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Original Article

Simultaneous estimation of hydrochlorothiazide, amlodipine,


and losartan in tablet dosage form by RP-HPLC
Anandkumar R. Tengli*, B.M. Gurupadayya, Neeraj Soni
Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS University, S.S. Nagar, Mysore 570015, India
a r t i c l e i n f o
Article history:
Received 23 February 2013
Accepted 8 March 2013
Available online 29 March 2013
Keywords:
HPLC
Hydrochlorothiazide
Amlodipine
Losartan and telmisartan
Simultaneous estimation
a b s t r a c t
A simple, sensitive and specic liquid chromatographic method with UV detection (230 nm)
was developed for the simultaneous estimation of hydrochlorothiazide, amlodipine and
losartan in tablet dosage form and telmisartan as an internal standard. Separation was
achieved witha phenomenex luna 5m CN100R, 250 4.60 mm5 micronsize column, ambient
temperature with a low pressure gradient mode with mobile phase containing acetonitrile,
water and 0.4% of potassium dihydrogen phosphate buffer pH 2.7 adjusted with ortho-
phosphoric acid (45:35:20). The ow rate was 1 mL min
1
and eluent was monitored at
230 nm. The selected chromatographic conditions were found to effectively separate hy-
drochlorothiazide, amlodipine and losartan with retention time of 3.9, 4.9 and 5.8 min
respectively. The linearity range of hydrochlorothiazide, amlodipine and losartan found in
the range of 12.5e62.5 mg ml
1
, 2.5e12.5 mg ml
1
and 50e250 mg ml
1
respectively. The pro-
posed method was found to be accurate, precise, reproducible and specic and it can also be
used for routine quality-control analysis of these drugs in combination tablets.
Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.
1. Introduction
Hydrochlorothiazide (HCT), 6-chloro-3,4-dihydro-2H-1,2,4-
benzothiadiazine-7- sulphonamide-1,1- dioxide is the potent
orally diuretic and antihypertensive agent related to chloro-
thiazide. This inhibits active chloride reabsorption and thus
increases the excretion of sodium chloride and water. There
are many published determination methods for HCT in tablets
using spectrophotometric,
1e3
uorodensistometric,
4
gas and
liquid chromatographic
5e8
polarographic techniques.
9
Amlodipine(AMLO) chemically, 2-[(2-aminoethoxy)methyl]-
4-(2-chlorophenyl)-1, 4- dihydro-6 methyl-3, 5-pyridine dicar-
boxylic acid 3-ethyl, 5-methyl ester, is an antihypertensive and
an antianginal agent in the formof the besylate salt. Its activity
resides mainly in the () isomer, that inhibits transmembrane
inux of calcium ions into vascular smooth muscle. Literature
review reveals various methods for estimation of amlodipine
alone and its combination are by liquid chromatography
coupled with UV,
10,11
voltametric,
12
mass spectrophoto-
metric,
13,14
HPLC,
15e19
HPTLC.
20
Losartan potassium is monopotassium salt of 4-butyl-4-
chloro-1- [[2

-(1H-tetrazole-5-yl)[1,1

-biphenyl]-4-yl]methyl]-
1H-imidazole-5-methanol. It is a selective, competitive
angiotensin II receptor type 1 (AT
1
) receptor antagonist. Los-
artan administration results in a decrease in total peripheral
resistance and cardiac venous return. However, several
methods have been described for the determination of los-
artan potassium drug substance in tablets. Various methods
* Corresponding author. Tel.: 91 (0) 8212548353, 91 (0) 9886658520 (mobile); fax: 91 (0) 8212548359.
E-mail addresses: anandrtengli@gmail.com, anandrtengli@rediffmail.com (A.R. Tengli).
Available online at www.sciencedirect.com
j ournal homepage: www. el sevi er. com/ l ocat e/ i j cas
i nt e r na t i o na l j our na l of c he mi c a l a nd a na l yt i c a l s c i e nc e 4 ( 2 0 1 3 ) 3 3 e3 8
0976-1209/$ e see front matter Copyright 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijcas.2013.03.003
developed are HPLC,
21e24
spectrophotometric,
25e27
capillary
electrophoresis (CE),
28
voltamatric,
29
capillary zone electro-
phoresis HPTLC and liquid chromatography electrospray
ionization tandem mass spectrometry.
A new combination dosage form of HCT, AMLO and LOSAT
is indicated for the treatment and management of hyperten-
sion. The focus of present study was to develop and validate
simple precise.
