Methotrexate determination in pharmaceuticals by enantioselective HPLC

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 37; no. 5; pp. 919 - 925
• Main Authors: El-Hady, D. Abd, El-Maali, N. Abo, Gotti, R., Bertucci, C., Mancini, F., Andrisano, V.
• Format: Journal Article Conference Proceeding
• Language: English
• Published: Amsterdam Elsevier B.V 29-04-2005; Elsevier Science
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chirobiotic T™ stationary phase; Chromatography, High Pressure Liquid - methods; Enantioselective HPLC; Fundamental and applied biological sciences. Psychology; General pharmacology; Medical sciences; Methotrexate; Methotrexate - analysis; Methotrexate - chemistry; Molecular Conformation; Pharmaceutical formulations; Pharmaceutical Preparations - analysis; Pharmaceutical Preparations - chemistry; Pharmacology. Drug treatments; Chirobiotic T™ stationary phase; Enantioselective HPLC; Methotrexate; Pharmaceutical formulations; Antifolate; Antineoplastic agent; Chirobiotic T# stationary phase; Antimetabolic; Enantiomer; HPLC chromatography; Dosage form; Quantitative analysis
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2004.07.046

A simple, sensitive and selective high performance liquid chromatographic method with UV detection for the chiral separation of racemic methotrexate (
rac-Mtx) and enantiomeric purity of
l-methotrexate in pharmaceutical formulations was developed and validated. The chiral separation was optimized studying both the nature of the stationary phase by using Chirobiotic T™, Chiracel OJ and human serum albumin columns and the effect of the mobile phase composition. The best results in terms of enantioresolution and enantioselectivity were achieved with a polar organic mobile phase on Chirobiotic T™ stationary phase. Essential steps in method validation such as precision, accuracy, suitability and stability were studied according to ICH guidelines. At wavelength 303
nm, the limit of detection (S/N
=
3) was found to be 0.9
μg/ml for
rac-Mtx. The separation of
d-Mtx at 0.2% (w/w) level (as limit of quantitation) from the main drug
l-Mtx was successfully obtained with 1.72 enantioresolution value. Enantiomeric purity of
l-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter- and intra-days relative standard deviation
≤
1.6%. Under the validated stereoselective HPLC conditions for methotrexate, folic acid was also analysed.