Hindawi Publishing Corporation
Journal of Analytical Methods in Chemistry
Volume 2012, Article ID 108281, 5 pages
SimultaneousAnalysisof Losartan Potassium, Amlodipine
Besylate,andHydrochlorothiazideinBulkand inTablets by
Department of Pharmaceutical Analysis, SRM College of Pharmacy, SRM University, Tamilnadu,
Kattankulathur 603 203, India
Correspondence should be addressed to Sivasubramanian Lakshmi, firstname.lastname@example.org
Received 22 November 2011; Accepted 23 January 2012
Academic Editor: Peter Stockwell
Copyright © 2012 K. Santhana Lakshmi and S. Lakshmi. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
A Simple high-performance thin layer chromatography (HPTLC) method for separation and quantitative analysis of losartan
potassium, amlodipine, and hydrochlorothiazide in bulk and in pharmaceutical formulations has been established and
validated. After extraction with methanol, sample and standard solutions were applied to silica gel plates and developed with
254nm. The Rfvalues of amlodipine besylate, hydrochlorothiazide, and losartan potassium were 0.35, 0.57, and 0.74, respectively.
Calibration plots were linear in the ranges 500–3000ng per spot for losartan potassium, amlodipine and hydrochlorothiazide, the
correlation coefficients, r, were 0.998, 0.998, and 0.999, respectively. The suitability of this method for quantitative determination
of these compounds was by validation in accordance with the requirements of pharmaceutical regulatory standards. The method
can be used for routine analysis of these drugs in bulk and in formulation.
(1H-tetrazol-5-yl) (biphenyl-4-yl) methyl] imidazole, potas-
sium is a strong nonpeptide antihypertensive agent which
exerts its action by specific blocking of angiotensin II
receptors . It has a gradual long-lasting effect as an anti-
hypertensive. Amlodipine (AML), 3-ethyl 5-methyl (RS) 2-
dihydropyridine-3,5-dicarboxylate is a calcium channel
blocker which inhibits the influx of extracellular calcium
across the myocardial and vascular muscle cell membranes
. Hydrochlorothiazide (HCZ), 6-chloro-3,4-dihydro-2H-
1,2,4-benzothiadiazine-7-sulphonamide 1,1-dioxide, which
is widely used in antihypertensive pharmaceutical prepa-
rations reduces active sodium reabsorption and peripheral
vascular resistance [1–3].
A literature survey reveals that a variety of spectropho-
tometric and chromatographic methods, including UV
derivative, the simultaneous equation method, colorimetric
determination, HPLC, ratio derivative and compensation
technique, and a stability indicating HPLC method, have
been reported for the determination of LOS in pharmaceuti-
cal dosage forms in combination with other drugs [4–14].
Spectrophotometric and chromatographic methods have
been reported for determination of AML, in combination
with other drugs, in bulk and pharmaceutical dosage forms
[15–24]. A variety of methods have been used for the
determination of hydrochlorothiazide [25–34]. No method
has been reported for the simultaneous estimation of LOS,
AML, and HCZ in the combined dosage form.
In recent years TLC has been improved to incorpo-
rate HPTLC-grade stationary phases, automated sample-
application devices, a controlled development environment,
2Journal of Analytical Methods in Chemistry
Table 1: Results from assay of losartan potassium, amlodipine
besylate, and hydrochlorothiazide in Trilopace∗H tablets.
(mg ± SD,
n = 3)
automated development, forced-flow techniques, computer-
controlled densitometry, quantitation, and fully validated
procedures. These features result in methods which are not
only convenient, rapid, robust, and cost effective but also
reproducible, accurate, and reliable. The objective of this
investigation was, therefore, to establish an HPTLC method
for simultaneous estimation of LOS, AML, and HCZ in bulk
and in tablets.
2.1. Materials and Reagents. Analytically pure samples of
losartan potassium, hydrochlorothiazide, and amlodipine
nai, as gift samples and used as working standards, methanol
of HPLC grade from Merck (Mumbai, India), and chloro-
form, acetone, and formic acid of analytical reagent grade
from S.D. Fine Chemicals were used, without purification to
prepare the mobile phase.
A solution containing 1mg/mL losartan potassium,
amlodipine besylate, and hydrochlorothiazide was prepared
by dissolving 10mg of each standard in 10mL methanol and
was used as working standard solution.
2.2. Sample Preparation. Twenty TRILOPACE∗tablets by
Akums Drugs & Pharmaceuticals Ltd containing 50mg
losartan potassium, 5mg amlodipine besylate, and 12.5mg
hydrochlorothiazide were weighed and powdered. An
and 12.5mg of HCZ was transferred to a 50mL volumetric
flask. After addition of 30mL of methanol and sonication
(30min), the solution was diluted to volume with the
same solvent and filtered through a 0.45µ filter (Millipore,
Milford, MA, USA). To this solution known amount of
amlodipine standard (20mg) was added (standard addition
method) as its content is very low in the formulation. This
solution (1.0, 2.0, and 3.0µL containing 1000, 2000, and
3000ng/spot of LOS, 500, 1000, and 1500ng/spot of AML,
and 250, 500, and 750ng/spot of HCZ) was used for assay of
losartan potassium, amlodipine, and hydrochlorothiazide in
2.3. Chromatography. Chromatography was performed on
10cm×10cm aluminium HPTLC plates coated with 0.2mm
layers of silica gel 60 F254(Merck). Samples were applied as
6mm bands by means of a CAMAG (Muttenz Switzerland)
Linomat V automatic sample applicator equipped with a
Table 2: Results from evaluation of precision.
