Method Development and Validation of Stability Indicating RP-HPLC Method for Simultaneous Estimation of Escitalopram Oxalate and Clonazepam in Bulk and its Pharmaceutical Formulations
This article refers to simple isocratic reverse-phase high-performance liquid chromatographic method (RP-HPLC) developed for the simultaneous quantification of Escitalopram Oxalate (EST) and Clonazepam (CZP) in active pharmaceutical ingredient and pharmaceuticals. The separation of the two drugs was attained using a C₁₈ column (250mm×4.6mm, 5µ) as a stationary phase. The mobile phase was used as a mixture of methanol; acetonitrile; and 0.05M potassium dihydrogen orthophosphate buffer (pH 4 adjusted by orthophosphoric acid) with an isocratic ratio of 40:20:40 v/v. Detection was made by using PDA detector at 210 nm. Escitalopram Oxalate (RT= 4.428 minutes) and Clonazepam (RT= 6.532 minutes) were separated in a single chromatographic run with resolution of 8.719. The calibration plot indicated good linear relationship with r2 = 0.998 for Escitalopram Oxalate in concentration range of 32 µg/ml - 48 µg/ml and r2 = 0.999 for Clonazepam in concentration range of 16 µg/ml - 24 µg/ml. The retrievals for Escitalopram Oxalate and Clonazepam were found to be 99.75% and 99.00%, respectively. The established analytical method was validated and found acceptable as per ICH guidelines for linearity, precision, accuracy, specificity, limit of detection, limit of quantification, robustness and stability. Escitalopram Oxalate and Clonazepam individually as well as in combination were exposed to different stress conditions like acid, base, thermal, photolytic and oxidation degradation and peaks of a degraded product were well determined from peaks of pure drug. This method is modest, quick and appropriate for routine quality control analysis.
Keywords: Reverse Phase – HPLC; Escitalopram Oxalate; Clonazepam; Validation; Degradation study.
2. Kasper S, Stein DJ, Loft H, Nil R, Escitalopram in the treatment of social anxiety disorder-Randomized, placebo-controlled, flexible-dosage study, British Journal of Psychiatry, 2005; 186(3):222-226.
3. Rao N, The clinical pharmacokinetics of escitalopram, Clinical Pharmacokinetics, 2007; 46(4):281–290.
4. Singh S, Shah H, Gupta S, Jain M, Sharma K, Thakkar P, Shah R, Liquid chromatography–electrospray ionisation mass spectrometry method for the determination of escitalopram in human plasma and its application in bio-equivalence study, Journal of Chromatography B, 2004; 811:209–215.
5. Martindale KP, The Extra Pharmacopoeia ‐ Martindale, The Pharmaceutical Press, London, 1999.
6. British Pharmacopoeia, 3 Vol, Medicines and Health Care Products Regulatory Agency London; 2018:383-384.
7. United States Pharmacopoeia, 41-NF 37, Rockville: The United States Pharmacopoeial Convention; 2018:1021-1021.
8. Monti JM, Pandi-Perumal SR, Möhler H, GABA and Sleep, Molecular, Functional and Clinical Aspects, Springer, Basel. 2010.
9. García-Fernández MA, Fernández-Abedul MT, Costa-García A, Determination of buprenorphine in biological samples by high performance liquid chromatography with electrochemical detection, Chromatographia, 2001; 53(11-12):704-708.
10. Panchal JA, Maheshwari DG, Review on chromatographic and spectrophotometric estimation of escitalopram oxalate and eszopiclone in bulk and in different dosage form, European Journal of Pharmaceutical and Medical Research, 2017; 4(1):183-191.
11. Kumar V, Singh HP, Rathore RPS, Method development & validation of escitalopram oxalate & etizolam by UV- spectrophotometry, International Journal of Institutional Pharmacy and Life Sciences, 2015; 5(2):12-42.
12. Kakde RB, Satone DD, Spectrophotometric Method for Simultaneous Estimation of Escitalopram Oxalate and Clonazepam in Tablet Dosage Form. Indian Journal of Pharmaceutical Sciences, 2009; 71(6):702-705.
13. Liu SY, Liu KS, Kuei CH, Tzeng JI, Ho ST, Wang JJ, Simultaneous determination of buprenorphine and its prodrug, buprenorphine propionate, by high-performance liquid chromatography with fluorescence detection: application to pharmacokinetic studies in rabbits, Journal of Chromatography. B, 2005; 818:233-239.
14. Everhart ET, Cheung P, Shwonek P, Zabel K, Tisdale EC Jacob, P, Mendelson J, Jones RT, Subnanogram-concentration measurement of buprenorphine in human plasma by electron-capture capillary gas chromatography: application to pharmacokinetics of sublingual buprenorphine Clinical Chemistry, 1997; 43:2292-2302.
15. Lisi AM, Kazlauskas R, Trout GJ, Gas chromatographic-mass spectrometric quantitation of urinary buprenorphine and norbuprenorphine after derivatization by direct extractive alkylation, Journal of Chromatography B, Biomedical Sciences and Applications, 1997; 692(1):67-77.
16. Rajamanickam V, Santhosam D, Sridharan D, Thenmozhi A, Simultaneous determination of risperidone and Trihexhyphenidyl hydrochloride from bulk and tablet dosage form by RP-HPLC, Asian Journal of Research in Chemistry, 2010; 3 (3):549–551.
17. Dabir J, Mary Mathew E, Moorkoth S, Analytical method development and validation of RP-HPLC method for simultaneous estimation of N-acetyl cysteine and cefexime from its fixed dose combination, Research Journal of Pharmacy and Technology, 2016; 9(7):835-842.
18. Vinyas M, Velivela S, Yadav G, Pati NB, Gupta VRM, Analytical Method Development and Validation of alogliptin by RP-HPLC Method, Research Journal of Pharmacy and Technology, 2016; 9(7):775-778.
19. Patel RB, Patel MR, Dubey N, Dubey NN, and Patel BG, HPTLC method development and validation: Strategy to minimize methodological failures, Journal of Food and Drug Analysis, 2012; 20:561–571.
20. Kumar M, Rao JR, Yadav SS, Sathiyanarayanan L, Vikas, Development and validation of a stability-indicating HPTLC method for analysis of bumetanide in the bulk drug and tablet dosage form, Research Journal of Pharmacy and Technology, 2010; 3(1): 239-243.
21. International Conference on Harmonization (ICH), Validation of analytical procedures: text and methodology Q2(R1)., Geneva, Switzerland, 2005.
22. Reddy Thumma PK, Deepthi PN, Sudheer B, Reddy PP, Kandukuri S, Banji D, Formulation, evaluation and stability studies for the sustained release mucoadhesive microcapsules of gliclazide, Research Journal of Pharmacy and Technology, 2013; 6(11):1242-1246.
23. Agrawal H, Kaul N, Paradkar AR, Mahadik KR, The ICH guidance in practice: stress degradation studies on indinavir sulphate and development of a validated specific stability-indicating HPTLC assay method. Farmacopia, 2004; 59(9):729–738.
24. Mahalingam V, Vijayabaskar S, Kalaivani RA, Somanathan T, Analytical method development and validation for the analysis of donepezil hydrochloride and its related substances using ultra performance liquid chromatography, Research Journal of Pharmacy and Technology, 2017; 10(8):2743-2749.
25. Shetti P, Venkatachalam A, Stability indicating HPLC method for simultaneous quantification of trihexyphenidyl hydrochloride, trifluoperazine hydrochloride and chlorpromazine hydrochloride from tablet formulation. Journal of Chemistry, 2010; 7:299-313.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. that allows others to share the work with an acknowledgment of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).