A method for determining the concentration of rivaroxaban based on anti-factor xa activity, a reagent for the determination, and a method for preparing the same
By optimizing the formulation of bovine Xa factor and chromogenic substrate, combined with sample dilution and segmented standard curves, the high cost and narrow linear range of existing anti-Xa factor activity detection kits have been solved, achieving accurate detection and cost-effectiveness for high-concentration samples.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE AFFILIATED HOSPITAL OF QINGDAO UNIV
- Filing Date
- 2026-03-13
- Publication Date
- 2026-05-29
AI Technical Summary
Existing anti-Xa factor activity assay kits are expensive, have a narrow linear range, and are difficult to meet the needs of high-concentration sample detection. Furthermore, there is a lack of high-quality domestically produced reagents, which affects the consistency and accuracy of the test results.
By employing an optimized formulation of bovine Xa factor, chromogenic substrate, and sample diluent, combined with a segmented standard curve strategy, the detection linear range was extended to 1500 ng/mL, reducing costs and improving detection sensitivity and specificity.
It enables accurate detection of high-concentration samples, reduces detection costs, and improves the consistency and accuracy of test results. It is suitable for conventional coagulation analyzers.
Smart Images
Figure CN122108722A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a method for determining rivaroxaban concentration based on anti-Xa factor activity, reagents for determination, and preparation methods thereof. Background Technology
[0002] Rivaroxaban is an oral direct factor Xa inhibitor that exerts its anticoagulant effect by selectively and reversibly inhibiting coagulation factor Xa. It is widely used in clinical fields such as the prevention and treatment of venous thromboembolism and stroke prevention in patients with non-valvular atrial fibrillation. Although routine use usually does not require monitoring, in special clinical situations such as bleeding or thrombotic events, perioperative period, renal insufficiency, extreme weight, suspected overdose, or drug interactions, it is still necessary to accurately measure the patient's plasma rivaroxaban concentration to assess its anticoagulant strength and guide clinical decision-making.
[0003] Currently, the primary clinical method for assessing the anticoagulant activity of rivaroxaban is the chromogenic substrate-based assay for anti-Xa factor activity. This method utilizes the specific binding of exogenous Xa factor to rivaroxaban in the test sample. The remaining free Xa factor catalyzes the formation of a colored product from the chromogenic substrate. By measuring the rate of change in absorbance at a specific wavelength, the concentration of rivaroxaban can be quantitatively calculated. International guidelines also recommend this method as the preferred option for monitoring rivaroxaban.
[0004] However, existing anti-Xa factor activity assay kits still have several significant drawbacks: First, the market is mainly monopolized by imported brands, and the high price of reagents leads to high costs per test, limiting the widespread application of this method in medical institutions at all levels; second, the detection linear range of existing reagents is generally narrow, with the upper limit typically only 500-800 ng / mL, making it difficult to meet the accurate detection requirements of high-concentration samples such as high-dose treatments and drug overdoses; third, the development of domestically produced high-quality reagents is lagging behind, lacking stable performance, good correlation with gold standard methods (such as liquid chromatography-tandem mass spectrometry), and controllable costs as alternative products; in addition, the comparability of detection results between different brand reagent systems is poor, affecting the consistency of clinical judgment. Therefore, there is an urgent need to develop a rivaroxaban concentration assay reagent and method with a wide detection linear range, high sensitivity, good specificity, low cost, and compatibility with conventional coagulation analyzers. Summary of the Invention
[0005] Based on this, the purpose of this invention is to provide a method for determining the concentration of rivaroxaban based on anti-Xa factor activity, the reagents for determination, and the preparation method thereof, so as to solve the technical problems of high detection cost, narrow linear range, inaccurate detection of high concentration samples, and lack of domestically produced high-performance reagents in the prior art.
[0006] To achieve the above objectives, the present invention provides the following technical solution: a reagent for determining rivaroxaban concentration based on anti-Xa factor activity, comprising reagent R1, reagent R2, and sample diluent; reagent R1 contains Xa factor, buffer, stabilizer, and inorganic salt; reagent R2 contains chromogenic substrate, buffer, polyethylene glycol, and inorganic salt; the sample diluent contains buffer, stabilizer, and inorganic salt, and is used for 1:2 dilution of samples with a concentration range of 650-1500 ng / mL.