(ng per spot)
n = 3)
n = 3)
between the bands of 14.0mm and the spraying rate of
50nl per second were maintained. Ascending development
of the plate up to a distance of 85mm was performed at
25 ± 2◦C, with chloroform-methanol-acetone-formic acid
7.5:1.3:0.5:0.03 (v/v/v/v) as mobile phase. The develop-
ment was carried out in a CAMAG twin-trough chamber
previously saturated with mobile phase vapour for 20min.
The average development time was 30min. Densitometric
scanning at 254nm was performed with a CAMAG TLC
scanner 3 equipped with CAMAG Wincats software version
1.4.4 using deuterium light source. During scanning process,
the slit dimensions were fixed at 4.00mm ×0.30mm.
3.Results and Discussion
3.1. Validation of the Method. The method was validated in
accordance with ICH guidelines .
3.1.1. Linearity. Different aliquots of standard solution
equivalent to 0.5–3µg of LOS, AML, and HCZ per band
were applied on the precoated TLC plates. The plates were
then developed, dried, and scanned as described above.
Calibration plots were constructed by plotting peak areas
against the corresponding concentration of drugs (ng per
spot). For all three drugs, the detector response was found to
be a linear function of amount in the range 500–3000ng per
spot. The correlation coefficients of all the three drugs were
found to be 0.9988 for LOS, 0.9985 for AML, and 0.9990 for
HCZ, respectively. The average linear regression equations
wereY = 4571.56X+416.48 forLOS,Y = 3229.40X+300.24
for AML, and Y = 5917.03X +422.70 for HCZ.
3.1.2. Sensitivity. The sensitivity of measurement of LOS,
AML, and HCZ was estimated in terms of the limit of
quantitation (LOQ). The smallest amount of each drug
was also detected under the chromatographic conditions in
terms of the limit of detection (LOD). LOQ and LOD were
calculated by use of the equations LOD = 3 × N/B and
LOQ = 10 × N/B, where N is the standard deviation of the
Journal of Analytical Methods in Chemistry3
Table 3: Results from recovery studies.
Brand nameDrug Recovery level (%)
Initial amount (ng)
Amount added (ng)
Losartan potassium (50mg)
Amlodipine besylate (5mg)
peak areas of the drugs, taken as a measure of the noise and
B is the slope of the corresponding calibration plot. LOQ
and LOD for losartan potassium were found to be 0.382 and
0.121µg/spot, respectively. For amlodipine they were 0.584
and 0.188µg/spot, respectively, and for hydrochlorothiazide
they were 0.497 and 0.162µg/spot, respectively.
3.2. Evaluation of Precision for Assay of the Pharmaceutical
Preparation. The amount of losartan potassium, amlodip-
ine, and hydrochlorothiazide in the pharmaceutical prepa-
ration were determined by replicate analysis (n = 3). The
results are reported in Table 1.
Precision was determined by analysis of standard solu-
tions containing concentrations of LOS, AML, and HCZ
covering the entire calibration range. The precision of the
method as intraday variation (CV, %) was determined by
analysis of these solutions three times on the same day.
Interday precision (CV, %) was assessed by analysis of these
solutions on three different days over a period of one week.
The results of the precision studies are shown in Table 2.
3.2.1. Accuracy. An accuracy of the method was determined
by analysis of standard additions at three different levels, that
is, multiple-level recovery studies. The preanalyzed sample
solution (2, 0.5, and 0.2µg/mL of LOS, HCZ, and AML)
was spiked with amounts equivalent to 80, 100, and 120%
of standard drugs. These solutions were reanalysed, and the
recoveries were found to be within the acceptable limits
3.2.2. Specificity. The mobile phase used was found to be
effective in resolving the drugs (Figure 1). The RF values
of losartan potassium, amlodipine, and hydrochlorothiazide
were 0.74, 0.35, and 0.57, respectively. Typical overlaid
absorption spectra of LOS, AML, and HCZ is shown in
Figure 2. Peak purity of the drugs was tested by acquiring
spectra at the peak start (S), peak apex (A), and peak end
losartan potassium r(S,M) = 0.9996 and r(M,E) = 0.9994,
for amlodipine r(S,M) = 0.9994 and r(M,E) = 0.9996, and
for hydrochlorothiazide r(S,M) = 0.9998 and r(M,E) =
0.9997. The results of peak purity ensure the specificity and
Figure 1: Typical densitogram obtained from losartan potassium
(RF = 0.74), amlodipine besylate (RF = 0.35), and hydrochloroth-
iazide (RF = 0.57). Detection was at 254nm and the mobile phase
was chloroform:methanol:acetone:formic acid 7.5:1.3:0.5:0.03
can conclude that no impurities or degradation products
3.2.3. Repeatability. The repeatability of sample preparation
times on a HPTLC plate. After development of plate, peak
height and peak area were recorded for the zones. The CV
(%) of peak height and area were calculated and found to be
0.45 and 0.56, respectively, for LOS, 0.34 and 0.43 for AML,
and 0.67 and 0.32 for HCZ.
The proposed HPTLC method for simultaneous analysis of
losartan potassium, amlodipine, and hydrochlorothiazide in
pharmaceutical dosage forms has been established for the
first time. Use of HPTLC enables analysis of several samples
4 Journal of Analytical Methods in Chemistry
200 220 240 260 280300
320 340 360380
Figure 2: Typical absorption spectra of losartan potassium,
amlodipine besylate, and hydrochlorothiazide.
at the same time. The method is very simple, rapid, and
provides accurate and precise results.
The authors express their gratitude to the Madras Phar-
maceuticals Ltd, Chennai, for the generous gifts of pure
samples of losartan potassium, amlodipine besylate, and
hydrochlorothiazide. The authors thank the management for
providing the facilities to carry out the work.
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 ICH Guidelines Q2B, Validation of Analytical Procedures—