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.