[0007] The present invention is further configured such that, in reagent R1, the Xa factor is bovine Xa factor with a purity ≥95% and a concentration of 0.8-1.2 IU / mL; the buffer is Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L; the stabilizer is bovine serum albumin with a concentration of 0.2-0.8% (w / v); and the inorganic salt is NaCl with a concentration of 0.1-0.5 mol / L.
[0008] The present invention is further configured such that, in reagent R2, the chromogenic substrate is selected from one of S-2732, S-2222 or S-2765, and the concentration is 0.1-0.4 mmol / L; the buffer is Tris-HCl buffer, with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L; and the polyethylene glycol is PEG-6000, with a concentration of 0.05-0.3% (w / v).
[0009] The present invention is further configured such that, in the sample diluent, the buffer is Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L; the stabilizer is bovine serum albumin with a concentration of 0.2-0.8% (w / v); and the inorganic salt is NaCl with a concentration of 0.1-0.5 mol / L.
[0010] The present invention is further configured such that the preparation method of the reagent for determining the concentration of rivaroxaban based on anti-Xa factor activity includes the following steps: S1. Preparation of reagent R1: Bovine Xa factor, Tris-HCl buffer, bovine serum albumin and NaCl are mixed at a preset concentration, the pH value is adjusted to 7.4-8.5, and after aliquoting, it is stored in the dark at 2-8℃; S2. Preparation of reagent R2: Chromogenic substrate S-2732, Tris-HCl buffer and PEG-6000 are mixed at a preset concentration, the pH value is adjusted to 7.4-8.5, and after aliquoting, it is stored in the dark at 2-8℃; S3. Preparation of sample dilution: Tris-HCl buffer, bovine serum albumin and NaCl are mixed at a preset concentration, the pH value is adjusted to 7.4-8.5, and it is aliquoted for use.
[0011] The present invention is further configured such that the method for determining rivaroxaban concentration based on anti-Xa factor activity includes the following steps: S1, preparing a series of rivaroxaban calibrators covering a first concentration range and a second concentration range; for the calibrators in the first concentration range, direct measurement is performed; for the calibrators in the second concentration range, dilution is performed using the sample diluent before measurement; linear standard curves for the first concentration range and the second concentration range are established with the calibrator concentration as the abscissa and the measured absorbance change rate ΔOD / min as the ordinate; S2, determining the estimated concentration range of the sample to be tested; if the estimated concentration belongs to the first concentration range, then directly taking... The sample to be tested is then tested; if the estimated concentration falls within the second concentration range, the sample to be tested is quantitatively diluted using the sample diluent, and the diluted sample is then tested; the testing process is as follows: the sample, preheated reagent R1 and reagent R2 are added to the reaction system in sequence, incubated for a predetermined time, and the absorbance change is monitored at a wavelength of 405 nm for a specific time period to calculate ΔOD / min; S3, based on the concentration range determined in the sample pretreatment and testing, the measured ΔOD / min of the sample to be tested is substituted into the corresponding segmented standard curve to calculate the rivaroxaban concentration value; if the sample has been diluted, the result is multiplied by the corresponding dilution factor.
[0012] The present invention is further configured such that, in establishing the segmented standard curve, the first concentration range is 30-650 ng / mL and the second concentration range is 650-1500 ng / mL; for the calibrator or sample in the second concentration range, a sample diluent is used for 1:2 dilution.
[0013] The present invention is further configured such that, in the pretreatment and detection of the sample to be tested, the reaction conditions of the detection process are as follows: reaction temperature 37°C, volume ratio of sample, reagent R1, and reagent R2 is 10:50:50; after adding reagent R1, incubate for 180±20 seconds, then add reagent R2; after adding reagent R2, continuously monitor absorbance within a time interval of 60 to 90 seconds.
[0014] The present invention is further configured such that, in the preprocessing and detection of the sample to be tested, the determination of the estimated concentration range of the sample to be tested is based on clinical medication information, sample source, and rapid screening through preliminary experiments.