2. Experimental
2.1. Chemicals and reagents
Working standards, amlodipine and losartan were from Aur-
bindo Laboratories, Hyderabad, India. HCT and TEL were from
Aristo Pharmaceutical Ltd. Mumbai, India. Potassium dihy-
drogen phosphate, Orthophosphoric acid AR grade purchased
from Merck. HPLC solvents like, Acetonitrile, Methanol and
water from Ranchem, Mumbai. The pharmaceutical dosage
formcontaining 12.5 mg HCT, 2.5 mg AMLOand 50 mg LOSAT,
Nusar-AMH 20 tablets (Emcure Pharmaceuticals Ltd.) pur-
chased from a local drug store. Telmisartan which was
employed as an internal standard (IS) was obtained from
Ranbaxy Laboratories, New Delhi.
2.2. Instrumentation
The development and validation of the assay was performed
on a Shimadzu LC2010 integrator a 4-liquid gradient HPLC
system (Kyoto Japan), provided with high speed auto sampler,
column oven, degasser and UV detector to provide a compact
and convenient for LC with LC solution soft ware chromato-
graphic analysis were performed on phenomenex luna 5m CN
100R, 250 4.60 mm 5 micron size column. The ow rate was
1 mL min
1
and the injection volume 20 ml, UV detection was
performed at 230 nm. Peak identity was conrmed by both
retention time comparison and comparison spectra obtained
from the UV detector.
2.3. Standard preparation
Separate stock solutions of 1000 mg mL
1
of Hydrochlorothia-
zide, amlodipine and losartan were prepared in Acetonitrile,
then 1 mL of stock solution into a 10 mL of standard volu-
metric ask and diluted with mobile phase. The prepared
stock solutions were stored at 4

C protected from light.
2.4. Preparation of calibration plot
Standard solutions were freshly prepared from the stock solu-
tion by diluting with mobile phase as 2, 4, 6, 8, 10 mg mL
1
Hy-
drochlorothiazide, 5, 10, 15, 20, 25mgmL
1
amlodipineand3, 6, 9,
12, 15 mg mL
1
losartan and IS (internal standard), respectively.
Each solution was injected in triplicate and chromatographed
under the chromatographic conditions specied above. Telmi-
sartan (8 mg mL
1
) was used as internal standard for determi-
nation of mixtures of Hydrochlorothiazide, amlodipine and
losartan with Telmisartan. Linear relationships were obtained
when drug to- internal standard peak area ratios were plotted
against the corresponding concentrations for each drug.
2.5. Sample preparation
Average weight was calculated by weighing 20 tablets. The
tablets were crushed into homogenous powder. A quantity of
powder equivalent to one tablet containing 12.5 mg of hy-
drochlorothiazide, 2.5 mg of amlodipine and 50 mg of losartan
was transferred into a 100 mL volumetric ask. To this ask,
50 mL of Methanol were added, and the solution was soni-
cated for 25 min with intermittent shaking. The solution was
cooled to ambient temperature. Then the volume was made
up with Acetonitrile and centrifuged at 10,000 rpm for 10 min.
The centrifuged solution ltered through a 0.45 mm Nylon l-
ters (Millipore, Milford, MA, USA). From the ltered solution,
aliquots of appropriate volume were transferred to 10 mL
volumetric asks and diluted to volume with mobile phase to
furnish the concentration range listed in Table 1
3. Method validation
3.1. Linear range
The linearity of the method was evaluated by analyzing
different concentration of the drugs. According to ICH rec-
ommendations,
35
at least ve concentrations must be used. In
this study ve concentrations were chosen, in the ranges
12.5e62.5, 2.5e12.5 and 50e250 mg mL
1
hydrochlorothiazide,
amlodipine and losartan, respectively.
3.2. Accuracy and precision
The accuracy of the method was determined by recovery ex-
periments using the standard addition method. Each solution
was injected in triplicate and percentage recovery was
Table 1 e Results of system suitability study.
Hydrochlorothiazide Amlodipine Losartan Telmisartan (IS)
Retention
time (min)
Peak area Retention
time (min)
Peak area Retention
time (min)
Peak area Retention
time (min)
Peak area
Mean 3.99 2,439,961 4.86 2,788,379 5.83 3,363,027 7.24 6,519,518
SD 0.016 1,308,330 0.040 1,293,304 0.031 1,637,458 0.0609 15910
RSD 0.408 53.620 0.827 46.381 0.537 48.689 0.853 0.24403
i nt e r na t i o na l j o ur na l o f c he mi c a l a nd a na l y t i c a l s c i e nc e 4 ( 2 0 1 3 ) 3 3 e3 8 34
calculated. The precision of the method was assessed by
studying intra-day and inter-day variation. In the intra-day
studies, standard and sample solutions were analyzed in
triplicate on the same day and percentage RSDwas calculated.
In the inter-day studies, standard and sample solutions were
analysed in triplicate on three consecutive days and per-
centage RSD were calculated.
