Quantification of intracellular homocysteine by stable isotope dilution liquid chromatography/tandem mass...

A precise and accurate stable isotope dilution liquid chromatography/tandem mass spectrometry method for the analysis of intracellular homocysteine has been developed. An internal standard, [(2)H(8)]-homocystine, was added to cell pellets from EA.hy 926 cells grown in culture under low and high folate concentrations. D,L-dithiothreitol was used to reduce cellular homocystine to homocysteine. Cellular proteins were precipitated by the addition of formic acid in acetonitrile. After centrifugation, a portion of the supernatant was analyzed by liquid chromatography/tandem mass spectrometry. Using a Supelcosil cyano column with an Applied Biosystems API 4000 triple quadrupole mass spectrometer, the SRM transitions for homocysteine (m/z 136 to m/z 90) and [(2)H(4)]-homocysteine (m/z 140 to m/z 94) were monitored. The method was validated by conducting five replicate analyses on three different days at four different concentrations (concentrations at the lower limit of quantitation and expected lower quartile, mid-range and upper quartile). The limit of detection was 2 ng/10(6) EA.hy 926 cells. Using this method, the intracellular homocysteine concentration in EA.hy 926 cells ranged from 10 to 36 ng/10(6) cells.