[0015] The present invention is further configured to include step S4, quality control: in each batch test or within a predetermined time interval, a quality control product of known concentration is used for synchronous testing, and when the test result of the quality control product falls within a preset range, the test result of that batch or time period is determined to be valid.
[0016] In summary, the present invention has the following main beneficial effects: The reagents, their preparation methods, and corresponding detection methods provided by this invention significantly reduce costs by using optimized bovine-derived factors and domestically produced raw materials. By utilizing proprietary sample diluents and a segmented standard curve strategy, the detection linear range is effectively broadened to 1500 ng / mL, ensuring the detection accuracy of high-concentration samples. At the same time, this scheme has excellent sensitivity, specificity, and stability, achieving detection performance highly correlated with the gold standard method. Thus, it successfully overcomes the technical bottlenecks of high detection costs, limited linear range, inaccuracy in high-value samples, and lack of high-quality domestically produced reagents in existing technologies. Attached Figure Description
[0017] Figure 1 This is a schematic diagram of the linear fitting curve of rivaroxaban concentrations from 30 to 650 ng / mL according to the present invention; Figure 2 This is a schematic diagram of the basic linear fitting curve of rivaroxaban at a concentration of 650-1500 ng / mL according to the present invention. Figure 3 This is a schematic diagram of the linear fitting curve between the measured and theoretical values of rivaroxaban detected in this invention; Figure 4 This invention provides a schematic diagram of the linear fitting curve between the measured and theoretical values of rivaroxaban. Figure 5 This is a schematic diagram of the fitting curve between the measured and theoretical values of rivaroxaban at high concentrations of 650-1500 ng / mL according to the present invention. Figure 6 This is a schematic diagram of the stability evaluation curve of the reagent under thermal damage conditions at 37°C as determined by the present invention. Figure 7 To illustrate the linear fitting curve between the measured and theoretical values of rivaroxaban in this invention, a scatter plot showing the correlation between the reagents of this invention and the concentration of rivaroxaban detected by LC-MS / MS is provided. Figure 8 Bland-Altman consistency analysis of the reagents of this invention with LC-MS / MS detection of rivaroxaban concentration. Detailed Implementation
[0018] like Figures 1 to 8 As shown, this invention provides a reagent for determining rivaroxaban concentration based on anti-Xa factor activity, comprising reagent R1, reagent R2, and a sample diluent. Reagent R1 contains Xa factor, buffer solution, stabilizer, and inorganic salt; reagent R2 contains a chromogenic substrate, buffer solution, polyethylene glycol, and inorganic salt; the sample diluent contains buffer solution, stabilizer, and inorganic salt, used to dilute samples with concentrations higher than a preset threshold.
[0019] In reagent R1, factor Xa is bovine factor Xa with a purity ≥95% and a concentration of 0.8-1.2 IU / mL, preferably 0.9 IU / mL. Bovine factor Xa exhibits high specificity and stability, and demonstrates excellent binding ability to rivaroxaban, making it the core component of this invention. The purity of bovine factor Xa is determined using high-performance liquid chromatography (HPLC) to ensure it meets the requirement of ≥95%.
[0020] The buffer solution is a Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L, preferably a Tris-HCl buffer with a pH of 8.0 and a concentration of 20 mmol / L. This buffer system can maintain the pH stability of the reaction system, ensuring the activity of factor Xa and the specificity of the reaction. The Tris-HCl buffer is precisely prepared to ensure good buffering capacity at a reaction temperature of 37°C.
[0021] The stabilizer is bovine serum albumin (BSA), with a concentration of 0.2-0.8% (w / v), preferably 0.5% (w / v). As a stabilizer, BSA effectively prevents the denaturation and inactivation of factor Xa, improving the stability and reproducibility of the reagent. The concentration of BSA is ensured to be within the range of 0.2-0.8% through precise weighing and dissolution.
[0022] The inorganic salt is NaCl, with a concentration of 0.1-0.5 mol / L, preferably 0.35 mol / L. As an inorganic salt component, NaCl can simulate the physiological saline environment, maintain the ionic strength of the reaction system, and ensure the activity of factor Xa and the specificity of the reaction. The concentration of NaCl is precisely controlled to ensure it remains within the range of 0.1-0.5 mol / L.
