A Simple Internally-Standardised Isocratic HPLC Assay for Vitamin B₆ in Human Serum

Abstract

A simple method which is practical for use in a routine clinical chemistry laboratory, for assay of vitamin B₆ vitamers in serum is presented. The procedure uses a C₁₈ (ODS) analytical column and fluorimetric detection (325 nm excitation, 400 nm emission) after post column derivatisation with pH 7.5 phosphate buffer containing lg/1 sodium sulphite. The mobile phase is pH 2.5, 0.067 mol/1 KH₂PO₄ adjusted with concentrated orthophosphoric acid and containing 125 umol/1 sodium hexanesulphonate as an ion pairing agent. Pyridoxamine 5′-phosphate is suggested for use as an internal standard when assaying human serum.