VALIDATED STABILITY-INDICATING RP-HPLC ASSAY METHOD FOR AZATHIOPRINE IN PHARMACEUTICAL DOSAGE FORM ACCORDING TO ICH GUIDELINES

Authors

  • BANDI RAMACHANDRA P. SUPRIYA Department of Chemistry, S. V. University, Tirupati-517502, A. P. India
  • C. Ramanjulu Department of Chemistry, S. V. University, Tirupati-517502, A. P. India
  • N. Venkatasubba Naidu Department of Chemistry, S. V. University, Tirupati-517502, A. P. India

Keywords:

Azathioprine, Assay HPLC, Validation, Stability-indicating, Degradation profile

Abstract

Objective: A simple, sensitive, precise and accurate stability-indicating HPLC method has been developed and validated for determination of Azathioprine in bulk drug and in pharmaceutical dosage form in the presence of degradation products.

Methods: An isocratic, reversed phase HPLC method was developed to separate the drug from the degradation products, using a Hypersil C8, 150 X 4.6 mm, 5µ, Thermostat column compartment connected with Waters (alliance) Empower software. Mobile phase consists mixture of Potassium dihydrogen phosphate(A) and Acetonitrile(B) in the ratio (pH 6.50±0.1, ratio 60:40 v/v) at a flow rate of 1.0 mL /min. The linear regression analysis data for the calibration curve showed a good linear relationship with regression coefficient 1. The detection was carried out at a wavelength of 254 nm.

Results: The linearity of the method were excellent over the range 2.5-15 μg/ml, the linear regression equation was Y=76621×+12515. The Azathioprine was subjected to stress conditions of hydrolysis (acid, base), photolysis and thermal degradation. Degradation was observed for Azathioprine in acid, base, heat and UV. The degradation products were well resolved from the main peak. The percentage recovery of Azathioprine was from (98.0 to 102.0 %.) in the pharmaceutical dosage form.

Conclusion: The results demonstrated that the method would have a great value when applied in quality control and stability studies. The developed method was validated with respect to linearity, accuracy (recovery), precision, system suitability, specificity and robustness. The forced degradation studies prove the stability indicating power of the method.

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References

American society of health-system pharmacists azathioprine, azathioprine sodium ahfs drug information american society of health-system pharmacists. ISBN 2012;978(1):58528-267.

Elion G. The purine path to chemotherapy. Sci 1989;244:41–7.

Patel AA, Swerlick RA, McCall CO. Azathioprine in dermatology: the past, the present, and the future. J Am Acad Dermatol 2006;55(3):369-89.

B Elion G, GH Hitchings, AG Johns, DG Eds. Handbook of Experimental Pharmacology: Sartorelli, Springer Verlag; Berlin 1975;38:404-25.

Gorski A, Korszak-Kowalska A, Nowaczyk G, Paczek L. Gaciong. Immunopharmacol 1983;6:259-66.

Jianbo W, Pan Z, Suqin H. Direct determination of Azathioprine inhuman fluids and pharmaceutical formulation using flow injection chemiluminescence analysis. J Chin Chem Soc 2011;58(6):1-7.

Spectrometric determination and validation of Azathioprine in API and pharmaceutical dosage form. J Optoele Biomed Mate 2010;2:213-6.

Prakash M Davadra, Vivek V Mepal, Mukul R Jain, Chhelshanker G Joshiband Atul H. Bapodra A validated UPLC method for the determination of process-related impurities in azathioprine bulk drug Anal. Methods, 2011;199(3):198–204.

US Pharmacopoeia, USP 2007;30:1471–2.

TT Fazio, AK Singh, ER Maria, Kedor-Hackmann, MI Rocha Santoro. Quantitative determination and sampling of azathioprine residues for cleaning validation in production area. J Pharm Biomed Anal 2007;43:1495–8.

Christiana Stefan, Warren Walsh, Tibor Banka, Khosrow Adeli, Zulfika Verjee. Improved HPLC methodology for monitoring thiopurinemetabolites in patients on therapy. Clini Biochem 2004:37(9):764-71.

Binscheck T, Meyer H, Wellhoner HH. HPLC assay for the measurement of Azathioprine in human serum sample. J Chromatogr B Biomed Appl 1996; 26(675):287-94.

Jain PS, Thakre PD, Chaudhari PJ, Chavan ML, Surana SJ. Determination of Azathioprine in bulk and pharmaceutical dosage form by HPTLC. J Pharm Bio Allied Sci 2012;4(4):318-21.

Smita S, Mukesh S. Spectrophotometric and atomic absorption spectrometric Determination and validation of azathioprine in API and pharmaceutical dosage form. J Optoele Biomed Mate 2010;2(4):213–6.

Anstey A, Lennard L, Mayou SC, Kirby JD. Pancytopenia related toazathioprine an enzyme deficiency caused by a common genetic polymorphism. J R Soc Med 1992;85:752– 6.

