GC-FID-based quantification of the sum of the three forms of vitamin B3 from animal liver

Vitamin B-3 (nicotinic acid, nicotinamide) is an essential water-soluble vitamin and cellular energy metabolism depends on the vitamin B-3-derived cofactors. Inaccessible covalently-linked nicotinic acid in food such as maize can cause vitamin B-3 deficiency in animals since maize is also deficient in tryptophan, the precursor of nicotinic acid. A sensitive and reproducible GC-FID-based method for the quantification of the sum of the three forms of vitamin B3 from animal liver was developed. Free nicotinic acid, free nicotinamide and nicotinamide moiety of NAD(+)/NADP(+) (and their riboside precursors) were simultaneously derivatized as methyl nicotinate. Reaction time and temperature and the extraction procedure for methyl nicotinate were optimized. Starting from wild boar liver, removal of proteins, solvent exchange, derivatization, and chloroform extraction resulted in sufficient enrichment and baseline separation of methyl nicotinate. The within-laboratory reproducibility of the full procedure was determined with RSD < 10%. On-column limit of detection and lower limit of quantification for methyl nicotinate were both sub-picomole. The accuracy of the method was determined from the recoveries of the pre-extraction spiked-in vitamin B-3 standards. The overall recovery for the full procedure was 16% but very consistent (RSD = 7%), enabling determination of apparent vitamin B-3 concentrations for relative quantitative comparison.