DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR SIMULTANEOUS ESTIMATION OF AMITRIPTYLINE HYDROCHLORIDE AND METHYLCOBALAMIN IN THEIR TABLET DOSAGE FORM BY UV SPECTROPHOTOMETRIC METHOD

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ABOUT AUTHORS:
Chetna V. Karchaliya*, Parula B. Patel
Department of Quality Assurance,
S. J. Thakkar Pharmacy College,
Rajkot, Gujarat, India
*sonichetna158@gmail.com

ABSTRACT:
The simple, accurate and precise Absorption Correction Method has been developed for the simultaneous estimation of Amitriptyline hydrochloride and Methylcobalamin in combined tablet dosage form. The method utilizes distilled water as solvent and λmax of Amitriptyline hydrochloride and Methylcobalamin selected for analysis were found to be 239 nm and 351 nm respectively. The method was validated as per International Conference on Harmonization (ICH) guidelines. The Linearity range lies between 20-60 µg/ml (R² 0.9998) for Amitrityline hydrochloride and 3-9 µg/ml (R² 0.9990) for methylcobalamin. The accuracy and precision were determined and found to comply with ICH guidelines. The method showed good reproducibility and recovery with %RSD in desired range. The proposed method can be applied for routine analysis of both drugs.

REFERENCE ID: PHARMATUTOR-ART-2343

INTRODUCTION:
Amitriptyline Hydrochloride (ATH) is chemically, 3- (10, 11- Dihydro- 5H- dibenzo [a, d] cyclohepten- 5- ylidene)- N, N- dimethyl-1- propanamine¹. It is a tricyclic antidepressant used in case of anxiety and also exerts an anticholinergic activity¹. AMI is official in IP, BP and USP. The IP¹, BP² and USP³ describe HPLC, non-aqueous titration and titrimetric methods, respectively for estimation of AMI.

Methylcobalamin (MCA) is chemically, (1R,2R,4S,7S)-7-{[(2S)-3-hydroxy-2-phenylpropanol]oxy}-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.02,4]nonane⁴. It isa form of Vitamin B12 used in the treatment of trigeminal neuralgia, megaloplastic anaemia, diabetic neuropathy and facial paralysis in Bell’s palsy syndrome. It is official in Japanese Pharmacopoeia.JP⁴ describe HPLC method for estimation of MCA.

The Extensive review of Literature revealed that many analytical methods like UV spectrometry^[5-8] ,RP-HPLC^[9-10] and HPTLC^[11-12] have been reported for estimation of Amitriptyline hydrochloride and Methylcobalamin in individual and combined ( with other drugs) dosage form. But no any spectrophotometric or RP-HPLC method was available for simultaneous estimation of Amitriptyline hydrochloride and Methylcobalamin in their combined dosage form.

So, it was thought of interest to develop simple, rapid, accurate and precise spectrophotometric and RP-HPLC methods for simultaneous estimation of Amitriptyline hydrochloride and Methylcobalamin in bulk and in tablet dosage form. The developed methods were validated for its linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) according to the ICH guidelines (Q2R1).

Figure 1: Chemical Structure of Amitriptyline hydrochloride

Figure 2: Chemical Structure of Methylcobalamin

MATERIALS AND METHODS:
An UV-Visible double beam spectrophotometer (Shimadzu, Model - 1800) having two matched quartz cells with 10 mm light path. All weighing were done on electronic balance (Shimadzu, Model-AUX-220). Distilled water was used as solvent. Amitriptyline hydrochloride and Methylcobalamin reference standard was provided as gift sample by La Pharmaceuticals, Ahemdabad, Gujarat and Athene Chemical Pvt. Ltd., Ahemdabad, Gujarat respectively. The commercial fixed dose combination product (AMNURITE 10) tablet was procured from the local market.Each tablet containing 10 mg ATH and 1500mcg MCA.

Preparation of working standard solution for ATH: Accurately weighed 10 mg of ATH was transferred to 10 ml volumetric flask, dissolved and diluted up to mark with Distilled water to obtain final concentration of 1000 μg/ml ATH. Solution was further diluted with Distilled water to obtained working standard solutions of ATH.

Preparation of working standard solution for MCA: Accurately weighed 10 mg of MCA was transferred to 10 ml volumetric flask, dissolved and diluted up to mark with Distilled water to obtain final concentration of 1000 μg/ml MCA. Solution was further diluted with Distilled water to obtained working standard solutions of MCA.

Selection of suitable wavelengths for analysis: Solutions containing appropriate concentration of ATH (10 µg/ml) and MCA (10 µg/ml) in Distilled water were scanned using UV spectrophotometer in “Spectrum mode” in the range of 400 – 200 nm and their spectra were overlaid. From overlaid spectra of both the drugs analytical wavelengths for detection were selected.

Detection Wavelengths: 239 nm (λ₂) and 351 nm (λ₁).

Preparation of calibration curves: Absorbance of prepared standard solutions having concentration 20, 30, 40, 50 and 60 μg/ml for ATH and 3, 4.5, 6, 7.5 and 9 μg/ml for MCA were measured at 239 nm and 351 nm. Standard calibration curves of absorbance against concentration were plotted. Absorptivity coefficients were determined using calibration curves at both the wavelengths.

