Improved GC-MS methods for analysis of homocysteine, methionine and methylmalonate in cultured cells
Purpose: The aim of the present study is to establish a new gas chromatography mass spectrometry (GC-MS) method using a triple quadrupole which would allow for more sensitive quantification of changes in homocysteine, methionine and methylmalonate in small numbers of cultured cells and brain tissue. Homocysteine, methionine and methylmalonate are established markers of cobalamin deficiency. Methods: Cultured cells (1 x 106) and 5mg of human brain were incubated with 10 mg/ml dithiothreitol in methanol for one hour at room temperature to reduce homocysteine disulfide bonds. Lipids were removed using methyl-tert-butyl ether and the aqueous phase derivatized with N-Methyl- N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA). Samples were subsequently analysed with a 7000 Agilent triple-quadrupole GC-MS. Multiple reaction monitoring was performed on the compounds in electron ionisation mode at 70eV. Corresponding deuterated internal standards were used to quantify compounds. Results: We identified the following transitions: 420->392, 318 424 ->396, 322; 320->292, 218, 323->295, 221; 289->189, 147 292->192, which allowed for sensitive quantification of homocysteine, methionine and methylmalonate and their deuterated standards respectively. The lowest limits of detection for homocysteine, methionine and methylmalonate were found to be 0.001 pM in three different cell lines and brain tissue. Sample preparation and analysis was complete in 4 h. Conclusion: Homocysteine, methionine and methylmalonate were successfully quantified in low cell numbers. This method is widely applicable for studies of cobalamin function in cells and has the potential advantage of conserving precious human tissue samples.
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