High-performance liquid chromatographic determination of an arginine-containing octapeptide antagonist of vasopressin in human plasma by means of a selective post-column reaction with fluorescence detection.

A novel high-performance liquid chromatographic method was developed for the determination in plasma samples of the synthetic octapeptide SK&F 105494 (O-ethyl-D-tyrosyl-L-phenylalanyl-L-valyl-L-asparaginyl-5-[1- (carboxymethyl)cyclohexyl]-L-norvalyl-L-arginyl-D-arginamide , cyclic (5-1) peptide), an active aquaretic agent which significantly increases water excretion in experimental animal models through competitive antagonism of renal epithelial vasopressin receptors. The method involves isolation of Sk&F 105494 from plasma samples by solid phase extraction prior to chromatographic analysis. Following chromatographic separation, in-line fluorescence detection was accomplished via a selective-post-column reaction of the guanidino group of the arginine moiety of the peptide with alkaline ninhydrin to generate a highly fluorescent product. Optimization of the post-column reaction conditions and the use of high-performance liquid chromatography columns of reduced internal diameter (2.1 mm), resulted in an on-column detection limit of 50 pg (45 fmol). The limit of sensitivity in plasma was 0.5 ng/ml. The assay was linear over the range 0.5-100 ng/ml. Precision and accuracy were within 11% across the calibration range. The assay was shown to be suitable to study the pharmacokinetics of SK&F 105494 in human subjects and animals. In addition, the methodology developed has general applicability in the detection of arginine-containing peptides in biological matrices.