[0023] In reagent R2, the chromogenic substrate is selected from S-2732, S-2222, or S-2765, with a concentration of 0.1-0.4 mmol / L, preferably S-2732 at a concentration of 0.25 mmol / L. S-2732 (SucIleGlu(γPip)GlyArgpNA·HCl) is the preferred chromogenic substrate of this invention, as it has a moderate reaction rate with factor Xa and can produce a stable colored product, facilitating absorbance measurement. The concentration of the chromogenic substrate is ensured to be within the range of 0.1-0.4 mmol / L through precise weighing and dissolution.
[0024] The buffer solution is a Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L, preferably a Tris-HCl buffer with a pH of 8.0 and a concentration of 20 mmol / L. This buffer system is consistent with the buffer system of reagent R1 to ensure the pH stability of the reaction system.
[0025] The polyethylene glycol used is PEG-6000, with a concentration of 0.05-0.3% (w / v), preferably 0.1% (w / v). PEG-6000, as a preferred type of polyethylene glycol, improves the solubility and stability of the chromogenic substrate, and enhances the sensitivity and repeatability of the reaction. The concentration of PEG-6000 is precisely controlled to ensure it remains within the range of 0.05-0.3%.
[0026] The sample dilution buffer is a Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L, preferably a Tris-HCl buffer with a pH of 8.0 and a concentration of 20 mmol / L. This buffer system is consistent with the buffer systems of reagents R1 and R2 to ensure the pH stability of the diluted sample.
[0027] The stabilizer is bovine serum albumin (BSA), with a concentration of 0.2-0.8% (w / v), preferably 0.5% (w / v). As a stabilizer, BSA prevents the degradation and adsorption of rivaroxaban in the diluted sample, ensuring the accuracy of the test results.
[0028] The inorganic salt is NaCl, with a concentration of 0.1-0.5 mol / L, preferably 0.10 mol / L. As an inorganic salt, NaCl maintains the ionic strength of the diluted sample, ensuring accurate detection. The concentration of NaCl is precisely controlled to ensure it remains within the range of 0.1-0.5 mol / L.
[0029] The reagent preparation method of the present invention includes the following steps: Preparation of Reagent R1: Mix bovine factor Xa, Tris-HCl buffer, bovine serum albumin, and NaCl at the preset concentrations, adjust the pH to 7.4-8.5, aliquot, and store at 2-8℃ protected from light. Specific preparation steps are as follows: Select 0.9 IU / mL bovine factor Xa, 20 mmol / L Tris-HCl buffer (pH 8.0), 0.5% bovine serum albumin (w / v), 0.35 mol / L NaCl, and 0.05% sodium azide. After preparing the above reagents, aliquot into 3 ml bottles and store at 2-8℃ protected from light. Shelf life is 7 days.
[0030] Preparation of reagent R2: Mix chromogenic substrate S-2732, Tris-HCl buffer, and PEG-6000 at the preset concentrations, adjust the pH to 7.4-8.5, aliquot, and store at 2-8℃ protected from light. Specific preparation steps are as follows: Select 0.25 mmol / L chromogenic substrate S-2732, 20 mmol / L Tris-HCl buffer at pH 8.0, and 0.1% (w / v) PEG-6000. After preparing the above reagents, aliquot into 3 ml vials and store at 2-8℃ protected from light. Shelf life is 14 days.
[0031] Preparation of sample diluent: Mix Tris-HCl buffer, bovine serum albumin, and NaCl at the preset concentrations, adjust the pH to 7.4-8.5, and dispense into vials. Specific preparation steps are as follows: Prepare 20 mmol / L Tris-HCl buffer (pH 8.0), 0.5% bovine serum albumin (w / v), and 0.10 mol / L NaCl. After preparing the above reagents, dispense 2 ml vials into vials for use. This sample diluent is used for 1:2 dilution of high-concentration samples with a concentration range of 650-1500 ng / mL.
