Effect of various anticoagulants on the bioanalysis of drugs in rat blood: implication for pharmacokinetic studies of anticancer drugs

Background: Pharmacokinetic studies are vital in development and optimization of drugs. While blood samples can be collected either in EDTA, heparin or citrate containing tubes for the estimation of drug levels in plasma, EDTA tubes are more commonly used. The purpose of this study was to evaluate the effects of anticoagulants on bioanalysis of drugs. Six drugs used extensively in cancer therapy were selected. Albino wistar rats (N = 6 per drug) were dosed with one of the following drugs intraperitoneally-pemetrexed (50 mg/kg), imatinib (50 mg/kg), erlotinib (25 mg/kg), meropenem (60 mg/kg), 6-mercaptopurine (20 mg/kg) and voriconazole (6 mg/kg). Blood samples were collected 2 h after dosing (1 h in 6-mercaptopurine group due to short half-life) by terminal bleeding from the retro-orbital plexus. Blood was collected in each of Disodium ETDA, heparin, trisodium citrate (TSC) and no anticoagulant (plain) tubes. Drug levels in these samples were determined by validated HPLC assays. ANOVA with Tukey's post hoc test was performed to identify statistically significant differences in drug concentrations in anticoagulant tubes. p < 0.05 was considered statistically significant. Results: Significant differences in concentration between anticoagulant tubes was observed in case of erlotinib (p = 0.013) and meropenem (p = 0.00), while borderline statistical significance for pemetrexed (p = 0.076). TSC tubes overestimated erlotinib levels, heparin tubes underestimated meropenem concentrations and EDTA tubes overestimated pemetrexed concentrations. Conclusions: Careful selection of anti-coagulant is necessary for accurate characterization of pharmacokinetics of drugs. Routine use of EDTA tubes may lead to erroneous interpretation of pharmacokinetic data.