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) o r fluorescence (HPLC-FL) detection are described for leucine-enkephalin (LE ) in cerebrospinal fluid (CSF). Both approaches involve the hydroxylation o f the Tyr(1)-moiety of LE by mushroom tyrosinase. Production of a. catechol permitted the selective clean-up of the analyte using boronate gels and pr oduced species which were more readily oxidizable than the LE. The controll ed 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. (C) 1999 Elsevier Science B.V. Al l rights reserved.