Volumetric absorptive microsampling meets electromembrane extraction for the first time: Case example of doxorubicin and its metabolite in whole blood samples.

Background: Microsampling of biological fluids followed by innovative sample pre-treatment reflects trends in bioanalytical chemistry. Volumetric absorptive microsampling (VAMS) enables exact whole blood volume collection and reduces the impact of hematocrit on the assay. In animal studies, it complies with the 3R principles (refine, reduce, replace). It allows for a gentle bleeding technique and a reduction in the number of laboratory animals by enabling ethically acceptable repeated blood collection from a single animal. Treating VAMS tips with electromembrane extraction (EME) in 96-well format offers a smart combination of non-invasive, low-volume blood collection with effective, environmentally friendly sample clean-up.

Results: This study introduces the first application of EME in 96-well format for direct isolation of analytes from 10 μL of whole blood collected onto a VAMS device. Doxorubicin, a clinically used anticancer drug also utilized in cancer/cardio-oncology research involving rodents, where microsampling offers important advantages, and its metabolite doxorubicinol, were selected as relevant analytes. The optimized EME yielded reproducible recoveries for both analytes regardless of hematocrit levels, different anticoagulants, or free multivalent ions in the sample. Compared to conventional VAMS tips treatment, EME reduced matrix effects, increased throughput, and an environmental friendliness of the extraction. The EME followed by the UHPLC-MS/MS assay was validated for both analytes in whole blood absorbed onto VAMS tips. The same protocol was implemented to treat plasma to determine the blood-to-plasma ratio of the analytes in the same experiments. The practical utility was demonstrated by analyzing real samples collected from the doxorubicin-treated nude mice.

Significance: The study offers a novel assay combining whole blood microsampling and sample clean-up in microextraction scale for preclinical pharmacokinetic studies with doxorubicin in rodents and for pharmacokinetic/pharmacodynamic modeling. This advancement in bioanalytical chemistry promotes scalable environmentally friendly procedures compatible with the 3R ethical principles in animal studies. Moreover, the concept of direct VAMS tips treatment with EME may also be easily translatable to clinical settings.