Determination of selected water-soluble vitamins using hydrophilic chromatography: A comparison of photodiode array, fluorescence, and coulometric detection, and validation in a breakfast cereal matrix

Water-soluble vitamins are an important class of compounds that require quantification from food sources to monitor nutritional value. In this study we have analysed six water-soluble B vitamins ([thiamine (B1), riboflavin (B2), nicotinic acid (B3, NAc), nicotinamide (B3, NAm), pyridoxal (B6), folic acid (B9)], and ascorbic acid (vit C) with hydrophilic interaction liquid chromatography (HILIC), and compared UV, fluorescent (FLD) and coulometric detection to optimise a method to quantitate the vitamins from food sources. Employing UV/diode array (DAD) and fluorimetric detection, six B vitamins were detected in a single run using gradient elution from 100% to 60% solvent B [10mM ammonium acetate, pH 5.0, in acetonitrile and water 95:5 (v:v)] over 18min. UV detection was performed at 268nm for B1, 260nm for both B3 species and 284nm for B9. FLD was employed for B2 at excitation wavelength of 268nm, emission of 513nm, and 284nm/317nm for B6. Coulometric detection can be used to detect B6 and B9, and vit C, and was performed isocratically at 75% and 85% of solvent B, respectively. B6 was analysed at a potential of 720mV, while B9 was analysed at 600mV, and vit C at 30mV. Retention times (0.96 to 11.81min), intra-day repeatability (CV 1.6 to 3.6), inter-day variability (CV 1.8 to 11.1), and linearity (R 0.9877 to 0.9995) remained good under these conditions with limits of detection varying from 6.6 to 164.6ngmL(-1), limits of quantification between 16.8 and 548.7ngmL(-1). The method was successfully applied for quantification of six B vitamins from a fortified food product and is, to our knowledge, the first to simultaneously determine multiple water-soluble vitamins extracted from a food matrix using HILIC.