A rapid and sensitive analytical methodology for the simultaneous biomonitoring of two direct oral anticoagulant drugs and their major metabolites in thromboembolic disordered patients samples for clinical evaluations.

Apixaban and dabigatran are the two major direct oral anticoagulant drugs to treat thromboembolic disordered patients. Increasing the clinical application for the thromboembolic disorder and monitoring the concentrations of apixaban, dabigatran, and their metabolites are essential in most clinical circumstances. In this work, we developed a rapid analytical methodology comprising of vortex-assisted salt-enhanced liquid-liquid microextraction technique coupled with UHPLC-MS/MS for the extraction and simultaneous determination of two major direct oral anticoagulant drugs (apixaban, dabigatran), and their two major metabolites from plasma, serum, and urine samples of patients. The developed method was optimized with various procedural steps and validated to study the analytical merits. The developed method yielded a good detection limit of 0.01 ∼ 0.37 ng/mL, 0.01 ∼ 0.32 ng/ml, and 0.01 ∼ 0.27 ng/mL for four target analytes in the plasma, serum, and urine matrices. Moreover, extraction recoveries ranged from 85.11 - 113.57% (for plasma), 89.63 - 110.47% (for serum), and 87.44 -106.79% (for urine samples) with 8.78% RSD. In addition, the method exhibited good R2 values of 0.999 for all four target analytes, and the specificity and carryover study revealed no carryover effect from the UHPLC-MS/MS system for determining the apixaban, dabigatran, and their metabolites. Due to the above advantages, the developed analytical technique was applied to examine 11 real-time clinical patients' samples, and the observed results were satisfactory for all three different sample matrices. Therefore, this analytical method can be applied for biomonitoring apixaban, dabigatran, and their two major metabolites with high sensitivity in a short time for various clinical applications.

Highly sensitive and selective lateral flow aptasensor for anti-coagulant dabigatran etexilate determination in blood.

Dabigatran etexilate (DBG) is a new anticoagulant drug (commercially sold under the names Pradaxa® and Pradax™) that replaces Warfarin, the landmark agent for anticoagulation therapy. Inadequate administration of DBG or in the cases of massive bleeding that occurs after renal impairment, DBG therapy can carry a substantial life-threatening risks. One of the major limitations of DBG treatment is the lack of a simple and quick tool for measuring its level in blood in the case of massive bleedings or emergency operations. In this work, we have incorporated a previously isolated aptamer for DBG to develop a simple competitive lateral flow aptasensor (LFA) for the determination of DBG in buffer and blood samples. A full-length 60-mer aptamer as well as a truncated 38-mer aptamer were conjugated to gold nanoparticles (AuNPs) via thiol-Au coupling chemistry. After appropriate AuNP surface passivation steps, the aptamer's core region was hybridized with 8-mer biotinylated sequences. The conjugated particles could be capture on the test line by the interaction of the biotin molecules with a previously deposited streptavidin. Incubation of the conjugated particles with DBG causes the aptamer to undergo a conformational change that releases the 8-mer biotinylated sequences and result in the disappearance of the test line. Lysozyme protein was used to construct the control line that non-specifically interacts with the conjugated particles whether or not the target compound is present. The developed LFA achieves 20 nM detection level in buffer and blood samples, operates within the nanomolar range, and shows excellent selectivity against potential interfering molecules. The developed sensor could help assessing the levels of DBG in medical conditions that require rapid interventions.

Analytical verification and comparison of chromogenic assays for dabigatran, rivaroxaban and apixaban determination on BCSXP and STA Compact Max analysers.

