A new comprehensive technique of catheterisation, blood sampling, sample preparation and sample analysis by means of high-pressure liquid chromatography for pharmacokinetic studies with estradiol-linked nitrosoureas and their metabolites
Betsch, B.; Berger, M.R.; Spiegelhalder, B.
Arzneimittel-Forschung 40(9): 1022-1025
1990
ISSN/ISBN: 0004-4172 PMID: 2080936 Document Number: 357380
Estradiol-linked nitrosoureas are offering new perspectives in the antineoplastic chemotherapy of estradiol-receptor positive mammary carcinomas. In such a molecule estradiol has the function of a carrier which brings about a specific accumulation of the anticancer drug in estradiol-receptor containing tumor cells. However, there is only little knowledge about the pharmacokinetic behavior of this new group of anticancer agents. For that reason a new comprehensive technique of catheterisation, blood sampling, sample preparation and sample analysis with high-pressure liquid chromatography (HPLC) for preclinical pharmacokinetic studies with estradiol-linked nitrosoureas and their metabolites has been developed. N-(2-Chloroethyl)-N-nitroso-carbamoyl-L-alanine-estradiol-17-ester (CNC-alanine-estradiol-17-ester) and N-(2-chloroethyl)-N-nitroso-carbamoyl-L-alanine (CNC-alanine) were used as test compounds. The drugs were tested in female Sprague-Dawley rats with chemically induced mammary carcinomas. The laboratory animals were supplied with two catheters prior to the pharmacokinetic experiments. The blood samples were drawn from the vena cava catheter after the drug had been applied through a vena jugularis catheter. The compounds were extracted from plasma with C18 silicagel reversed phase cartridges. The clean-up technique delivered clear samples only slightly contaminated with the biological matrix. The recovery from plasma was 75 .+-. 5% for the hormone-linked CNC-alanine-estradiol-17-ester and 70 .+-. 5% for the unlinked CNC-alanine. The analysis was carried out by means of HPLC. To allow a simultaneous detection of CNC-alanine-estradiol-17-ester and its metabolite CNC-alanine being released after breakdown of the esterbond the unlinked compound (CNC alanine) was derived with diazomethane prior to analysis. The compounds were detected and quantified by measuring the UV-absorption. The separation was performed with a C18 reversed-phase column and a solvent gradient of methanol (M), acetonitrile (A) and water (W). HPLC-analysis was started with a polar solvent (begin: M/A/W = 10/20/70) and then shifted to a lipophilic solvent (end: M/A/W = 80/0/20). The limit of detection was 1 nmol/ml. In cases of high sample contamination the UV-spectrum was assessed as a further criteria of identification.sbd.in addition to the retention time. The technique of double catheterisation allows a precise timing of drug application and continuous blood sampling from a single animal. The use of laboratory animals can be reduced by 75% with the described method. The technique of sample preparation and sample analysis provides a selective, highly sensitive, rapid and well reproducible procedure for pharmacokinetic studies with estradiol-linked nitrosoureas and their metabolites. Furthermore, the described method seems to be suitable for future studies with new, similar anticancer agents.