3.3. Limits of detection (LOD) and limit quantitation
(LOQ)
In accordance with ICH recommendations, the approach
based on the standard deviation of the response and the slope
of the calibration plots was used to determine detection and
quantication limits. LOD and LOQ values were estimated as
[(standard deviation of repeatability)/(Slope of the regression
equation)] by multiplying with 3.3 and 10 respectively. The
values obtained are given in Table 1.
3.4. Selectivity
The selectivity of the method was evaluated by assessing
whether excipients present in the pharmaceutical formula-
tions interfered with the analysis. A placebo for each tablet
was prepared by mixing the respective excipients, and solu-
tions were prepared by following the procedure described in
the section on sample preparation. The commonly used tablet
excipients did not interfere with the method.
3.5. Robustness
Robustness is a measure of capacity of analytical methods to
remain unaffected by small but deliberate variation of the
operating conditions. This was tested by studying the effect of
changing mobile phase pHby 0.2, the amount of buffer in the
mobile phase by 2%, and detector wavelength by 2 nm.
4. Results and discussion
To establish and validate an efcient method for analysis of
these drugs in pharmaceutical formulations, preliminary tests
were performed with the objective of selecting optimum
chromatographic conditions. The separation was tried using
either columns described previously in the literature or alter-
native stationary phases. The main problems encountered
during these investigations were lack of resolution between
hydrochlorothiazide, Amlodipine, Losartan and IS. To solve
these problems, three columns, C18, C8, and CN, were used for
simultaneous determination of the drugs. The best resolution
and peak shape, without excessive tailing, were obtained by
use of the CN column. The effect of both mobile phase
composition, ow rate and pH were also studied. The best
resolution with reasonable retention time was obtained with
mobile phase containing Acetonitrile, water and 0.4% of po-
tassium dihydrogen phosphate buffer pH 2.7 adjusted with
orthophosphoric acid (45:35:20) with 1.0 mL min
1
ow rate.
To avoid multiple peaks of peptides on reversed phase col-
umns, the pH must be controlled with buffers, for example
potassium dihydrogen phosphate. A major reason for using a
concentration of 0.5 mM was to achieve maximum sensitivity
of UV detection at low wavelengths. Members of this class of
ACE inhibitor contain weak benzene chromophores and are
characterized by low molar absorptivity, so the detector was
set at 230 nm to increase the sensitivity of the method. The
specicity of the method is illustrated in (Figs. 1 & 2), which
indicates separationof the compounds was complete. Average
retention times standard deviation for IS, HCT, AMLO and
LOSAT were 3.9 0.016, 4.9 0.040, 5.8 0.031, and
7.2 0.060 min, respectively, for six replicate analyses. In
determination of accuracy and precision, recovery was
100 2%, which indicates the method is accurate, and intra-
day and inter-day variation, as RSD, were no more than
1.25%, indicating the method is precise. In determination of
the robustness of the method, slight variation of mobile phase
pH, amount of buffer, in the mobile phase, and detector
wavelength had no signicant effect on chromatographic
resolution.
4.1. Method validation
4.1.1. System suitability
TheRSDvalues of peakareaandretentiontimefor drugs andIS
are within 2%indicating the suitability of the system(Table 2).
Fig. 1 e HPLC Chromatogram of pure drug.
Fig. 2 e HPLC Chromatogram of tablet formulation.
i nt e r na t i o na l j our na l of c he mi c a l a nd a na l yt i c a l s c i e nc e 4 ( 2 0 1 3 ) 3 3 e3 8 35
4.1.2. Linearity
The calibration curves were prepared by plotting the peak
areas of the drug to IS which were linear in the range of
12.5e62.5, 2.5e12.5 and 50e250 mg mL
1
HCT, AMLO and
LOSAT, respectively. Peak area ratios and concentrations were
subjectedto least square linear regressionanalysis to calculate
the calibration equations and correlation coefcients. The
mean regression equations were found as y 0.125x 0.126
(R
2
0.990, n 6), y 0.132x 0.106 (R
2
0.990, n 6) and
y 0.163x 0.143 (R
2
0.991, n 6) for HCT, AMLOand LOSAT,
respectively. y ax b where y is the peakarea ratioof drugs,
a is the slope, b is the intercept and x is the concentration
of the measured solution in mg mL
1
. The result shows that
there is an excellent correlation between the peak area ratios
and the concentrations of drugs in the range tested.
4.1.3. LOD and LOQ
The LODwas 0.03 mg mL
1
for HCT, 0.03 mg mL
1
for AMLOand
0.108 mg mL
1
for LOSAT at a signal to noise ratio of 3.1. The
limit of quantication was determined as 0.1 HCT, 0.1 mg mL
1
for AMLOand 0.228 mg mL
1
for LOSATat a signal to noise ratio
of 10:1.
