High Performance Liquid Chromatography Assay for Alanine Amino Transferase

An improved method of alanine-amino transferase determination is proposed. The reaction is carried out with alanine and 2-oxoglutarate as substrates and analysis is by HPLC on a reversed-phase chromatographic system using a Ca8 column and tetrabutylammonium phosphate in Phosphate buffer (pH 7.0)-acetonitrile as mobile phase. The enzyme activity was determined by directly following the formation of pyruvic acid without employing any sec- Ondary reaction, which is necessary in the spectrophotometric method. The detection limit of pyruvic acid is 10 Pmole/al-I and the standard deviation for the enzymatic activity of standard solutions is 5.4%. Furthermore under the chromatographic conditions selected it is possible to detect the presence of some intermediate species.