Determination of Methionine and Selenomethionine in Yeast by Species-Specific Isotope Dilution GC/MS

Abstract

A method for the simultaneous determination of methionine (Met) and selenomethionine (SeMet) in yeast using species-specific isotope dilution (ID) gas chromatography/mass spectrometry (GC/MS) is described. Samples were digested by refluxing for 16 h with 4 M methanesulfonic acid. Analytes were derivatized with methyl chloroformate and extracted into chloroform for GC/MS analysis. In addition to use of commercially available ¹³C-enriched Met and SeMet spikes for species specific ID analysis, a ⁷⁴Se-enriched SeMet spike was also available for comparison of results. In selective ion monitoring mode, the intensities of ions at m/z 221, 222, 269, 270, and 263 were used to calculate the 221/222, 269/270, and 269/263 ion ratios for quantification of Met and SeMet. Concentrations of 5959 ± 33 and 3404 ± 12 μg g^-1 (one standard deviation, n = 6) with relative standard deviations of 0.55 and 0.36% for Met and SeMet, respectively, were obtained using ¹³C-enriched spikes. A concentration of 3417 ± 8 μg g^-1 (one standard deviation, n = 6) was obtained using the ⁷⁴Se-enriched SeMet spike. The concentration of SeMet measured in the yeast is equivalent to 66.43 ± 0.24% of total Se and 30.31 ± 0.11% of total Met is in the form of SeMet. Method detection limits (three times the standard deviation) of 3.4 and 1.0 μg g^-1 were estimated for Met and SeMet, respectively, based on a 0.25-g subsample of yeast with 1 mL of extract used for derivatization. A similar concentration of 5930 ± 29 μg g^-1 (one standard deviation, n = 4) for Met and a lower concentration of 2787 ± 49 μg g^-1 (one standard deviation, n = 4) for SeMet were obtained for this yeast sample using species-specific ID analysis based on GC/MS with ¹³C-enriched Met and SeMet spikes when a 2-h open microwave digestion approach using 8 M methanesulfonic acid was used.