High-performance Liquid Chromatographic Determination of Intrathecally Administered [d-Ala²-d-Leu⁵]-enkephalin Concentrations in Canine Cerebrospinal Fluid

Sherry Cox, University of Tennessee, Knoxville
Ralph C. Harvey, University of Tennessee, Knoxville
Donita Frazier, University of Tennessee, Knoxville

Abstract

A rapid and useful method for high-performance liquid chromatographic analysis of exogenous [d-Ala²-d-Leu⁵]-enkephalin (DADLE) in cerebrospinal fluid (CSF) is described. CSF (0.5 ml) samples were filtered using a 0.22-μm Co-Star filter. Chromatography was performed on a μBondapak C18 column using a mobile phase of A, 0.05 M sodium phosphate (monobasic, pH 6.0) and B, 60% acetonitrile in 0.05 M sodium phosphate (pH 6.0) with a flow-rate of 1 ml/min. Absorbance at 210 nm was measured. The procedure produced a linear curve for the concentration range 1–10 μg/ml. The development of the assay produced rapid, repeatable and accurate results for CSF analysis of DADLE at concentrations achieved with therapeutic administration of the peptide. This method could also be used in the future for analysis of compounds like DADLE.