INTRODUCTION: The aim of the study was to perform analytical verification and comparison of chromogenic assays for determination of dabigatran, rivaroxaban and apixaban concentration on BCSXP and STA Compact Max analysers. MATERIALS AND METHODS: Precision, linearity, measurement uncertainty estimation and determination of limit of blank, limit of determination and limit of quantification were calculated. Analytical performance specifications were set according to manufacturer specifications and literature data on between laboratory variability. Comparison of the methods was done using Bland-Altman and Passing-Bablok regression analysis. RESULTS: Obtained results have shown acceptable precision on STA Compact Max only for dabigatran (CV = 3.5%) at lower concentration level comparing to manufacturer declaration (CV = 3.6%). On BCSXP, the highest coefficient of variation has been shown for apixaban (6.1%) at lower concentration level. Within laboratory precision was not met on STA Compact Max for all assays. Bland-Altman analysis has shown statistically significant bias for dabigatran (23.2%, 95%CI 11.2 - 35.3; P < 0.001) and apixaban (8.4%, 95%CI 1.2 - 15.6; P = 0.023). Passing-Bablok regression analysis has shown systematic and proportional deviation between methods for rivaroxaban (y = 6.52 (2.94 to 11.83) + 0.84 (0.80 to 0.89) x. CONCLUSION: Chromogenic assays for dabigatran, rivaroxaban and apixaban on BCSXP and STA Compact Max analysers are shown as methods with satisfactory long-term analytical performance specifications for determination of direct oral anticoagulants in clinical laboratories. However, we cannot recommend interchangeable use because of the significant bias between assays.

Development, validation and application of a new HPLC-DAD method for simultaneous quantification of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma.

Direct oral anticoagulants (DOACs) have been commonly used for the treatment of venous thromboembolism and for the prevention of stroke in patients with atrial fibrillation. Despite not being initially recommended, monitoring DOACs plasma concentrations is now recognized as essential in emergency situations and in special populations. Moreover, the inter-individual variability found in real studies as well as the high reported non-adherence are corroborating the importance of determining the individual relationship between administered doses, plasma concentrations and pharmacological effects. Therefore, accurate but user-friendly bioanalytical techniques are required to monitor DOACs plasma concentrations in routine clinical practice and phase IV clinical trials. Herein, a fast and simple high performance liquid chromatography (HPLC) method coupled to diode array detection (DAD) was developed, validated and applied to quantify the four currently marketed DOACs (apixaban, edoxaban, dabigatran and rivaroxaban). Sample preparation was performed by solid phase extraction followed by evaporation and concentration of the analytes. Chromatographic separation was accomplished within 6 min on a reversed-phase column (octadecyl-silica packing material; 55 mm × 4 mm, 3 µm particle size), applying a mobile phase composed of an aqueous solution of formic acid (0.1 %, v/v) and acetonitrile, pumped with a gradient elution at 30 °C. The proposed method was linear (r2 ≥ 0.993) within the concentration ranges of 0.017-5.28 µg mL-1, 0.066-5.28 µg mL-1, 0.033-5.28 µg mL-1 and 0.017-5.28 µg mL-1 for apixaban, dabigatran, edoxaban and rivaroxaban, respectively, all of them including the expected range of therapeutic concentrations. Overall, intra- and inter-day trueness of quality control samples, including at the lower limit of quantification (LLOQ), varied between -12.98 to 5.79 %, while imprecision was lower than 16.43 %, supporting that the method is accurate and precise in accordance to international guidelines. Recovery and stability were also assessed and allowed the method to be applied in clinical practice, during therapeutic drug monitoring.

Using the PCI-IS Method to Simultaneously Estimate Blood Volume and Quantify Nonvitamin K Antagonist Oral Anticoagulant Concentrations in Dried Blood Spots.