[0032] The bovine Xa factor is a commercially available high-purity formulation with a purity ≥95% and a specific activity of not less than 100 IU / mg. It can be purified from bovine plasma by anion exchange chromatography combined with gel filtration, and its activity is confirmed by parallel determination with international standards.
[0033] The chromogenic substrate S-2732 (HD-Ile-Glu-Gly-Arg-pNA·2AcOH) is the preferred substrate of this invention; although S-2222 and S-2765 have similar structures, comparative experiments in this system have verified that only S-2732 can maintain a good linear response (R0) over a wide concentration range (30–1500 ng / mL). 2 ≥0.995) and intra-batch coefficient of variation (CV<5%). If other chromogenic substrates are used, their suitability in terms of linearity, sensitivity, and immunity must be re-verified.
[0034] The method for determining rivaroxaban concentration of the present invention includes the following steps: A series of rivaroxaban calibrators covering a first concentration range and a second concentration range were prepared. The first concentration range was 30-650 ng / mL, and the second concentration range was 650-1500 ng / mL. Calibrators in the first concentration range were measured directly; calibrators in the second concentration range were diluted 1:2 with sample dilution buffer before measurement. Linear standard curves were established for the first and second concentration ranges, with calibrator concentration on the x-axis and the measured absorbance change rate ΔOD / min on the y-axis.
[0035] The specific steps for establishing the standard curve are as follows: Rivaroxaban calibrator is measured twice at eight concentration points: 30, 50, 250, 450, 650, 950, 1250, and 1500 ng / mL. For calibrators with concentrations below 650 ng / mL, the measurement is performed directly; for calibrators with concentrations between 650 and 1500 ng / mL, they are first diluted 1:2 with sample diluent before measurement. The measured ΔOD / min value is plotted as the ordinate, and the calibrator concentration as the abscissa to construct the standard curve. The regression equation for the standard curve in the first concentration range (30-650 ng / mL) is Y = 0.97698X + 2.66501, R0 = 0.97698X + 2.66501. 2 =0.989; the standard curve regression equation for the second concentration range (650-1500 ng / mL) is Y=0.98708X+16.16742, R 2 =0.9991.
[0036] The method for establishing segmented standard curves is universally applicable: First, prepare a series of calibrators covering the expected detection range (e.g., 30–1500 ng / mL); then, fit two independent four-parameter logistic regression or linear regression equations using data from the low concentration range (30–650 ng / mL) and the high concentration range (500–1500 ng / mL), respectively; the two curves overlap in the 500–650 ng / mL range for cross-validation of consistency. This strategy is not dependent on specific instruments or reagent batches; any laboratory equipped with an ELISA reader and anti-Xa activity detection capabilities can use this procedure to reconstruct a segmented calibration model suitable for its own system.
[0037] Determine the estimated concentration range of the sample to be tested. The estimated concentration range can be determined through clinical medication information, sample source, or rapid screening through preliminary experiments. If the estimated concentration falls within the first concentration range (30-650 ng / mL), the sample to be tested is taken directly; if the estimated concentration falls within the second concentration range (650-1500 ng / mL), the sample to be tested is quantitatively diluted 1:2 using sample diluent, and the diluted sample is taken for testing.
[0038] The detection process was as follows: the sample, preheated reagent R1, and reagent R2 were added sequentially to the reaction system. After incubation for a predetermined time, the absorbance change was monitored at a wavelength of 405 nm over a specific time period, and ΔOD / min was calculated. Specific detection conditions were: reaction temperature 37℃, volume ratio of sample, reagent R1, and reagent R2 10:50:50; incubation for 180±20 seconds after adding reagent R1, followed by the addition of reagent R2; and continuous monitoring of absorbance for 60 to 90 seconds after adding reagent R2. The absorbance change ΔOD showed a good linear relationship within this time interval, accurately reflecting the rivaroxaban concentration.
[0039] Based on the concentration range determined during sample pretreatment and detection, the measured ΔOD / min of the sample is substituted into the corresponding piecewise standard curve to calculate the rivaroxaban concentration value. If the sample has been diluted, the result is multiplied by the corresponding dilution factor.
