Cutting through the matrix: Analysis of antithrombotic medications in plasma

Antithrombotic medications (Figure 1) are pharmaceutical agents critical to modern cardiovascular healthcare^1-3. Utilising SPE-LC-MS/MS provided a reliable, sensitive and selective clean-up for the simultaneous quantification of anticoagulants and antiplatelets in the plasma.

Figure 1 - Chemical structures of anticoagulants: apixaban, dabigatran etexilate, edoxaban, rivaroxaban and warfarin and antiplatelets: clopidogrel, dipyridamole and prasugrel

Analysis of antithrombotic medications is essential in the growing clinical sector where rapid, reliable and comprehensive measurements are required⁴. Furthermore, with the approval of specific reversal agents, such as idarucizumab for dabigatran⁵ and andexanet alfa for rivaroxaban⁶, determining the exact antithrombotic concentration within plasma has become imperative for safe and effective treatment.

Several coagulation-based, phenotypic and platelet function assays have been developed⁷, including diluted thrombin time, ecarin clotting time, and drug-specific anti-factor Xa assays^8-10. However, these techniques lack the versatility to quantify multiple medications simultaneously and possess poor accuracy at low plasma concentrations¹¹. Subsequently, LC-MS/MS methods emerged as the gold standard for the quantification of anticoagulants and antiplatelets in biological matrices¹². 

While several methods have been reported for separately quantifying anticoagulants and antiplatelets in plasma¹³, few methods combine quantification of both into a single method. Plasma represents a highly complex biological matrix containing proteins, phospholipids, salts, and endogenous metabolites¹⁴. These are capable of causing ion suppression, reduced sensitivity, and contamination of LC-MS ion sources¹⁵. Traditional approaches frequently employ protein precipitation or phospholipid removal sample preparation, which can leave behind interfering compounds, reducing sensitivity and method flexibility. SPE (solid phase extraction) provides a cleaner and targeted extract by effectively and selectively removing endogenous interferences whilst retaining the target analyte. 

A sensitive SPE-LC-MS/MS method was developed and validated for the simultaneous quantification of major anticoagulant and antiplatelet medications in animal plasma (Figure 2). 

Figure 2 - Chromatographic separation of 50 ng/mL antithrombotic analyte QC sample using LC–MS/MS method.

The method demonstrated excellent linearity (R² > 0.98), low MQLs (< 5.75 ng/mL) and good reproducibility (< 15% RSD) without clogging. The method was stress-tested using five different unfiltered animal plasmas (mouse, rat, rabbit, beagle, monkey) the results of which showed… 

Read the full application note here - Cutting Through the Matrix

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