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Simultaneous analysis of 13 C‐glutathione as its dimeric form GSSG and its precursor [1‐ 13 C]glycine using liquid chromatography/isotope ratio mass spectrometry
Author(s) -
Schierbeek Henk,
Rook Denise,
te Braake Frans W. J.,
Dorst Kristien Y.,
Voortman Gardi,
Godin JeanPhilippe,
Fay LaurentBernard,
van Goudoever Johannes B.
Publication year - 2009
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4200
Subject(s) - chemistry , mass spectrometry , chromatography , isotope ratio mass spectrometry , isotope , glutathione , glycine , glutathione disulfide , high performance liquid chromatography , quantitative analysis (chemistry) , analytical chemistry (journal) , stable isotope ratio , volume (thermodynamics) , sample preparation , biochemistry , amino acid , enzyme , physics , quantum mechanics
Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine 13 C‐glutathione as its dimeric form (GSSG) and its precursor [1‐ 13 C]glycine in a small volume of erythrocytes in one single analysis. After having transformed 13 C‐glutathione into its dimeric form GSSG, we determined both the intra‐erythrocytic concentrations and the 13 C‐isotopic enrichment of GSSG and glycine in 150 µL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of µmol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 µmol/mL. The 13 C‐isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3‰) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants. Copyright © 2009 John Wiley & Sons, Ltd.