Journal of Thrombosis and Haemostasis, 12: 1636–1646
DOI: 10.1111/jth.12702
ORIGINAL ARTICLE
Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants E. M. H. SCHMITZ,*†‡ K. BOONEN,* D. J. A. VAN DEN HEUVEL,* J. L. J. VAN DONGEN,†‡ M . W . M . S C H E L L I N G S , § J . M . A . E M M E N , § F . V A N D E R G R A A F , ‡ § L . B R U N S V E L D † ‡ and D. VAN DE KERKHOF*‡ *Clinical Laboratory, Catharina Hospital, Eindhoven; †Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven; ‡Expert Center Clinical Chemistry Eindhoven, Eindhoven; and §Clinical Laboratory, Maxima Medical Center Veldhoven, Veldhoven, the Netherlands
To cite this article: Schmitz EMH, Boonen K, van den Heuvel DJA, van Dongen JLJ, Schellings MWM, Emmen JMA, van der Graaf F, Brunsveld L, van de Kerkhof D. Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatrography – tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants. J Thromb Haemost 2014; 12: 1636–46.
Abstract. Background: Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. Objective: To develop and validate a reference ultra-performance liquid chromatography – tandem mass spectrometry (UPLCMS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. Methods: The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. Results: The UPLC-MS/MS
Correspondence: Daan van de Kerkhof, Catharina Hospital Eindhoven, Clinical Laboratory, Michelangelolaan 2, 5602 EJ Eindhoven, the Netherlands. Tel.: +31 40 2398641; fax: +31 40 2398637. E-mail: [email protected] Received 26 November 2013 Manuscript handled by: S. Kitchen Final decision: P. H. Reitsma, 10 August 2014
method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay. Conclusions: Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal. Keywords: apixaban; dabigatran; drug monitoring; mass spectrometry; rivaroxaban.
Introduction Since 2010, three novel direct oral anticoagulants (DOACs) have been registered by the Food and Drug Administration (FDA) and European Medicines Agency Commission (EMA) for the prevention of deep vein thrombosis in patients undergoing knee or hip replacement surgery, and for the prevention of stroke in patients with non-valvular atrial fibrillation: dabigatran, rivaroxaban, and apixaban. DOACs, previously known as novel oral anticoagulants (NOACs), are direct inhibitors of coagulation factors. Dabigatran inhibits thrombin (FIIa), and rivaroxaban and apixaban inhibit FXa. DOACs have © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1637
more predictable pharmacokinetics and pharmacodynamics than the traditionally used coumarin-derived anticoagulants [1,2], and fewer food and drug–drug interactions have been reported [3]. It was therefore advocated that dosing based on laboratory testing would be obsolete [4]. These advantages have led to an increasing number of patients receiving these drugs. However, the current consensus is that laboratory monitoring of DOACs is important in specific situations, e.g. in patients with a very high or low body mass index or in those with diminished renal function, or in emergency (bleeding) situations [5–7]. Unfortunately, conventional laboratory assays such as prothrombin time (PT) and activated partial thromboplastin time (APTT) assays are not suitable for accurate quantification of DOACs [8]. Insufficient sensitivities and non-linear dose responses of the PT and APTT assays can lead to only slightly elevated coagulation times with high DOAC concentrations [9–12]. To fulfil this unmet need for accurate quantification of DOACs, several companies have developed new coagulation assays or new protocols and calibrators for existing coagulation assays. For quantification of direct thrombin inhibitors (DTIs), the diluted thrombin time (dTT) assay is advocated as the preferable test [13–15]. This is a quantitative clotting antiFIIa assay in which a defined amount of thrombin is added to diluted plasma. The Hemoclot DTI clotting assay is the most studied dTT assay for analysis of direct thrombin inhibitors [14]. Alternatively, snake venombased assays can be used, such as the Ecarin Clotting Time (ECT) assay, the Ecarin Chromogenic Assay (ECA), or the Prothrombinase-induced Clotting Time (PiCT) assay. In the ECA, prothrombin is converted by ecarin into chromogenically detected meizothrombin [13,16]. The PiCT assay is based on selective FV activation by Russell’s viper venom, and is sensitive for both DTIs and FXa inhibitors [17]. The current preferred assay for quantification of FXa inhibitors is the anti-FXa assay, for which numerous commercial kits are available [18–20]. Liquid chromatography (LC)–mass spectrometry (MS) is widely applied for quantification of drugs. This technique has much better selectivity than coagulation activity-based assays, enabling specific detection and quantification of coagulation inhibitors [21–24]. As coagulation assays measure activities of various coagulation factors, they are prone to interference by other coagulant drugs or endogenous or preanalytical changes in coagulation factors. This may result in inaccurate quantification of the anticoagulant’s concentration in plasma. Therefore, LC-MS detection is expected to be superior to coagulation assays for accurate quantification of DOACs. However, many clinical laboratories do not have (24-h) access to LC-MS analysis, which makes this technique unsuitable for routine measurement of DOACs. Coagulation assays are better employed for routine measurement, but at present it is unclear which coagulation assays are most appropriate for quantification of DOACs. Therefore, we © 2014 International Society on Thrombosis and Haemostasis
developed and validated a reference ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS) method for quantification of dabigatran, rivaroxaban, and apixaban. The UPLC-MS/MS and coagulation assays were validated by the use of drugenriched plasma. For dabigatran and rivaroxaban, the UPLC-MS/MS method was also compared with coagulation methods by the use of patient samples. Materials and methods UPLC-MS/MS sample preparation
Citrated plasma pools were spiked with dabigatran, rivaroxaban and apixaban (Alsachim, Illkirch Graffenstaden, France) for a six-point calibration (calibration range 23–750 ng mL 1) and quality control (QC) (50, 275 and 500 ng mL 1). For evaluation of adsorption to erythrocytes, full citrated blood samples were spiked with the DOACs and centrifuged at 2683 9 g for 5 min to obtain spiked plasma. Isotopically labeled internal standards of the DOACs were added for quantification (100 ng mL 1) ([13C6]dabigatran, [13C6]rivaroxaban, and [13C,2H7]apixaban; Alsachim). Sample clean-up was performed by protein precipitation with acetonitrile (dilution of the sample at a ratio of 1 : 3). The supernatant was diluted with mobile phase A (dilution of 1 : 4). UPLC-MS/MS method
UPLC-MS/MS analysis of the samples was performed with an Acquity UPLC binary solvent manager, sample manager, and column manager, and an Acquity TQ Detector (Waters, Milford, MA, USA). Chromatographic separation was achieved by injection of 10 lL of sample on an Acquity UPLC BEH C8 column (100 9 2.1 mm, 1.7 lm; Waters) at 30 °C. The flow rate was 300 lL min 1, with the mobile phases 2.5 mM ammonium formate at pH 3.0 (A) and acetonitrile (B). A 4.75-min gradient run was performed: 0–1.0 min, 15% B; 1.0–3.0 min, linear to 75% B; 3.0–3.5 min, linear to 95% B; 3.5–4.0 min, 95% B; and 4.0–4.75 min, return to initial conditions. Mass analysis was performed with a two-step multiple reaction monitoring (MRM) method (Table 1). Peak integration was performed with MASSLYNX software V4.1 SCN 714 (Waters, Milford, MA, USA) with ApexTrack Peak Integration. The peak area ratio between the drug and its internal standard (response) was used to quantify the amount of drug in the plasma and to correct for recovery loss. UPLC-MS/MS validation
To determine within-run and between-run imprecision and accuracy (bias), three concentration levels of QC samples were each analyzed five times on five different days. For recovery determination, spiked plasma samples (A) and
1638 E. M. H. Schmitz et al Table 1 Mass spectrometry settings for single-run quantification of dabigatran, rivaroxaban, and apixaban
Compound Period 1 (0–2 min) Dabigatran [13C6]Dabigatran Period 2 (2–4.75 min) Rivaroxaban [13C6]Rivaroxaban Apixaban [13C–2H7]Apixaban
Q1 mass
Q3 mass
Dwell (s)
Cone (V)
CE (eV)
472.2 478.2
289.1 295.2
0.078 0.078
42 44
30 30
436.1 442.2 460.2 468.2
144.9 144.9 77.0 107.1
0.036 0.036 0.036 0.036
54 58 66 56
28 28 64 52
CE, collision energy.
