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, specificmethodology to determine the amount of total selenium in selenium-enriched biomass of Saccharomyces cerevisiae was designed and implementedusing 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 tothe ICH Q2 (R1) requirements. The linearity was established over the dynamic range of 25-700 µg L-1 (r2=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.

Keywords:

HPLC-UV , 4 , 5-Diamino-o-xylene (DAX) , Se(IV) , Se(VI)