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Development and Validation of a Spectrophotometric method for estimation of Amitriptyline Hydrochloride in Bulk and Tablet Dosage Form

JAYKUMAR H. GOR, HEMANT KUMAR JAIN* and K. N. GUJAR

Department of Quality Assurance Techniques, STES’s Sinhgad College of Pharmacy, Vadgaon (Bk), Off. Sinhgad Road, Pune - 411041, Maharashtra, India.

Corresponding Author:
Hemant Kumar Jain
Department of Quality Assurance techniques Sinhgad College of Pharmacy
Vadgaon (Bk), Off Sinhgad Road
Pune- 411041, Maharashtra, India
E-mail: [email protected]

Received Date: 11-06-2013; Accepted Date: 24-06-2013

Citation: Gor JH, Jain HK, Gujar K. Development and Validation of a Spectrophotometric method for estimation of Amitriptyline Hydrochloride in Bulk and Tablet Dosage Form. Int. J. Drug Dev. &
Res., July-September 2013, 5 (3): 356-360

Copyright: © 2013 Hemant Kumar Jain et al, publisher and licensee IYPF. This is an Open Access article which permits unrestricted noncommercial use, provided the original work is properly cited.

 

Abstract

Amitriptyline Hydrochloride (AMI) is a tricyclic antidepressant drug. The aim of this work is to develop a simple, accurate, reproducible and cost effective spectrophotometric method for determination of Amitriptyline Hydrochloride in a pharmaceutical dosage. The absorption maximum of this drug was at 239.0 nm and method has followed linearity in the concentration range 4-24 μg/mL. The generated regression equation was ABS = 0.043 C + 0.035 with R2 value as 0.9995. The limit of detection (LOD) and limit of quantification (LOQ) were 0.1094μg/ml & 0.3315μg/ml, respectively. Percent relative standard deviation values for the intra-day and inter-day precision were 0.215 and 0.452, respectively, indicated good precision. Results of the recovery studies showed good accuracy of the method. Statistical analysis of results suggests that the developed method is suitable for routine estimation of Amitriptyline Hydrochloride in pharmaceutical formulations.

Keywords

Amitriptyline Hydrochloride, Estimation, Validation, UV spectrophotometery.

Introduction

Amitriptyline Hydrochloride (AMI) is a tricyclic antidepressant.[1] It is chemically known as 3- (10,11-Dihydro-5H-dibenzo [a,d] cyclohepten-5- ylidene)-N,Ndimethyl-1-propanamine[2] (Figure 1). This drug is used mainly in the treatment of depression. AMI is official as drug substance as well as Tablet dosage form in Indian Pharmacopoeia (IP), British Pharmacopoeia (BP) and United State Pharmacopoeia (USP). The non aqueous titration method isdescribed in IP[3] and USP[4] andacidbase titration method is described in BP[5] for assay of AMI. Other methods viz. fluorescence polarization immunoassay[6], Ultra-violet (UV) spectrometry[7] have been reported for determination of AMI. Whereas chemometric[8] and chromatographic methods[9-10] have been reported for determination of AMI in combination with drugs. Another literature suggested that determination of AMI withits major metabolites in blood[11] using HPLC. There are only few papers available for estimation of AMI using UV spectrophotometery, but most of these techniques require a sophisticated data processing facility. In this context, we wish to further explore UV spectrophotometery using simple and routinely used spectrophotometer without data processing facility. The present paper describes development and validation of a simple UV spectrophotometric method for assay of AMI in bulk and tablet dosage form.

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Figure 1: Amitriptyline Hydrochloride

Materials and Methods

Instruments

Shimadzu UV 1800 (Japan) with 1 cm matched quartz cells, connected to computer loaded with UV Prob Software, was employed for this work. Shimadzu AX200 (Japan) digital balance and Spectrolab UCB 40 (Germany) ultrasonicator, were used.

Materials

A gift sample of active pharmaceutical drug (API) of AMI was procured from Astron Research Ltd. (Ahemedabad, Gujarat). Commercially available tablets (Triptomar: containing 10 mg AMI) were obtained from local pharmacy. All the solvents used for this study were of AR grade.

Spectrophotometric method

The standard stock solution of AMI was prepared by transferring, accurately weighed, 100 mg of API to 100 mL of volumetric flask. It was suitably dissolved and volume was made up to the mark by using methanol. This stock solution of the drug was further diluted with the same solvent to obtain 10 μg/mL. The resultant solution was scanned in spectrum mode within 400-200 nm after baseline correction. The spectra of the drug obtained (Figure 2), was used to determine the absorption maxima (λmax). Two different λmax were found, but λmax at 239.0 nm wavelength was selected for this study. Using the stock solution, series of standard working solutions were prepared for AMI at five different levels between 4-24 μg/mL. The absorbances of these solutions were recorded at 239 nm.