blank plasma samples (B) were prepared as described above. Samples B were spiked with dabigatran, rivaroxaban and apixaban after sample preparation. To calculate recovery, the peak area in sample A was divided by the peak area found in sample B for each DOAC. Carry-over was determined by performing an EP10 protocol [25], in which low-, medium- and high-concentration samples were analyzed in a predetermined alternate order. Matrix effects were studied by injecting six prepared blank plasma samples onto the chromatographic column, during continuous infusion of dabigatran, rivaroxaban or apixaban in the mass spectrometer. Specificity was considered to be adequate if no interfering peaks were present in drug-naive samples. A signal-to-noise ratio (SNR) was determined in diluted samples. The concentration corresponding to an SNR of 3 was established as the lower limit of detection (LLOD). Stability was studied by repeated analysis of spiked QC samples after three freeze–thaw cycles ( 20 °C to room temperature and 20 °C to 37 °C), and after 24 h of storage at room temperature and at 4 °C. Long-term stability during storage at 80 °C was investigated after 72 days. The stability of prepared samples (after protein precipitation and in the final solution for analysis) was determined after storage for 24 h at room temperature and at 4 °C. The range of acceptance for imprecision and accuracy (bias) was defined as < 15% and 85–115%, respectively, based on current FDA criteria for drug analysis [26]. Stability was accepted in the range of 80–120%. The method robustness was tested by a 10% deliberate adjustment of flow rate, buffer concentration, gradient settings, and column temperature. Furthermore, the pH of mobile phase A was adjusted by 0.1, and ammonium formate was replaced by ammonium acetate. The responses of dabigatran, rivaroxaban and apixaban were calculated for all conditions to determine whether these adjustments influenced quantification of the DOACs. The significance of response deviations was tested with one-way ANOVA (P < 0.05). Coagulation assay methods
All analyses were performed on the STA-R Evolution Analyzer (Stago, Asnieres sur Seine, France). The assays
evaluated for dabigatran were the Hemoclot dTT assay (Hyphen Biomed, Neuville-sur-Oise, France), LD dTT assay (laboratory-developed dTT, for which Stago’s thrombin reagent was used and eight-fold sample dilution with normal plasma was applied), the HaemoSys ECA (JenAffin GmbH, Jena, Germany), and the Pefakit PiCT assay (Pentapharm, Basel, Switzerland). For rivaroxaban and apixaban, the Liquid anti-Xa (Stago), Biophen Heparin anti-Xa (LRT; Hyphen Biomed), Biophen DiXal antiXa (Hyphen Biomed) and Pefakit PiCT assays were evaluated. Reagents were stored at 4 °C between measurements, as described in the package inserts. Plasma pools were spiked with dabigatran, rivaroxaban or apixaban for calibration (750 ng mL 1) and QC (92 and 375 ng mL 1 for dabigatran; 82 and 308 ng mL 1 for rivaroxaban; 87 and 386 ng mL 1 for apixaban). The in-house calibrators were used for all tests, except for the Hemoclot dTT assay, and diluted on the STA-R Evolution Analyzer with normal plasma to obtain a five-point calibration. The calibration line for the LD dTT was linear (calibration range 38–500 ng mL 1). The calibration curves for the ECA and PiCT assays were secondorder polynomial, as specified by the manufacturers (calibration range: ECA, 38–500 ng mL 1; PiCT-IIa, 25–250 ng mL 1; PiCT-Xa, 19–250 ng mL 1). Calibration curves for the Stago Liquid anti-Xa and Biophen Heparin anti-Xa assays were log-linear, as specified by the manufacturer (calibration range: Liquid anti-Xa, 30– 500 ng mL 1; Biophen Heparin anti-Xa, 0–490 ng mL 1). The calibration line for the Biophen DiXal anti-Xa assay was linear (calibration range: 0–490 ng mL 1). For the Hemoclot dTT assay, the manufacturer’s calibrators were used to obtain a linear, three-point calibration line (calibration range: 40–500 ng mL 1). Coagulation assay validation
Within-run and between-run imprecision and accuracy of the coagulation assays was determined by performing duplicate QC measurements in 10 separate runs, each being preceded by calibration. Accuracy was calculated as the determined concentration divided by the concentration determined with UPLC-MS/MS. Imprecision was accepted when this was < 15%, and accuracy was accepted when it was between 85% and 115% [26]. The LLOD was calculated as the mean plus two standard deviations of normal plasma, which was measured five times. The validity of the studied coagulation assays for quantification of dabigatran and rivaroxaban was evaluated by agreement of the coagulation assay findings with the UPLC-MS/MS findings obtained with patient samples. The differences in DOAC concentrations were visualized in a modified Bland–Altman plot, depicting the reference method’s results on the horizontal axis. A paired-samples t-test was performed to determine whether there was a © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1639
fixed bias between the coagulation assay and the UPLCMS/MS method (confirmed when P < 0.05). Plasma was collected from patients taking dabigatran or rivaroxaban as part of routine care (37 samples for dabigatran and 27 for rivaroxaban). The obtained plasma was anonymized for the study. The study was conducted in accordance with the local medical ethical protocol as prescribed by Dutch law. The collection of blood samples was not timed, and the patient population used was undefined regarding clinical background (including both cardiologic and orthopedic patients).
73%, 78%, and 104%, respectively. Recovery was thus not complete for dabigatran and rivaroxaban, but quantification was not impaired, owing to the addition of the labeled internal standards. The within-run and betweenrun imprecision of the UPLC-MS/MS method was < 15%, and the accuracy met the acceptance criteria, except for rivaroxaban at the low concentration (Table 2). Robustness testing showed a change of retention time with changing flow rate, as expected. However, the responses of all three DOACs were not significantly affected by any of the deliberate changes to the UPLCMS/MS method (P ≥ 0.112).
Results Coagulation assay validation UPLC-MS/MS validation
For imprecision and accuracy determined with the spiked QC samples, see Table 2. The LD dTT assay showed acceptable within-run and between-run imprecision (≤ 9.9%) and accuracy (107–112%) for both QC levels (92 and 375 ng mL 1), in contrast to the Hemoclot dTT assay, which showed acceptable within-run imprecision (≤ 10.1%) but inacceptable between-run imprecision (≥ 33.0%) and accuracy (125–131%). The ECA and the PiCT-IIa assay showed acceptable within-run imprecision (ECA, ≤ 4.4%; PiCT-IIa, ≤ 4.3%). The between-run imprecision and accuracy were only acceptable for the high QC level (ECA, 5.8% and 108%, respectively; PiCTIIa, 10.8% and 99.3%, respectively). All evaluated assays for quantification of rivaroxaban showed good imprecision (within-run ≤ 6.4%; between-run ≤ 12.6%) at both QC levels (82 and 308 ng mL 1). Accuracy at the high QC level was acceptable for all assays, except for the Liquid anti-Xa assay (119%). At the low QC level, this was the case for the PiCT-Xa assay (79.6%) and the Biophen DiXal anti-Xa assay (132%). For apixaban, a reproducible PiCT-Xa assay could not be developed, and the Liquid anti-Xa assay showed unacceptable between-run imprecision (18.3% for the low QC level [87 ng mL 1] and 15.4% for the high QC level [386 ng mL 1]).
A UPLC-MS/MS method for single-run quantification of dabigatran, rivaroxaban, and apixaban, with isotopically labeled standards, was successfully developed (Fig. 1). R2 of the fitted linear calibration lines was ≥ 0.99 for all three DOACs. No significant difference in response could be seen between samples prepared from plasma and those prepared from whole blood, indicating that the studied DOACs are not adsorbed to erythrocytes. Spiked citrated plasma can thus be used for QC and calibration samples. Stability tests showed adequate stability during three freeze–thaw cycles, 24 h of plasma storage at room temperature and 4 °C, and 72 days of plasma storage at a temperature lower than 80 °C. Prepared samples (after protein precipitation and preparation of the final solution for UPLC-MS/MS analysis) were stable for at least 24 h at room temperature and at 4 °C. Detailed results of stability analysis are provided in the tables of the Supporting Information. No interfering peaks were present in drug-naive samples, and the LLOD of the UPLC-MS/MS method was ≤ 0.025 ng mL 1 for all three DOACs. Matrix effects were absent (Fig. 2) and the maximum determined carryover was 1.0%. For dabigatran, rivaroxaban, and apixaban, the rates of recovery after sample preparation were 1.52
100
Dabigatran
Apixaban
%
100
%
0
0.50 1.00
1.50
100
3.50 4.00
2.00 2.50 3.00
Apixaban
4.50
0
3.16 100
Apixaban IS
%
3.24
0
Rivaroxaban
%
0
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10
Fig. 1. Mass chromatogram of the multiple reaction monitoring (MRM) method for the single-run detection of dabigatran, rivaroxaban, apixaban and their isotopically labeled standards ([13C6]dabigatran, [13C6]rivaroxaban and [13C–2H7]apixaban). Elution times of the direct oral anticoagulants and its labeled standard are similar, but they can be separately analyzed and quantified, owing to their mass difference. IS, internal standard; UPLC-MS/MS, ultra-performance liquid chromatography–mass spectrometry/mass spectrometry. © 2014 International Society on Thrombosis and Haemostasis
Hemoclot dTT LD dTT ECA PiCT-IIa
89.3
Liquid anti-Xa
103
Accuracy (%)
79.6 106.7 105 132
Accuracy (%)
125 107 83.9 169
Accuracy (%)
106 116 94.6
Accuracy (%)
3.3
Imprecision Within-run (%)
6.4 4.0 1.5 4.3
Imprecision Within-run (%)
8.3 3.7 4.4 4.3
Imprecision Within-run (%)
5.9 6.4 3.5
Imprecision Within-run (%)
18.3
Imprecision Between-run (%)
12.6 3.5 5.1 7.7
314
Mean (target: 386 ng mL 1)
314 368 272 324
3.9 2.9 3.8
93.2
Accuracy (%)
102 119 88.3 105
Accuracy (%)
131 112 108 99.3
1.1
Imprecision Within-run (%)
6.2 2.4 1.6 1.8
Imprecision Within-run (%)
10.1 1.5 1.9 3.5
Imprecision Within-run (%)
Imprecision Within-run (%)
Accuracy (%)
103 114 92.0
Accuracy (%)
Mean (target: 308 ng mL 1)
491 419 406 372
Mean (target: 375 ng mL 1)
284 314 253
Mean (target: 250 ng mL 1)
Imprecision Between-run (%)
33.0 9.9 16.4 16.9
Imprecision Between-run (%)
2.8 4.2 9.5
Imprecision Between-run (%)
QC level 2
The CV was calculated from duplicate measurements obtained over a period of 10 days.
Mean (target: 87 ng mL 1)
Coagulation assays apixaban
65.1 87.2 85.6 108
116 99.2 77.4 156
Coagulation assays dabigatran
PiCT-Xa Liquid anti-Xa Heparin anti-Xa DiXal anti-Xa
Mean (target: 92 ng mL 1)
Dabigatran Rivaroxaban Apixaban
Mean (target: 82 ng mL 1)
52.8 58.3 47.3
UPLC-MS/MS
Coagulation assays rivaroxaban
Mean (target: 50 ng mL 1)
QC level 1
15.4
Imprecision Between-run (%)
8.2 3.3 2.8 2.5
Imprecision Between-run (%)
34.3 7.9 5.8 10.8
Imprecision Between-run (%)
0.0 7.8 9.1
Imprecision Between-run (%) 505 569 457
Mean (target: 500 ng mL 1)
QC level 3
101 114 91.4
Accuracy (%)
4.6 4.6 3.8
Imprecision Within-run (%)
0.0 7.1 7.5
Imprecision Between-run (%)
Table 2 Mean concentration of quality control (QC) samples (QC level no.) with their accuracy and within-run and between-run imprecision for the ultra-performance liquid chromatography/ tandem mass spectrometry (UPLC-MS/MS) method and coagulation assays
1640 E. M. H. Schmitz et al
© 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1641 100 A
4.624.65 4.55
%
4.21 0.81 0.87
Dabigatran 4.15
3.93 3.57 2.78
0
3.36
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
100 B
Time
4.55
4.22
Rivaroxaban
0.87
4.43
0.79
%
3.73
4.12
0.74 1.02
0.190.350.56 0.62
0
1.361.491.74 1.85
2.07
2.49 2.692.77 2.98 3.323.35 3.56 2.26 2.87
3.99
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Time
4.55
100 C
4.43 4.22
Apixaban
0.87
4.32 0.83 0.78
%
4.13
0.73 0.35 0.530.63 0.13
1.00
1.33 1.47
4.06 3.96 3.55 3.58 2.66 2.73 2.78 3.213.36 3.68
0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Time
Fig. 2. Illustration of determination of the matrix effects. The direct oral anticoagulant (DOAC) is continuously infused in the mass spectrometer: (A) dabigatran; (B) rivaroxaban; (C) apixaban. After sample preparation, blank samples (n = 6) are injected (t = 0) and separated over the ultra-performance liquid chromatography column. The elution of matrix components from plasma causes deviation of the mass spectrometry/mass spectrometry trace, as a result of ionization competition by dabigatran and the matrix. At the retention time of the DOACs (arrows), no deviating response was observed.
