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.
Larsimont
V
V
Department of Pharmaceutics, University of Florida, Gainesville 32610, USA.
Bogaards
M
M
Mulleneers
P
P
Hochhaus
G
G
D000363:Agaricales / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004151:Dipeptides / Q000032:analysis; D004563:Electrochemistry / Q000379:methods; D004743:Enkephalin, Leucine / Q000032:analysis* / Q000737:chemistry; D005029:Ethylenediamines / Q000737:chemistry; D006900:Hydroxylation; D014442:Monophenol Monooxygenase / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods
D004151:Dipeptides; D005029:Ethylenediamines; C009870:meso-1,2-diphenylethylenediamine; D004743:Enkephalin, Leucine; D014442:Monophenol Monooxygenase