4.1.4. Precision
Intra-day precision was performed by relative standard devi-
ation of ve repeated assayes of samples at the three
concentration levels. Inter-day precision was determined by
analyzing the same set of samples of ve different days. The
RSD values were found to be 0.117e0.945% for HCT,
0.337e1.599% for AMLO and 0.128e1.310% for LOSAT respec-
tively, indicating good precision (Table 2).
4.1.5. Recovery
To examine the accuracy of the method, recovery studies
were carried out by standard addition method. The percent
recovery of the added standard to the assay samples was
calculated from:
Recovery % C
1
C
u
=C
a
100
Were C
1
is the total concentration of analyte found; C
u
is the
concentration analyte present in the formulation; and C
a
is
the concentration added to the formulation. The average
percent recoveries recoveries obtained as 99.03e99.90% indi-
cate good accuracy of the method (Table 3)
4.1.6. Specicity
The specicity of the RP-HPLC method was determined by the
complete separation of HCT, AMLO, LOSAT and IS as show in
(Figs. 1 & 2) with parameters like retention time (t
R
), resolu-
tion (R
s
) and tailing factor (T ). The peaks obtained for HCT,
AMLO, LOSAT and IS were sharp and have a clear baseline
separation.
Table 2 e Intra-day and inter-day precision and accuracy of HCT, RPL and TEL.
Name of
the drug
Actual
concentration
(mg mL
1
)
Intra-day Inter-day
Found concentration
(mg mL
1
) SD
RSD (%) RME (%) Found concentration
(mg mL
1
) SD
RSD (%) RME (%)
HCT 13 12.77 0.119 0.933 0.417 12.788 0.083 0.648 0.290
25.5 25.39 0.097 0.385 0.172 25.562 0.241 0.945 0.423
37.5 37.42 0.063 0.168 0.075 37.444 0.044 0.117 0.052
AMLO 2.5 2.458 0.038 1.560 0.698 2.482 0.008 0.337 0.151
5 4.876 0.031 0.642 0.287 4.876 0.078 1.599 0.715
7.5 7.452 0.043 0.580 0.260 7.442 0.071 0.952 0.426
LOSAT 50 49.598 0.377 0.760 0.340 49.584 0.650 1.310 0.586
100 99.678 0.231 0.232 0.104 99.792 0.139 0.140 0.062
150 149.698 0.191 0.128 0.057 149.574 0.375 0.251 0.112
Table 3 e Results of recovery studies by standard addition method.
Name of the drug Amount of drug
in tablet (mg)
a
Amount of pure
drug added (mg)
Total found
(mg)
b
(mean SD
c
)
RSD (%) Recovery of pure
drug added (%)
HCT 37.5 37 74.39 0.045 0.061 99.70
37.5 37.5 74.948 0.046 0.061 98.37
37.5 38 75.45 0.0391 0.051 99.88
AMLO 7.5 7 14.45 0.026 0.179 99.31
7.5 7.5 14.92 0.049 0.326 98.96
7.5 8 15.45 0.023 0.148 99.47
LOSAT 150 145 294.98 0.132 0.045 99.89
150 150 299.9 0.079 0.026 99.93
150 155 304.92 0.061 0.020 99.94
a Nusar-AMH tablet (12.5 mg of HCT, 2.5 mg AMLO, 50 mg LOSAT).
b Five independent analyses.
c Standard deviation.
i nt e r na t i o na l j o ur na l o f c he mi c a l a nd a na l y t i c a l s c i e nc e 4 ( 2 0 1 3 ) 3 3 e3 8 36
4.1.7. Robustness
To ensure the insensitivity of the HPLC method to minor
changes in the experimental conditions it is important to
demonstrate robustness of the method. None of the modi-
cations caused a signicant change in the resolution between
the drugs and IS, peak area RSD, USP tailing factor, peak width
or theoretical plates.
HPLC Chromatograms.
5. Conclusion
A simple, rapid, and reliable LC method has been established
for simultaneous determination of HCT, AMLO and LOSAT
either alone or in their ternary formulations. The method has
several advantages, including rapid analysis, a simple mobile
phase, simple sample preparation, and improved sensitivity.
It is suitable for analysis of these antihypertensive agents in
their ternary formulations in a single isocratic run, in contrast
with previous methods. This makes the method suitable for
routine analysis in quality-control laboratories.
Conicts of interest
All authors have none to declare.
Acknowledgement
The authors would like to thank JSS University Mysore, India,
Principal, administrative ofcer JSS college of Pharmacy
Mysore, for providing all facilities to complete this research
work.
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