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.

Development of a LC-MS/MS method using stable isotope dilution for the quantification of individual B6 vitamers in fruits, vegetables, and cereals

“…In a survey regarding the separation of various substrates—among others PN, PL, and PLP—on different HILIC stationary phases, LOQs of 0.006 to 0.03 mg/kg for PN and 0.019 to 0.068 mg/kg for PL were determined using stock solutions [ 67 ].…”

A Novel Two-Dimensional Liquid Chromatography Combined with Ultraviolet Detection Method for Quantitative Determination of Pyridoxal 5′-Phosphate, 4-Pyridoxine Acid and Pyridoxal in Animal Plasma

“…For separating PLP, PA, and PL from endogenous substances, the stationary phase was selected for reversed-phase C18 and C8 column [ 29 , 46 , 47 , 48 , 49 , 50 , 51 ], and the mobile phase was isocratic elution, which requires a suitable buffer added to adjust the pH and the elimination of overlapping peaks, tail peaks, and shoulder peaks. A Waters Summetry ® C18 column was selected as the 2D column, and the Aston TM ® SPY column (phenyl hexyl column, Hunan Demite instrument Co., Ltd., Changsha, China) was chosen as the 1D column, which had simultaneous retention of three analytes, and the mobile phases of the two dimensions had to be compatible.…”

Recent Developments in High-Performance Liquid Chromatography Stationary Phases

“…Spagou et al conducted metabolic profiling of rat urine in a hepatotoxicity study of galactosamine and the protective effect of glycine . Several other reports of interest involve metabolites. − …”