The LLODs of the coagulation assays are shown in Table 3. The PiCT assay was omitted, because of its high imprecision. The Hemoclot dTT assay showed a higher LLOD (43.5 ng mL 1) than the LD dTT assay and the ECA (≤ 17.4 ng mL 1). The Liquid anti-Xa assay showed a higher LLOD (24.0 ng mL 1) than the Biophen © 2014 International Society on Thrombosis and Haemostasis
Heparin and Biophen DiXal anti-Xa assays (≤ 2.8 ng mL 1). Agreement between the UPLC-MS/MS method and coagulation assays was determined by the use of modified Bland–Altman plots (see Fig. 3). Drug concentrations lower than the calculated LLODs were removed for all assays except the PiCT. A paired-samples t-test showed that that there was only a statistically significant bias in dabigatran concentration between the ECA and the UPLC-MS/ MS method (29.4 ng mL 1; P < 0.000). This could not be shown for the LD dTT, Hemoclot dTT and PiCT-IIa assays (P = 0.067, P = 0.450, and P = 0.143, respectively). In the case of rivaroxaban, the Bland–Altman plots showed that a statistically significant bias in drug concentration could be found only between the PiCT-Xa and UPLC-MS/MS assays (36.6 ng mL 1, P = 0.022; Fig. 4). This bias was not statistically significant for the Liquid anti-Xa, Biophen DiXal anti-Xa and Biophen Heparin anti-Xa assays (≤ 3 ng mL 1, P ≥ 0.061). The limits of agreement for the PiCT-Xa assay ( 88.0 to 161) were much wider than for the other coagulation assays (≥ 24 to ≤ 26), indicating much larger imprecision for the PiCT-Xa assay. Discussion There is general consensus that, although routine monitoring of DOACs is not recommended, laboratory analysis is required in specific situations. An increasing number of chromogenic or clotting-based assays have become available to quantify thrombin and/or FXa inhibitors. However, standardization is lacking, and the potential of non-dTT-based and non-anti-FXa-based assays is unclear. Therefore, we developed and validated a UPLC-MS/MS method for quantification of dabigatran, rivaroxaban, and apixaban. The method was shown to be precise, accurate, sensitive, specific, and robust [27]. The UPLCMS/MS method was therefore used as the reference method for comparison with the various coagulation assays. During sample preparation, dabigatran and rivaroxaban are not completely recovered from the plasma, but this was compensated for by the labeled internal standard. Quantification was therefore not impaired. We compared this UPLC-MS/MS method with several essentially different coagulation assays, which were developed for quantification of DOACs. Studies have already shown that routine coagulation assays, such as PT, thrombin time and APTT assays, are not suitable for accurate quantification of DOACs [1,2,12,28]. They were therefore omitted from our research. For dabigatran, we included the clotting-based dTT assay, the ecarin-induced meizothrombin formation-dependent ECA, and the prothrombinase complex-dependent PiCT-IIa assay. For rivaroxaban and apixaban, different chromogenic anti-FXa assays and the PiCT-Xa assay were selected. Besides the commercially available assays, we also used an in-house-
1642 E. M. H. Schmitz et al Table 3 Lower limits of detection (LLODs) of the different coagulation assays, calculated as mean plus two standard deviations (SDs) of five blank measurements
Dabigatran Hemoclot dTT LD dTT ECA Rivaroxaban Liquid anti-Xa Biophen Heparin anti-Xa Biophen DiXal anti-Xa Apixaban Liquid anti-Xa
Mean (ng mL 1)
SD (ng mL 1)
LLOD (ng mL 1)
18.8 6.9 15.3
12.3 5.2 0.0
43.5 17.4 15.3
18.3 1.7 2.7
2.9 0.0 0.0
24.0 1.7 2.8
19.7
4.7
29.1
developed method (LD dTT). This assay was based on the protocol of the Hemoclot dTT assay, for which we used Stago’s thrombin reagent and in-house normal plasma. To calibrate the coagulation assays, in-house calibrators were used for all assays except the Hemoclot dTT assay. A disadvantage of this strategy is that the manu-
B
150
Bias (–8.97) P-value: 0.450
100
95% Limits of agreement (– 105 to 87.5)
Difference (UPLC-MS/MS – LD dTT) (ng mL–1)
Difference (UPLC-MS/MS – Hemoclot dTT) (ng mL–1)
A
facturer kits may not perform to the manufacturer’s specifications unless the calibrator defined by the manufacturer is used. However, the PiCT and ECA kits did not have a dedicated calibrator at the time when this study was performed. Also, test results of the different kits are more comparable when the same calibrators are used. For quantification of dabigatran, the modified LD dTT assay was analytically superior to the Hemoclot dTT assay, ECA, and PiCT-IIa assay, because it was the only coagulation assay that showed acceptable imprecision (within-run, ≤ 3.7%; between-run, ≤ 9.9%) and accuracy (≤ 112%) at both low and high dabigatran concentrations. Freyburger et al. [29] also satisfactorily used a ‘homemade’ dTT assay for a study investigating the effect of dabigatran on orthopedic patients. We experienced reproducibility problems with the Hemoclot dTT assay, which caused unacceptable accuracy and imprecision. This was attributable to instability of the reagents, despite the fact that they were used according to the manufacturer’s recommendations. Within-run and between-run variability caused by the use of human thrombin rather than the more stable bovine thrombin might also have contributed. Inter-
50 0 – 50 – 100
150
Bias (8.51) P-value: 0.067
100 50 0 – 50
– 100
– 150
– 150 0
50
100
150
200
250
0
300
50
100
150
200
250
300
UPLC-MS/MS (ng mL–1)
UPLC-MS/MS (ng mL–1)
D
C Bias (29.4) P-value: < 0.000
100
150
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Fig. 3. Modified Bland–Altman plot showing the agreement of dabigatran coagulation assays with ultra-performance liquid chromatography– mass spectrometry/mass spectrometry (UPLC-MS/MS) in random patient samples. Samples with drug concentrations lower than the lower limit of detection were removed from analysis, except for the PiCT-IIa assay. (A) Hemoclot dTT (n = 18). (B) LD dTT (n = 28). (C) ECA (n = 37). (D) PiCT-IIa (n = 37). © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1643
B
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– 100
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Fig. 4. Modified Bland–Altman plot showing the agreement of rivaroxaban coagulation assays with ultra-performance liquid chromatography– mass spectrometry/mass spectrometry (UPLC-MS/MS) in random patient samples. Samples with drug concentrations lower than the lower limit of detection were removed from analysis, except for the PiCT-Xa assay. (A) Liquid anti-Xa (n = 25). (B) Biophen DiXal anti-Xa (n = 18). (C) Biophen Heparin anti-Xa (n = 22). (D) PiCT-Xa (n = 27).
