Chemicals and reagents
Rivaroxaban was provided by Bayer HealthCare AG (Wuppertal, Germany) (Fig. 1a). Rivaroxaban-d4, used as an internal standard (IS), was purchased from Toronto Research Chemicals (Canada) (Fig. 1b). Methanol and acetonitrile of HPLC grade were purchased from Thermo Fisher (MA, USA). LCMS-grade formic acid and dimethyl sulfoxide (DMSO) of analytical grade were purchased from JK Chemical (Beijing, China) and Sigma–Aldrich (France), respectively. Water was purified with a Milli-Q system (Millipore Waters, Darmstadt, Germany).
Calibrator and quality control sample preparation
The powdered compound of rivaroxaban and rivaroxaban-d4 were dissolved in DMSO to prepare stock solutions at 100 μg/mL and then the stock solutions stored at − 20 °C. Further dilutions were made in methanol to obtain series of intermediate and final working solutions. Then the appropriate amount of working solutions were added in blank human plasma to get the calibration curve with concentrations 0.5, 1, 2, 10, 20, 100, 200, and 400 ng/mL and quality control (QC) samples with concentrations 1.5, 15, and 300 ng/mL.
Instrument and analytical conditions
An Acquity UPLC system (WATERS, Milford, MA, USA) with an autosampler temperature of 10 °C and Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm particle size; Waters, Milford, MA, USA) was applied to determine the samples in this study. The mobile phase consisted of acetonitrile (A) and ultrapure water (B), and the gradient programme of the mobile phase was as follows: 32% A at 0–1.5 min; 32–90% A at 1.5–1.51 min; 90–32% A at 2.5–2.51 min; and 32% A at 2.51–3 min. The flow rate and the column temperature were set at 0.4 mL/min and 35 °C, respectively.
The analytes were detected in the Acquity tandem quadrupole detector (Waters Xevo TQ-S, Milford, MA, USA) with positive electrospray ionization (ESI+) and multiple-reaction monitoring (MRM) mode. And the mass transitions were m/z 437 → 145.0 and m/z 440.1 → 145.0 for rivaroxaban and rivaroxaban-d4, respectively. The operating parameters were as follows: cone voltage, 35 V; collision voltage, 30 V; collision gas flow, 0.16 mL/min; nebulizer gas pressure, 7.0 bar; and desolvation temperature, 500 °C. The retention times were 1.03 min for both rivaroxaban and rivaroxaban-d4.
Sample pretreatment
Sample preparation was performed by protein precipitation with acetonitrile. A 50 μL aliquot of plasma sample and 150 μL of acetonitrile containing the IS at a concentration of 10 ng/mL were mixed and vortexed for 2 min, then the mixture was centrifuged at 13000 r/min for 10 min at 25 °C.The supernatant was dried under nitrogen at normal temperature, redissolved with 32% acetonitrile and 68% ultrapure water containing 0.2% formic acid and vortexed. After filtering through a 0.22 μm micro-membrane filter, the supernatant was transferred to an autosampler vial and 10 μL was injected into the UPLC system automatically.
Study design and patients
The study population consisted of adult subjects with deep venous thrombosis (DVT) from Peking Union Medical College Hospital. Eligible subjects were those treated with rivaroxaban and aged 18 or older. Subjects were ineligible if they had severe damage to liver and kidney function, severe cardiopulmonary insufficiency, or they combined other anticoagulants, such as CYP450 3A4 and strong P-glycoprotein inhibitors.
The clinician conducted drug administration based on the patients’ condition. There were some patients who did not have very severe clots or bleeding after taking 20 mg rivaroxaban. Clinicians typically consider giving these patients 10 mg bid or 15 mg qd rivaroxaban. Rivaroxaban (Bayer HealthCare AG, Wuppertal, Germany) was taken with food. When concentrations of rivaroxaban reached a steady state (day seven or later), blood samples were taken 2 h after administration and before the next dose. To ensure patient adherence, we sent text messages to patients to remind them to take the medication and asked patients to fill out medication record forms. Patients continued to take rivaroxaban for at least 3 months. All samples were centrifuged for 10 min at 3000×g, and the plasma was then stored at − 80 °C until analysis.
Sample analysis
Rivaroxaban plasma concentrations were determined by UPLC-MS/MS. PT, aPTT and anti-factor Xa activity were determined at the same time in the clinical laboratory of Peking Union Medical College Hospital.
Statistical analyses were performed using SPSS software (SPSS for Windows, version 20.0, IBM Corp, Armonk, NY, USA). The arithmetic mean was calculated, and the results are presented as the mean ± standard deviation (SD). The association between PT, aPTT, anti-factor Xa activity and rivaroxaban plasma concentrations by UPLC-MS/MS was determined by Spearman correlation.