[0040] Within each batch test or a predetermined time interval, quality control samples of known concentrations were simultaneously tested. The test results for that batch or time period were considered valid if the quality control sample results fell within a preset range. The quality control sample concentrations were low (184.57 ng / mL), medium (338.18 ng / mL), and high (434.56 ng / mL), with each having a CV value ≤ 5%.
[0041] The performance of the reagents of this invention has been fully validated, including imprecision assessment, linear range assessment, stability assessment, and correlation analysis with the gold standard method.
[0042] Intra-assay imprecision assessment: Three levels of quality control plasma (low 184.57 ng / mL, medium 338.18 ng / mL, and high 434.56 ng / mL) were measured 20 times consecutively using the reagent of this invention, and the CV value was calculated. The results showed that the intra-assay CV value was 1.19%-1.69%, all ≤5%.
[0043] Inter-day imprecision assessment: Three levels of quality control plasma were collected, aliquoted into 40 portions, and stored at -80°C. One portion was taken out each day and measured twice daily using the assay reagent for a total of 20 days. The CV value was calculated. The results showed that the inter-day CV value was 1.56%-2.27%, all ≤5%.
[0044] Linearity range assessment was performed using low-value clinical plasma samples, with eight concentration gradients prepared. Within the concentration range of 30-650 ng / mL, the measured values showed a good linear relationship with the theoretical values, and the regression equation was Y = 0.97698X + 2.66501, R0. 2 =0.989. Within the concentration range of 650-1500 ng / mL, the measured values also showed a good linear relationship with the theoretical values, with the regression equation being Y=0.98708X+16.16742, R0. 2 =0.9991. The linear range of this detection method is 30-1500 ng / mL, covering the range of common clinical concentrations.
[0045] Three levels of plasma were used for stability assessment, and the concentration of rivaroxaban in the anti-Xa factor activity assay was determined using this reagent. The reagent was subjected to thermal destruction at 37°C. The concentrations of rivaroxaban in the three plasmas were measured on days 1, 3, 7, 10, and 14 after thermal destruction. The results showed that, 14 days after thermal destruction, the relative deviations of the rivaroxaban concentrations in the high, intermediate, and low-value plasmas from the initial values were 2.38%, 2.14%, and 2.76%, respectively, all less than 3%. This reagent exhibits good thermal stability and remains stable for at least 14 days after thermal destruction.
[0046] Eighty plasma samples were tested using this assay reagent, and the results were analyzed using LC-MS / MS. The correlation coefficient between the two methods was 0.9945, and the regression equation was Y = 0.97698X + 2.66501, R0. 2 =0.989. Bland-Altman analysis showed that the detection results of the two detection systems were in good agreement.
[0047] The assay reagents of this invention are applicable to coagulation analyzers of various brands and models available on the market, such as the Beijing Zhongchi XL-3200c coagulation analyzer, the Stago STA-Compact RMax fully automated coagulation analyzer, the Werfen TOP700 fully automated coagulation analyzer, and the Beckman Coulter ACL7000 fully automated coagulation analyzer. For different coagulation analyzers, specific reaction parameters, such as reaction time and monitoring time, can be adjusted appropriately to ensure the accuracy and repeatability of the test results.
[0048] The reagent of this invention requires a small sample volume (only 10 μL), saving clinical specimens. It is particularly suitable for special populations with limited blood volume, such as infants and critically ill patients, and is suitable for promotion in primary healthcare institutions. The reagent has a simple composition, is easy to operate during testing, has a total reaction time of less than 5 minutes, and has a significantly lower testing cost than imported reagents, thus having broad market application prospects.
[0049] The steps of the methods or algorithms described in this application can be directly embedded in hardware, software units executed by a processor, or a combination of both. Exemplarily, a storage medium can be connected to a processor so that the processor can read information from and write information to the storage medium. Optionally, the storage medium can also be integrated into the processor. Optionally, the processor and the storage medium can also be located in different components within a terminal. These computer program instructions can also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable apparatus for implementing the process. Figure 1One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.
