Reversed-phase high-performance liquid chromatography method with coulometric electrochemical and ultraviolet detection for the quantification of vitamins B1 (thiamine), B6 (pyridoxamine, pyridoxal and pyridoxine) and B12 in animal and plant foods

A rapid and sensitive high-performance liquid chromatography (HPLC) method with coulometric electrochemical and ultraviolet detections for analysis of vitamin B (B1, B6 and B12) in animal and plant foods has been developed. A combination of acid digestion and enzymatic extraction to release protein bound and phosphorylated vitamins followed by HPLC analysis was applied. The analyses were carried out on a LC18 column 5 μm (25 cm × 4.6 mm), using the mobile phase consisting of methanol–phosphate buffer (10:90) and 0.018 M trimethylamine, pH 3.55, following at 1.0 ml min−1. The method offers excellent linearity with regression coefficient r > 0.998, good repeatability and reproducibility and relatively short analysis time (17 min). The relative standard deviation (RSD) of the intermediate precision was satisfactory for all the vitamins studied and amounted to <7.3%. The repeatability of the method was <4.7%. The limits of quantification (LOQ) for pyridoxamine, pyridoxal, pyridoxine, vitamins B1 and B12 in seafood products were as follows: 2.1, 2.01, 0.99, 62.0 and 0.11 ng ml−1, respectively. The mean recovery values from a food products spiked with all five vitamins ranged from 92.3 to 101.3%, with a relative standard deviation less than 3.4%. The proposed separation and detection procedures were successfully applied for the simultaneous determination of vitamins B1, B6 and B12 in fruit juices and vitamins B6 and B12 in seafood.