title: Use of different stationary phases for separation of isoniazid, its metabolites and vitamin B6 forms.
abstract: The ability of different stationary phases developed for the analysis of polar compounds (ZIC-HILIC, ZIC-pHILIC and Zorbax SB-Aq) to separate isoniazid, its metabolites (acetylisonazid, pyridoxal isonicotinoyl hydrazone, pyridoxal isonicotinoyl hydrazone 5-phosphate), pyridoxine, pyridoxal and pyridoxal 5-phosphate under MS compatible conditions was systematically investigated using HPLC-UV. The mobile phase strength, pH and buffer concentration were modified to assess their impact on the retention of these compounds. The best available separation of the compounds was achieved using 1 mM ammonium formate (pH≈6) and ACN (20:80, v/v) on ZIC-HILIC and employing 5 mM ammonium formate (pH 3.0) and ACN (40:60, v/v) on ZIC-pHILIC. A gradient profile using 0.5 mM ammonium formate (pH≈6) and MeOH (0-12 min: 10% MeOH, 12-15 min: 10-50% MeOH, 15-35 min: 50% MeOH, 35.0-35.2 min: 50-10% MeOH, 35.2-45.0 min: 10% MeOH) provided the best separation of the compounds on Zorbax SB-Aq. Subsequent LC-MS analysis demonstrated that ZIC-HILIC is useful for the analysis of pyridoxine, pyridoxal and pyridoxal isonicotinoyl hydrazone. However, the chromatographic conditions developed for the analysis of the compounds on Zorbax SB-Aq are capable of achieving the best separation of all compounds in this study with the higher sensitivity for most of the analytes.
authors.affiliation: Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského, Hradec Králové, Czech Republic.
authors.forename: Ivana
authors.initials: I
authors.lastname: Pasáková
authors.forename: Marcela
authors.initials: M
authors.lastname: Gladziszová
authors.forename: Jana
authors.initials: J
authors.lastname: Charvátová
authors.forename: Ján
authors.initials: J
authors.lastname: Stariat
authors.forename: Jiří
authors.initials: J
authors.lastname: Klimeš
authors.forename: Petra
authors.initials: P
authors.lastname: Kovaříková
mesh_terms: D000995:Antitubercular Agents / Q000032:analysis* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D007538:Isoniazid / Q000032:analysis* / Q000378:metabolism; D025101:Vitamin B 6 / Q000032:analysis*
chemical_list: D000995:Antitubercular Agents; D025101:Vitamin B 6; D007538:Isoniazid