estingly, these problems were not reported by other studies using this assay [12,14]. The PiCT assay also showed reproducibility problems, similar to the findings of Douxfils et al. [12], making this assay unsuitable for monitoring purposes. Ecarin-based assays have often been used in pharmacologic studies. ECT assays were shown to be sensitive and to have a good linear response to increasing dabigatran concentrations [1,2], although interlot variability was an issue [12]. The ECA was shown to have good sensitivity and linearity for higher dabigatran concentrations, but non-linearity for samples with a concentration of < 50 ng mL 1 was demonstrated [12]. This was supported by the findings of this study. Optimizing the calibration, e.g. by addition of a blank sample to the calibration line, might improve those results. The Hemoclot dTT assay has a higher LLOD than the LD dTT assay and the ECA, making the latter two more suitable for dabigatran quantification in the low concentration range. The Hemoclot dTT assay applies only three calibrators, whereas the LD dTT assay and ECA use five, © 2014 International Society on Thrombosis and Haemostasis
possibly making the fitted lines of the LD dTT assay and ECA more accurate than the Hemoclot dTT assay’s fitted line. Another explanation for the Hemoclot dTT assay’s higher LLOD could be its higher imprecision. For rivaroxaban, the Biophen Heparin and Biophen DiXal assays have lower LLODs than the Liquid anti-Xa assay, which could be explained by the incorporation of a blank sample in the calibration. We were not able to adequately determine an LLOD for the PiCT assays, owing to their high imprecision. Agreement between the dabigatran coagulation assays and UPLC-MS/MS showed that, on average, the coagulation assays gave higher dabigatran concentrations than the reference method. This bias could be explained by dabigatran’s pharmacologically active metabolites, which are not measured by the UPLC-MS/MS method. It was shown by Blech et al. [30] that these dabigatran acylglucuronides represent ~ 20% of the total drug exposure in plasma. Dabigatran metabolism can therefore cause discrepancies between this UPLC-MS/MS method and the
1644 E. M. H. Schmitz et al
coagulation assays. It is not yet known whether these discrepancies are clinically relevant, as the relationship between dabigatran concentration and bleeding risk is poorly understood. The bias between coagulation assays and the reference method was statistically significant only for the ECA (P < 0.000). For the LD dTT, Hemoclot dTT and PiCT-IIa assays, this was not significant. In the case of the Hemoclot dTT and PiCT-IIa assays, this could be explained by the higher imprecision of these assays. We therefore believe that the modified LD dTT assay is the most suitable coagulation assay for quantification of dabigatran. Validation of the coagulation assays for quantification of rivaroxaban showed better precision of the anti-FXa assays than of the PiCT-Xa assay. The PiCT-Xa assay showed reproducibility problems, which were possibly caused by the one-step procedure that is needed for quantification for FXa antagonists, as opposed to the two-step procedure for thrombin inhibitors. All three studied antiFXa assays for rivaroxaban showed good agreement with the UPLC-MS/MS method. This was striking, as different chromogenic anti-FXa specific substrate assays were previously shown to behave differently on plasma samples spiked with rivaroxaban [20]. The PiCT-Xa assay showed poor agreement with the reference method when patient samples were measured. The method was shown to be both inaccurate and imprecise, and this was also demonstrated during validation with spiked plasma samples. We therefore conclude that the anti-FXa assays tested are suitable for quantification of rivaroxaban, whereas the PiCT-Xa assay is not. The Liquid anti-Xa assay was adequate for quantification of apixaban regarding accuracy, but the imprecision was too high. The protocol of the PiCT-Xa assay for measurement of rivaroxaban concentrations was not suitable for apixaban measurements, owing to the higher potency of apixaban [31]. Modifications of the PiCT-Xa method were applied, but none of them resulted in a reproducible assay (data not shown). It has been shown in the APPRAISE-1a clinical trial that adequate quantification of apixaban is possible with a Stago Rotachrom assay [32]. However, this assay is no longer available. Therefore, adequate determination of apixaban with coagulation methods remains an area to be explored further. For both dabigatran and rivaroxaban, two different dosing regimes are applied, depending on the indication. Van Ryn et al. [4] observed peak and trough dabigatran concentrations of 183 ng mL 1 (range 62–447 ng mL 1) and 37 ng mL 1 (range 10–96 ng mL 1), respectively, in patients receiving 220 mg once daily. Rivaroxaban peak and trough concentrations, as observed by Buller et al. [33], were 244 ng mL 1 (range 175–360 ng mL 1) and 32 ng mL 1 (range 19–60 ng mL 1), respectively, in patients taking 20 mg once daily. The drug concentrations used for calibration of the UPLC-MS/MS method (23–750 ng mL 1) therefore cover the expected drug
range in patient samples. Also, the LLOD was ≤ 0.025 ng mL 1, enabling quantification in the high and low concentration ranges. Although the clinical value of reporting low concentrations of DOACs is unproven, it can be of value for assay comparison, as specifications of coagulation assays usually also extend to the low concentration range. One of the strengths of this study is that, in addition to spiked plasma, blood samples obtained from patients treated with dabigatran or rivaroxaban were tested. This is especially important for dabigatran, as some of its metabolites are expected to be pharmacologically active [30]. Moreover, this is the first study to describe an extensive validation of a UPLC-MS/MS method in which dabigatran, rivaroxaban and apixaban are quantified in a single run, combining high analytical quality with high sample throughput possibilities. One of the problems concerning DOACs is the lack of evidence that established drug concentrations provide information on the risk of bleeding or thrombotic events. However, as many hospital laboratories are currently adopting different coagulation methods for DOAC quantification in clinical practice in emergency cases, we believe that achieving assay quality and harmonization of analytical outcome is an issue of importance. We used the commonly applied FDA criteria for drug analysis [26] as acceptance criteria for our validation. These criteria were developed for drug analysis with chromatographic techniques and not for coagulation assays, which are useful for clinical decision-making but are analytically inferior regarding precision and specificity. This was also shown during this study: many coagulation assays did not fulfil these criteria, whereas the UPLC-MS/MS method did. We chose to use these criteria because there is, as yet, no consensus regarding analytical acceptance criteria for application of coagulation assays. In conclusion, our results support the consensus that a dTT assay should be used for quantification of dabigatran and an anti-FXa assay for quantification of rivaroxaban. Laboratories should be aware that the various coagulation assays differ significantly from one another. Although it is currently unknown whether these differences are clinically relevant, comparison with a reference method and proficiency testing should be pursued. UPLC-MS/MS is a very suitable technique for assisting in this. An important clinical concern is that the relationship between DOAC plasma concentrations and bleeding risk is unclear. Moreover, as antidotes are still under development, patients using DOACs who are at risk of bleeding or present with a major bleeding event cannot be effectively treated. Now that appropriate laboratory tests are becoming available, clinical studies should be focused on those issues. Addendum E. M. H. Schmitz, K. Boonen, D. J. A. van den Heuvel, J. L. J. van Dongen, M. W. M. Schellings, J. M. A. Emmen, © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1645
F. van der Graaf, L. Brunsveld, and D. van de Kerkhof contributed to the design of the study, to the collection and analysis of the data, revision of the intellectual content of the manuscript, and approval of the final version to be published. E. M. H. Schmitz, K. Boonen, and D. van de Kerkhof were responsible for the concept of the study and critically writing the intellectual content of the manuscript. Acknowledgements The work in this paper was financially supported by a grant from the Noyon Foundation, granted by the Netherlands Society for Clinical Chemistry and Laboratory Medicine (NVKC). Disclosure of Conflict of Interests The authors state that they have no conflict of interest. Supporting Information Additional Supporting Information may be found in the online version of this article: Data S1. Stability of DOACs during storage. Table S1. Stability data of dabigatran. Table S2. Stability data of rivaroxaban. Table S3. Stability data of apixaban. References 1 Stangier J, Rathgen K, St€ahle H, Gansser D, Roth W. The pharmacokinetics, pharmacodynamics and tolerability of dabigatran etexilate, a new oral direct thrombin inhibitor, in healthy male subjects. Br J Clin Pharmacol 2007; 64: 292–303. 2 Stangier J. Clinical pharmacokinetics and pharmacodynamics of the oral direct thrombin inhibitor dabigatran etexilate. Clin Pharmacokinet 2008; 47: 285–95. 3 Bassand J. Review of atrial fibrillation outcome trials of oral anticoagulant and antiplatelet agents. Europace 2012; 14: 312–24. 4 van Ryn J, Stangier J, Haertter S, Liesenfeld K-H, Wienen W, Feuring M. Dabigatran etexilate – a novel, reversible, oral direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity. Thromb Haemost 2010; 103: 1116–27. 5 Ageno W, Gallus AS, Wittkowsky A, Crowther M, Hylek EM, Palareti G. Oral anticoagulant therapy: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines. Chest 2012; 141: e44S–88S. 6 Huisman MV, Lip GY, Diener HC, Brueckmann M, van Ryn J, Clemens A. Dabigatran etexilate for stroke prevention in patients with atrial fibrillation: resolving uncertainties in routine practice. Thromb Haemost 2012; 107: 838–47. 7 Camm AJ, Lip GY, De Caterina R, Savelieva I, Atar D, Hohnloser SH, Hindricks G, Kirchhof P; ESC Committee for Practice Guidelines (CPG). 2012 focused update of the ESC guidelines for the management of atrial fibrillation: an update of the 2010 ESC guidelines for the management of atrial fibrillation. Eur Heart J 2012; 33: 2719–47. © 2014 International Society on Thrombosis and Haemostasis
8 Samama MM, Guinet C. Laboratory assessment of new anticoagulants. Clin Chem Lab Med 2011; 49: 761–72. 9 Hawes EM, Deal AM, Funk-Adcock D, Gosselin R, Jeanneret C, Cook AM, Taylor JM, Whinna HC, Winkler AM, Moll S. Performance of coagulation tests in patients on therapeutic doses of dabigatran: a cross-sectional pharmacodynamic study based on peak and trough plasma levels. J Thromb Haemost 2013; 8: 1493–502. 10 Hapgood G, Butler J, Malan E, Chunilal S, Tran H. The effect of dabigatran on the activated partial thromboplastin time and thrombin time as determined by the Hemoclot thrombin inhibitor assay in patient plasma samples. Thromb Haemost 2013; 2: 308–15. 11 Barrett YC, Wang Z, Knabb RM. A novel prothrombin time assay for assessing the anticoagulant activity of oral factor Xa inhibitors. Clin Appl Thromb Hemost 2012; 5: 552–8. 12 Douxfils J, Chatelain C, Chatelain B, Dogne JM, Mullier F. Impact of dabigatran on routine and specific coagulation assays: a practical laboratory guide. Thromb Haemost 2013; 2: 283–94. 13 Curvers J, van de Kerkhof D, Stroobants AK, van den Dool E-J, Scharnhorst V. Measuring direct thrombin inhibitors with routine and dedicated coagulation assays. Which assay is helpful? Am J Clin Pathol 2012; 138: 551–8. 14 Stangier J, Feuring M. Using the HEMOCLOT direct thrombin inhibitor assay to determine plasma concentrations of dabigatran. Blood Coagul Fibrinolysis 2012; 23: 138–43. 15 Douxfils J, Mullier F, Robert S, Chatelain C, Chatelain B, Dogne J-M. Impact of dabigatran on a large panel of routine or specific coagulation assays. Laboratory recommendations for monitoring of dabigatran etexilate. Thromb Haemost 2012; 107: 985–97. 16 Lange U, Nowak G, Bucha E. The Ecarin Chromogenic Assay – a new method for quantification of direct thrombin inhibitors like hirudin. Pathophysiol Haemost Thromb 2003; 33: 184– 91. 17 Calatzis A, Peetz D, Haas S, Spannagl M, Rudin K, Wilmer M. Prothrombinase-induced clotting time assay for determination of the anticoagulant effects of unfractionated and low-molecularweight heparins, fondaparinux, and thrombin inhibitors. Am J Clin Pathol 2008; 130: 446–54. 18 Asmis LM, Alberio L, Angelillo-Scherrer A, Korte W, Mendez A, Reber G, Seifert B, Stricker H, Tsakiris DA, Wuillemin WA. Rivaroxaban: quantification by anti-FXa assay and influence on coagulation tests: a study in 9 Swiss laboratories. Thromb Res 2012; 129: 492–8. 19 Mani H, Kasper A, Lindhoff-Last E. Measuring the anticoagulant effects of target specific oral anticoagulants – reasons, methods and current limitations. J Thromb Thrombolysis 2013; 2: 187–94. 20 Harenberg J, Kr€amer R, Giese C, Marx S, Weiss C, Wehling M. Determination of rivaroxaban by different factor Xa specific chromogenic substrate assays: reduction of interassay variability. J Thromb Thrombolysis 2011; 32: 267–71. 21 Douxfils J, Dogne JM, Mullier F, Chatelain B, R€ onquist-Nii Y, Malmstr€ om RE, Hjemdahl P. Comparison of calibrated dilute thrombin time and aPTT tests with LC-MS/MS for the therapeutic monitoring of patients treated with dabigatran etexilate. Thromb Haemost 2013; 3: 543–9. 22 Antovic JP, Skeppholm M, Eintrei J, Boija EE, S€ oderblom L, Norberg EM, Onel€ ov L, R€ onquist-Nii Y, Pohanka A, Beck O, Hjemdahl P, Malmstr€ om RE. Evaluation of coagulation assays versus LC-MS/MS for determination of dabigatran concentration in plasma. Eur J Clin Pharmacol 2013; 11: 1875–81. 23 Rohde G. Determination of rivaroxaban – a novel, oral, direct factor Xa inhibitor – in human plasma by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2008; 872: 43–50.
