INTRODUCTION:
Trandolapril {(2S, 3aR, 7aS)-1-[(2S)-2-[[(1S)-1(ethoxycarbonyl) -3-phenylpropyl] amino]-1-oxopropl] octahydro-1H-indole-2-carboxylic acid}1, a prodrug, is rapidly metabolized to the active compound trandolaprilat, which binds with high affinity and long duration to the angiotensin converting enzyme. Trandolapril, thus, modulates the renin-angiotensin-aldosterone system that plays a major part in regulating blood pressure. In hypertensive patients, this drug causes a reduction of systolic and diastolic blood pressure 2.
Literature survey reveals some methods reported viz. High Performance Liquid Chromatographic (HPLC) methods for determination of Trandolapril in human plasma as single and in combination with other drugs3-5, Quantification of trandolapril and its metabolite trandolaprilat in human plasma by liquid chromatography/tandem mass spectrometry using solid-phase extraction6, Determination of Related Compounds and Degradates of Trandolapril using a Stability Indicating Reverse Phase High Performance Liquid Chromatographic Method7, Simultaneous high-performance thin-layer chromatography densitometric assay of trandolapril and verapamil in the combination preparation8 and HPTLC method for Trandolapril using a ternary solvent system9.
No reports were found for the stability indicating assay method as per ICH guidelines, for Trandolapril by HPTLC method. This paper describes a simple, accurate, sensitive and validated stability indicating HPTLC method for Trandolapril as the bulk drug. The drug stability test guidelines Q1A (R2) issued by International Conference on Harmonization (ICH) requires that analytical test procedures for stability samples should be fully validated and the assay should be stability indicating. The aim of the present study accordingly was to establish inherent stability of the Trandolapril through stress studies under a variety of ICH recommended test conditions10
MATERIAL AND METHODS:
Working standard of Trandolapril (assay 99. 63 % w/w) was provided by Cipla Pharmaceuticals Pvt. Ltd., Mumbai, India and was used as such without further purification. Ethyl methyl ketone, Glacial Acetic acid, Conc. HCl, NaOH and H2O2 (all from S. D. fine Chem. Laboratories Pvt. Ltd., Mumbai, India) used were of analytical reagent grade.
Instruments.
For stability indicating HPTLC method development, Camag HPTLC system consisting of Linomat-5 applicator, Camag TLC Scanner 3 and WinCATS software V 1. 4. 3 were used.
For photo-degradation studies, Photostability Chamber was used. (Make - Newtronic)
All the weighing was done on Shimadzu balance (Model AY-120).
Method:
Selection of Detection Wavelength:
After chromatographic development, the bands were scanned over the range of 200-400 nm. It was observed that drug showed considerable absorbance at 220 nm. So, 220 nm was selected as the wavelength for detection.
Preparation of standard solution:
Standard stock solution of Trandolapril was prepared by dissolving 25 mg of drug in 25 ml of methanol to get concentration of 1000 mcg/mL. The solution was then further diluted with methanol to get working standard solution of Trandolapril having concentration of 100 mcg/ml.
Stress degradation studies:
Degradation under alkali catalysed hydrolytic condition:
2. 5ml of working standard solution was mixed with 2. 5ml of 0. 1N NaOH. The solution was diluted to 2.5 ml with methanol and kept for 2 hours. After 2 hours 15ul of the solution was spotted on TLC plate.
Degradation under acid catalysed hydrolytic condition:
25ml of working standard solution was mixed with 2. 5ml of 5N HCL. The solution was diluted to 25 ml with methanol and kept for 1 hr. 15ul of solution was spotted.
Degradation under neutral hydrolytic condition:
5ml of working standard solution were mixed with 5ml water. The solution was diluted to 50 ml with methanol and refluxed for 1 hour. Cool the solution to room tempt. 15 ul of the solution was then spotted.
Degradation under oxidative condition:
2. 5ml of working standard solution was mixed with 2. 5ml 30% solution of H2O2. The solution was diluted to 25 ml with methanol and was kept overnight. 15ul of the solution was then spotted.
Degradation under dry heat:
Dry heat studies were performed by keeping drug sample in oven (1000 C) for a period of 48 hours. Samples were withdrawn at appropriate time, dissolved in methanol and diluted to get 100μg/ml as final conc. 15ul of the solution was spotted.
Photo-degradation studies:
Photolytic studies were also carried out by exposure of drug to UV light up to 200 watt hours/square meter and subsequently to cool fluorescent light to achieve an illumination of 1. 2 million Lux. Hr. Sample was weighed, dissolved and diluted get 100 mcg/ml. 15ul of both UV light sample and Fluorescence light was spotted.
Method Validation:
Linearity and Range:
The linearity of an analytical procedure is its ability (within a given range) to obtain test result which are directly proportional to the concentration (amount) of analyte in the sample. It was studied by analyzing five concentrations of the drug and process was repeated five times. It was done over the range of 500-2500 ng/ band.
Precision:
Precision of the system was evaluated by analyzing six independent standard preparations and % RSD value obtained was calculated to determine any intra-day variation. These studies were also repeated on different days to determine inter-day variation.
Accuracy:
To check accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 80, 100 and 120 %. Mean percentage recovery was determined.
Limit of detection and limit of quantitation
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value.
Based on the Standard Deviation of the Response and the Slope, detection limit (DL) may be expressed as:
3.3
DL =
S
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. Based on the Standard Deviation of the Response and the Slope, The quantitation limit (QL) may be expressed as:
10
QL =
S
Where,
σ = the standard deviation of the response for the lowest conc. in the range
S = the slope of the calibration curve.
Specificity:
The specificity of the method was ascertained by peak purity profiling studies. Purity of the drug peak was ascertained by analyzing the spectrum at peak start, middle and at peak end. The peak purity was determined on WinCATS software V 1. 4. 3.
RESULTS:
Development of the optimum mobile phase:
Chromatographic separation studies were carried out on the working standard solution of Trandolapril (100μg/ml). Initially, trials were carried out using methanol and various other solvents.