Table 1.
Multiple Reaction Monitoring (MRM) transitions monitored (m/z) with cone and collision energy.
Table 2.
Validation results of LOD, LLOQ, precision, recovery and accuracy.
Fig 1.
MRM chromatograms of plasma samples with and without DOACs.
MRM chromatograms of a plasma sample without any DOACs are depicted on the left (A, B, E, F), as well as MRM chromatograms of a plasma sample containing both dabigatran and rivaroxaban are depicted on the right (I, J, M, N). MRM chromatograms of the internal standard [13C6]-dabigatran (C, D, K, L) concerning dabigatran, as well as the internal standard [13C6]-rivaroxaban (G, H, O, P) concerning rivaroxaban are also shown. The first mass transition which was used for quantification of the drug, as well as the second mass transition which was used for verification is plotted. Data were normalized to largest peak in the plots. An estimated peak height is shown in the plots below the transition remark.
Fig 2.
MRM chromatograms of patients’ plasma samples.
MRM chromatograms of a plasma sample from a patient which have been treated with dabigatran are depicted on the left (A, B, E, F), as well as MRM chromatograms a plasma sample from a patient which have been treated with rivaroxaban are depicted on the right (I, J, M, N). MRM chromatograms of the internal standard [13C6]-dabigatran (C, D, K, L) concerning dabigatran, as well as the internal standard [13C6]-rivaroxaban (G, H, O, P) concerning rivaroxaban are also shown. The first mass transition which was used for quantification of the drug, as well as the second mass transition which was used for verification is plotted. Data were normalized to largest peak in the plots. An estimated peak height is shown in the plots below the transition remark.
Fig 3.
Product ion spectra of dabigatran (A) and rivaroxaban (C), as well as its internal standards [13C6]-dabigatran (B) and [13C6]-rivaroxaban (D) are depicted. The MH+ precursor ions and the fragment ions are shown. The chemical structures of the molecules are depicted. In addition, the positions of the 13C6 atoms of the internal standards [13C6]-dabigatran (B) and [13C6]-rivaroxaban (D) are shown.
Fig 4.
Ion suppression profiles of DOACs.
Ion suppression profile for dabigatran is depicted on the left and for rivaroxaban is depicted on the right, respectively, performed with a post-column flow injection of 0.1 mg/L drug into the UPLC elute of drug-free samples. In addition, the mass transition of the corresponding internal standard is shown. Data were normalized to largest peak in the plots. An estimated peak height is shown in the plots below the transition remark.
Table 3.
LC-MS/MS method comparison for the determination of DOACs.
Fig 5.
Method comparison using Passing-Bablok regression and Bland-Altman plot.
(A) Comparison of dabigatran results obtained by the LC-MRM MS assay and the DTI assay used for dabigatran measurement from CoaChrom Diagnostica performed on the ACL-TOP analyzer by Passing-Bablok regression: DTI assay = 0.86 (LC-MRM MS)– 1.03 [μg/L] (r = 0.99; n = 55; 95% CI for slope, 0.84–0.89; 95% CI for intercept, -6.10 μg/L—4.35 μg/L). (B) Bland-Altman plot for the comparison of LC-MRM MS assay vs. DTI assay from CoaChrom Diagnostica. The mean value (n = 55) of the two methods is plotted against the difference between the two values (LC-MRM MS assay–DTI assay from CoaChrom Diagnostica). The mean difference between the two methods was 32.1 μg/L. The mean (–) and ± 2 SD lines (---) are plotted for references. (C) Comparison of rivaroxaban results obtained by LC-MRM MS assay and the anti-Xa assay used for rivaroxaban from Chromogenix performed on the ACL-TOP analyzer by Passing-Bablok regression: anti-Xa assay = 1.07 (LC-MRM MS) + 0.00 [μg/L] (r = 0.98; n = 55; 95% CI for slope, 1.00–1.12; 95% CI for intercept, -7.10 μg/L– 7.80 μg/L). (D) Bland-Altman plot for the comparison of LC-MRM MS assay vs. anti-Xa assay used for rivaroxaban from Chromogenix. The mean value (n = 55) of the two methods is plotted against the difference between the two values (LC-MRM MS assay–anti-Xa assay from Chromogenix). The mean difference between the two methods was -14.4 μg/L. The mean (–) and ± 2 SD lines (---) are plotted for references.
Fig 6.
Effects of dabigatran and rivaroxaban on blood clotting.
Quick values and aPTT of dabigatran or rivaroxaban spiked blood samples. The mean and standard deviation of two measurements of each case are depicted.
Fig 7.
Mutual influences of DOAC measurement.
(A) Measurement of rivaroxaban in rivaroxaban and dabigatran spiked plasma. Concentrations were quantified using our UPLC-MRM MS assay (free squares), as well as the automated anti-Xa assay used for rivaroxaban measurement from Chromogenix on the ACL-TOP analyzer (filled circles). For clarification, the measuring points were connected (solid line). The line of identity X = Y is also shown (dotted line). (B) Measurement of dabigatran in rivaroxaban and dabigatran spiked plasma. Concentrations were quantified using our UPLC-MRM MS assay (free squares), as well as the automated DTI assay for dabigatran measurement from CoaChrom Diagnostica performed on the ACL-TOP analyzer. For clarification, the measuring points were connected (solid line). The line of identity X = Y is also shown (dotted line).