1646 E. M. H. Schmitz et al 24 Delavenne X, Moracchini J, Laporte S, Mismetti P, Basset T. UPLC MS/MS assay for routine quantification of dabigatran – a direct thrombin inhibitor – in human plasma. J Pharm Biomed Anal 2012; 58: 152–6. 25 EP10-A3: Preliminary Evaluation of Quantitative Clinical Laboratory Measurement Procedures; Approved Guideline – Third edition. Wayne, PA: Clinical and Laboratory Standards Institute, 2006. 26 Guidance for industry. Bioanalytical method validation. Rockville, MD: US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Vetenary Medicine (CVM), 2001. 27 Schmitz EMH, van den Heuvel D, Boonen K, van Dongen JLJ, Brunsveld L, van de Kerkhof D. Determination of dabigatran, rivaroxaban and apixaban using UPLC-MS/MS and comparison with coagulation assays for therapy monitoring. Ned Tijdschr Klin Chem Labgeneesk 2013; 38: 142–4. 28 Liesenfeld K-H, Sch€afer HG, Troc oniz IF, Tillmann C, Eriksson BI, Stangier J. Effects of the direct thrombin inhibitor dabigatran on ex vivo coagulation time in orthopaedic surgery patients: a population model analysis. Br J Clin Pharmacol 2006; 62: 527–37. 29 Freyburger G, Macouillard G, Labrouche S, Sztark F. Coagulation parameters in patients receiving dabigatran etexilate or
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© 2014 International Society on Thrombosis and Haemostasis
DOI: 10.1111/jth.12702
ORIGINAL ARTICLE
Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants E. M. H. SCHMITZ,*†‡ K. BOONEN,* D. J. A. VAN DEN HEUVEL,* J. L. J. VAN DONGEN,†‡ M . W . M . S C H E L L I N G S , § J . M . A . E M M E N , § F . V A N D E R G R A A F , ‡ § L . B R U N S V E L D † ‡ and D. VAN DE KERKHOF*‡ *Clinical Laboratory, Catharina Hospital, Eindhoven; †Laboratory of Chemical Biology and Institute of Complex Molecular Systems, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven; ‡Expert Center Clinical Chemistry Eindhoven, Eindhoven; and §Clinical Laboratory, Maxima Medical Center Veldhoven, Veldhoven, the Netherlands
To cite this article: Schmitz EMH, Boonen K, van den Heuvel DJA, van Dongen JLJ, Schellings MWM, Emmen JMA, van der Graaf F, Brunsveld L, van de Kerkhof D. Determination of dabigatran, rivaroxaban and apixaban by ultra-performance liquid chromatrography – tandem mass spectrometry (UPLC-MS/MS) and coagulation assays for therapy monitoring of novel direct oral anticoagulants. J Thromb Haemost 2014; 12: 1636–46.
Abstract. Background: Three novel direct oral anticoagulants (DOACs) have recently been registered by the Food and Drug Administration and European Medicines Agency Commission: dabigatran, rivaroxaban, and apixaban. To quantify DOACs in plasma, various dedicated coagulation assays have been developed. Objective: To develop and validate a reference ultra-performance liquid chromatography – tandem mass spectrometry (UPLCMS/MS) method and to evaluate the analytical performance of several coagulation assays for quantification of dabigatran, rivaroxaban, and apixaban. Methods: The developed UPLC-MS/MS method was validated by determination of precision, accuracy, specificity, matrix effects, lower limits of detection, carry-over, recovery, stability, and robustness. The following coagulation assays were evaluated for accuracy and precision: laboratory-developed (LD) diluted thrombin time (dTT), Hemoclot dTT, Pefakit PiCT, ECA, Liquid anti-Xa, Biophen Heparin (LRT), and Biophen DiXal anti-Xa. Agreement between the various coagulation assays and UPLC-MS/MS was determined with random samples from patients using dabigatran or rivaroxaban. Results: The UPLC-MS/MS
Correspondence: Daan van de Kerkhof, Catharina Hospital Eindhoven, Clinical Laboratory, Michelangelolaan 2, 5602 EJ Eindhoven, the Netherlands. Tel.: +31 40 2398641; fax: +31 40 2398637. E-mail: [email protected] Received 26 November 2013 Manuscript handled by: S. Kitchen Final decision: P. H. Reitsma, 10 August 2014
method was shown to be accurate, precise, sensitive, stable, and robust. The dabigatran coagulation assay showing the best precision, accuracy and agreement with the UPLC-MS/MS method was the LD dTT test. For rivaroxaban, the anti-factor Xa assays were superior to the PiCT-Xa assay with regard to precision, accuracy, and agreement with the reference method. For apixaban, the Liquid anti-Xa assay was superior to the PiCT-Xa assay. Conclusions: Statistically significant differences were observed between the various coagulation assays as compared with the UPLC-MS/MS reference method. It is currently unknown whether these differences are clinically relevant. When DOACs are quantified with coagulation assays, comparison with a reference method as part of proficiency testing is therefore pivotal. Keywords: apixaban; dabigatran; drug monitoring; mass spectrometry; rivaroxaban.
Introduction Since 2010, three novel direct oral anticoagulants (DOACs) have been registered by the Food and Drug Administration (FDA) and European Medicines Agency Commission (EMA) for the prevention of deep vein thrombosis in patients undergoing knee or hip replacement surgery, and for the prevention of stroke in patients with non-valvular atrial fibrillation: dabigatran, rivaroxaban, and apixaban. DOACs, previously known as novel oral anticoagulants (NOACs), are direct inhibitors of coagulation factors. Dabigatran inhibits thrombin (FIIa), and rivaroxaban and apixaban inhibit FXa. DOACs have © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1637
more predictable pharmacokinetics and pharmacodynamics than the traditionally used coumarin-derived anticoagulants [1,2], and fewer food and drug–drug interactions have been reported [3]. It was therefore advocated that dosing based on laboratory testing would be obsolete [4]. These advantages have led to an increasing number of patients receiving these drugs. However, the current consensus is that laboratory monitoring of DOACs is important in specific situations, e.g. in patients with a very high or low body mass index or in those with diminished renal function, or in emergency (bleeding) situations [5–7]. Unfortunately, conventional laboratory assays such as prothrombin time (PT) and activated partial thromboplastin time (APTT) assays are not suitable for accurate quantification of DOACs [8]. Insufficient sensitivities and non-linear dose responses of the PT and APTT assays can lead to only slightly elevated coagulation times with high DOAC concentrations [9–12]. To fulfil this unmet need for accurate quantification of DOACs, several companies have developed new coagulation assays or new protocols and calibrators for existing coagulation assays. For quantification of direct thrombin inhibitors (DTIs), the diluted thrombin time (dTT) assay is advocated as the preferable test [13–15]. This is a quantitative clotting antiFIIa assay in which a defined amount of thrombin is added to diluted plasma. The Hemoclot DTI clotting assay is the most studied dTT assay for analysis of direct thrombin inhibitors [14]. Alternatively, snake venombased assays can be used, such as the Ecarin Clotting Time (ECT) assay, the Ecarin Chromogenic Assay (ECA), or the Prothrombinase-induced Clotting Time (PiCT) assay. In the ECA, prothrombin is converted by ecarin into chromogenically detected meizothrombin [13,16]. The PiCT assay is based on selective FV activation by Russell’s viper venom, and is sensitive for both DTIs and FXa inhibitors [17]. The current preferred assay for quantification of FXa inhibitors is the anti-FXa assay, for which numerous commercial kits are available [18–20]. Liquid chromatography (LC)–mass spectrometry (MS) is widely applied for quantification of drugs. This technique has much better selectivity than coagulation activity-based assays, enabling specific detection and quantification of coagulation inhibitors [21–24]. As coagulation assays measure activities of various coagulation factors, they are prone to interference by other coagulant drugs or endogenous or preanalytical changes in coagulation factors. This may result in inaccurate quantification of the anticoagulant’s concentration in plasma. Therefore, LC-MS detection is expected to be superior to coagulation assays for accurate quantification of DOACs. However, many clinical laboratories do not have (24-h) access to LC-MS analysis, which makes this technique unsuitable for routine measurement of DOACs. Coagulation assays are better employed for routine measurement, but at present it is unclear which coagulation assays are most appropriate for quantification of DOACs. Therefore, we © 2014 International Society on Thrombosis and Haemostasis
developed and validated a reference ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS) method for quantification of dabigatran, rivaroxaban, and apixaban. The UPLC-MS/MS and coagulation assays were validated by the use of drugenriched plasma. For dabigatran and rivaroxaban, the UPLC-MS/MS method was also compared with coagulation methods by the use of patient samples. Materials and methods UPLC-MS/MS sample preparation
Citrated plasma pools were spiked with dabigatran, rivaroxaban and apixaban (Alsachim, Illkirch Graffenstaden, France) for a six-point calibration (calibration range 23–750 ng mL 1) and quality control (QC) (50, 275 and 500 ng mL 1). For evaluation of adsorption to erythrocytes, full citrated blood samples were spiked with the DOACs and centrifuged at 2683 9 g for 5 min to obtain spiked plasma. Isotopically labeled internal standards of the DOACs were added for quantification (100 ng mL 1) ([13C6]dabigatran, [13C6]rivaroxaban, and [13C,2H7]apixaban; Alsachim). Sample clean-up was performed by protein precipitation with acetonitrile (dilution of the sample at a ratio of 1 : 3). The supernatant was diluted with mobile phase A (dilution of 1 : 4). UPLC-MS/MS method
UPLC-MS/MS analysis of the samples was performed with an Acquity UPLC binary solvent manager, sample manager, and column manager, and an Acquity TQ Detector (Waters, Milford, MA, USA). Chromatographic separation was achieved by injection of 10 lL of sample on an Acquity UPLC BEH C8 column (100 9 2.1 mm, 1.7 lm; Waters) at 30 °C. The flow rate was 300 lL min 1, with the mobile phases 2.5 mM ammonium formate at pH 3.0 (A) and acetonitrile (B). A 4.75-min gradient run was performed: 0–1.0 min, 15% B; 1.0–3.0 min, linear to 75% B; 3.0–3.5 min, linear to 95% B; 3.5–4.0 min, 95% B; and 4.0–4.75 min, return to initial conditions. Mass analysis was performed with a two-step multiple reaction monitoring (MRM) method (Table 1). Peak integration was performed with MASSLYNX software V4.1 SCN 714 (Waters, Milford, MA, USA) with ApexTrack Peak Integration. The peak area ratio between the drug and its internal standard (response) was used to quantify the amount of drug in the plasma and to correct for recovery loss. UPLC-MS/MS validation
To determine within-run and between-run imprecision and accuracy (bias), three concentration levels of QC samples were each analyzed five times on five different days. For recovery determination, spiked plasma samples (A) and
1638 E. M. H. Schmitz et al Table 1 Mass spectrometry settings for single-run quantification of dabigatran, rivaroxaban, and apixaban
Compound Period 1 (0–2 min) Dabigatran [13C6]Dabigatran Period 2 (2–4.75 min) Rivaroxaban [13C6]Rivaroxaban Apixaban [13C–2H7]Apixaban
Q1 mass
Q3 mass
Dwell (s)
Cone (V)
CE (eV)
472.2 478.2
289.1 295.2
0.078 0.078
42 44
30 30
436.1 442.2 460.2 468.2
144.9 144.9 77.0 107.1
0.036 0.036 0.036 0.036
54 58 66 56
28 28 64 52
CE, collision energy.
