MSACL 2022 Abstract

Introduction
Direct oral anticoagulants (DOACs) have been increasingly prescribed for stroke prevention and treatment of thromboembolic disease (deep vein thrombosis and pulmonary embolism). An advantage of DOACs over other anticoagulants, such as Warfarin, is that routine laboratory monitoring is not recommended. Despite the ease of dosing, DOACs introduce a risk for hemorrhages and life threatening bleeding in high-risk patient populations. Laboratory testing may be useful for ruling out DOACs as a cause of major bleeds or in guiding the administration of DOAC reversal agents in patients with DOAC concentrations that exceed recommended levels. Unfortunately, standard coagulation assays, including prothrombin time (PT) and activated partial thromboplastin time (aPTT), correlate poorly with plasma concentrations of DOACs. The chromogenic anti-factor Xa assay is a reliable predictor of DOAC concentration, however the utility of this test in emergency settings is limited by turnaround times of approximately 1 hour, heparin interference, and the inability to screen for multiple DOACs in one test.

Coated blade spray (CBS) is a solid-phase microextraction-based technology that enables rapid qualitative and quantitative analysis of complex matrices through direct coupling with a mass spectrometer. CBS mass spectrometry (CBS-MS) has the potential to address the unmet clinical need of rapidly assessing and managing life threating bleeding caused by DOACs. A quantitative CBS-MS assay was developed and validated to measure clinically relevant plasma concentrations of a panel of anticoagulants including 3 DOACs (Apixaban, Edoxaban, and Rivaroxaban) and the direct thrombin inhibitor Argatroban.

Methods
Calibrators were prepared in citrated plasma at 10 concentrations ranging from 0.5 1000 ng/mL for DOACs (Apixaban, Edoxaban, and Rivaroxaban) and 1 2000 ng/mL for Argatroban. Quality controls were prepared separately from the calibrators at 0.8, 8.0, and 80 ng/mL for DOACs and 1600, 160, and 3 ng/mL for Argatroban. Apixaban-13C-d3, 2H6 Edoxaban, Rivaroxaban-d4, and 2H3 Argatroban were used as internal standards.

Citrated plasma samples were applied to stainless steel blades that were coated with hydrophilic-lipophilic balance (HLB) polymer. Blades were then incubated with internal standards in Acetonitrile: Phosphate-buffered saline. A solution of 0.1% formic acid in 95:5 Methanol:Water was sprayed onto blades for elution of analytes. CBS-MS/MS data was acquired with a Sciex QTRAP6500 system in positive ion mode with a voltage of 3.25 kV. Data were analyzed using Sciex MultiQuant software.

Results
For all analytes (N=4) tested at 10 concentrations; the intra-day accuracy ranged from 91-107% and the intra-day imprecision (CV) ranged from 1-11%, and the inter-day imprecision ranged from 3-8%. The LOQ for each analyte was: Apixaban 10 ng/mL (CV 10%, Accuracy 94%); Argatroban 20 ng/mL (CV 11%, Accuracy 89%); Edoxaban 10 ng/mL (CV 3%, Accuracy 111%); Rivaroxaban 10 ng/mL (CV 9%, Accuracy 102%). R2 values greater than 0.99 for all analytes. No carryover was observed. A comparison study of CBS-MS/MS to LC-MS/MS for anticoagulant quantitation in patient samples in patient samples is currently underway.

Conclusions
Method validation data confirmed the capacity of CBS-MS to quantitate clinically relevant concentrations for a panel of direct acting anticoagulants. With a total analysis time of < 1 minute per sample this technology has the potential to provide results to emergency departments at a faster turnaround time than anti-factor Xa assays and LC MS/MS.