Analysis of leucine enkephalin by high-performance liquid chromatography using enzymatic derivatization by tyrosinase and electrochemical or fluorescence detection.

Two tyrosine specific, HPLC methods with either electrochemical (HPLC-ED) or fluorescence (HPLC-FL) detection are described for leucine-enkephalin (LE) in cerebrospinal fluid (CSF). Both approaches involve the hydroxylation of the Tyr1-moiety of LE by mushroom tyrosinase. Production of a catechol permitted the selective clean-up of the analyte using boronate gels and produced species which were more readily oxidizable than the LE. The controlled oxidation of the catechol to corresponding chinones enabled the reaction with 1,2-diamino-1,2-diphenylethane (DPE) and subsequent quantification by HPLC-FL. The HPLC-ED and HPLC-FL yielded limits of detection for LE in CSF of 360 and 500 fmol per injection, respectively. Inter-day variability of calibration curve samples was lower than 20% for the HPLC-ED with slightly higher variability for the HPLC-FL assay.