title: Optimization of a reversed-phase-high-performance thin-layer chromatography method for the separation of isoniazid, ethambutol, rifampicin and pyrazinamide in fixed-dose combination antituberculosis tablets.
abstract: This paper presents the development of a new RP-HPTLC method for the separation of pyrazinamide, isoniazid, rifampicin and ethambutol in a four fixed-dose combination (4 FDC) tablet formulation. It is a single method with two steps in which after plate development pyrazinamide, isoniazid and rifampicin are detected at an UV wavelength of 280 nm. Then ethambutol is derivatized and detected at a VIS wavelength of 450 nm. Methanol, ethanol and propan-1-ol were evaluated modifiers to form alcohol-water mobile phases. Systematic optimization of the composition of each alcohol in the mobile phase was carried out using the window diagramming concept to obtain the best separation. Examination of the Rf distribution of the separated compounds showed that separation of the compounds with the mobile phase containing ethanol at the optimal fraction was almost situated within the optimal Rf-values region of 0.20-0.80. Therefore, ethanol was selected as organic modifier and the optimal mobile phase composition was found to be ethanol, water, glacial acetic acid (>99% acetic acid) and 37% ammonia solution (70/30/5/1, v/v/v/v). The method is new, quick and cheap compared to the actual method in the International Pharmacopoeia for the assay of the 4 FDC tablets, which involves the use of two separate HPLC methods.
authors.affiliation: Directorate of Laboratory Services, Tanzania Food and Drugs Authority, P.O. Box 77150, Dar es Salaam, Tanzania.
authors.forename: D H
authors.initials: DH
authors.lastname: Shewiyo
authors.forename: E
authors.initials: E
authors.lastname: Kaale
authors.forename: P G
authors.initials: PG
authors.lastname: Risha
authors.forename: B
authors.initials: B
authors.lastname: Dejaegher
authors.forename: J
authors.initials: J
authors.lastname: Smeyers-Verbeke
authors.forename: Y
authors.initials: Y
authors.lastname: Vander Heyden
mesh_terms: D000995:Antitubercular Agents / Q000032:analysis / Q000302:isolation & purification; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D002855:Chromatography, Thin Layer / Q000379:methods*; D004977:Ethambutol / Q000032:analysis / Q000302:isolation & purification*; D000431:Ethanol / Q000737:chemistry; D007538:Isoniazid / Q000032:analysis / Q000302:isolation & purification*; D011718:Pyrazinamide / Q000032:analysis / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012293:Rifampin / Q000032:analysis / Q000302:isolation & purification*; D013607:Tablets / Q000737:chemistry
chemical_list: D000995:Antitubercular Agents; D013607:Tablets; D011718:Pyrazinamide; D000431:Ethanol; D004977:Ethambutol; D007538:Isoniazid; D012293:Rifampin