NDLI: Determination of free methionine in human blood plasma by species-specific isotope dilution HPLC-ICP-MS using <sup>34</sup>S-labelled methionine

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Journal of Analytical Atomic Spectrometry Vol - 31 Issue - 8

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Determination of free methionine in human blood plasma by species-specific isotope dilution HPLC-ICP-MS using ³⁴S-labelled methionine

Content Provider	Royal Society of Chemistry (RSC)
Author	Blas, Oscar Galilea San Espílez, P. Herrero Alonso, José Ignacio García García, Belen Prieto Marchante-Gayón, Juan Manuel Sanz, F. Moreno Menéndez, Francisco V. Álvarez
Copyright Year	2016
Abstract	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 34S-labelled methionine as species-specific spike. The 34S-labelled methionine was obtained from yeasts grown in a medium enriched with 34S (>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 34S-labelled methionine standard solutions obtained were characterised both in terms of 34S isotope enrichment (82.7 ± 0.6%) and total sulfur concentration (396 ± 6 μg 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 34S-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 34S-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 32S and 34S 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.
Starting Page	1885
Ending Page	1894
Page Count	10
File Format	HTM / HTML PDF
ISSN	02679477
Volume Number	31
Issue Number	9
Journal	Journal of Analytical Atomic Spectrometry
DOI	10.1039/c6ja00125d
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Methionine Blood plasma Chromatography Protease High-performance liquid chromatography Isotope Sulfur Natural abundance Gas chromatography\u2013mass spectrometry Acid Centrifugation Ammonium PH Methanol
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Analytical Chemistry

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