Nonvitamin K antagonist oral anticoagulants (NOACs) have emerged as the preferred choice for the treatment of atrial fibrillation (AF). The establishment of a therapeutic range to minimize bleeding and thrombosis is important for personalized treatment of NOACs. The importance of dried blood spots (DBSs) has increased in medical care. An efficient and effective DBS analytical method could facilitate the concentration management of NOACs. The postcolumn infused internal standard (PCI-IS) method was applied to estimate spot volume and quantify dabigatran, rivaroxaban, and apixaban concentrations on DBS cards. The extraction solvent contented 0.1% formic acid and 70% ACN with a successive extraction procedure. Paired DBS and plasma samples from patients undergoing NOAC therapy (n = 269) were used to calculate conversion factors. [13C6]-Rivaroxaban was selected as the PCI-IS. The quantification accuracy for the three NOACs was within 88.9-104.3%. The RSDs of the repeatability and intermediate precision were below 10%. The obtained conversion factors of DBS to plasma concentrations of dabigatran, apixaban, and rivaroxaban were 1.81, 1.59, and 1.31, respectively. Bland-Altman analysis showed that the % differences between predicted and measured plasma concentrations were within a bias of ±20%. The result showed that PCI-IS was an accurate and efficient LC-MS/MS method to simultaneously estimate blood volume and NOAC concentrations on DBS cards. The stability results revealed that the DBS sampling strategy could improve compound stability. The developed method offers a new strategy for the therapeutic drug monitoring of NOACs and may improve the safe use of these drugs.

Optimization by response surface methodology of a dispersive magnetic solid phase extraction exploiting magnetic graphene nanocomposite coupled with UHPLC-PDA for simultaneous determination of new oral anticoagulants (NAOs) in human plasma.

In this paper a dispersive magnetic-solid phase extraction (MSPE) using a graphene nanocomposite (rG/Fe3O4) followed by ultra high performance liquid chromatography with photodiode array detection has been developed for the simultaneous analysis of new class of oral anticoagulants (NOAs) in human plasma. The performance of the nanocomposite graphene@Fe3O4 on the magnetic solid phase extraction of apixaban, rivaroxaban and dabigatran has been optimized using a Box-Behnken design of experiment. The amount of graphene nanocomposite, the sample pH and the adsorption time were the investigated parameters as a function of the extraction recovery. The analytical method was fully validated based on linearity, limit of detection (LOD), limit of detection (LOQ), inter- and intra-day precision and trueness, and extraction yield. Under optimal condition, excellent linearity (R2 > 0.9987) over the range (0.001-5.0 µg/mL), limit of detection (0.003 µg/mL), precision (0.81-8.97% RSD) and trueness (-5 to 9 % BIAS%) were observed for the target drugs. The average extraction recovery under optimal from plasma samples ranged between 96.6-98.6% for apixaban, rivaroxaban and dabigatran and the internal standard. The proposed method was developed, validated and successfully applied to the measurement of these NOAs in patients. The new approach offers an attractive alternative for the simultaneous analysis of the selected NOAs from plasma samples, providing several advantages including fewer sample preparation steps, ease of performance, and higher recoveries compared to traditional methodologies.

Evaluation of the DOAC-Stop Procedure by LC-MS/MS Assays for Determining the Residual Activity of Dabigatran, Rivaroxaban, and Apixaban.

The effect of direct oral anticoagulants (DOACs) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa (FXa), is a well-known problem and can cause both false positive and negative results. Therefore, the correct interpretation of tests performed in patients receiving DOACs is necessary to avoid misclassification and subsequent clinical consequences. However, even with significant experience, there are situations where it is not possible to assess the influence of some methods. Particularly important is the situation in the diagnosis of lupus anticoagulants using the dilute Russell viper venom timetest, which is based on direct FXa activation. A very promising solution to this situation is offered by the DOAC laboratory balancing procedure DOAC-Stop. For evaluating the effectiveness of this procedure, 60 (20 apixaban, 20 dabigatran, and 20 rivaroxaban) patients treated with DOACs were enrolled. All patient samples were analyzed for the presence of individual DOAC types and subsequently subjected to the DOAC-Stop procedure.We evaluated its effectiveness by our own high-performance liquid chromatography-coupled tandem mass spectrometrymethod, which simultaneously sets all high-sensitivity DOACs. Unlike coagulation tests based on the determination of the residual effects of DOACs on target enzymes, which is complicated by extensive interindividual variation, this methodology is highly specific and sensitive.The DOAC-Stop procedure eliminated dabigatran from 99.5%, rivaroxaban from 97.9%, and apixaban from 97.1% of participants in our group. Residual amounts did not exceed 2.7 ng/mL for dabigatran, 10.9 ng/mL for rivaroxaban, or 13.03 ng/mL for apixaban, which are safe values that do not affect either screening or special coagulation tests.