blank plasma samples (B) were prepared as described above. Samples B were spiked with dabigatran, rivaroxaban and apixaban after sample preparation. To calculate recovery, the peak area in sample A was divided by the peak area found in sample B for each DOAC. Carry-over was determined by performing an EP10 protocol [25], in which low-, medium- and high-concentration samples were analyzed in a predetermined alternate order. Matrix effects were studied by injecting six prepared blank plasma samples onto the chromatographic column, during continuous infusion of dabigatran, rivaroxaban or apixaban in the mass spectrometer. Specificity was considered to be adequate if no interfering peaks were present in drug-naive samples. A signal-to-noise ratio (SNR) was determined in diluted samples. The concentration corresponding to an SNR of 3 was established as the lower limit of detection (LLOD). Stability was studied by repeated analysis of spiked QC samples after three freeze–thaw cycles ( 20 °C to room temperature and 20 °C to 37 °C), and after 24 h of storage at room temperature and at 4 °C. Long-term stability during storage at 80 °C was investigated after 72 days. The stability of prepared samples (after protein precipitation and in the final solution for analysis) was determined after storage for 24 h at room temperature and at 4 °C. The range of acceptance for imprecision and accuracy (bias) was defined as < 15% and 85–115%, respectively, based on current FDA criteria for drug analysis [26]. Stability was accepted in the range of 80–120%. The method robustness was tested by a 10% deliberate adjustment of flow rate, buffer concentration, gradient settings, and column temperature. Furthermore, the pH of mobile phase A was adjusted by 0.1, and ammonium formate was replaced by ammonium acetate. The responses of dabigatran, rivaroxaban and apixaban were calculated for all conditions to determine whether these adjustments influenced quantification of the DOACs. The significance of response deviations was tested with one-way ANOVA (P < 0.05). Coagulation assay methods
All analyses were performed on the STA-R Evolution Analyzer (Stago, Asnieres sur Seine, France). The assays
evaluated for dabigatran were the Hemoclot dTT assay (Hyphen Biomed, Neuville-sur-Oise, France), LD dTT assay (laboratory-developed dTT, for which Stago’s thrombin reagent was used and eight-fold sample dilution with normal plasma was applied), the HaemoSys ECA (JenAffin GmbH, Jena, Germany), and the Pefakit PiCT assay (Pentapharm, Basel, Switzerland). For rivaroxaban and apixaban, the Liquid anti-Xa (Stago), Biophen Heparin anti-Xa (LRT; Hyphen Biomed), Biophen DiXal antiXa (Hyphen Biomed) and Pefakit PiCT assays were evaluated. Reagents were stored at 4 °C between measurements, as described in the package inserts. Plasma pools were spiked with dabigatran, rivaroxaban or apixaban for calibration (750 ng mL 1) and QC (92 and 375 ng mL 1 for dabigatran; 82 and 308 ng mL 1 for rivaroxaban; 87 and 386 ng mL 1 for apixaban). The in-house calibrators were used for all tests, except for the Hemoclot dTT assay, and diluted on the STA-R Evolution Analyzer with normal plasma to obtain a five-point calibration. The calibration line for the LD dTT was linear (calibration range 38–500 ng mL 1). The calibration curves for the ECA and PiCT assays were secondorder polynomial, as specified by the manufacturers (calibration range: ECA, 38–500 ng mL 1; PiCT-IIa, 25–250 ng mL 1; PiCT-Xa, 19–250 ng mL 1). Calibration curves for the Stago Liquid anti-Xa and Biophen Heparin anti-Xa assays were log-linear, as specified by the manufacturer (calibration range: Liquid anti-Xa, 30– 500 ng mL 1; Biophen Heparin anti-Xa, 0–490 ng mL 1). The calibration line for the Biophen DiXal anti-Xa assay was linear (calibration range: 0–490 ng mL 1). For the Hemoclot dTT assay, the manufacturer’s calibrators were used to obtain a linear, three-point calibration line (calibration range: 40–500 ng mL 1). Coagulation assay validation
Within-run and between-run imprecision and accuracy of the coagulation assays was determined by performing duplicate QC measurements in 10 separate runs, each being preceded by calibration. Accuracy was calculated as the determined concentration divided by the concentration determined with UPLC-MS/MS. Imprecision was accepted when this was < 15%, and accuracy was accepted when it was between 85% and 115% [26]. The LLOD was calculated as the mean plus two standard deviations of normal plasma, which was measured five times. The validity of the studied coagulation assays for quantification of dabigatran and rivaroxaban was evaluated by agreement of the coagulation assay findings with the UPLC-MS/MS findings obtained with patient samples. The differences in DOAC concentrations were visualized in a modified Bland–Altman plot, depicting the reference method’s results on the horizontal axis. A paired-samples t-test was performed to determine whether there was a © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1639
fixed bias between the coagulation assay and the UPLCMS/MS method (confirmed when P < 0.05). Plasma was collected from patients taking dabigatran or rivaroxaban as part of routine care (37 samples for dabigatran and 27 for rivaroxaban). The obtained plasma was anonymized for the study. The study was conducted in accordance with the local medical ethical protocol as prescribed by Dutch law. The collection of blood samples was not timed, and the patient population used was undefined regarding clinical background (including both cardiologic and orthopedic patients).
73%, 78%, and 104%, respectively. Recovery was thus not complete for dabigatran and rivaroxaban, but quantification was not impaired, owing to the addition of the labeled internal standards. The within-run and betweenrun imprecision of the UPLC-MS/MS method was < 15%, and the accuracy met the acceptance criteria, except for rivaroxaban at the low concentration (Table 2). Robustness testing showed a change of retention time with changing flow rate, as expected. However, the responses of all three DOACs were not significantly affected by any of the deliberate changes to the UPLCMS/MS method (P ≥ 0.112).
Results Coagulation assay validation UPLC-MS/MS validation
For imprecision and accuracy determined with the spiked QC samples, see Table 2. The LD dTT assay showed acceptable within-run and between-run imprecision (≤ 9.9%) and accuracy (107–112%) for both QC levels (92 and 375 ng mL 1), in contrast to the Hemoclot dTT assay, which showed acceptable within-run imprecision (≤ 10.1%) but inacceptable between-run imprecision (≥ 33.0%) and accuracy (125–131%). The ECA and the PiCT-IIa assay showed acceptable within-run imprecision (ECA, ≤ 4.4%; PiCT-IIa, ≤ 4.3%). The between-run imprecision and accuracy were only acceptable for the high QC level (ECA, 5.8% and 108%, respectively; PiCTIIa, 10.8% and 99.3%, respectively). All evaluated assays for quantification of rivaroxaban showed good imprecision (within-run ≤ 6.4%; between-run ≤ 12.6%) at both QC levels (82 and 308 ng mL 1). Accuracy at the high QC level was acceptable for all assays, except for the Liquid anti-Xa assay (119%). At the low QC level, this was the case for the PiCT-Xa assay (79.6%) and the Biophen DiXal anti-Xa assay (132%). For apixaban, a reproducible PiCT-Xa assay could not be developed, and the Liquid anti-Xa assay showed unacceptable between-run imprecision (18.3% for the low QC level [87 ng mL 1] and 15.4% for the high QC level [386 ng mL 1]).
