Application of Validated HPTLC Method for Dissolution
Study of FDC Tablets Containing Telmisartan and Hydrochlorothiazide
Hamid Khan1* Mushir Ali1, Alka
Ahuja1, Javed Ali1, Sayeed Ahmad2
1Associate
Professor, JK College of Pharmacy, Bilaspur, CG, India-495001
1Department
of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi,
India-110062
2Department
of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Hamdard University,
New Delhi-110062, India.
*Corresponding Author E-mail: khanhamid770@gmail.com
ABSTRACT:
A
simple, selective, precise and HPTLC method for simultaneous determination of
telmisartan and hydrochlorothiazide in the bulk drugs and in their fixed dose
combination tablets (FDC) was developed and validated. The method was further
applied in the dissolution study of formulated and marketed tablets containing
these two drugs. The method employed HPTLC aluminium plates precoated with silica
gel 60 F254 as the stationary phase. The solvent system consisted of
toluene-ethyl acetate-methanol-glacial acetic acid, 6: 3: 1: 0.5 (v/v).
The detection was performed at 272 nm. The RF values were
0.70 ± 2 for telmisartan and 0.44 ± 2 for hydrochlorothiazide. The linear
relationships were obtained between peak area and amount of drugs in the range
of 1-1000 ng spot-1 with correlation coefficient of 0.9992 for
telmisartan and 0.9994 for hydrochlorothiazide. The method was validated for
precision, robustness and recovery. The limits of detection and quantification
were 15.54 and 48.44 ng spot-1 for telmisartan and 10.22 and 35.68
ng spot-1 for hydrochlorothiazide, respectively. In-vitro drug
release from prepared tablets showed better drug release when compared with
marketed tablets.
KEYWORDS: Telmisartan,
Hydrochlorothiazide, HPTLC, Dissolution study.
INTRODUCTION:
Telmisartan
is 4-{[4-Methyl-6-(1-methyl-2-benzimidazolyl)-2-propyl-1-benzimidazolyl]
methyl}-2-biphenylcarboxylic acid and an antihypertensive drug [1].
Hydrochlorothiazide is 6-chloro-3, 4-dihydro-2H-1, 2,
4-benzothiazine-7-sulphonamide-1, 1-dioxide and widely used thiazide diuretic
[2]. The fixed dose combination tablets containing telmisartan and
hydrochlorothiazide are used for treatment of mild to moderate hypertension.
Literature
survey revealed that few analytical methods have been reported for analysis of
telmisartan as individual drugs in pharmaceutical preparations or biological
fluids by various methods such as HPLC [3-5], HPTLC [6], and LC-MS [7-9].
Hydrochlorothiazide as individual drug is determined by UV Spectrophotometry
[10], HPLC [11, 12], and LC-MS [13, 14]. Simultaneous determination of
telmisartan and hydrochlorothiazide in tablets and human plasma has been
reported by derivative Spectrophotometry, TLC-densitometry and spectrofluorimetry
[15], HPLC [16, 17], and LC-MS [18]. However one HPTLC method has been reported
for simultaneous estimation of telmisartan and hydrochlorothiazide in tablet
dosage form. In the reported method the linear ranges were comparatively very
narrow with higher limit and lesser separation factor of two drugs [19]. Hence
it was aimed to develop a simple, economic and fully validated HPTLC method
with better resolution, linearity, and sensitivity for simultaneous
determination of telmisartan and hydrochlorothiazide in FDC tablets. The
developed method was applied for dissolution study of formulated and marketed
tablets.
EXPERIMENTAL:
Materials:
Telmisartan
and hydrochlorothiazide standard powders were kindly supplied as a gift by M/S
Systopic Laboratories, New Delhi, India. HPMC K100, HPMC K100M, Carbopol 71G,
polyvinyl pyrrolidone (PVP), sodium starch glycollate (SSG), and magnesium
stearate was purchased from S.D. Fine Chemicals Ltd., Mumbai, India. Tablet
formulations were obtained commercially within their shelf lives with the
labeled amounts of 80 mg telmisartan and 25 mg hydrochlorothiazide. All
chemicals used were of analytical grade.
