Microwave-assisted acid digestion protocol for the determination of methionine and selenomethionine in selenium-enriched yeast by species specific isotope dilution GC-MS

Abstract

A quantitative and fast microwave assisted protein digestion method is described for the simultaneous determination of methionine (Met) and selenomethionine (SeMet) in yeast. Extraction of Met and SeMet from the selenized yeast was performed in a focused microwave system using methanesulfonic acid (MSA). The effects of parameters such as extraction time, temperature, power and sample mass on the extraction efficiencies of Met and SeMet were investigated. Species specific isotope dilution (ID) calibration using ¹³C enriched Met and SeMet spikes was employed to obtain accurate results. Analytes were derivatized with methyl chloroformate and extracted into chloroform prior to species specific ID GC-MS analysis. Using a 20 minute extraction time at 165 °C and 6 ml of 4 M MSA was found to be efficient for both analytes based on a 50 mg sample mass. Under these conditions, concentrations of 5862 ± 32 and 3366 ± 60 μg g⁻¹ (one standard deviation, n = 3) for Met and SeMet, respectively, were obtained in SELM-1 yeast certified reference material (CRM). The obtained results are in good agreement with the certified values of 5758 ± 277 and 3448 ± 146 μg g⁻¹ (expanded uncertainty, k = 2). Compared to previous MSA reflux digestion, this newly proposed method offers dramatic reduction in extraction time from 8–16 hours of the conventional MSA reflux to 20 minutes by microwave extraction, significantly improving the sample throughput. Additionally, the microwave extraction is fully automated and uses 75% less reagent (MSA) than the conventional acid reflux setup. The developed method is suitable for quasi real time production monitoring of Met and SeMet in Se enriched yeast and other food products.