Development and Validation of a QuEChERS–LC–MS/MS Method for Simultaneous Determination of Four Direct Oral Anticoagulants in Citrate Plasma and Quantitative Dried Plasma Spots

Direct oral anticoagulants (DOACs), including apixaban, dabigatran, edoxaban, and rivaroxaban, may require quantitative measurement in selected clinical situations despite fixed‐dose administration. This study developed and validated a quick, easy, cheap, effective, rugged, and safe (QuEChERS)‐assisted liquid chromatography‐tandem mass spectrometry method for their simultaneous determination in citrate plasma and quantitative dried plasma spot (qDPS) samples. Chromatographic separation was achieved on a Kinetex PS C18 column using a 4.0 min gradient, followed by positive electrospray ionization and scheduled multiple reaction monitoring. Sample preparation was optimized by comparing acetonitrile (ACN) protein precipitation, unbuffered and citrate‐buffered QuEChERS extraction salts, and dispersive solid‐phase extraction clean‐up using primary‐secondary amine, C18, EMR Lipid, Z‐Sep+, or their combinations. The final procedure comprised ACN extraction with MgSO 4 , NaCl, and Bond Elut EMR Lipid, followed by clean‐up with MgSO 4 , primary‐secondary amine, and Z‐Sep+. ACN provided the most consistent recovery across analytes, concentration levels, and matrices. The method was validated in accordance with International Council for Harmonization M10 using commercial plasma calibrators and independently prepared quality‐control samples. Accuracy and precision met the predefined acceptance criteria in both matrices. Mean recovery ranged from 84.2% to 91.2% in citrate plasma and from 79.6% to 87.8% in dried plasma samples. Internal standard‐normalized matrix factors ranged from 0.87 to 0.98, indicating limited ion suppression after the optimized clean‐up procedure. External verification using materials from two independent proficiency‐testing providers showed satisfactory agreement with assigned target values, with all results falling within the providers’ acceptance ranges. The method provides a robust QuEChERS‐assisted workflow for the quantitative determination of DOACs in conventional citrate plasma and qDPS samples. Its clinical interchangeability between matrices requires further evaluation using paired patient samples.