科研之友
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摘要
A species-specific Isotope Dilution (ID) method is described for the determination of free methionine in human blood plasma by High Performance Liquid Chromatography - Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS) using S-34-labelled methionine as species-specific spike. The S-34-labelled methionine was obtained from yeasts grown in a medium enriched with S-34 (>91%) in the form of sulphate. Methionine was extracted from yeasts using enzymatic digestion with protease XIV followed by isolation using preparative reverse phase HPLC. Two separate batches of the labelled methionine standard were obtained. The S-34-labelled methionine standard solutions obtained were characterised both in terms of S-34 isotope enrichment (82.7 +/- 0.6%) and total sulfur concentration (396 +/- 6 mu g g(-1)) by reverse Isotope Dilution HPLC-ICP-MS using a natural abundance methionine standard solution and a multicollector instrument working in the pseudo-high resolution mode to avoid spectral interference. Additionally, the identity of the S-34-labelled methionine isolated by preparative HPLC and its isotope enrichment was confirmed by Gas Chromatography-Mass Spectrometry (GC-MS). Human blood plasma samples were spiked with the S-34-labelled methionine spike. Then, plasma proteins were precipitated with trifluoroacetic acid and separated by centrifugation. Methionine was separated from the rest of sulfur containing compounds by reversed phase chromatography in isocratic mode using a mobile phase of 75 mM ammonium acetate (pH 7.4) containing 2% of methanol. The retention time of methionine (8.3 minutes) was confirmed by fortifying the samples with natural abundance methionine and also by collecting the methionine peak and further analysis by GC-MS. Finally, methionine in human blood plasma samples was determined by measuring the signals for S-32 and S-34 in a double focusing ICP-MS instrument working at medium resolution (R = 4000). Concentrations were calculated by integrating the methionine peak for both masses and applying the isotope dilution equation after mass bias correction. The recoveries for samples fortified at different concentration levels ranged between 98.4 and 100.5%. Additionally, good agreement was obtained between the results found with this method and those reported by the clinical laboratory using a validated routine method.
- 出版日期2016
