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 competit ive 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 deprotein ized and separated by reversed-phase HPLC. Fractions for the HPLC elua te were measured by a RIA and two immunoreactive components were found in dog plasma. One fraction was assumed to be unchanged CLT and the o ther was the (1-6) fragment peptide of CLT [CLT(1-6)] in accordance wi th the retention times. For the simultaneous determination by the comb ined 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 CL T and 14-20 pg ml-1 for the CLT(1-6) peptide. Diluted urine samples fr om dogs were directly injected into the HPLC and the immunoreactivitie s of the fractions were measured. More than 98% immunoreactivity was f ound 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 estimat ed 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.