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.
Kanzakigawa Laboratory, Shionogi & Co. Ltd. Osaka, Japan.
G
G
Kominami
H
H
Okabe
K
K
Imoda
K
K
Mizojiri
D000818:Animals; D002108:Ceruletide / Q000008:administration & dosage / Q000097:blood* / Q000493:pharmacokinetics / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D007273:Injections, Intramuscular; D010446:Peptide Fragments / Q000097:blood / Q000652:urine; D011863:Radioimmunoassay; D015203:Reproducibility of Results
D010446:Peptide Fragments; D002108:Ceruletide