Determination of 13 Vitamin B and the Related Compounds Using HPLC with UV Detection and Application to Food Supplements

Abstract

A high-performance liquid chromatographic method was developed for the separation and determination of 13 vitamin B compounds, including thiamin, riboflavin, riboflavin 5′-monophosphate, pyridoxine, hydroxocobalamin, cyanocobalamin, adenosylcobalamin, methylcobalamin, niacin, niacinamide, pantothenic acid, folic acid, and biotin. The method consisted of an ODS column, a gradient elution using pH 3.0 phosphate buffer–acetonitrile, ion-pairing reagent as mobile phase at 1.0 mL min−1, and UV detection at 210 nm. As a result, the 13 vitamin B compounds were separated in 60 min, and the specificity, linearity, range, limit of detection, limit of quantitation, intra- and inter-day precision, and recovery were satisfactory. The limit of detections ranged from 0.03 to 0.41 µg mL−1. Intra- and inter-day precision of peak area, expressed as RSD, were 0.542–5.144% and 0.216–6.367%, respectively. The method was used to evaluate vitamin B compounds in energy drinks and multivitamin pills. In case of energy drink analysis, the method identified and evaluated not only vitamin B compounds but also major ingredients, such as caffeine, vanillin, and benzoic acid. The vitamin B compounds in energy drinks and multivitamin pills were also quantitated and compared with values listed on each product label. Each quantitative value was close to the label values, suggesting high quantitative ability of the developed method.