Semi-automated system for analysis of vitamin B6 complex by ion-exchange column chromatography

A simple system for semi-automatic analysis of pyridoxal, pyridoxine and pyridoxamine has been developed using diazide of 5-chloroaniline 2,4-disulfonyl chloride as a color-producing reagent in combination with desoxypyridoxine as an internal standard. It employs Aminex A-5 column, 10x0.6ø cm, as an adsorbent. Adsorbates were eluted successively with 3 discrete phosphate buffers (0.4 N Na+). The effluent is mixed continuously with capillary streams of the diazide reagent and of sodium acetate. The mixture, maintained at 65 degrees C in a heating bath, then passes through a spiral of Teflon tubing with a residence time of 2 min. Characteristic orange colored products formed by a diazo coupling reaction are continuously monitored at 440 nm in a flow photometer. The individual peaks on the recorded chromatogram are manually integrated by a conventional HW method. The elution position and recovery of desoxypyridoxine permits correction for sensitivity changes or mechanical losses which might occur during a series of analyses. The analyzer system described allow quantitation from 2 to 25 mug of pyridoxal, pyridoxine and pyridoxamine in a single sample within 2 hr and with a precision of 100 +/- 4%. It is also found suitable as a routine procedure for the analysis of varied biological samples.