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J. Adv. Pharm. Tech. Res. Vol. 1 (2), Apr-Jun, 2010
ISSN 0976-2094
HPTLC METHOD DEVELOPMENT AND VALIDATION OF TRANDOLAPRIL IN BULK AND PHARMACEUTICAL DOSAGE FORMS N. Sreekanth*1, Bahlul Z.Awen2, Ch. Babu Rao2 1. Department of Pharmacy, College of Public Health and Medical sciences, Jimma University, Jimma, (Ethiopia) 2. Faculty of Pharmacy, 7th April University, Zawia, (Libya) Corresponding Author’s E-mail: -
[email protected] Received: 01st April 2010
Revised: 28th May 2010
Accepted: 06th June 2010
ABSTRACT A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and completely validated for the estimation of trandolapril in bulk and pharmaceutical dosage forms. Quantification of trandolapril was carried out with percolated silica gel 60F 254 as stationary phase using mobile phase consisting of
Chloroform:
Methanol:
Acetic
acid
(8:1.5:0.5
v/v/v)
and
scanned
in
Absorbance/Reflectance mode at 212 nm using Camag TLC scanner 3 with WinCAT software. The Rf value of trandolapril was found to be 0.54 (±0.03).
The proposed
method has permitted the quantification of trandolapril over the linearity range of 25150 ng/spot and its percentage recovery was found to 99.7%. The intra day and inter day precision were found to be 1.26% and 1.4%, respectively. The limit of detection and the limit of quantification were found to be 18 ng/spot and 54 ng/spot, respectively. The proposed method can be successfully applied for the estimation of drug content of different marketed formulations simultaneously on a single plate and provides a faster and cost effective quality control tool for routine analysis of trandolapril as bulk drug and in tablet dosage forms.
Key words: HPTLC, Validation, Trandolapril.
INTRODUCTION
of trandolapril was shown in Fig. 1. Trandolapril is a nonsulphydryl prodrug
Trandolapril, chemically, it is (2S, 3aR,
that is hydrolysed to the active diacid
7aS)-1-[(S)-N-[(S)-1-carboxy-3-
trandolaprila. Trandolapril is an orally
phenylpropyl]
administered
alanyl]
hexahydro-2-
angiotensin
converting
indolinecarboxylic acid, 1-ethyl ester [1]
enzyme inhibitor that has been used in
and
the
is
not
official
in
any
pharmacopoeia. The chemical structure
© JAPTR, All Rights Reserved
treatment
hypertension
172
and
of
patients
with
congestive
heart
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J. Adv. Pharm. Tech. Res. Vol. 1 (2), Apr-Jun, 2010
ISSN 0976-2094
failure, and myocardial infarction [2-3].
Tablet
Literature
few
amount 2.0 mg of trandolapril (Zetpril-
HPLC methods were reported for the
2, Hetero Drugs Pvt. Ltd. and Mavik-2,
estimation
Abott
survey
of
revealed
trandolapril
that
in
the
biological fluids [4-10]. The present study
illustrates
development
formulations
with
Pharmaceuticals
labeled
Ltd)
were
purchased from the local market.
and
validation [11] of a simple, accurate,
Apparatus
precise and specific HPTLC method for
A CAMAG HPTLC system (Switzerland)
the estimation of trandolapril tablet
comprising
dosage forms.
semiautomatic
a
CAMAG sample
Linomat
IV
applicator,
a
CAMAG TLC Scanner 3, A CAMAG twintrough chamber (10 × 10 cm), CAMAG CATS 4 software, A Hamilton syringe (100 µl), A Shimadzu libror AEG-220 weighing balance and A ultra sonicator (Frontline FS-4, Mumbai) was used during the study.
Chromatographic conditions The chromatographic conditions were Fig.
1:
Chemical
structure
of
optimized
trandolapril
and
estimations
were
performed on a stationary phase, pre coated silica gel 60 F254 aluminum
EXPERIMENTAL
sheets (10×10 cm) which were pre-
Reagents
washed with methanol and dried in air,
Pure working standard of trandolapril
with
was procured as a gift sample from
Methanol: Acetic acid (8:1.5:0.5 v/v/v) .
Ranbaxy Ltd., Himachal Pradesh. All
The
chemicals and reagents used were of
plate was allowed to saturate for about
analytical grade. A Silica gel 60F 254 TLC
30 min and the migration distance
pre coated aluminum plates (10×10 cm,
allowed was 72 mm. The wavelength
layer thickness 0.2 mm, E. Merck,
scanning was performed at 212 nm
Mumbai) were used as a stationary
keeping the slit dimension 5×0.45 mm.
phase. Chloroform: Methanol: Acetic
The source of radiation was deuterium
acid (8:1.5:0.5 v/v/v) was used as
lamp
mobile phase and methanol was used
spectrum
as solvent.
standard solutions of trandolapril was
Commercially available
© JAPTR, All Rights Reserved
173
mobile
phase
of
chromatographic
emitting
Chloroform:
chamber
and
a
continuous
UV
between
190-400nm.
