Application of gas chromatography-mass spectrometry with selected ion monitoring to the urinanalysis of 4-pyridoxic acid.

An analytical protocol has been developed for the analysis of urinary 4-pyridoxic acid (4-PA) by gas chromatography-mass spectrometry (GC-MS) for use in metabolic studies. Aliquots of urine were deproteinised and fractionated by isocratic reversed-phase high-performance liquid chromatography. The eluent fraction containing the 4-PA was collected, freeze-dried and silylated using N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide. Derivatisation produced the mono-tert.-butyldimethylsilyl derivative of 4-PA lactone. This derivative was readily amenable to GC-MS analysis in the electron ionisation (70 eV) mode, yielding a prominent fragment ion at m/z 222 ([M-57]+; base peak). A heavy isotope-labelled derivative of pyridoxine [dideuteriated pyridoxine; 3-hydroxy-4-(hydroxymethyl)-5-[hydroxymethyl-2H2]-2-methylpyridine] has been synthesised and is being employed to determine the kinetics of labelling of the body pools of vitamin B6. Kinetic measurements are based on the determination of the relative proportions of metabolically produced deuterium-labelled and non-labelled 4-PA in urine, obtained from stable isotope ratios determined by low-resolution selected ion monitoring using a bench-top quadrupole GC-MS system.