Two-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 microns, 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.

A comparative study of the reversed-phase HPLC retention behaviour of S-adenosyl-L-methionine and its related metabolites on Hypersil ODS and Supelcosil LC-ABZ stationary phases.

S-Adenosyl-L-methionine (SAM) and its metabolites S-adenosyl-L-homocysteine (SAH) and methyl-thioadenosine (MTA) are endogenous compounds that are heavily involved in a variety of biochemical processes, and have therefore been the target for several assays in body fluids and tissues. Reversed-phase chromatographic behaviour of SAM and its metabolites has been studied by using Supelcosil LC-ABZ column, specially designed for analysis of acidic, basic, zwitterionic and neutral compounds, and on a Hypersil ODS column as a function of mobile phase pH. The retentions of the compounds, expressed by the capacity ratio (k'), are measured on both column with mobile phases comprised of 10% acetonitrile and 10 mM ammonium formate buffer with pH values ranging from 2 to 9. Higher selectivity is observed on Supelcosil LC-ABZ within pH range 4-6. Different retention properties are observed at very low pH and seemed as if the Supelcosil LC-ABZ column reduced the effect of the mobile phase pH by about 1 pH unit. Whilst the Supelcosil column can be recommended for the routine analysis of SAM and its related metabolites in biological fluids by using mobile phase pH 5, the Hypersil ODS column may be suggested for use with mobile phase pH values of 3-4.