The
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J. Adv. Pharm. Tech. Res. Vol. 1 (2), Apr-Jun, 2010
spotted
and
developed
at
constant
ISSN 0976-2094
air. Photometric measurements were
temperature of 25 ± 2ºC.
performed
at
212
nm
in
absorbance/reflectance mode with the CAMAG TLC scanner 3 using CATS 4
Preparation of mobile phase Chloroform: (8:1.5:0.5
Methanol:
v/v/v)
was
Acetic employed
acid
software incorporating track optimizing
as
option. The standard plot of trandolapril was established by plotting the peak
mobile phase.
area
Vs
concentration
(ng/ml)
corresponding to each spot.
Preparation of standard solution of trandolapril A
working
standard
of
trandolapril
Estimation
about 2.5 mg was accurately weighed
of
trandolapril
in
marketed tablet formulation
and transferred in to 100 ml volumetric
Twenty tablets were accurately weighed
flask. A volume of methanol about 25
and finely powdered. The powder which
ml was added and sonicated for about
is equivalent to 2.5 mg of trandolapril
20 min; finally the volume was made up
was weighed, mixed with 25 ml of
to 100ml with methanol to obtain the
methanol and sonicated for 15 min. The
concentration about 25 µg/ml. From
solution of tablet was filtered through
this stock solution 0.1 ml was taken
Whatman filter paper No. 41 and the
and the volume made up to 100ml to
residue was thoroughly washed with
get concentration about 25ng/ml.
methanol. The filtrate and washings were combined in a 100 ml volumetric
Preparation of calibration curve
flask and diluted to the mark with the
Aliquots (1, 2, 3, 4, 5 and 6 µl) of
methanol to get the final concentration
standard solution of trandolapril were
of 25 µg/ml of trandolapril. From this
spotted on pre coated TLC plates using
stock solution 0.1 ml was taken and the
semi automatic spotter under nitrogen
volume
stream. The plate was dried in air and
concentration about 25ng/ml. Three
developed up to 72 mm at constant
micro liters of sample solution was
temperature
of
applied on a TLC plate under a nitrogen
acid
stream using a semi automatic spotter.
(8:1.5:0.5 v/v/v) as mobile phase in a
The amount of trandolapril present in
CAMAG twin through chamber which
the sample solution was determined by
was previously saturated with mobile
fitting
phase for about 30 min. the plate was
corresponding to trandolapril into the
removed from the chamber and dried in
equation of the line representing the
Chloroform:
with
a
Methanol:
mixture Acetic
© JAPTR, All Rights Reserved
174
made up to
the
area
100ml
values
of
to get
peaks
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J. Adv. Pharm. Tech. Res. Vol. 1 (2), Apr-Jun, 2010
ISSN 0976-2094
calibration curve of trandolapril. All
Validation of method
determinations
The
were
performed
in
triplicate.
Linearity
for
the
detection
of
trandolapril was 25-150 ng/ml with
R2=
0.998; Y=21.07x + 21.71. The results RESULTS AND DISCUSSION
were
Method development
precision
Trandolapril was soluble in methanol,
reproducibility)
there fore methanol was selected as the
spotting 3 µl of drug solution six times
solvent. A solvent system consisting of
on a TLC plate, followed by development
Chloroform:
acid
of plate and recording the peak area for
(8:1.5:0.5 v/v/v) was selected as mobile
6 spots. The % RSD for peak area
phase,
values of trandolapril was found to be
that
Methanol:
would
Acetic
give
dense
and
shown
in
of
the
the
Table-1.
method
was
The
(System
assessed
by
compact spot with appropriate Rf values
1.04%.
was
Table-2a. The method reproducibility
selected
for
Trandolapril
in
formulations.