HPTLC Instrumentation:
Camag
(Muttenz, Switzerland) HPTLC instrument with winCATS software was used for
analysis. The samples were spotted in the form of bands of width 5.5 mm with
Camag microlitre syringe on precoated silica gel aluminium plate 60 F254 (20
× 10 cm with 0.2 mm thickness, E. Merck, Darmstadt, Germany) using a Camag
Linomat sample applicator. A constant application rate of 150 nls-1
was employed and space between two bands was 10 mm. The mobile phase consisted
of toluene-ethyl acetate-methanol-glacial acetic acid, 6: 3: 1: 0.5 (v/v).
Ascending development was carried out in twin trough glass chamber saturated
with mobile phase. The optimized chamber saturation time for mobile phase was
10 min at room temperature. The length of chromatogram run was 85 mm. After
development, plates were dried and scanned by Camag TLC scanner in the
absorbance mode at 272 nm.
Validation
of the Method:
The developed method was
validated according to ICH validation guidelines [20]. The
validation parameters addressed were linearity and range, limit of detection
and quantization, precision, and accuracy.
Calibration
Plots :
A
stock solution of 250 µg ml-1 of telmisartan and hydrochlorothiazide
was prepared in methanol. Different volumes of stock solution, 0.1, 0.2, 0.4,
0.8, 1.6, 2.0 and 4.0 µl were spotted in triplicate on the plate to obtain
concentrations of 1, 25, 50, 100, 200, 400, 500 and 1000 ng spot-1
of both the drugs. The plate was developed, dried, and scanned as described
above. Calibration plots were constructed by plotting peak area against the
corresponding amount of each drug.
Precision:
The
intraday precision was assessed by performing six analysis of the drugs in the
tablet formulation under the same experimental conditions. Interday precision
was assessed by performing replicate analysis of drugs in tablet formulation on
three days over a period of one week. The results of precision were expressed
in terms of RSD (%).
Robustness:
Robustness
was assessed to evaluate the effect of small variation in the chromatographic
conditions on the results from analysis of the drugs. The experiment was
conducted by scanning the plates at two different wavelength (+2, -2) at
concentration level of 500 and 100 ng spot-1 for telmisartan and
hydrochlorothiazide, respectively in the tablet formulation. The RSD (%) of
peak area was calculated from six replicates.
LOD
and LOQ:
Lower
parts of the linear range of the calibration plot were used to determine limit
of detection (LOD) and limit of quantification (LOQ) by applying the equations,
LOD = 3.3 × SD / S and LOQ = 10 × SD / S). Where S
is the slope of the calibration plot and SD is standard deviation of the
blank sample.
Recovery
Study:
The
accuracy of the method was determined by addition of standard solution of drugs
at three different levels. Sample solution of tablet formulation containing 500
ng µL-1 for telmisartan and 100 ng µL-1 for
hydrochlorothiazide was prepared. This solution was spiked with extra 25%, 50%,
and 75% of the standard solution of telmisartan and hydrochlorothiazide, and
the mixtures were reanalyzed by the proposed method.
Analysis
of drugs in tablets:
Twenty
tablets were weighed accurately and powdered. Powder equivalent to 80 mg of
telmisartan and 25 mg of hydrochlorothiazide was taken and transferred to a 50
ml volumetric flask. The powder was dissolved with approximately 25 ml of
methanol and ultrasonicated for 10 min. The final volume was made up with
methanol. The solution was filtered through Whatman No. 41 filter paper and 1µl
of the filtrate was applied to HPTLC plate to furnish 500 ng spot-1
for telmisartan and 100 ng spot-1 for hydrochlorothiazide. The
amount of each drug in each tablet was determined from the respective
calibration plot.
Preparation
of Tablets:
Wet
granulation method was applied for preparation of granules for tablets.
Telmisartan is poorly water soluble and hydrochlorothiazide is highly water
soluble drug. Hence the tablets were prepared by formulating telmisartan as
sustained release and hydrochlorothiazide as immediate release. The tablets
were composed of two separate layers and both the layers were prepared
separately by applying the following methods.
Granulation
of the SR Layer:
The
SR layers were prepared by mixing the ingredients in different compositions as
presented in Table 1. HPMC K100 and co-polymer carbopol 71G at different ratios
were blended with telmisartan in a planetary mixer for 5 min. The granules were
prepared from the mixed powders using sieve of mesh 20 screen, and dried at 30
ºC for 1 h. After drying the granules were again sized by a mesh 20 screen and
mixed with magnesium stearate for 2 min. The final weight of each trial
formulation was kept at 150 mg.
