PB0099 - Homogeneous Enzyme Immunoassay for the Direct Determination of Rivaroxaban in Human Plasma

Background: Direct oral anticoagulants (DOACs) are now an alternative therapy to vitamin K
antagonists (VKAs) for prevention of blood clots and stroke in patients at risk of deep venous thrombosis,
atrial fibrillation, pulmonary embolism, and post-surgical clots. Indirect time based or factor Xa activity measurements are usually prone to interferences while hyphenated mass spectrometry techniques require skilled personnel and specialized equipment. A novel enzyme immunoassay to determine rivaroxaban (Xarelto) concentrations in human plasma is described.

Aims: To develop a homogeneous enzyme immunoassay to determine rivaroxaban concentrations in plasma on automated clinical analyzers

Methods: The ARK Rivaroxaban Assay is a liquid stable homogeneous enzyme immunoassay, consisting of two reagents, 6 calibrators covering the range of 0-500 ng/mL and 3 controls (10, 30 and 300 ng/mL). The performance of the ARK assay was evaluated on the Beckman AU680 Automated Clinical Chemistry Analyzer. Precision, limit of quantitation (LOQ), recovery, specificity and method comparison were studied.

Results: Total precision ranged from 3.5 to 7.8 %CV while within-run precision ranged from 3.3 to 6.9 %CV during a 5-day precision study (CLSI guideline EP15-A2) that evaluated precision for the 3 quality controls samples. The assay accurately recovered spiked rivaroxaban samples at 7 concentrations spanning the range from 5.0 to 400.0 ng/mL with percent recovery ranging from 95.2 to 108.6. LOQ was 3.0 ng/mL. Fifty specimens at concentrations ranging from 3.2 to 343.0 ng/mL were assayed and gave the following Passing Bablock regression results when compared to LC-MS/MS values: ARK = 0.97 LC-MS/MS + 6.2 (r2 = 0.98).

Conclusion(s): The ARK Rivaroxaban Assay measures rivaroxaban in human plasma with excellent precision at very low concentrations which is essential for emergency situations. The ability to measure trough levels of rivaroxaban directly with high accuracy and fast turn-around time makes this method clinically useful for the determination of rivaroxaban concentration in human plasma.