The simultaneous determination of neurotensin and its major fragments by on-line trace enrichment HPLC

An HPLC assay using on-line cation exchange trace enrichment and acetonitrile gradient elution, ion pair reverse phase separation with electrochemical detection (EC) is described for the simultaneous determination of the tridecapeptide neurotensin (NT) and six of its fragments. Cyclic voltammetric analysis indicated that the oxidative electrochemical 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-arginine pair at position 8-9). Using the described method, NT_1-6, NT_1-8, NT_1-10, NT_1-11, NT_8-13, NT_9-13 and NT_1-13, were baseline resolved within 20 min with a limit of detection varying from 1 to 5 ng peptide/injection. Other structurally similar or quantitatively significant neuropeptides (e.g. substance P, somatostatin, bombesin) did not interfere. Initial application of this on-line trace enrichment HPLC-EC assay to the question of the molecular nature of NT in unprocessed human CSF indicated the predominance of NT_1-13 with an apparent formation of NT_1-8 and NT_9-13 resulting from more vigorous sample preparation techniques. The improvements in assay specificity, signal-to-noise ratios, biomatrix compatibility and assayable sample volume compared to non-enrichment HPLC-EC are discussed.