REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY COUPLED WITH 2ND-ORDER DERIVATIVE SPECTROSCOPY FOR THE QUANTITATION OF AROMATIC-AMINO-ACIDS IN PEPTIDES - APPLICATION TO HEMORPHINS

The characterization of aromatic amino acid-containing peptides in bio logical fluids or protein hydrolysates is commonly achieved using clas sical size-exclusion (SE) and reversed-phase (RP) high-performance liq uid chromatography (HPLC) coupled with direct ultraviolet (UV) spectro metry. Here, a non-destructive quantitative determination of aromatic amino acids in peptides is developed using second-order derivative spe ctra obtained during RP-HPLC coupled with photodiode array detection. In this method, the free aromatic amino acids were used as standards. Sensitivity and accuracy were verified using some peptides, including bioactive hemorphins. The method was applied to determine the amounts of hemorphins present in a complex peptic bovine hemoglobin hydrolysat e.