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Figure 2: UV spectrum ofAmitriptyline Hydrochloride

Preparation of sample solution

Triptomar tablets (Label claim: 10 mg of Amitriptyline HCl per tablet) were selected for the present work. Twenty tablets were weighed and average weight was calculated. These tablets were crushed and powdered in glass mortar. The tablet powder equivalent to 10 mg of AMI was accurately weighed, transferred to a 100 mL of volumetric flask and diluted up to mark with methanol. This solution was further diluted to obtain 15μg/mL solution with same solvent.

Method Validation

Linearity and range

The working standard solutions were prepared by dilution of the stock solution with Methanol in the range of 4-24 μg/ml. The Absorbances of these solutions were measured at 239.0 nm. The relationship between absorption (as a dependant variable) and standard working solution (as an independent variable) were established by simple linear regression method. The regression equation was obtained and this relationship is presented in the calibration curve (Figure 3). The range of solution has been decided according to statistical analysis of regression equation.

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Figure 3: Calibration curve of Amitriptyline Hydrochloride

Precision

Intra and inter-day precision studies were performed by measuring the absorbance of standard solution at three different times during the single day and on three subsequent days, respectively. The percentage relative standard deviation (%RSD) was calculated (Table 2).

Sr. No. Sample solution concentration (μg/ml) Amount Found (%) Mean Amount Found (%) % RSD*
1 15 100.29 100.36 0.168
2 15 100.35
3 15 100.59

Table 1: Results of Assay of Tablet dosage form

Sr. No. Parameters Intra-day precision Inter-day precision
1 Sample solution concentration (μg/ml) 15 15
2 % Assay* 100.41%±0.39 00.59%±0.63
3 % RSD 0.215 0.452

Table 2: Precision data of Amitriptyline Hydrochloride

Limit of Detection (LOD) and Limit of Quantitation (LOQ)

Six sets of known concentrations (4-24 μg/ml) were prepared. Calibration curves were plotted for each set. LOD and LOQ were calculated using the formulae as

image

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Where, S is value of slopes of calibration plot and SD is calculated using values of y intercepts of regression equations.

Accuracy

Recovery studies were carried out by applying the developed method to drug sample at which Amitriptyline Hydrochloride corresponding to 80, 100, 120% of label claim was present. Three determinations at each level were performed and results were expressed as % RSD (Table 3).

Sr. No . Accuracy Level Amount added (μg/ml) %Recovery * Mean % Recover y % RSD
1 I (80%) 12 101.11±0.27 100.29% 0.52 3
2 II (100%) 15 100.09±0.22
3 III (120%) 18 100.45±0.31

Table 3: Accuracy data of Amitriptyline Hydrochloride

Results and Discussion

The generated regression equation was ABS = 0.043 C + 0.035 with R2 value as 0.9995. Where, ABS is absorbance, C is concentration and R is correlation coefficient. Here, value of R2 is very close to 1 that suggests that the method is following linearity in the concentration range 4-24 μg/mL of AMI. Results obtained by assay of Amitriptyline Hydrochloride tablet formulation suggest that applicability of developed methods to the tablets, as an average amount found was 100.29% with low % RSD (0.168). Percent relative standard deviation (%RSD) values for the intra-day and inter-day precision were 0.215 and 0.452, respectively, which is under acceptable range. This indicates good precision. LOD and LOQ values were 0.1094 μg/ml & 0.3315 μg/ml, respectively. This suggests that lowest amount of drug that can be detected using this analytical procedure is 0.1094 μg/ml and lowest amount of drug in a sample that can be quantitatively determined with suitable precision and accuracy is 0.3315 μg/ml. Percent (%) recovery was calculated as amount of drug found/drug added X 100. The range of % recovery was 100.11 to 100.45% (with mean 100.20%). Results of the recovery studies indicated good accuracy of the method.

There was no interference from the excipients of tablet formulation. The summary of validation parameters is presented in Table 4.

Parameters Amitriptyline Hydrochloride
λmax (nm) 239.0
Linearity range 4-24 μg/ml
Correlation coefficient (r2) 0.9995
Slope (m) ± SD* 0.043
Intercept (c) ± SD* 0.035
Regression equation (y = mx+c) ABS = 0.043 C + 0.035 Simple linear equation
LOD (μg/ml ) 0.1094
LOQ (μg/ml ) 0.3315
Precision % RSD*
Intraday
Interday

0.215
0.452
% Recovery 100.29%

Table 4: Summary of Validation Parameters

Conclusion

A simple, accurate and precise UV Spectrophotometric method for estimation of Amitriptyline Hydrochloride was developed. This method was validated as per ICH guidelines. Results suggest that the developed method is suitable for routine estimation of Amitriptyline Hydrochloride in pharmaceutical formulations.

Acknowledgements

The authors are thankful to Astron Research Ltd. (Ahmedabad) for pure Amitriptyline Hydrochloride gift sample, President of Sinhgad Technical Education society and Principal of Sinhgad College of Pharmacy, Vadgaon (Bk), Pune, for providing required facilities for research work.

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