Table 1. Composition for
telmisartan SR layer
|
Ingredients (mg)
|
Formulation Code
|
|
F1
|
F2
|
F3
|
|
Telmisartan
|
80
|
80
|
80
|
|
HPMC
K 100
|
45
|
35
|
25
|
|
Carbopol
71G
|
5
|
10
|
15
|
|
Polyvinyl
Pyrrolidone
|
10
|
15
|
20
|
|
Magnesium
Stearate
|
10
|
10
|
10
|
|
Total
weight
|
150
|
150
|
150
|
Granulation of the IR
Layer:
The IR layers were prepared
by mixing the ingredients in different compositions as presented in Table 2.
HPMC K100, PVP and SSG at different ratios were blended with
hydrochlorothiazide in a planetary mixer for 5 min. The granules were prepared
from the mixed powders using sieve of mesh 20 screen, and dried at 30 ºC for 1
h. After drying the granules were again sized by a mesh 20 screen and mixed
with magnesium stearate for 2 min. The final weight of each trial formulation
was kept at 80 mg.
Table 2. Composition for hydrochlorothiazide IR layer
|
Ingredients (mg)
|
Formulation Code
|
|
F1
|
F2
|
F3
|
|
Hydrochlorothiazide
|
25
|
25
|
25
|
|
HPMC
K100
|
25
|
20
|
15
|
|
Sodium
Starch Glycollate
|
10
|
15
|
20
|
|
Polyvinyl
Pyrrolidone
|
10
|
10
|
10
|
|
Magnesium
Stearate
|
10
|
10
|
10
|
|
Total
weight
|
80
|
80
|
80
|
Compression
and Evaluation of Bilayer Tablets:
Bilayered
tablets were prepared by compressing the granules to total weight of 230 mg to
an average hardness of 7 Kg/cm2 using double rotary tablet punching
machine (Cadmach, Mumbai, India). Finally the formulation codes for bilayered
tablets were named as F1, F2, and F3, where composition of F1 is SR1 for the SR
layer and IR1 for the IR layer. The various quality control tests were carried
out for the prepared tablets such as weight variation, friability, Hardness,
and thickness test.
Dissolution
Study:
The
proposed method was applied for the dissolution study of formulated and
marketed tablets as per the USFDA recommended method [21]. Drug release from
the tablet was determined using USP paddle apparatus, Veego VDR-8 DR (Veego
Instruments, Mumbai, India). The dissolution medium consisted of distilled
water 900 ml and phosphate buffer, pH 7.5, maintained at 37 ± 0.5 °C with a
rotation speed of 75 rpm. Each dissolution test was performed on total number
of six tablets. A single tablet was added to the dissolution medium in each
vessel. Five ml of sample was taken in to 10 ml volumetric flask at 10, 15, 20,
30, 45, and 60 min. An equivalent amount of fresh medium was added to the
dissolution medium after each sampling. The samples were filtered through
Whatman No. 41 filter paper and directly applied on to the plate. The plates
were developed, scanned and chromatograms recorded. The amount of drugs
dissolved in different time intervals were calculated from the corresponding
peak areas.
RESULTS
AND DISCUSSION:
Validation
of the Method:
The
mobile phase toluene-ethyl acetate-methanol-glacial acetic acid in the ratio of
6: 3: 1: 0.5 (v/v) resolved telmisartan and hydrochlorothiazide with RF
values of 0.70 ± 0.02 and 0.44 ± 0.02, respectively, as shown in Figure 1.
Linear regression data for the calibration plots of both the drugs were
indicative of good linear relationship between peak area and concentration over
the range of 1-1000 ng spot.-1 The LOD and LOQ were 0.5 and 1 ng
spot-1 respectively, for telmisartan and 1 and 2 ng spot-1
respectively, for hydrochlorothiazide which indicated the adequate sensitivity
of the method. The low values of RSD for intraday and interday precision
suggested an excellent precision of the method. The method was used for
estimation of both the drugs in tablet dosage forms after spiking with 25, 50,
and 75 % additional standard drug solutions. The recovery of both the drugs was
found within 98-101 %, indicated that accuracy of the method was adequate. The
low values of RSD of the results obtained after plates were scanned at two
different wavelengths (+2, -2) with no significant difference (ANOVA, P>0.05),
indicated the robustness of the method. The results of validation parameters
are summarized in Table 1.