A UPLC-MS/MS method for single-run quantification of dabigatran, rivaroxaban, and apixaban, with isotopically labeled standards, was successfully developed (Fig. 1). R2 of the fitted linear calibration lines was ≥ 0.99 for all three DOACs. No significant difference in response could be seen between samples prepared from plasma and those prepared from whole blood, indicating that the studied DOACs are not adsorbed to erythrocytes. Spiked citrated plasma can thus be used for QC and calibration samples. Stability tests showed adequate stability during three freeze–thaw cycles, 24 h of plasma storage at room temperature and 4 °C, and 72 days of plasma storage at a temperature lower than 80 °C. Prepared samples (after protein precipitation and preparation of the final solution for UPLC-MS/MS analysis) were stable for at least 24 h at room temperature and at 4 °C. Detailed results of stability analysis are provided in the tables of the Supporting Information. No interfering peaks were present in drug-naive samples, and the LLOD of the UPLC-MS/MS method was ≤ 0.025 ng mL 1 for all three DOACs. Matrix effects were absent (Fig. 2) and the maximum determined carryover was 1.0%. For dabigatran, rivaroxaban, and apixaban, the rates of recovery after sample preparation were 1.52
100
Dabigatran
Apixaban
%
100
%
0
0.50 1.00
1.50
100
3.50 4.00
2.00 2.50 3.00
Apixaban
4.50
0
3.16 100
Apixaban IS
%
3.24
0
Rivaroxaban
%
0
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
2.10 2.20 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10
Fig. 1. Mass chromatogram of the multiple reaction monitoring (MRM) method for the single-run detection of dabigatran, rivaroxaban, apixaban and their isotopically labeled standards ([13C6]dabigatran, [13C6]rivaroxaban and [13C–2H7]apixaban). Elution times of the direct oral anticoagulants and its labeled standard are similar, but they can be separately analyzed and quantified, owing to their mass difference. IS, internal standard; UPLC-MS/MS, ultra-performance liquid chromatography–mass spectrometry/mass spectrometry. © 2014 International Society on Thrombosis and Haemostasis
Hemoclot dTT LD dTT ECA PiCT-IIa
89.3
Liquid anti-Xa
103
Accuracy (%)
79.6 106.7 105 132
Accuracy (%)
125 107 83.9 169
Accuracy (%)
106 116 94.6
Accuracy (%)
3.3
Imprecision Within-run (%)
6.4 4.0 1.5 4.3
Imprecision Within-run (%)
8.3 3.7 4.4 4.3
Imprecision Within-run (%)
5.9 6.4 3.5
Imprecision Within-run (%)
18.3
Imprecision Between-run (%)
12.6 3.5 5.1 7.7
314
Mean (target: 386 ng mL 1)
314 368 272 324
3.9 2.9 3.8
93.2
Accuracy (%)
102 119 88.3 105
Accuracy (%)
131 112 108 99.3
1.1
Imprecision Within-run (%)
6.2 2.4 1.6 1.8
Imprecision Within-run (%)
10.1 1.5 1.9 3.5
Imprecision Within-run (%)
Imprecision Within-run (%)
Accuracy (%)
103 114 92.0
Accuracy (%)
Mean (target: 308 ng mL 1)
491 419 406 372
Mean (target: 375 ng mL 1)
284 314 253
Mean (target: 250 ng mL 1)
Imprecision Between-run (%)
33.0 9.9 16.4 16.9
Imprecision Between-run (%)
2.8 4.2 9.5
Imprecision Between-run (%)
QC level 2
The CV was calculated from duplicate measurements obtained over a period of 10 days.
Mean (target: 87 ng mL 1)
Coagulation assays apixaban
65.1 87.2 85.6 108
116 99.2 77.4 156
Coagulation assays dabigatran
PiCT-Xa Liquid anti-Xa Heparin anti-Xa DiXal anti-Xa
Mean (target: 92 ng mL 1)
Dabigatran Rivaroxaban Apixaban
Mean (target: 82 ng mL 1)
52.8 58.3 47.3
UPLC-MS/MS
Coagulation assays rivaroxaban
Mean (target: 50 ng mL 1)
QC level 1
15.4
Imprecision Between-run (%)
8.2 3.3 2.8 2.5
Imprecision Between-run (%)
34.3 7.9 5.8 10.8
Imprecision Between-run (%)
0.0 7.8 9.1
Imprecision Between-run (%) 505 569 457
Mean (target: 500 ng mL 1)
QC level 3
101 114 91.4
Accuracy (%)
4.6 4.6 3.8
Imprecision Within-run (%)
0.0 7.1 7.5
Imprecision Between-run (%)
Table 2 Mean concentration of quality control (QC) samples (QC level no.) with their accuracy and within-run and between-run imprecision for the ultra-performance liquid chromatography/ tandem mass spectrometry (UPLC-MS/MS) method and coagulation assays
1640 E. M. H. Schmitz et al
© 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1641 100 A
4.624.65 4.55
%
4.21 0.81 0.87
Dabigatran 4.15
3.93 3.57 2.78
0
3.36
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
100 B
Time
4.55
4.22
Rivaroxaban
0.87
4.43
0.79
%
3.73
4.12
0.74 1.02
0.190.350.56 0.62
0
1.361.491.74 1.85
2.07
2.49 2.692.77 2.98 3.323.35 3.56 2.26 2.87
3.99
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Time
4.55
100 C
4.43 4.22
Apixaban
0.87
4.32 0.83 0.78
%
4.13
0.73 0.35 0.530.63 0.13
1.00
1.33 1.47
4.06 3.96 3.55 3.58 2.66 2.73 2.78 3.213.36 3.68
0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50
Time
Fig. 2. Illustration of determination of the matrix effects. The direct oral anticoagulant (DOAC) is continuously infused in the mass spectrometer: (A) dabigatran; (B) rivaroxaban; (C) apixaban. After sample preparation, blank samples (n = 6) are injected (t = 0) and separated over the ultra-performance liquid chromatography column. The elution of matrix components from plasma causes deviation of the mass spectrometry/mass spectrometry trace, as a result of ionization competition by dabigatran and the matrix. At the retention time of the DOACs (arrows), no deviating response was observed.
The LLODs of the coagulation assays are shown in Table 3. The PiCT assay was omitted, because of its high imprecision. The Hemoclot dTT assay showed a higher LLOD (43.5 ng mL 1) than the LD dTT assay and the ECA (≤ 17.4 ng mL 1). The Liquid anti-Xa assay showed a higher LLOD (24.0 ng mL 1) than the Biophen © 2014 International Society on Thrombosis and Haemostasis
Heparin and Biophen DiXal anti-Xa assays (≤ 2.8 ng mL 1). Agreement between the UPLC-MS/MS method and coagulation assays was determined by the use of modified Bland–Altman plots (see Fig. 3). Drug concentrations lower than the calculated LLODs were removed for all assays except the PiCT. A paired-samples t-test showed that that there was only a statistically significant bias in dabigatran concentration between the ECA and the UPLC-MS/ MS method (29.4 ng mL 1; P < 0.000). This could not be shown for the LD dTT, Hemoclot dTT and PiCT-IIa assays (P = 0.067, P = 0.450, and P = 0.143, respectively). In the case of rivaroxaban, the Bland–Altman plots showed that a statistically significant bias in drug concentration could be found only between the PiCT-Xa and UPLC-MS/MS assays (36.6 ng mL 1, P = 0.022; Fig. 4). This bias was not statistically significant for the Liquid anti-Xa, Biophen DiXal anti-Xa and Biophen Heparin anti-Xa assays (≤ 3 ng mL 1, P ≥ 0.061). The limits of agreement for the PiCT-Xa assay ( 88.0 to 161) were much wider than for the other coagulation assays (≥ 24 to ≤ 26), indicating much larger imprecision for the PiCT-Xa assay. Discussion There is general consensus that, although routine monitoring of DOACs is not recommended, laboratory analysis is required in specific situations. An increasing number of chromogenic or clotting-based assays have become available to quantify thrombin and/or FXa inhibitors. However, standardization is lacking, and the potential of non-dTT-based and non-anti-FXa-based assays is unclear. Therefore, we developed and validated a UPLC-MS/MS method for quantification of dabigatran, rivaroxaban, and apixaban. The method was shown to be precise, accurate, sensitive, specific, and robust [27]. The UPLCMS/MS method was therefore used as the reference method for comparison with the various coagulation assays. During sample preparation, dabigatran and rivaroxaban are not completely recovered from the plasma, but this was compensated for by the labeled internal standard. Quantification was therefore not impaired. We compared this UPLC-MS/MS method with several essentially different coagulation assays, which were developed for quantification of DOACs. Studies have already shown that routine coagulation assays, such as PT, thrombin time and APTT assays, are not suitable for accurate quantification of DOACs [1,2,12,28]. They were therefore omitted from our research. For dabigatran, we included the clotting-based dTT assay, the ecarin-induced meizothrombin formation-dependent ECA, and the prothrombinase complex-dependent PiCT-IIa assay. For rivaroxaban and apixaban, different chromogenic anti-FXa assays and the PiCT-Xa assay were selected. Besides the commercially available assays, we also used an in-house-
1642 E. M. H. Schmitz et al Table 3 Lower limits of detection (LLODs) of the different coagulation assays, calculated as mean plus two standard deviations (SDs) of five blank measurements
Dabigatran Hemoclot dTT LD dTT ECA Rivaroxaban Liquid anti-Xa Biophen Heparin anti-Xa Biophen DiXal anti-Xa Apixaban Liquid anti-Xa
Mean (ng mL 1)
SD (ng mL 1)
LLOD (ng mL 1)
18.8 6.9 15.3
12.3 5.2 0.0
43.5 17.4 15.3
18.3 1.7 2.7
2.9 0.0 0.0
24.0 1.7 2.8
19.7
4.7
29.1
developed method (LD dTT). This assay was based on the protocol of the Hemoclot dTT assay, for which we used Stago’s thrombin reagent and in-house normal plasma. To calibrate the coagulation assays, in-house calibrators were used for all assays except the Hemoclot dTT assay. A disadvantage of this strategy is that the manu-
B
150
Bias (–8.97) P-value: 0.450
100
95% Limits of agreement (– 105 to 87.5)
Difference (UPLC-MS/MS – LD dTT) (ng mL–1)
Difference (UPLC-MS/MS – Hemoclot dTT) (ng mL–1)
A
facturer kits may not perform to the manufacturer’s specifications unless the calibrator defined by the manufacturer is used. However, the PiCT and ECA kits did not have a dedicated calibrator at the time when this study was performed. Also, test results of the different kits are more comparable when the same calibrators are used. For quantification of dabigatran, the modified LD dTT assay was analytically superior to the Hemoclot dTT assay, ECA, and PiCT-IIa assay, because it was the only coagulation assay that showed acceptable imprecision (within-run, ≤ 3.7%; between-run, ≤ 9.9%) and accuracy (≤ 112%) at both low and high dabigatran concentrations. Freyburger et al. [29] also satisfactorily used a ‘homemade’ dTT assay for a study investigating the effect of dabigatran on orthopedic patients. We experienced reproducibility problems with the Hemoclot dTT assay, which caused unacceptable accuracy and imprecision. This was attributable to instability of the reagents, despite the fact that they were used according to the manufacturer’s recommendations. Within-run and between-run variability caused by the use of human thrombin rather than the more stable bovine thrombin might also have contributed. Inter-
50 0 – 50 – 100
150
Bias (8.51) P-value: 0.067
100 50 0 – 50
– 100
– 150
– 150 0
50