Lennard L, Van Loon J, Lilleyman J, Weinshilboum R. Thiopurine pharmacogenetics in leukemia: correlation of erythrocyte thiopu-rine methyl transferase activity and 6 thioguanine nucleotides concentrations. Clin Pharmacol Ther 1987;41:18 –25.

Boulieu R, Lenoir A, Mornex JF, Bertocchi M. Intracellular thiopu-rine nucleotides and azathioprine myelotoxicity in organ transplant patients. Br J Clin Pharmacol 1997;43:116 –8.

Vogt M, Stet E, De Abreu R, Bokkering J, Lambooy L, Trijbels J. The importance of methylthio-IMP for methyl mercaptopurine ribonucleoside (Me-MPr) cytotoxicity in Molt F4 human malignant Tlymphoblasts. Biochim Biophys Acta 1993:1181:189–94.

Anstey AV, Wakelin S, Reynolds NJ. British association of dermatologists therapy, guidelines and audit subcommittee. guidelines for prescribing azathioprine in dermatology. Br J Dermatol 2004;151:1123-32.

Demirtas-Ertan G, Rowshani AT, ten Berge IJ. Azathioprine-induced shock in a patient suffering from undifferentiated erosive oligoarthritis. Neth J Med 2006;64:124-6.

Kaushal K, Manchanda Y. Long-term safety and toxicity of azathioprine in patients with air-borne contact dermatitis. Indian J Dermatol Venereol 2001;67:75-7.

Bhaskar M, Manohara YN, Gayasuddin M, Balaraju M, Bharath Kumar T. Spectrophotometric determination of azathioprine in bulk and pharmaceutical dosage forms. Int J Chem Tech Res 2010;2:376-8.

Smita S, Mukesh S. Spectrophotometric and atomic absorption spectrometric determination and validation of azathioprine in API and pharmaceutical dosage form. J Optoele Biomed Mate 2010;2:213-6.

Jianbo W, Pan Z, Suqin H. Direct determination of azathioprine in human fluids and pharmaceutical formulation using flow injection chemiluminescence analysis. J Chin Chem Soc 2011;58:1-7.

Binscheck T, Meyer H, Wellhoner H. High-performance liquid chromatographic assay for the measurement of azathioprine in human serum samples. J Chromatogr B Biomed Appl 1996;675:287-94.

Lakshmi C, Reddy M. Spectrophotometric determination of azathioprine in pharmaceutical formulations. Talanta 1998;47:1279-86.

Boulieu R, Lenoir A. High performance liquid chromatographic determination of thiopurine metabolites of azathioprine in biolog-ical fluids. J Chromatogr 1993;615:352-6.

Erdmann G, France L, Bostrom B, Canafax M. A reversed phase high performance liquid chromatographic approach in determining total red blood cell concentrations of 6 thioguanine, 6-mercapto-purine, methylthioguanine and methylmercaptopurine in a patient receiving thiopurine therapy. Biomed Chromatogr 1990;4:47–51.

Lennard L, Singleton H. High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6mercaptopurine: quantification of red blood cell 6 thioguanine nucleotide, 6thioinosinic acid and methylmercaptopurine metabolites in a single sample. J Chromatogr 1992:58:383–90.

Janine EP, Friedman JM. Teratogen update: Azathioprine and 6-mercaptopurine. Tetral 2002;65 (5):240-61.

Fell F, Plag SM. Stability-indicating assay for azathioprine and 6-mercaptopurine by reversed-phase high-performance liquid chromatography. J Chromatogr 1979;186(30):691-704.

Hofmann U, Heinkele G, Angelberger S, Schaeffeler E, Lichtenberger C, Jaeger S. Simultaneous quantification of eleven thiopurine nucleotides by liquid chromatography-tandem mass spectrometry. Anal Chem 2012;84(3):1294-301.

El-Yazigi A, Wahab FA. Expedient liquid chromato-graphic analysis of azathioprine in plasma by use of silica solid phase extraction. Ther Drug Mon 1992;14(4):312-6.

Albertioni F, Pettersson B, Ohlman S, Peterson C. Analysis of azathioprine and 6-mercaptopurine in plasma in renal transplant recipients after administration with oral azathioprine. J Liq Chromatogr Relat Technol 1995;18:(20):3991-4005.

Maddocks JL. Assay of azathioprine, 6-mercaptopurine and a novel thiopurine metabolite in human plasma. Br J Clin Pharmacol 1979;8(3):273–8.

S Singh, M Bakshi. J Pharm Biomed Anal 2002;28:1011-40.

http://private.ich.org/cache/compo/363-272-1.html, accessed in September, 2009;15.

Published

01-10-2014

How to Cite

SUPRIYA, B. R. P., C. Ramanjulu, and N. V. Naidu. “VALIDATED STABILITY-INDICATING RP-HPLC ASSAY METHOD FOR AZATHIOPRINE IN PHARMACEUTICAL DOSAGE FORM ACCORDING TO ICH GUIDELINES”. International Journal of Pharmacy and Pharmaceutical Sciences, vol. 6, no. 10, Oct. 2014, pp. 301-7, https://mail.innovareacademics.in/journals/index.php/ijpps/article/view/2747.

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