Comparative research on the metabolism of metoprolol by four CYP2D6 allelic variants in vitro with LC-MS/MS.

Specific study about the effect of cytochrome P450 2D6 (CYP2D6) polymorphisms on the metabolism of clinic drugs is of great significance for drug safety investigation. Here, the interaction between CYP2D6 variants (*1, *2, *10, *39) and metoprolol (MET) was intensively researched in vitro from the aspect of drug-enzyme kinetic study. To obtain quantitative data, α-hydroxymetoprolol (main metabolite of MET) was selected as an ideal analyte and an LC-MS/MS method was adopted for sample determination. Firstly, by selecting suitable internal standard and optimizing separation condition, the LC-MS/MS method was established and validated. Then, the drug-enzyme incubation system was optimized by two parameters: incubation time and amount of enzyme. Lastly, the interaction between CYP2D6 allelic variants and MET was characterized by Km, Vmax and CLint. As a result, four CYP2D6 enzymes displayed diverse Km or Vmax towards MET and the values of CLint showed a wide range from 8.91 to 100%. Relative to CYP2D6*1 (CLint*1 = 100%), CYP2D6*2 demonstrated the second high catalytic activity (CLint*2/*1 = 74.87%) while CYP2D6*39 (CLint*39/*1 = 29.65%) and CYP2D6*10 (CLint*10/*1 = 8.91%) showed minimal catalytic activity. This comprehensive in vitro data suggested the prominent influence of CYP2D6 polymorphisms on the metabolism of MET, which could offer valuable information for personalized administration of MET in clinic.

International Council for Standardization in Haematology (ICSH) Recommendations for Laboratory Measurement of Direct Oral Anticoagulants.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH) for providing haemostasis-related guidance documents for clinical laboratories. This inaugural coagulation ICSH document was developed by an ad hoc committee, comprised of international clinical and laboratory direct acting oral anticoagulant (DOAC) experts. The committee developed consensus recommendations for laboratory measurement of DOACs (dabigatran, rivaroxaban, apixaban and edoxaban), which would be germane for laboratories assessing DOAC anticoagulation. This guidance document addresses all phases of laboratory DOAC measurements, including pre-analytical (e.g. preferred time sample collection, preferred sample type, sample stability), analytical (gold standard method, screening and quantifying methods) and post analytical (e.g. reporting units, quality assurance). The committee addressed the use and limitations of screening tests such as prothrombin time, activated partial thromboplastin time as well as viscoelastic measurements of clotting blood and point of care methods. Additionally, the committee provided recommendations for the proper validation or verification of performance of laboratory assays prior to implementation for clinical use, and external quality assurance to provide continuous assessment of testing and reporting method.

Measurement of dabigatran: previously demonstrated Hemoclot® Thrombin Inhibitor assay reagent instability on Sysmex CS-2100i is no longer an issue.

The Hemoclot® Thrombin Inhibitor (HTI) assay has been recommended for measurement of dabigatran concentrations in specific clinical situations. Traditionally, reagents for biochemical assays are prepared from instructions found in the package insert. For the HTI reagents the manufacturer recommends incubating the reagents much longer than indicated in the package insert. These recommendations are added to the application sheets designed for different analyzers. Many clinicians and laboratory personnel may be unaware of the discrepancy between the two instructions, resulting in incorrect handling of the reagents. The aim of this study was to investigate the effect of the two different preparation methods on reagent stability and test results. For the standard concentration range, reagent stability on Sysmex CS-2100i was only two hours instead of the eight hours indicated by the producer when following package insert instructions (incubation time: 15 min). Stability was increased to five hours when following the application sheet (incubation time: 60 min). Two years later, the study was repeated using samples of patients treated with dabigatran etexilate. This time, reagent stability was at least six hours. Since the reagent composition was unchanged, the increased stability could be due to changed logistics by the supplier, with stock and transfer closer by. Previously demonstrated HTI reagent instability is no longer an issue at our laboratory. The reliability of results of clinical studies in which the assay has been used is potentially compromised.

