Introduction – Sulphur-containing metabolites play an important role in metabolism and homeostasis. Determination of
these metabolites is challenging owing to their low concentrations and the interference in mass spectrometry analysis.
Objective – To develop a sensitive and accurate method based on liquid chromatography, electrospray ionisation, tandem
mass spectrometry (LC-ESI-MS/MS) and 34S-metabolic labelling for quantification of methionine, reduced glutathione, oxidised
glutathione in Arabidopsis thaliana.
Methodology – A hydroponic set-up was used for the in vivo 34S-metabolic labelling of A. thaliana. The 34S-labelled metabolites
biosynthesised in plant were extracted and used as internal standards. Tissue was extracted with perchloric acid (PCA) or
PCA containing a known amount of the analytes for recovery analysis. Tissue extract mixed with extract of 34S-labelled A.
thaliana in an appropriate ratio was subjected to a LC system and electrospray ionisation-mass spectrometric (ESI-MS)
analysis. Quantification of metabolites was measured by comparing the 32S/34S ratios obtained for samples with the calibration
curves.
Results – Calibration curves showed linearity with regression coefficients in the range of 0.9994–0.9999. Analyte recoveries
were approximately 100%. The coefficients of variation of intra-assay and inter-assay were less than 4.2% and 5%, respectively.
The ranges for the limits of detection determined for Met, GSSG and GSH were 10 fmol, <10 fmol and 1.12 fmol and
the limits of quantification determined for Met, GSSG and GSH were 0.44 pmol, 0.16 pmol and 34 fmol, respectively.
Conclusion – The validated method for determination of methionine, reduced glutathione and oxidised glutathione was
effectively applied to measure metabolite dynamics of sulphur-containing metabolites at the whole-plant level.
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