Analysis of enkephalins, beta-endorphins and small peptides in their sequences by highly sensitive high-performance liquid chromatography with electrochemical detection: implications in opioid peptide metabolism.
Sensitivity in the 10-100 pg range for enkephalins, beta-endorphin, tyrosine (T), 12 tyrosylglycine (T-G) and tyrosylglycylglycine (T-G-G) was attained by using a high-performance liquid chromatographic (HPLC) method with electrochemical detection which is at least 100 times more sensitive than HPLC with UV detection. The chromatographic conditions on a reversed-phase C18 silica column were 50 mM sodium phosphate buffer (pH 2.1) (A) in acetonitrile-methanol (1:1) (B), isocratic mixture, flow-rate 0.6-1 ml/min, UV detection at 205 nm, electrochemical oxidation potential + 1.25 V. The separation of T, T-G and T-G-G was obtained by using 10% B while the separation of the pentapeptide, enkephalins required 40% B. Separation of enkephalins from beta-endorphin was attained at a shorter retention times did not exceed 15 min. This method can be used to determine tissue levels and pharmacodynamics of enkephalins and beta-endorphin. A highly specific measurement of the different enzymes involved in the metabolism of enkephalin has been achieved.
D000305:Adrenal Cortex Hormones / Q000494:pharmacology; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000097:blood; D000818:Animals; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D010450:Endopeptidases / Q000378:metabolism; D004723:Endorphins / Q000302:isolation & purification*; D004745:Enkephalins / Q000302:isolation & purification*; D008297:Male; D010455:Peptides / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D001615:beta-Endorphin
D000305:Adrenal Cortex Hormones; D004723:Endorphins; D004745:Enkephalins; D010455:Peptides; D001615:beta-Endorphin; D010450:Endopeptidases; D000626:Aminopeptidases