Quantification of direct‐acting oral anticoagulants: Application of a clinically validated liquid chromatography‐tandem mass spectrometry method to forensic cases

Abstract

In certain forensic cases, a quantification of direct‐acting oral anticoagulants (DOACs) can be necessary. We evaluate the applicability of a previously described liquid chromatography–tandem mass spectrometry (LC‐MS/MS) methodology for the determination of DOACs in plasma to postmortem specimen. Postmortem internal quality control (PIQC) samples were prepared in pooled blank postmortem heart blood, femoral blood, cerebrospinal fluid (CSF), and urine as well in plasma. To examine the application of the clinical method to forensic cases, the main validation parameters were reinvestigated using PIQC samples. Postmortem samples of 12 forensic cases with evidence of previous rivaroxaban intake and unknown bleeding disorders were analyzed. Interday variability remained within the acceptance criterion of ±15%. Matrix effects were comparable in blank plasma and postmortem matrix extracts. After 4 weeks of storage in the refrigerator, no relevant decrease of DOACs was evident. After 96 h of storage at room temperature, a slight decrease in edoxaban concentration was observed in CSF and urine, while plasma edoxaban decreased by about 50%. Median (range) rivaroxaban concentrations determined in specimen of forensic cases were as follows: heart blood (n = 6), 17.2 ng/ml (<LOQ, 56.6 ng/ml); femoral blood (n = 12), 27.6 ng/ml (<LOQ, 110.5 ng/ml); CSF (n = 7), 11.7 ng/ml (<LOQ, 17.5 ng/ml); urine (n = 6), 275.7 ng/ml (14.5–870.9 ng/ml). The median heart/femoral blood rivaroxaban ratio was 1.2 (n = 5). Exemplary, a forensic case with detection of edoxaban in femoral blood, CSF, and urine, is presented. DOACs can be detected in postmortem heart and femoral blood, CSF, and urine specimen by LC‐MS/MS. Based on limited forensic cases, no significant redistribution was evident for rivaroxaban, which was found at highest concentrations in urine.

Parameters Optimization of Edoxaban Extraction from Dried Plasma Spots

“…An important step in this work was the choice of the solvent providing the highest recovery, since there are no data on the extraction of edoxaban from DPS, but for extraction from DBS, authors used 30:70 isopropanol/methyl t-butyl ether (v/v) in the work [11]. Other authors used precipitation of proteins with acetonitrile for extraction [14].…”

“…Dried plasma spots (DPS) can be used to solve this problem. There are works [11,14], in which the authors used an internal standard with LC-MS / MS method to assess the concentration of edoxaban in DBS, plasma, whole blood, but there are no studies for the quantitative determination of edoxaban in dry plasma spot. One of the first steps in determining the concentration of edoxaban is to extract it from the DPS.…”

Development and validation of capillary zone electrophoresis and high‐performance liquid chromatography methods for the determination of oral anticoagulant edoxaban in pharmaceutical tablets

“…EDX is not an official drug in any Pharmacopoeia. However, the literature reports different analytical methods, especially chromatographics, for determining EDX in biological fluids [5][6][7][8][9][10][11][12][13][14]. Gous et al [5] developed and validated a method for monitoring of EDX in human plasma using turbulent flow liquid chromatography with high-resolution mass spectrometry.…”

“…Gous et al [5] developed and validated a method for monitoring of EDX in human plasma using turbulent flow liquid chromatography with high-resolution mass spectrometry. Wiesen et al [11] have recently developed a method for quantifying direct-acting oral anticoagulants by liquid chromatography-tandem mass spectrometry for forensic cases. Reddy et al [15] developed a stability-indicating assay method for highperformance liquid chromatography (HPLC) to quantify EDX in bulk and tablet form, using a C18 column and an isocratic mobile phase using K 2 HPO 4 : methanol.…”

“…EDX is not an official drug in any Pharmacopoeia. However, the literature reports different analytical methods, especially chromatographics, for determining EDX in biological fluids [5–14]. Gous et al.…”