the m/z 409.2 precursor ion to the of water followed by 1 ml of 5% methanol. The
m/z 238 product ion for AMD, and m/z 413/238 analytes were slowly eluted using 0.5 ml of mobile
for AMD-d4 . ATL was monitored with m/z 267 phase and 5 µl was injected onto LC–MS/MS
precursor ion to the m/z 190.1 product ion and system for ana­lysis.
m/z 274/190.1 for ATL- d7. Quadrupole Q1 and
Q3 were set on unit resolution. The dwell time „„Method validation
was 200 msec. The analytical data were processed A full validation according to the US FDA and
by Analyst software (version 1.5.2). European Medicines Agency guidelines was
performed for the assay in human plasma [28,101] .
„„Standard solutions
AMD, ATL and their corresponding IS were „„Specificity & selectivity
weighed accurately into volumetric flasks using The specificity of the method was evaluated by
an analytical micro balance. They were then analyzing human plasma samples from at least six
diluted with methanol. The primary stock solu- different lots to investigate the potential interfer-
tions of AMD and ATL, and both IS solutions ences at the LC peak region for analytes and IS.
were prepared at 0.5 mg/ml. The stock solutions The acceptance criterion for experiment was that
of AMD, ATL and ISs were stored at 4°C, which at least four out of six lots should have <20% area
were found to be stable for 28 days (data not response to that of the LLOQ level response in
shown) and successively diluted with methanol the same matrix.
to prepare secondary stocks and working solutions
to prepare calibration curve (CC) for AMD and „„Recovery
ATL. Another set of secondary and working stock The efficiency of AMD, ATL and IS extraction
solutions of AMD and ATL were made in metha- from human plasma was determined by com-
nol (from primary stock) for preparation of QC paring the responses of the analytes extracted
samples. Working stock solutions were stored at from replicate QC samples (n = 6) with those of
approximately 4°C for a week (data not shown). neat standard solutions spiked in post-extracted
Working stocks were used to prepare plasma plasma blank sample at equivalent concentra-
calibration standards. A working IS solution of tions by SPE. Recoveries of AMD and ATL were
AMD-d4 (3000 pg/ml) and ATL-d7 (400 ng/ml) determined at LQC, MQC and HQC concen-
was prepared in methanol. Calibration samples trations; that is, for AMD: 150 pg/ml (LQC),
were prepared by spiking 5 µl of appropriate 3000 pg/ml (MQC) and 6500 pg/ml (HQC);
working solution (pooled for both analytes) into for ATL: 30 ng/ml (LQC), 300 ng/ml (MQC)
495 µl of control human plasma on the day of and 650 ng/ml (HQC); whereas the recovery
ana­lysis. Samples for the determination of pre- of AMD-d4 and ATL-d7 (IS) were determined
cision and accuracy were prepared by spiking at a single concentration of 3000 pg/ml and
control human plasma in bulk with AMD and 400 ng/ml, respectively.
ATL at appropriate concentrations – for AMD:
50 pg/ml (LLOQ), 150 pg/ml (low quality con- „„Matrix effect
trol [LQC]), 3000 pg/ml (medium QC [MQC]) The matrix effect has been investigated to ensure
and 6500 pg/ml (high QC [HQC]); for ATL: that precision and accuracy of the method is not
10 ng/ml (LLOQ), 30 ng/ml (LQC), 300 ng/ml 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 stability for 24 h at 10°C), freeze–thaw sta-
isotopes (AMD-d 4 and ATL-d7 ) employed in bility (three cycles) and long-term stability
the method are expected to nullify the matrix (30 days at -65 ± 15°C) were performed at
effects. However, a quantitative matrix effect LQC and HQC levels using six replicates at
experiment was performed at the LQC, MQC each level. Samples were considered stable if
and HQC levels. Matrix effect was determined assay values were within the acceptable limits
by comparing the response of the neat analytes of accuracy (i.e., 85–115% from fresh samples)
spiked into post-extracted plasma blanks (n = 4) and precision (i.e., ±15% RSD) [28] .
with that of neat solutions prepared in mobile
phase at equivalent concentrations. Chro- „„Dilution integrity
matographic method and sample preparation Dilution integrity was investigated to ensure
techniques were fine tuned in such a way that that samples could be diluted with blank
the amount of matrix effect should be ±15% matrix without affecting the final concentra-
deviation from the nominal concentration tion. Dilution integrity experiment will be
values. performed for study sample concentrations
crossing the ULOQ. AMD and ATL spiked
„„Calibration curve human plasma samples prepared at twofold
The seven-point CC for AMD (50, 100, 200, above the ULOQ concentration for each of
500, 2000, 5000 and 8000 pg/ml) and ATL the analyte; that is, 16000 pg/ml for AMD;
(10, 20, 50, 100, 200, 500 and 800 ng/ml) 1600 ng/ml for ATL, and diluted fivefold with
was constructed by plotting the peak area ratio human blank plasma to obtain the final test
of each analyte:IS against the nominal con- concentrations of 3200 pg/ml and 320 ng/ml
centration of calibration standards in human (n = 4) for AMD and ATL, respectively. The
plasma. Following the evaluation of different back-calculated standard concentrations had
weighing factors, the results were fitted to lin- to comply to have both precision of <15% and
ear regression ana­lysis with the use of 1/X 2 accuracy of 100 ± 15% similar to other QC
weighing factor. The CC had to have a cor- samples [28] .
relation coefficient (r) of 0.99 or better. The
acceptance criteria for each back-calculated PK
„„ study
standard concentration were ±15% deviation A PK study was performed in healthy male
from the nominal value except at LLOQ, which human volunteers (n = 6). The study proto-
was set at ±20% [28] . col was approved by the Wellquest Clinical
Research Laboratory’s Institutional ethics com-
„„Precision & accuracy mittee and the volunteers provided informed
The intra-assay precision and accuracy were written consent. Following oral co-administra-
estimated by analyzing six replicates contain- tion of Adopin-AT tablet to human volunteers,
ing AMD and ATL at four different QC levels; blood samples were collected at predose and 1,
that is, for AMD: 50 (LLOQ ), 150 (LQC), 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 24, 36, 48, 72
3000 (MQC) and 6500 pg/ml (HQC); for and 96 h post-dose samples for both AMD and
ATL: 10 (LLOQ), 30 (LQC), 300 (MQC) and ATL in K 2EDTA vacutainer collection tubes
650 ng/ml (HQC) in plasma. The inter-assay (BD, NJ, USA). The tubes were centrifuged
precision was determined by analyzing the four at 4000 rpm for 10 min and the plasma was
level QC samples on five different runs. The collected. The collected samples were stored at
acceptance criteria for each back-calculated -65 ± 15°C until ana­lysis. An aliquot of 200 µl
standard concentration were 85–115% accu- of thawed plasma samples were spiked with
racy from the nominal value except at LLOQ, both IS and processed as mentioned in the
which was set at 80–120%. A precision of sample preparation section. Along with clini-
within ±15% RSD, except for the LLOQ, cal samples, QC samples at LQC, MQC and
where it should not exceed ±20% of RSD [28] . HQC concentrations were assayed in triplicate,
and were distributed among unknown samples
„„Stability experiments in the analytical run.
Stability tests were conducted to evaluate the The criteria for acceptance of the analytical
AMD and ATL stability in plasma samples runs encompassed the following: 67% of the
under different conditions. Bench-top stability QC samples accuracy must be within 85–115%
(8 h), processed samples stability (auto­sampler of the nominal concentration and not less than

