Kenji Mizojiri

Combined high-performance liquid chromatography and radioimmunoassay for ceruletide and its metabolites in dog plasma and urine

A combined high-performance liquid chromatographic (HPLC) and competitive radioimmunoassay (RIA) method for ceruletide (CLT), an analogue of cholecystokinin-8, was developed to investigate the behaviour of CLT in dogs. Dog plasma samples after administration of CLT were deproteinized and separated by reversed-phase HPLC. Fractions for the HPLC eluate were measured by a RIA and two immunoreactive components were found in dog plasma. One fraction was assumed to be unchanged CLT and the other was the (1-6) fragment peptide of CLT [CLT(1-6)] in accordance with the retention times. For the simultaneous determination by the combined method in dog plasma, the assay recoveries of both compounds were 100% and the values for assay precision (RSD intra-assay) were 8-19% for the CLT assay and 2-17% for the CLT(1-6) peptide assay. The lower limit of quantitation in dog plasma was estimated as 18 pg ml-1 for CLT and 14-20 pg ml-1 for the CLT(1-6) peptide. Diluted urine samples from dogs were directly injected into the HPLC and the immunoreactivities of the fractions were measured. More than 98% immunoreactivity was found at the retention time of CLT(1-6) peptide. Thus only the CLT(1-6) peptide was measured in dog urine by the RIA without HPLC separation. Urine samples could be assayed directly only after dilution 1:20. The assay recovery for urine was 100% and the precision (RSD) was estimated to be < 10%. The lower limit of quantitation for dog urine was 70 pg ml-1. The combined method of HPLC and immunoassay is extremely useful for peptide analyses.