2-DIMENSIONAL HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR ASSAYING S-ADENOSYL-L-METHIONINE AND ITS RELATED METABOLITES IN TISSUES

S-Adenosyl-L-methionine (SAM) is a methyl-donor compound which is actively involved in a variety of biochemical reactions. An assay has been developed permitting the quantitative measurement of SAM and its related metabolites (S-adenosylhomocysteine, decarboxylated SAM, methylthioadenosine, adenosine and adenine) in liver and cell cultures. As gradient reversed-phase chromatographic or cation-exchange chromatographic methods often resulted in overlapping peaks, a two-dimensional high-performance liquid chromatographic (HPLC) procedure was developed involving gradient reversed-phase chromatographic separation followed by ion-exchange chromatography. After precipitating large molecules in the sample by perchloric acid, gel permeation was carried out on a Sephadex G 25 column to separate small water-soluble metabolites from proteins and membrane fragments. The freeze-dried sample was injected onto an ODS column and a 0-10% acetonitrile gradient in 10 mM ammonium formate buffer (pH 2.9) (20 min, linear) was applied. The relevant fractions were collected and injected onto a cation-exchange column (Partisil SCX, 10 mum. 250 mm x 4.6 mm I.D.). Elution and quantification were carried out using ammonium formate buffers of various concentration (15-400 mM), pH 2.9. The detector response (254 nm) as a function of concentration was linear over the concentration range 30-500 pmol. The detection limits of the compounds after the two-dimensional chromatographic procedure ranged from 10 to 60 pmol and the recovery was higher than 70%. The reproducibility of the results obtained from given samples was within 9-22% for rat liver and 6-24% for mast cells.