HPTLC Method Development and Validation for the Estimation of Trandolapril in Bulk and Its Formulations

 

M Rama kotaiah1*,  B Ganesh1,  KB Chandra Sekhar1, Shaik Harun Rasheed1, Y Venkateswarlu2 and B Dhandapani3

1Department of Chemistry, Jawaharlal Nehru Technological University, Anantapur – 515002, Andhra Pradesh.

2Dept.of Pharmaceutical Analysis, Donbosco College of Pharmacy, Pulladi Gunta, Etukuru, Guntur, (A. P.)

3Dept.of Pharmaceutical Analysis, A. M. Reddy Memorial College of Pharmacy, Narasaraopet, Guntur District – 522601, (A.P.)

*Corresponding Author E-mail: mrk.pharma@gmail.com, dhandapanirx@gmail.com

 

ABSTRACT:

A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the estimation of Trandolapril in bulk and pharmaceutical dosage forms. Quantification was carried out with percolated silica gel 60F 254 as stationary phase and mobile phase consisting of chloroform: methanol: aceticacid (8:1.5:0.5 v/v/v). The detection of spot was carried out at 212 nm using Camag TLC scanner 3 with WinCAT software. The method was validated in terms of linearity, accuracy, precision and specificity. The Rf value of trandolapril was found to be 0.54 (±0.03). The calibration curve was found to be linear between 25 to 150 ng/spot for trandolapril. The limit of detection and the limit of quantification for the trandolapril were found to be 10 ng/spot and 24 ng/spot. The proposed method can be successfully used 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 dosages forms.

 

KEYWORDS: HPTLC, Validation, Trandolapril

 

 

INTRODUCTION:

Trandolapril, chemically it is (2S, 3aR, 7aS)-1-[(S)-N-[(S)-1-carboxy-3-phenylpropyl] alanyl] hexahydro-2-indolinecarboxylic acid, 1-ethyl ester1 and is not official in any pharmacopoeia. Trandolapril is a nonsulphydryl prodrug that is hydrolysed to the active diacid trandolaprila. Trandolapril is an orally administered angiotensin converting enzyme inhibitor that has been used in the treatment of patients with hypertension and congestive heart failure, and myocardial infarction2-3. Literature survey revealed that few HPLC methods were reported for the estimation of trandolapril in the biological fluids4-10. The present study illustrates development and validation of a simple, accurate, precise and specific HPTLC method for the estimation of trandolapril tablet dosages forms.

 

MATERIALS AND METHODS:

Instrumentation:

A Camag HPTLC system comprising of Camag Linnomat V automatic sample applicator, Hamilton syringe (100 µl), Camag TLC Scanner 3 with Camag Win CATS software, Camag twin-trough chamber (10×10 cm) and ultrasonicator were used during the study.

 

Reagents and Materials:

Trandolapril working standard was procured as a gift sample from Ranbaxy Ltd., Himachal Pradesh. Silica gel 60F 254 TLC plates (10×10 cm, layer thickness 0.2 mm, E. Merck, Mumbai) were used as a stationary phase. All chemicals and reagents used were of analytical grade. Chloroform: methanol: aceticacid (8:1.5:0.5 v/v/v) was used as mobile phase. Methanol was used as solvent.  Tablets containing 2.0 mg trandolapril were purchased (Zetpril-2, Hetero Drugs Pvt Ltd. and Mavik-2, Abott Pharmaceuticals Ltd).

 

Chromatographic Conditions:

TLC plates were pre-washed with methanol. The chromatographic conditions were maintained on precoated silica gel 60F 254 aluminum sheets (10×10 cm) as stationary phase, chloroform: methanol: acetic acid (8:1.5:0.5 v/v/v) as mobile phase, chamber and plate saturation time of 30 min, migration distance allowed was 72 mm, wavelength scanning was done at 212 nm keeping the slit dimension at 5×0.45 mm. A deuterium lamp provided the source of radiation. Three microlitres of the standard solution of trandolapril was spotted and developed at constant temperature. Photometric measurements were performed at 212 nm (Fig – 1) in reflectance mode with Camag TLC scanner 3 using Win CATS software.

 

Fig – 1 Overlay Spectrum of Trandolapril at 212 nm

 

Preparation of Standard Solution and Calibration Curve:

Working standard of trandolapril (10 mg) was weighed accurately and transferred to a 100 ml volumetric flask and further dilution was made to get a concentration of 25 µg/ml. Aliquots of 1, 2, 3, 4, 5 and 6 µl of working standard solution of trandolapril were applied on the TLC plate (25 µg/ml of drug) to obtain concentration of 25, 50, 75, 100 and 125 ng per spot of trandolapril respectively. TLC plate was dried, developed with chloroform: methanol: aceticacid (8:1.5:0.5 v/v/v) as mobile phase and analyzed photometrically at 212 nm in reflectance mode with Camag TLC scanner 3 using Win CATS software. The densitogram was developed, sprayed, scanned and quantitatively evaluated. The data of peak area Vs drug concentration was treated by linear least-square regression analysis.

