A sensitive non-radioactive method for determination of the stereospecificity of the C-4' hydrogen transfer on the coenzymes (pyridoxal phosphate, PLP; and pyridoxamine phosphate, PMP) of aminotransferases has been developed. Aminotransferase of unknown stereospecificity in its PLP form was incubated in (H2O)-H-2 with a substrate amino acid resulted in PMP labeled with deuterium at C-4' in the pro-S or pro-R configuration according to the stereospecificity of the aminotransferase tested. The [4'-H-2]PMP was isolated from the enzyme protein and divided into two portions. The first portion was incubated in aqueous buffer with apo-aspartate aminotransferase (a reference si-face specific enzyme), and the other was incubated with apo-branched-chain amino acid aminotransferase (a reference re-face specific enzyme) in the presence of a substrate 2-oxo acid. The H-2 at C-4' is retained with the PLP if the aminotransferase in question transfers C-4' hydrogen on the opposite face of the coenzyme compared with the reference aminotransferase, but the H-2 is removed if the test and reference aminotransferases catalyze hydrogen transfer on the same face. PLP formed in the final reactions was analyzed by LC-MS/MS for the presence or absence of H-2. The method was highly sensitive that for the aminotransferase with ca. 50 kDa subunit molecular weight, only 2 mg of the enzyme was sufficient for the whole test. With this method, the use of radioactive substances could be avoided without compromising the sensitivity of the assay. (C) 2011 Elsevier Inc. All rights reserved.