Liquid-Liquid Extraction with 'Inter-Well Volume Replacement' Sample Dilution for Quantification of Apixaban in Human Dried Blood Spots by UHPLC-MS/MS

摘要

1. A uHPLC-MS/MS assay with automated liquid-liquid extraction and "inter-well volume replacement" sample dilution was developed for the analysis of apixaban in human dried blood spots. 2. This assay utilized extraction screening results and existing liquid-liquid extraction knowledge to maximize the assay sensitivity. 3. The novel "inter-well volume replacement" approach for DBS sample dilution was proven to be efficient and accurate for sample dilution with precision of < 5.5% CV and accuracy of ±3.9% for dilution QC (at 2500 ng/mL) with 10-fold dilution (n=12). 4. The assay was linear from 0.500 to 500 ng/mL for the quantification of apixaban in human DBS. The intra- and inter-assay precisions were within 4.4% CV and 2.6% CV, respectively. The assay accuracy was within ±5.4% of the nominal concentrations. 5. This robust and sensitive LC-MS/MS assay has been applied to an apixaban oral solution bioavailability study to evaluate the DBS technology as an alternative PK sampling technique.