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

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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 IS), human blank plasma spiked with AMD at
solution spiked into post-extracted blank sam- LLOQ, and an in vivo plasma sample obtained
ple at equivalent concentration were estimated at 5 h after oral administration of Adopin-AT,
for AMD and ATL. The recovery at LQC and respectively. Similarly, F igure  3A–3C show
HQC for AMD was 93 ± 13.0 and 92 ± 12.6%, chromatograms for the human blank plasma
whereas for ATL it was found to be 95 ± 7.43 (free of analytes and IS), human blank plasma
and 96 ± 11.21%, respectively. The recovery spiked with ATL at LLOQ, and an in vivo
of the stable-labeled IS were similar to their plasma sample showing the peak of ATL at
analytes at the tested single concentration level. 5 h following oral administration of Adopin-
AT, respectively. No interfering peaks from
„„Specificity & selectivity endo­genous compounds were observed at the
F igure   2A–2C show chromatogramsfor the retention times of AMD, ATL and IS in the
human blank plasma (free of analytes and 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 20
n = 6 samples at each time point
of Adopin-AT. The mean ± SD plasma concen- 15
tration versus time of AMD and ATL are shown 10
in Figure 4, which indicates the suitability of the 5
proposed method for PK studies of AMD and 0
ATL in humans. AMD and ATL were detect- -5
able in human plasma up to 96 and 36 h, respec- -10
tively. The maximum concentration (Cmax) in -15 4 5 6 24 36
plasma (2.88 ± 0.77 ng/ml) for AMD attained -20
at 8.17 ± 2.86 h (Tmax). The AUC 0–∞ (area Time (h)
under the plasma concentration–time curve
from 0 h to infinity) and t1/2 (half-life) values Figure 5. Graphical representation of results from incurred sample
for AMD were found to be 140 ± 3.47 ng.h/ml reana­lysis – percentage change of incurred sample reana­lysis value from
and 47.9 ± 17.4 h, respectively. Similarly for their initial mean value. (A) Amlodipine and (B) atenolol.
ATL the Cmax (0.33 ± 0.12 µg/ml) attained at
3.67 ± 0.81 h (Tmax), whereas the AUC0–∞ and extraction efficiency was not optimum for one of
t1/2 were found to be 3.31 ± 0.85 µg.h/ml and the analytes due to the differences in logP (logP
7.77 ± 0.90 h, respectively. The PK param- for AMD and ATL is 4.16 and 0.5, respectively)
eters obtained in this study for AMD and values. Protein precipitation technique was also
ATL following co-administration were in close tried where the recoveries were found to be less.
agreement with earlier reported values for AMD Finally, after few logical trials, the SPE tech-
and ATL individually [30,102] . nique gave us the required recoveries (>90%)
for analytes and IS. In order to avoid the matrix
„„Incurred sample reana­lysis effect and similar recovery efficiency for the IS,
For ISR we have selected plasma samples at five we have utilized corresponding deuterated ISs
different time points from each subject: three in the validation process. Validated methods
samples at Cmax range (4, 5 and 6 h) and two sam- are essential for the determination of AMD and
ples from the elimination phase (24 and 36 h). ATL concentrations in human plasma for bio-
A total of 30 samples were re-assayed under a equivalence studies. To the best of our knowl-
separate batch. As shown in Figure 5A & 5B, the edge, this is the first report on the simultaneous
obtained concentrations from ISR were found ana­lysis of AMD and ATL in human plasma.
within ±20% of their mean value, indicating The validated method is sensitive, specific,
good reproducibility of the current validated rugged and rapid, with a short run time of 3 min
method. for further application.

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