RESULT AND DISCUSSION:
Selection of Suitable Wavelength for Analysis:

Figure 3: Overlaid spectra of ATH (10 μg/ml) and MCA (10 μg/ml) in Distilled water

VALIDATION PARAMETERS:

Linearity:
Linearity study was carried at different concentration levels and from that according to dose ratio working range was selected to be 20-60 μg/ml and 3-9 μg/ml for ATH and MCA, respectively at both the wavelengths.

Table 1: Result of Calibration Curves of ATH and MCA for Absorption Correction method

Precision:
Precision of the method was determined in the terms of Repeatability, Intraday and Interday precision. Repeatability (% RSD) was assessed by analyzing test drug solution within the calibration range, six times on the same day. Intraday variation (% RSD) was determined by analysis of this solution three times on the same day. Interday variation (% RSD) was determined by analysis of this solution on three different day.

Limit of Detection (LOD) and Limit of Quantification (LOQ):
They were calculated as 3.3 σ/S and 10 σ/S respectively. Where σ is the standard deviation of the response (y-intercept) and S, is the mean of the slope of calibration plot.

Table 2 Result of LOD, LOQ and Precision by Absorption Correction method

Accuracy:
Accuracy was calculated by addition of standard drugs to preanalyzed sample at 3 different concentration levels. % Recovery was calculated from absorbance ratio. % Recovery was found to be between 98.34 – 100.21 for ATH and 98.73 – 102.11 for MCA.

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Table 3: Result of accuracy of ATH for absorption correction method

(n=3)

Table 4: Result of accuracy of MCA for absorption correction method

(n=3)

Analysis of Pharmaceutical formulation by Content uniformity:The method was successfully applied to determine the amounts of ATH and MCA present in the pharmaceutical formulation. The results obtained were in good agreement with the corresponding labelled amount.

Table 5: Content uniformity result by Absorption Correction Method: 

CONCLUSION:
Finally it concludes that all the parameters are within the limits and meet the acceptance criteria of ICH guidelines for method validation. The developed method is simple, accurate, precise and economical. Hence the method was a good approach for obtaining reliable results and found to be suitable for the routine analysis of Amitriptyline Hydrochloride and Methylcobalamin in their tablet dosage forms.

ACKNOWLEDGEMENT
The authors are thankful to S. J. Thakkar Pharmacy College, Rajkot, Gujarat, India for providing necessary facilities to carry out this work.

REFERENCES:
1. Indian Pharmacopoeia: The Indian Pharmacopoeia Commision, Ghaziabad, Volume II, 2010, pp 804.
2. British Pharmacopoeia: The Stationery Office on behalf of the Medicines and Healthcare products Regulatory Agency (MHRA), Volume I, 2010, pp 137, 264.
3. The United State Pharmacopoeia 27; United States Pharmacopoeial Convention, INC., 2004, pp 130, 378
4. Japanese Pharmacopoeia; Official monograph for part I; 15th Edn; The Ministry of Health and Welfare Ministerial Notification, 2006, pp 590.
5. Patel D and  Patel V; Simultaneous estimation of Amitriptyline Hydrochloride and Perphenazine by Absorption Ratio (Q- analysis) UV Spectrophotometric method in combined tablet dosage form; IJPSR; 2010; 1(12); 133-137.
6. Karia M and Gohel B; Q-absorbance ratio spectrophotometric method for simultaneous estimation of Amitriptyline HCL and Chlordiazepoxide in bulk drug and combined pharmaceutical dosage form; International Bulletin of Drug Research; 2013; 3(4), 29-36.
7. Galande VR, Baheti KG and Dehghan MH; UV-Visible spectrophotometric method for estimation of gabapentin and methylcobalamin in bulk and tablet; Int. J. Pharm Tech Res.; 2010; 2(1); 695-699.
8. Kalyankar TM, Panchakshari PP, Wadher SJ and  Pekamwar SS; Simultaneous Estimation of Duloxetine and Methylcobalamin in combined dosage form by Ultra-violet Spectrophotometry; Int. J. PharmTech Res.; 2013; 5(4); 1572-1580.
9. Sujatha N and Pavani KH; Analytical method development and validation of Amitriptyline hydrochloride and chlordiazepoxide in tablet by RP-HPLC; Indian Journal of Research in Pharmacy and Biotechnology; 2013; 1; 655-659.
10. Muthukumar S, Reddy S, Selvakumar D and David B; Development and validation of RP-HPLC method for the quantification of Piracetam and Mecobalamin; Int. J. Bio. Pharm. Res.; 2013; 4(10); 681-684.
11. Baheti KG and Galande VR; Validated simultaneous estimation of Gabapentin in the presence of methylcobalamin in tablet by HPTLC method; International Journal of Research in Pharmaceutical and Biomedical Sciences; 2011; 2(3); 1199- 1202
12. Patel D and Patel V; Simultaneous estimation of amitriptyline HCl and perphenazine in tablets by UV-Visible spectrophotometric and HPTLC; Int J Pharm Biomed Sci; 2010; 1(2); 20-23.

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