Isotope dilution method for determination of vitamin B2 in human plasma using liquid chromatography-tandem mass spectrometry.

Vitamin B2 (riboflavin) is an essential constituent of the coenzymes flavin mononucleotide and flavin adenine dinucleotide and is critical for human metabolism and energy production. Liquid chromatography separation, then tandem mass spectrometry detection (LC-MS/MS) is a highly specific technique that enables quick analysis. An isotope dilution method for determination of riboflavin in human plasma using LC-MS/MS has been developed and validated in this study. In this method, 100 μL of plasma were added of 75 μL of an internal standard and treated with 125 μL of 0.1 M zinc sulfate. The analysis was performed using an Agilent 6460C tandem mass spectrometer system operated in the positive-ion mode. Chromatography separation performance was obtained with an Agilent 1290 UHPLC equipped with a Poroshell 120 SB-Aq column (100 mm × 2.1 mm × 2.7 μm) at a flow rate of 350 μL·min. Riboflavin was measured within an instrument run time of 2.5 min. Riboflavin content was found to follow a linear trend in the 0.5 to 50.0 ng·mL range and dilution was validated for samples that exceed the last point of the calibration curve until two times. The intra-day relative standard deviation (RSD) was less than 10% and inter-day RSD was less than 11%. The average of recovery was 90.5-99.5%. A sensitive and selective method has been developed and validated successfully for quantitative analysis of riboflavin in human plasma with a simple sample extraction method and short execution time. The method is reliable and can be applied in clinical diagnosis of deficiency and supplementation monitored of this vitamin.