HPTLC METHOD DEVELOPMENT AND VALIDATION

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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

Available online on www.japtr.org



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


173

mobile

phase

of

chromatographic

emitting

Chloroform:

chamber

and

a

continuous

UV

between

190-400nm.

The




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


174

made up to

the

area

100ml

values

of

to get

peaks




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


175




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


176




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


177




ISSN 0976-2094

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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Human

of

Pharmaceutical formulations using Phase

for

on

Use.