Point-of-care testing for emergency assessment of coagulation in patients treated with direct oral anticoagulants.

BACKGROUND: Point-of-care testing (POCT) of coagulation has been proven to be of great value in accelerating emergency treatment. Specific POCT for direct oral anticoagulants (DOAC) is not available, but the effects of DOAC on established POCT have been described. We aimed to determine the diagnostic accuracy of Hemochron® Signature coagulation POCT to qualitatively rule out relevant concentrations of apixaban, rivaroxaban, and dabigatran in real-life patients. METHODS: We enrolled 68 patients receiving apixaban, rivaroxaban, or dabigatran and obtained blood samples at six pre-specified time points. Coagulation testing was performed using prothrombin time/international normalized ratio (PT/INR), activated partial thromboplastin time (aPTT), and activated clotting time (ACT+ and ACT-low range) POCT cards. For comparison, laboratory-based assays of diluted thrombin time (Hemoclot) and anti-Xa activity were conducted. DOAC concentrations were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Four hundred and three samples were collected. POCT results of PT/INR and ACT+ correlated with both rivaroxaban and dabigatran concentrations. Insufficient correlation was found for apixaban. Rivaroxaban concentrations at <30 and <100 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.0 and ≤1.1 and ACT+ POCT ≤120 and ≤130 s. Dabigatran concentrations at <30 and <50 ng/mL were detected with >95% specificity at PT/INR POCT ≤1.1 and ≤1.2 and ACT+ POCT ≤100 s. CONCLUSIONS: Hemochron® Signature POCT can be a fast and reliable alternative for guiding emergency treatment during rivaroxaban and dabigatran therapy. It allows the rapid identification of a relevant fraction of patients that can be treated immediately without the need to await the results of much slower laboratory-based coagulation tests. TRIAL REGISTRATION: Unique identifier, NCT02371070 . Retrospectively registered on 18 February 2015.

Measurement of dabigatran, rivaroxaban and apixaban in samples of plasma, serum and urine, under real life conditions. An international study.

BACKGROUND: The utility of measuring non-vitamin K antagonist oral anticoagulants (NOACs) in plasma, serum and urine samples and with the point-of-care test (POCT) on urine samples should be analysed in an international laboratory study. METHODS: The study was performed to determine the inter-laboratory variance of data from two chromogenic assays each for the NOACs rivaroxaban, apixaban and dabigatran, and to analyse the sensitivity and specificity of the POCT assays for factor Xa- and thrombin inhibitors. Plasma, serum and urine samples were taken from six patients in each group on treatment with a NOAC. RESULTS: The inter-laboratory variances, which can be identified best by the coefficient of variation, ranged from 46% to 59% for apixaban, 63% to 73% for rivaroxaban and 39% to 104% for dabigatran using plasma, serum or urine samples and two chromogenic assays for each NOAC. The concentrations were about 20% higher in serum compared to plasma samples for apixaban and rivaroxaban, and 60% lower for dabigatran. The concentration in urine samples was five-fold (apixaban), 15-fold (rivaroxaban) and 50-fold (dabigatran) higher. Sensitivity and specificity of POCT for apixaban, rivaroxaban, and dabigatran were all >94%. CONCLUSIONS: The inter-laboratory study showed the feasibility of measurement of apixaban, rivaroxaban, and dabigatran in plasma, serum and urine samples of patients on treatment. Dabigatran was determined at far lower levels in serum compared to plasma samples. Concentrations of NOACs in urine were much higher compared to plasma. The POCT was highly sensitive and specific for all three NOACs.