THE SIMULTANEOUS DETERMINATION OF NEUROTENSIN AND ITS MAJOR FRAGMENTSBY ONLINE TRACE ENRICHMENT HPLC WITH ELECTROCHEMICAL DETECTION

An HPLC assay using on-line cation exchange trace enrichment and aceto nitrile gradient elution, ion pair reverse phase separation with elect rochemical detection (EC) is described for the simultaneous determinat ion of the tridecapeptide neurotensin (NT) and six of its fragments. C yclic voltammetric analysis indicated that the oxidative electrochemic al properties of NT and its fragments is not merely a function of the sum of its electroactive amino acids (i.e. tyrosine) but reflects the presence and association of other amino acids (e.g. the arginine-argin ine pair at position 89). Using the described method, NT1-6, NT1-8, NT 1-10, NT1-11, NT8-13, NT9-13 and NT1-13 were baseline resolved within 20 min with a limit of detection varying from 1 to 5 ng peptide/inject ion. Other structurally similar or quantitatively significant neuropep tides (e.g. substance P, somatostatin, bombesin) did not interfere. In itial application of this on-line trace enrichment HPLC-EC assay to th e question of the molecular nature of NT in unprocessed human CSF indi cated the predominance of NT1-13 with an apparent formation of NT1-18 and NT9-13 resulting from more vigorous sample preparation techniques. The improvements in assay specificity, signal-to-noise ratios, biomat rix compatibility and assayable sample volume compared to non-enrichme nt HPLC-EC are discussed.