A Simplified, Specific HPLC Method of Assaying Thiamine and Riboflavin in Mushrooms

Mushrooms have been used as part of the average diet and as a nutraceutical for thousands of years due to their immense health benefits. The purpose of this study was to develop a simple, fast, accurate, specific, reproducible, and robust chromatographic method to identify and quantify two water-soluble vitamins: thiamine (B1) and riboflavin (B2) in mushrooms. The method employed for qualitative and quantitative analysis of these vitamins was Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) equipped with Ultraviolet–Visible (UV-Vis) Detector. The extraction process involved acid hydrolysis followed by enzymatic dephosphorylation with takadiastase enzyme. Chromatographic separation was achieved with a Shimadzu prominence HPLC system using isocratic elution mode on a Waters Xterra® MS C-18 column (4.6mm × 150mm, 5 μm) integrated with a XBridge® BEH C-18 Guard column (2.1mm × 5 mm, 5 μm). The mobile phase of this study consisted of buffer and methanol in the ratio of 80:20, where the buffer contained sodium-1-hexanesulfonate, glacial acetic acid, methanol, and pH adjusted to 3.0 with diethylamine. Vitamins were detected simultaneously at their lambda max wavelengths B1: 245nm and B2: 268nm using dual-wavelength UV detection technique to get their highest response. The proposed method was found to be specific, linear R>1.0, accurate, precise (% recovery ± SD; B1:104.45±4.5 and B2: 104.88±2.04), sensitive, (limit of detection for B1 and B2 was 0.043 and 0.029 μg/mL, respectively), and robust for mushrooms analysis. No coeluting peaks were observed at the retention time of the vitamins and all the peaks were spectrally homogenous. The standard and sample solutions were found to remain stable at cold temperature for 72 hours. In summary, our data suggest that the proposed method could be used in food industries to monitor the product quality during routine quality control purposes.