Figure
1 Typical HPTLC chromatogram of telmisartan and hydrochlorothiazide obtained
from mixed standard solution
Table
3 Results from Validation studies
|
Validation
Characteristics
|
Telmisartan
|
Hydrochlorothiazide
|
|
Linear range (ng spot -1),
(n = 3)
|
1-1000
|
1-1000
|
|
Correlation coefficient
|
0.9992
|
0.9994
|
|
Limit of detection (ng spot -1)
|
0.5
|
1
|
|
Limit of quantitation (ng
spot -1)
|
1
|
2
|
|
Intraday precision, RSD [%],
(n = 6)
Intraday precision, RSD [%],
(n = 6)
|
1.35
1.15
|
1.77
1.65
|
Analysis of drugs in tablets:
Spots
at RF 0.72 ± 0.02 for telmisartan and 0.44 ± 0.02 for
hydrochlorothiazide were observed in the chromatograms obtained from both the
drugs extracted from tablets. The telmisartan and hydrochlorothiazide content
were 98.25-99.86 % and 98.44-99.85 % (RSD 1.65 and 1.92 %, respectively), of
the label claimed. The low values of RSD indicated that method was suitable for
routine analysis of both the drugs in combined dosage forms.
Evaluation
of dissolution study:
Dissolution study was
carried out by validated HPTLC-UV method. The various experiments were carried
out by mixing the drugs with HPLC polymers. From the study it was observed that
when the content of HPMC K100 polymer increases in the formulation, it
dissolves slowly in the medium resulting provide the sustained release effect
over the extended period of time. Formulation F3 was found to release the
telmisartan in sustained manner up to 24 h. The in-vitro release profile of
telmisartan release from matrix tablets in F1, F2 and F3 is shown in Figure
2. Hence formulation F3 was selected the optimum formulation and compared
with the telmisartan marketed tablets. The HPMC K100 after mixing with sodium
starch glycollate (SSG) it leads to fast disintegration of hydrochlorothiazide
and shows its immediate release. Hence the tablets from formulation 3 were
provided the best immediate release effect of hydrochlorothiazide within 30
min. Hence formulation F3 was selected the optimum formulation and compared
with the marketed combination tablets. The optimized tablets showed the better
sustained release of telmisartan (99.75%) up to 20 h when compared to the
marketed combination tablets (95.95%) for 1 h as shown in Figure 3.
Well separated spots of both the drugs were observed in the chromatograms of
dissolution sample at RF 0.74 ± 0.02 for telmisartan and 0.45
± 0.02 for hydrochlorothiazide. The percentage releases after 60 min were 93.15
% for telmisartan and 96.74 % for hydrochlorothiazide in the tablets.
Figure
2: In-vitro release profile of Telmisartan from bilayer tablets in F1, F2, and
F3, (bars ± SD [n=3]).
Figure
3: Dissolution profiles of Telmisartan and Hydrochlorothiazide from marketed
tablets mean ± SD, (n = 3)
CONCLUSION:
The
developed HPTLC method was found to be simple, precise, specific, and accurate
with better range of linearity. The method was repeatable and selective for the
simultaneous determination of telmisartan and hydrochlorothiazide in the bulk
drugs and in their FDC tablets. The method was successfully applied in the
dissolution study of in-house formulated and marketed tablets. Bilayer FDC
tablets containing telmisartan as sustained release in one layer and
hydrochlorothiazide as immediate release from another layer was prepared by wet
granulation method. It was found that prepared tablets provided best sustained
release of telmisartan over a period of 20 h and immediate release of
hydrochlorothiazide within 30 min.
ACKNOWLEDGEMENT:
The authors are grateful to
Systopic Laboratories, New Delhi, India, for providing gift samples of
telmisartan and hydrochlorothiazide. The authors are also thankful to Dean and
In-charge of Instrumentation Facilities, Faculty of Pharmacy, Hamdard
University, New Delhi, India, for providing opportunities to work on HPTLC
system.
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