High-performance liquid chromatographic analysis of arginine-containing peptides in biological fluids by means of a selective post-column reaction with fluorescence detection.

Highly sensitive and specific high-performance liquid chromatographic methodology for the fluorescence detection of arginine-containing peptides in biological matrices is described. Following HPLC separation, a post-column reaction of the guanidino group of the arginine moiety with ninhydrin under basic conditions is utilized to generate a fluorescent peptide product which can be measured at high sensitivity. Careful optimization of the post-column reaction conditions, and the use of HPLC columns of reduced internal diameter, resulted in on-column detection limits as low as 50 fmol. Application to the determination of synthetic arginine-containing vasopressin antagonists in human plasma resulted in a quantitative response which is linear over the range 0.5-100 pmol/ml. The assay method is sufficiently sensitive, accurate, and precise for use in pharmacokinetic studies of these synthetic peptides. The methodology also has general applicability in the detection of naturally occurring arginine-containing peptides.