Method Development for Separation and Quantification of Niacin and its Metabolites in Human Blood Plasma

Abstract

A high performance liquid chromatography (HPLC) assay for the detection of Niacin (nicotinic acid/vitamin B3), nicotinamide, and nicotinuric acid in human blood plasma was developed. The method used a C18 stationary phase with a mobile phase of 0.1% formic acid in water with varied methanol for chromatographic separation. UV absorbance detection was used in conjunction with mass spectrometry. Samples were prepared by mixing with methanol containing niacin D-4 internal standard, vortexing under vacuum, adding an aqueous formic acid buffer to reconstitute the dried pellet, followed by centrifugation at 15000g. Linearity was assessed and LLD (lower limit of detection) was determined. The elution order was niacin, nicotinamide, and finally nicotinuric acid. No issues with the calibration for nicotinuric acid were encountered, however there was significant interference from nicotinuric acid on the nicotinamide calibration. Currently new methods to limit the interferences are being researched.