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250
0
300
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300
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D
C Bias (29.4) P-value: < 0.000
100
150
95% Limits of agreement (– 11.4 to 70.2)
Difference (UPLC-MS/MS – PiCT IIa) (ng mL–1)
150 Difference (UPLC-MS/MS – ECA) (ng mL–1)
95% Limits of agreement (– 37.6 to 54.7)
50 0 – 50 – 100
Bias (12.8) P-value: 0.143
100
95% Limits of agreement (– 75.3 to 101)
50 0 – 50 – 100 – 150
– 150 0
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150
200
UPLC-MS/MS (ng mL–1)
250
300
0
50
100
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300
UPLC-MS/MS (ng mL–1)
Fig. 3. Modified Bland–Altman plot showing the agreement of dabigatran coagulation assays with ultra-performance liquid chromatography– mass spectrometry/mass spectrometry (UPLC-MS/MS) in random patient samples. Samples with drug concentrations lower than the lower limit of detection were removed from analysis, except for the PiCT-IIa assay. (A) Hemoclot dTT (n = 18). (B) LD dTT (n = 28). (C) ECA (n = 37). (D) PiCT-IIa (n = 37). © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1643
B
200
Bias (0.53) P-value: 0.834
150
Difference (UPLC-MS/MS – Biophen DiXal anti-Xa) (ng mL–1)
Difference (UPLC-MS/MS – Liquid anti-Xa) (ng mL–1)
A
95% Limits of agreement (– 24.1 to 25.3)
100 50 0 – 50
– 100 40
80 UPLC-MS/MS (ng
120
95% Limits of agreement (– 16.0 to 26.2)
150 100 50 0 – 50
0
160
Bias (2.6) P-value: 0.170
80
120
UPLC-MS/MS (ng
D
200
40
mL–1)
95% Limits of agreement (– 16.7 to 21.9)
200
Difference (UPLC-MS/MS – PiCT-Xa) (ng mL–1)
Difference (UPLC-MS/MS – Biophen Heparin anti-Xa) (ng mL–1)
Bias (5.1) P-value: 0.061
– 100
0
C
200
150 100 50 0 – 50
– 100
Bias (36.6) P-value: 0.022
160
mL–1)
95% Limits of agreement (– 88.0 to 161)
150 100 50 0 – 50
– 100
0
40
80 UPLC-MS/MS (ng
120
160
mL–1)
0
40
80 UPLC-MS/MS (ng
120
160
mL–1)
Fig. 4. Modified Bland–Altman plot showing the agreement of rivaroxaban coagulation assays with ultra-performance liquid chromatography– mass spectrometry/mass spectrometry (UPLC-MS/MS) in random patient samples. Samples with drug concentrations lower than the lower limit of detection were removed from analysis, except for the PiCT-Xa assay. (A) Liquid anti-Xa (n = 25). (B) Biophen DiXal anti-Xa (n = 18). (C) Biophen Heparin anti-Xa (n = 22). (D) PiCT-Xa (n = 27).
estingly, these problems were not reported by other studies using this assay [12,14]. The PiCT assay also showed reproducibility problems, similar to the findings of Douxfils et al. [12], making this assay unsuitable for monitoring purposes. Ecarin-based assays have often been used in pharmacologic studies. ECT assays were shown to be sensitive and to have a good linear response to increasing dabigatran concentrations [1,2], although interlot variability was an issue [12]. The ECA was shown to have good sensitivity and linearity for higher dabigatran concentrations, but non-linearity for samples with a concentration of < 50 ng mL 1 was demonstrated [12]. This was supported by the findings of this study. Optimizing the calibration, e.g. by addition of a blank sample to the calibration line, might improve those results. The Hemoclot dTT assay has a higher LLOD than the LD dTT assay and the ECA, making the latter two more suitable for dabigatran quantification in the low concentration range. The Hemoclot dTT assay applies only three calibrators, whereas the LD dTT assay and ECA use five, © 2014 International Society on Thrombosis and Haemostasis
possibly making the fitted lines of the LD dTT assay and ECA more accurate than the Hemoclot dTT assay’s fitted line. Another explanation for the Hemoclot dTT assay’s higher LLOD could be its higher imprecision. For rivaroxaban, the Biophen Heparin and Biophen DiXal assays have lower LLODs than the Liquid anti-Xa assay, which could be explained by the incorporation of a blank sample in the calibration. We were not able to adequately determine an LLOD for the PiCT assays, owing to their high imprecision. Agreement between the dabigatran coagulation assays and UPLC-MS/MS showed that, on average, the coagulation assays gave higher dabigatran concentrations than the reference method. This bias could be explained by dabigatran’s pharmacologically active metabolites, which are not measured by the UPLC-MS/MS method. It was shown by Blech et al. [30] that these dabigatran acylglucuronides represent ~ 20% of the total drug exposure in plasma. Dabigatran metabolism can therefore cause discrepancies between this UPLC-MS/MS method and the
1644 E. M. H. Schmitz et al
coagulation assays. It is not yet known whether these discrepancies are clinically relevant, as the relationship between dabigatran concentration and bleeding risk is poorly understood. The bias between coagulation assays and the reference method was statistically significant only for the ECA (P < 0.000). For the LD dTT, Hemoclot dTT and PiCT-IIa assays, this was not significant. In the case of the Hemoclot dTT and PiCT-IIa assays, this could be explained by the higher imprecision of these assays. We therefore believe that the modified LD dTT assay is the most suitable coagulation assay for quantification of dabigatran. Validation of the coagulation assays for quantification of rivaroxaban showed better precision of the anti-FXa assays than of the PiCT-Xa assay. The PiCT-Xa assay showed reproducibility problems, which were possibly caused by the one-step procedure that is needed for quantification for FXa antagonists, as opposed to the two-step procedure for thrombin inhibitors. All three studied antiFXa assays for rivaroxaban showed good agreement with the UPLC-MS/MS method. This was striking, as different chromogenic anti-FXa specific substrate assays were previously shown to behave differently on plasma samples spiked with rivaroxaban [20]. The PiCT-Xa assay showed poor agreement with the reference method when patient samples were measured. The method was shown to be both inaccurate and imprecise, and this was also demonstrated during validation with spiked plasma samples. We therefore conclude that the anti-FXa assays tested are suitable for quantification of rivaroxaban, whereas the PiCT-Xa assay is not. The Liquid anti-Xa assay was adequate for quantification of apixaban regarding accuracy, but the imprecision was too high. The protocol of the PiCT-Xa assay for measurement of rivaroxaban concentrations was not suitable for apixaban measurements, owing to the higher potency of apixaban [31]. Modifications of the PiCT-Xa method were applied, but none of them resulted in a reproducible assay (data not shown). It has been shown in the APPRAISE-1a clinical trial that adequate quantification of apixaban is possible with a Stago Rotachrom assay [32]. However, this assay is no longer available. Therefore, adequate determination of apixaban with coagulation methods remains an area to be explored further. For both dabigatran and rivaroxaban, two different dosing regimes are applied, depending on the indication. Van Ryn et al. [4] observed peak and trough dabigatran concentrations of 183 ng mL 1 (range 62–447 ng mL 1) and 37 ng mL 1 (range 10–96 ng mL 1), respectively, in patients receiving 220 mg once daily. Rivaroxaban peak and trough concentrations, as observed by Buller et al. [33], were 244 ng mL 1 (range 175–360 ng mL 1) and 32 ng mL 1 (range 19–60 ng mL 1), respectively, in patients taking 20 mg once daily. The drug concentrations used for calibration of the UPLC-MS/MS method (23–750 ng mL 1) therefore cover the expected drug
range in patient samples. Also, the LLOD was ≤ 0.025 ng mL 1, enabling quantification in the high and low concentration ranges. Although the clinical value of reporting low concentrations of DOACs is unproven, it can be of value for assay comparison, as specifications of coagulation assays usually also extend to the low concentration range. One of the strengths of this study is that, in addition to spiked plasma, blood samples obtained from patients treated with dabigatran or rivaroxaban were tested. This is especially important for dabigatran, as some of its metabolites are expected to be pharmacologically active [30]. Moreover, this is the first study to describe an extensive validation of a UPLC-MS/MS method in which dabigatran, rivaroxaban and apixaban are quantified in a single run, combining high analytical quality with high sample throughput possibilities. One of the problems concerning DOACs is the lack of evidence that established drug concentrations provide information on the risk of bleeding or thrombotic events. However, as many hospital laboratories are currently adopting different coagulation methods for DOAC quantification in clinical practice in emergency cases, we believe that achieving assay quality and harmonization of analytical outcome is an issue of importance. We used the commonly applied FDA criteria for drug analysis [26] as acceptance criteria for our validation. These criteria were developed for drug analysis with chromatographic techniques and not for coagulation assays, which are useful for clinical decision-making but are analytically inferior regarding precision and specificity. This was also shown during this study: many coagulation assays did not fulfil these criteria, whereas the UPLC-MS/MS method did. We chose to use these criteria because there is, as yet, no consensus regarding analytical acceptance criteria for application of coagulation assays. In conclusion, our results support the consensus that a dTT assay should be used for quantification of dabigatran and an anti-FXa assay for quantification of rivaroxaban. Laboratories should be aware that the various coagulation assays differ significantly from one another. Although it is currently unknown whether these differences are clinically relevant, comparison with a reference method and proficiency testing should be pursued. UPLC-MS/MS is a very suitable technique for assisting in this. An important clinical concern is that the relationship between DOAC plasma concentrations and bleeding risk is unclear. Moreover, as antidotes are still under development, patients using DOACs who are at risk of bleeding or present with a major bleeding event cannot be effectively treated. Now that appropriate laboratory tests are becoming available, clinical studies should be focused on those issues. Addendum E. M. H. Schmitz, K. Boonen, D. J. A. van den Heuvel, J. L. J. van Dongen, M. W. M. Schellings, J. M. A. Emmen, © 2014 International Society on Thrombosis and Haemostasis
Determination of novel direct oral anticoagulants 1645
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