LC-MS analysis was performed using the liquid chromatography system (Dionex, Sunnyvale, CA, USA) coupled with a triple quadrupole 6500 tandem mass spectrometer (AB Sciex, Foster City, CA, USA) according to the previously published method [8 (link),9 (link)]. Dabigatran, rivaroxaban, and stable isotopically labelled D3-Dabigatran (Toronto Research Chemicals Inc, Toronto, ON, Canada) and apixaban (Pfizer Inc., New York, NY, USA) were dissolved in methanol (LC-MS quality, Sigma, St. Louis, MO, USA) at a final concentration of 1 mg/mL expressed as free substances. These stock solutions were used for the preparation of all other standards. For quantification, a series of calibration standards in methanol were prepared (concentrations 0, 10, 50, 100, and 500 ng/mL of DABI, API, and RIVA). The calibration standards were prepared in addition to drug-free plasma from healthy volunteers. All solutions were stored at −20 °C.
Rivaroxaban
Manufactured by LGC
Sourced in Canada, United States
Rivaroxaban is a laboratory-grade product designed for research and analytical purposes. It is a direct factor Xa inhibitor used to measure and analyze blood coagulation and related parameters in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Apixaban, by LGC (3 mentions)
Dabigatran, by LGC (2 mentions)
Apixaban, by Pfizer (1 mentions)
Rabbit anti human fibrinogen igg, by Agilent Technologies (1 mentions)
Alexa 594, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated mouse igg, by Beckman Coulter (1 mentions)
Ar c66096, by Bio-Techne (1 mentions)
Ppp reagent, by Diagnostica Stago (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Saxagliptin, by LGC (1 mentions)
Vildagliptin, by LGC (1 mentions)
Talinolol, by LGC (1 mentions)
Sitagliptin phosphate, by LGC (1 mentions)
14 c metformin, by Moravek Biochemicals (1 mentions)
Apixaban d3, by LGC (1 mentions)
Nexera uplc system, by AB Sciex (1 mentions)
Rivaroxaban d4, by LGC (1 mentions)
6 protocols using rivaroxaban
1
LC-MS Quantification of Anticoagulants
2
Evaluation of DrugSorb-ATR Device Performance
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Ticagrelor [purity 99.64%, high performance liquid chromatography (HPLC)] and ticagrelor-d7 (Purity: 96%, HPLC) were procured from MuseChem, Fairfield, NJ, USA. Apixaban (purity 98%, HPLC) and rivaroxaban (purity 99.7%, HPLC) were obtained from Toronto Research Chemicals, Toronto, Ontario, Canada and AK Scientific Inc, Union City, CA, USA, respectively. All other reagents were of standard analytical grade. The test and control circuit components were procured from McMaster–Carr, Elmhurst, IL, USA and Molded Products Inc., Harlan, IA, USA. Bovine whole blood (Lampire Biological Inc., Pipersville, PA, USA) with 4.5 U/mL Na-Heparin and total protein ranging between 6.6–8.8 g/dL, was used for all runs. Maxi–Therm Pediatric Blankets 56436-I (Cincinnati Sub Zero LLC, Cincinnati, OH, USA) connected with Polystat standard 3–6 L heat/cool bath (Cole–Parmer, Vernon Hills IL, USA) were used to maintain the temperature of the blood pool at 37°C throughout the runs. All tested DrugSorb-ATR devices were gamma sterilized and used the same filling, finishing and sterilization processes as commercially available devices. All instruments used for the measurement of run parameters were calibrated using instrumentation and standards from the United States National Institute of Standards and Technology (NIST) and are compliant with ISO-10 012:2003 and ANSI/NCSL Z540-3-2006.
3
Flow Chamber Assay Materials and Reagents
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The cover and capillary chips used in the flow chamber system (Fig. S1A
Dabigatran and rivaroxaban were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). AR-C66096, a specific P2Y12-receptor antagonist, was obtained from Tocris Bioscience (Bristol, UK). For the TG assay, PPP-Reagent (with phospholipids), PRP-Reagent (without phospholipids), and FluCa-reagent, a fluorogenic substrate (Z-Gly-Gly-Arg-AMC) dissolved in HEPES buffer and calcium chloride, were purchased from Diagnostica Stago (Parsippany, NJ). Recombinant TF (r-TF) was purchased from Mitsubishi Chemical Medience (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemicals (Osaka, Japan). Corn trypsin inhibitor (CTI) was prepared as reported previously [13] (link).
Dabigatran and rivaroxaban were obtained from Toronto Research Chemicals, Inc. (Toronto, Canada). AR-C66096, a specific P2Y12-receptor antagonist, was obtained from Tocris Bioscience (Bristol, UK). For the TG assay, PPP-Reagent (with phospholipids), PRP-Reagent (without phospholipids), and FluCa-reagent, a fluorogenic substrate (Z-Gly-Gly-Arg-AMC) dissolved in HEPES buffer and calcium chloride, were purchased from Diagnostica Stago (Parsippany, NJ). Recombinant TF (r-TF) was purchased from Mitsubishi Chemical Medience (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemicals (Osaka, Japan). Corn trypsin inhibitor (CTI) was prepared as reported previously [13] (link).
4
Quantification of Metformin and Antidepressants
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Unlabeled metformin was purchased from Wako Pure Chemical Industries (Osaka, Japan), and [ 14 C]metformin (100 mCi/mmol) was purchased from Moravek Biochemicals (Brea, CA, USA). Unlabeled almotriptan malate was purchased from Sigma (St. Louis, MO, USA).
Unlabeled alogliptin, naratriptan hydrochloride, rivaroxaban, saxagliptin, sitagliptin phosphate, sumatriptan succinate, talinolol and vildagliptin were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). All other chemicals and reagents were of analytical grade and are commercially available.
Unlabeled alogliptin, naratriptan hydrochloride, rivaroxaban, saxagliptin, sitagliptin phosphate, sumatriptan succinate, talinolol and vildagliptin were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). All other chemicals and reagents were of analytical grade and are commercially available.
5
Evaluating Anticoagulants' Antithrombotic Effects
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For the first part of the study, we obtained whole blood samples from 20 healthy volunteers who had not taken any medications (12 males and eight females; aged 29 - 54 years). All blood samples were numbered in order of registration. To confirm the in vitro antithrombotic effects of Rivaroxaban and apixaban at the expected clinical peak drug level, blood samples were prepared with drugs as follows. Rivaroxaban and apixaban were obtained from Toronto Research Chemicals (Toronto, Ontario, Canada). Rivaroxaban and apixaban were each dissolved in 100% dimethylsulfoxide to yield stock solutions with a concentration of 5 mM. Stock solutions were then diluted in blood samples at the time of assay (first 16 samples in the Rivaroxaban group and the rest of four samples in the apixaban group). The target final concentration of Rivaroxaban in this experiment was 800 μM, which corresponds to the expected peak concentration (Cmax-equivalent) of Rivaroxaban in blood after a dose of 15 mg/day [12 (link)]. Similarly, blood samples were spiked with apixaban stock solutions. The final concentration of apixaban was 450 μM, which corresponded to the peak concentration of apixaban in blood after a dose of 10 mg/day clinically [13 (link), 14 (link)]. We immediately evaluated the antithrombotic effects of these spiked blood samples in comparison with thrombus formation in control blood by the T-TAS®.
6
Quantitative LC-MS/MS Determination of DOACs
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