Author(s): Paskov I, Gladziszov M, Charvtov J, Stariat J, Klime J,
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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. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
This article was published in J Sep Sci
and referenced in Journal of Chromatography & Separation Techniques