Optimization of the Extraction Procedure of Apixaban from Dried Rat Plasma Spots

Aims: Apixaban is an anticoagulant used to treat and prevent blood clots, as well as to prevent stroke in people with atrial fibrillation. The dried spot analyses, including dried blood spots and dried plasma spots, are used to simplify techniques for determining drug concentrations in blood and plasma. In this case, equipment with highly sensitive detector is required, for example, mass spectrometer, as well as a high level of drug extraction from the dried spot. In this work, apixaban extraction from dried plasma spots (DPS) was studied in order to determine the optimal parameters of the extraction method.Study Design:Short Research Articles.Place and Duration of Study: Core Facility of Mass Spectrometric Analysis, Institute of Chemical Biology and Fundamental Medicine SB RAS, between September 2019 and February 2020.Methodology: The organic extraction method was chosen for evaluation as the most suitable for LC-MS assay. Several parameters: percentage of organic solvent, presence or absence of 0.1% formic acid (FA), time, volume and temperature of extraction were investigated to find the best combination for recovery of apixaban from DPS for further LC-MS analysis.Results: The results showed that the main influence on the extraction is the composition of the solvent, volume of solvent, as well as temperature and time of extraction. Pure acetonitrile is theworst solvent for extracting apixaban from DPS. Solvents: MeOH:H2O (100:0, v: v), MeOH: 0.1% FA in H2O(80:20, v:v), ACN: 0.1% FA in H2O (90:10, v:v) or ACN:MeOH (90:10, v:v) provide better recovery of apixaban. The optimum extraction parameters were as follows: 90% acetonitrile concentration, extraction temperature of 40°C, extraction time of 15 min, and solvent volume of 100L.Conclusion: For the extraction of apixaban from DPS, subject to further analysis by LC-MS, the most suitable solvent is 90% acetonitrile under the conditions described above.