The
method
for
quantification
of
(The
pharmaceutical present
the
The results were shown in
intra-day
determined
HPTLC
by
precision) analyzing
was
standard
quantification
solutions in the concentration range of
trandolapril in bulk and pharmaceutical
75 ng/spot to 100 ng/spot of drug for 3
dosage, revealed as simple, accurate
times on the same day and inter-day
value of 0.54. The
precision was determined by analyzing
typical densitogram of trandolapril was
corresponding standards daily for 3 day
shown in Fig.2.
over a period of one week. The intra-day
and precise with R
f
and inter-day coefficients of variation (%RSD) are in range of 0.39 to 1.26 and 0.17 to 1.4, respectively. The results were shown in Table-2b, 2c. Recovery studies were carried out to assess accuracy of the method. These studies were carried out at three levels. The percentage recovery was found to be within the limits and shown in Table-3. The assay for the marketed formulation was
established
with
the
present
chromatographic conditions developed and it was found to be more accurate Fig. 2: A typical Densitogram of
and reliable. The average drug content
Trandolapril
was found to be 99.15% of the labeled
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J. Adv. Pharm. Tech. Res. Vol. 1 (2), Apr-Jun, 2010
ISSN 0976-2094
claim. The results were shown in Table-
chromatographic conditions like mobile
4.
phase composition, Amount of mobile
Limits
of
Detection
Quantification detection
(LOQ),
and
(LOD)
the
and
limits
phase,
Plate
treatment,
Time
from
were
spotting to chromatography and time
calculated by the method based on the
from chromatography. The low value of
standard deviation of response (σ) and
% RSD indicates robustness of the
the slope of calibration plot (S), using
method. The results were shown in the
the formulae LOD = 3.3σ/S and LOQ =
Table-5. Specificity test of the proposed
10σ/S.
method demonstrated that there were
The
quantitation
of
LOD
and
LOQ
were
calculated and found to be 18 ng/Spot
no
and
Furthermore,
54
ng/Spot,
respectively.
Robustness was determined by altering
interference well
form
excipients.
shaped
peaks
indicate the specificity of the method.
Table 1: Linearity of Trandolapril S.
Track
Concentration
No.
No.
(ng/Spot)
Area
A1
A2
A3
Mean Peak Area ±SD
1
1
25
550.13
610.0
560.25
573.46±32.04
2
2
50
1079.6
1029.05
1076.53
1061.66±28.29
3
3
75
1595.1
1609.73
1565.0
1589.94±22.8
4
4
100
2110.0
2150.25
2175.25
2145.16±32.9
5
5
125
2709.1
2720.25
2729.25
2719.25±10.09
6
6
150
3100.6
3116.5
3157.63
3124.91±29.43
Table 2a: Precision of Trandolapril S. No.
Concentration (ng/spot)
Peak area
1
75
1595.1
2
75
1609.73
3
75
1566.00
4
75
1575.43
5
75
1598.05
6
75
1585.34
Mean Peak Area ± SD -1528.66± 15.95; %RSD- 1.04
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J. Adv. Pharm. Tech. Res. Vol. 1 (2), Apr-Jun, 2010
ISSN 0976-2094
INTRA-DAY PRECISION
Table 2b: Intra-day precision of Trandolapril S. No.
Concentration (ng/spot)
Area
1
Mean
S.D
% RSD
1595.366667
20.25594563
1.26
2717.85
10.6391964
0.39
3123.583333
20.33484284
0.65
1575.4
2
75
1615.9
3
1594.8
1
2715.7
2
100
2729.4
3
2708.45
1
3145.65
2
125
3105.6
3
3119.5
INTER-DAY PRECISION
Table 2c: Inter-day precision of Trandolapril S. No.
Concentration (ng/Spot)
1 2
Area
75
1608.5 1596.2
1
2705.69 100
2715.24
3
2710.56
1
3154.36
2
S.D
% RSD
1589.993333
22.26845602
1.4
2710.496667
4.775314998
0.17
3130.586667
22.12438775
0.7
1565.28
3
2
Mean
125
3
3110.6 3126.8
Table 3: Recovery studies of Trandolapril S.
Amount Present (mg)
Amount added (mg)
No.
(A)
(B)
1
2.0
2 3
A+B
Amount found
%Recovery
8.0
10.0
9.92
99.2
2.0
10.0
12.0
11.93
99.4
2.0
12.0
14.0
13.96
99.7
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Table 4: Assay of Trandolapril S. No.
Label claim (mg/tablet)
Amount of drug estimated* (mg)
% Purity
%RSD
1
2.0
1.983
99.15
0.13
*Mean of three values
Table 5: Robustness of Trandolapril S. No.
Parameter
%RSD
Mean %RSD
75
100
125
(n=3)
(n=3)
(n=3)
1.
Mobile phase Composition
0.56
0.48
0.32
0.45
2.
Amount of mobile phase
0.31
0.63
0.45
0.46
3.
Plate treatment
0.35
0.26
0.47
0.36
4.
Time from spotting to chromatography
0.33
0.57
0.48
5.
Time from chromatography to scanning
0.46
0.59
0.55
0.54
CONCLUSION The
developed
0.61
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