Simultaneous determination some of b group vitamins in nutritional products by liquid chromatography tamdem mass spectrometry following enzymatic hydrolysis

Abstract

Liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous qualification of relevant forms of B group vitamins including vitamin B1, B2, B3 and B6 in nutritional products. Samples are hydrolyzed by enzymatic digestion at 37ºC in the shaking water bath within approximately 12 to 14 hours. Papain and α-amylase enzymes were used to hydrolize the protein and complex carbohydrate. Acid phosphatase was used to cut phosphoryl links to form free vitamin forms. The separation was achieved on a C18 column (100mm×2.1mm×1.7μm). Analytes were eluted with the mobile phase of 10 mM ammonium formate and methanol by gradient program at a flow rate of 0.15 mL/min. The calibration curves ranged from 0.2 to 2000 ng/mL with the correlation coefficients > 0.998. Limits of detection and quantification of method ranged from 2.7 to 3.8 μg/100g and 9.1 - 12.6 μg/100g, respectively. The method was validated at three different concentrations with the recovery of 80 - 110%, relative standard deviation of repeatability, RSDr %, of 2.61 - 4.69%, and intermediate reproducibility, RSDR% of 3.40 - 9.69%. The method was applied to simultaneously determine the four water soluble vitamins in several of nutritional products.