 

Procedure for pharmaceutical formulations:

Twenty tablets were accurately weighed and finely powdered. The powder equivalent to 10mg of trandolapril (2mg) was accurately weighed, mixed with methanol (5 ml) and sonicated for 20 min. The solution was filtered through Whatman filter paper No. 41. The residue was thoroughly washed with methanol. The filtrate and washings were combined in a 10 ml volumetric flask and diluted to the mark with methanol. The filtrate was further diluted to get the concentrations with in the calibration range of trandolapril. The sample solution was applied on a TLC plate under a nitrogen stream using a semiautomatic spotter. The TLC plate was developed and photometrically analyzed as described under the Chromatographic conditions Fig – 2. The amounts of trandolapril present in the sample solution were determined by fitting the area values of peaks corresponding to trandolapril into the equation of the line representing the calibration curve of trandolapril. The results are shown in Table 1.

 

Method validation:

The developed method was validated in terms of linearity, accuracy, specificity, limit of detection, limit of quantification, intra-day and inter-day precision and repeatability of measurement as well as repeatability of sample application as per ICH guidelines11. The results are shown in Table 2and 3.

 

Fig – 2: HPTLC Chromatogram of Trandolapril in Formulation (75ng/spot)

 

RESULTS AND DISCUSSION:

A solvent system that would give dense and compact spot with appropriate Rf values was selected for quantification of Trandolapril in pharmaceutical formulations. The mobile phase consisting of chloroform: methanol: aceticacid (8:1.5:0.5 v/v/v) gave R f values of 0.54 [Figure - 1]. A 3-D View of Calibration curve densitogram of trandolapril at 212 nm [Figure-2]. The linear regression data (n=6, Table - 1) showed a good linear relationship over a concentration range of 25-150 ng/spot for trandolapril. The limit of detection and limit of quantification were found to be 18 ng/spot and 54 ng/spot, respectively.

 

The intra-day precision was determined by analyzing standard solutions in the concentration range of  75 ng/spot to 100 ng/spot of drug for 3 times on the same day while inter-day precision was determined by analyzing corresponding standards daily for 3 day over a period of one week. The intra-day and inter-day coefficients of variation (%RSD) are in range of 0.39 to 1.26 and 0.17 to 1.4, respectively.

 

Repeatability of sample application was assessed by spotting 3 µl of drug solution six times on a TLC plate followed by development of plate and recording the peak area for 6 spots. The % RSD for peak area values of trandolapril was found to be 1.04%.

 

Repeatability of measurement of peak area was determined by spotting 3 µl of trandolapril solution on a TLC plate and developing the plate. The separated spot was scanned six times without changing the position of the plate and % RSD for measurement of peak area of trandolapril was 1.09%.

Table 1: Analysis of trandolapril marketed formulation by HPTLC method

S. No.

Theoretical Content (mg/tablet) Label claim

Amount of drug recovered(mg)

%recovery

%RSD

1

2.0

1.984

99.2

 

0.13

2

2.0

1.985

99.25

3

2.0

1.980

99.0

 

Table – 2: Validation Parameters of the Proposed HPTLC Method

Parameters

Trandolapril

Linearity range (ng/spot)

25-150

Correlation coefficient

0.9954

Precision (%CV)

-                   Intra day (n = 3)

-                   Interday (n=5)

-                   Repeatability of Sample application (n=7)

-                   Repeatability of Peak area (n=7)

 

0.39 – 1.26

0.17 – 1.4

1.04

1.09

% Recovery

99.2 – 99.7

Limit of Detection (ng/spot)

10

Limit of Quantification (ng/spot)

24

 

Table 3:  Robustness data of Trandolapril

S. No.

Parameter

%RSD

Mean %RSD

 

 

75 (n=3)

100 (n=3)

125 (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

0.54

0.33

0.57

0.48

5.

Time from chromatography to scanning

0.61

0.46

0.59

0.55

 

Table 4: HPTLC recovery study data of Trandolapril

S. No.

Initial Amount (mg)  [A]

Addition of known quantity (mg)  [B]

A+B

Amount recovered (mg)

% Recovery

1.

2.0

8.0

10.0

9.92

99.2

2.

2.0

10.0

12.0

11.93

99.4

3.

2.0

12.0

14.0

13.96

99.7

 

To confirm the specificity of the proposed method, the solution of the formulation was spotted on the TLC plate, developed and scanned. It was observed that the excipients present in the formulation did not interfere with the peaks of trandolapril.

 

Recovery studies were carried out to assess accuracy of the method. These studies were carried out at three levels. Sample solution from tablet formulation was used to perform recovery studies. The percentage recovery was found to be within the limits. The low RSD value indicated the suitability of the method for routine analysis of trandolapril in pharmaceutical dosage forms. The results are shown in Table 4.

 

Robustness was determined by altering various experimental conditions like mobile phase composition, amount of mobile phase, plate treatment, time from spotting to chromatography and time from chromatography.  No significant changes were observed. The low value of % RSD indicated robustness of the method.

Hence the developed HPTLC technique is simple, precise, specific and accurate and the statistical analysis proved that method is reproducible and selective for the analysis of trandolapril in bulk drug and tablet formulations.

 

REFERENCES:

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Received on 22.10.2009        Modified on 29.11.2009

Accepted on 17.12.2009        © AJRC All right reserved

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 158-160