Analysis of Invertebrate Neuropeptides by RP-HPLC

Abstract

The application of reverse phase-high performance liquid chromatography to the analysis of four synthetic invertebrate neuropeptides is described. Proctolin (Arg-Try-Leu-Pro-Thr), locust adipokinetic hormone (p-Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH₂), crustacean erythrophore concentrating hormone (p-Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH₂) and mulluscan cardioexcitatory neuropeptide (Phe-Met-Arg-Phe-NH₂) were analyzed on several different reverse phase columns by means of gradient elution with 0.01M KH₂ PO₄, 0.1% H₃PO₄, 0.25N triethylammonium phosphate (TEAP), pH 2.20, or 0.1% trifluoroacetic acid (TFA) versus acetonitrile. Column effluents were monitored at both 254 and 195 nm except in the case of TFA where 254 and 210 nm were monitored. At the lower wavelength computerized background correction was sometimes necessary to correct excessive baseline drift during the course of the gradient run. Best results were obtained on the Supelcosil LC-18DB column with a concave gradient of 90° 40%B over 1 hr at 1.1 ml/min where B[dbnd]0.25N TEAP, pH 2.20, A[dbnd]acetonitrile. With this system less than 5 ng of peptide was detectable. The use of the volatile TFA buffer permitted recovery of peptides from the column effluent by lyophilization.