Determination of Picomole Levels of Neurotensin, Bombesin, and Related Peptide Fragments Using Gradient Elution High Performance Liquid Chromatography Coupled with Electrochemical Detection

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Abstract
A method for the separation and detection of two neuroactive peptides, neurotensin (NT) and bombesin (BB) as well as selected fragments of these peptides including NT 1–8, NT 1–10, NT 9–13, BB 1–8, BB 1–11 and BB 8–14 using gradient elution high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) is described. Several mobile phase compositions and gradients were tested and compared in terms of resolving power and sensitivity. The gradient method outlined here is 200 to 500 times more sensitive than HPLC-UV detection. A method for separating neurotensin isocratically is also described, and results indicate that the sensitivity of the assay is in the fentomolar range and approaches the sensitivity of radioimmuno-assay techniques. Means of ameliorating the sensitivity of the HPLC-ECD assay when using a gradient are discussed. Results of preliminary experiments using this technique to detect the presence of these peptides in various biological media are presented.