A hydrophilic interaction chromatography coupled to a mass spectrometry for the determination of glutathione in plant somatic embryos.
An electrospray ionization mass spectrometric (ESI-MS) determination of glutathione (GSH), a sulfur-containing tripeptide (gamma-Glu-Cys-Gly) with regulation and detoxication functions in metabolisms of most living organisms, from nanomolar to micromolar levels is described. A hydrophilic interaction chromatography (HILIC) with an isocratic elution using a mobile phase containing acetonitrile and an aqueous 0.00005% solution of trifluoroacetic acid (60/40%, v/v) was applied for the separation of GSH. The peptide detection was achieved in the presence of L-ascorbic acid which significantly enhanced the signal intensity of the molecular ion GSH [M+H]+ (m/z 308). The calibration curve was linear (R2=0.9995) in the concentration range from 2 nM to 10 microM with a detection limit (LOD, S/N=3) of 0.5 nM. The excellent detection limit, and the excellent selectivity and high reproducibility of this method enabled determination of GSH in a single plant somatic embryo of a Norway spruce (Picea abies). The average amount of GSH in the single somatic embryos (n=18) was 9 pmol per embryo. Owing to our results, it can be supposed that the proposed HILIC/ESI-MS analysis might be used for GSH determination in microscopic cell structures and in single cell analyses.
Vacek
Jan
J
Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 612 65 Brno, Czech Republic.
Klejdus
Borivoj
B
Petrlová
Jitka
J
Lojková
Lea
L
Kubán
Vlastimil
V
D000097:Acetonitriles / Q000737:chemistry; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D002138:Calibration; D002845:Chromatography / Q000379:methods; D005978:Glutathione / Q000032:analysis*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D028222:Picea / Q000737:chemistry* / Q000187:drug effects; D012639:Seeds / Q000737:chemistry / Q000187:drug effects; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014269:Trifluoroacetic Acid / Q000737:chemistry
D000097:Acetonitriles; D000975:Antioxidants; D010455:Peptides; D014269:Trifluoroacetic Acid; D005978:Glutathione; D001205:Ascorbic Acid; C032159:acetonitrile