Analysis of ascorbate and dehydroascorbate in plant extracts by high-resolution selected ion monitoring gas chromatography-mass spectrometry

Abstract

Here we report on the quantitative analysis of tert.-butyldimethylsilyl derivatives, of ascorbate and dehydroascorbate in plant extracts by high-resolution selected ion monitoring gas chromatography-mass spectrometry. The analysis involved an isotope dilution assay using [ 13C 1]ascorbate and [ 13C 1]dehydroascorbate. The limit of detection was approximately 0.5 pg for ascorbate and 5 pg for dehydroascorbate. The reproductibility of the whole quantification procedure, from extraction of Pinus sylvestris needles with 5% trichloroacetic acid to the final analysis, was very high: giving a relative standard deviation (R.S.D.) of 4% and 10% for ascorbate and dehydroascorbate, respectively. The reproducibility of the analysis of the redox ratio of ascorbate/(ascorbate + dehydroascorbate) was even higher (R.S.D. = 1%). The high-resolution selected ion monitoring gas chromatography-mass spectrometry methodology developed here with very high sensitivity and specificity for ascorbate and dehydroascorbate opens new perspectives for investigations on distribution patterns of these substances in plants.