Rapid Isolation of Nanogram Amounts of Crustacean Erythrophore Concentrating Hormone from Invertebrate Nerve Tissue by RP-HPLC

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Abstract
A reverse phase-high performance liquid chromatography (rp-hplc) method was developed for the rapid isolation of nanogram amounts of crustacean erythrophore concentrating hormone (CECH) from invertebrate nerve tissue. Tissue homogenates from the shrimp, Palaemoenetes pugio, were subjected to a multistep work-up to remove proteins and lipids prior to analysis by rp-hplc. Samples were eluted with a concave gradient of 0.1% trifluoroacetic acid (TFA) vs. acetonitrile. Detection at 210 and 254 nm combined with the use of highly efficient and end-capped columns permitted the determination of < 5 ng of CECH. Pure CECH was isolated from the columns by fraction collection followed by lyophilization of the volatile TFA buffer.