Rapid, Simple, Cost Effective and Validated Methodology for Total Selenium Determination in Saccharomyces Cerevisiae Using RP-HPLC-UV After Microwave Assisted Digestion

A facilitated, precise, specific methodology to determine the amount of total selenium in selenium-enriched biomass of Saccharomyces cerevisiae was designed and implemented using high-performance liquid chromatography technique and UV detection (HPLC-UV). In this research study, a novel chromogenic reagent of 4, 5-Diamino-o-xylene (DAX) was evaluated for off-line pre-column selenium (IV) complexation. The complex of Se (IV) was eluted isocratically on BRISA LC2 C18 (250×0.46 mm, 5 μm, Teknokroma) analytical column. The mobile phase used was a degassed solution of deionized water and acetonitrile solution in a ratio of 50:50 (v/v) at a flow rate of 1.0 mL/min. UV-Vis detector at 340nm was used for complex detection. The method parameters were validated according to the ICH Q2 (R1) requirements. The linearity was established over the dynamic range of 25-700 μg L^-1 (r^2=0.9994). The relative standard deviations (RSDs) were 1.1% for within-day determination (n=6) and 1.8% for between-day determination (n=6). The detection limit (LOD) and quantitation limit (LOQ) were found to be 8.0 and 25 μg L^-1, respectively.