[0050] Although this application has been described in conjunction with specific features and embodiments, it is obvious that various modifications and combinations can be made thereto without departing from the spirit and scope of this application. Accordingly, this specification and drawings are merely illustrative examples of this application and are considered to cover any and all modifications, variations, combinations, or equivalents within the scope of this application. Clearly, those skilled in the art can make various alterations and modifications to this application without departing from its scope. Thus, if such modifications and modifications fall within the scope of this application and its equivalents, this application intends to include such modifications and modifications.
Claims
1. A reagent for determining rivaroxaban concentration based on anti-factor Xa activity, characterized in that, Includes reagent R1, reagent R2, and sample diluent; The reagent R1 contains factor Xa, buffer solution, stabilizer and inorganic salt; The reagent R2 contains a chromogenic substrate, a buffer solution, polyethylene glycol, and an inorganic salt; The sample diluent contains a buffer, stabilizer, and inorganic salt, and is used for 1:2 dilution of samples with a concentration range of 650-1500 ng / mL.
2. The reagent for determination according to claim 1, characterized in that, In reagent R1, factor Xa is bovine factor Xa with a purity ≥95% and a concentration of 0.8-1.2 IU / mL; the buffer is Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L; the stabilizer is bovine serum albumin with a concentration of 0.2-0.8% (w / v); and the inorganic salt is NaCl with a concentration of 0.1-0.5 mol / L.
3. The reagent for determination according to claim 1, characterized in that, In reagent R2, the chromogenic substrate is selected from one of S-2732, S-2222 or S-2765, with a concentration of 0.1-0.4 mmol / L; the buffer is Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L; and the polyethylene glycol is PEG-6000 with a concentration of 0.05-0.3% (w / v).
4. The reagent for determination according to claim 1, characterized in that, The sample diluent contains Tris-HCl buffer with a pH of 7.4-8.5 and a concentration of 10-30 mmol / L; bovine serum albumin as the stabilizer with a concentration of 0.2-0.8% (w / v); and NaCl as the inorganic salt with a concentration of 0.1-0.5 mol / L.
5. A method for preparing a reagent for determining rivaroxaban concentration based on anti-Xa factor activity, characterized in that, Includes the following steps: S1. Preparation of reagent R1: Mix bovine factor Xa, Tris-HCl buffer, bovine serum albumin and NaCl at the preset concentration, adjust the pH to 7.4-8.5, dispense into containers and store at 2-8℃ in the dark; S2. Preparation of reagent R2: Mix the chromogenic substrate S-2732, Tris-HCl buffer and PEG-6000 at the preset concentration, adjust the pH value to 7.4-8.5, dispense into containers and store at 2-8℃ in the dark; S3. Preparation of sample dilution buffer: Mix Tris-HCl buffer, bovine serum albumin and NaCl at the preset concentration, adjust the pH to 7.4-8.5, and dispense for use.
6. A method for determining rivaroxaban concentration based on anti-factor Xa activity, characterized in that, Includes the following steps: S1. Prepare a series of rivaroxaban calibrators covering the first concentration range and the second concentration range; for the calibrators in the first concentration range, perform direct measurement; for the calibrators in the second concentration range, dilute them with the sample diluent before measurement; establish linear standard curves for the first concentration range and the second concentration range respectively, with the calibrator concentration as the abscissa and the measured absorbance change rate ΔOD / min as the ordinate. S2. Determine the estimated concentration range of the sample to be tested; if the estimated concentration belongs to the first concentration range, directly take the sample to be tested for detection; if the estimated concentration belongs to the second concentration range, use the sample diluent to quantitatively dilute the sample to be tested, and then take the diluted sample for detection; the detection process is as follows: add the sample, preheated reagent R1 and reagent R2 to the reaction system in sequence, incubate for a predetermined time, monitor the absorbance change at a wavelength of 405nm for a specific time period, and calculate ΔOD / min; S3. Based on the concentration range determined in the pretreatment and detection of the sample to be tested, substitute the measured ΔOD / min of the sample to be tested into the corresponding segmented standard curve to calculate the rivaroxaban concentration value. If the sample has been diluted, the result is multiplied by the corresponding dilution factor.
7. The determination method according to claim 6, characterized in that, In establishing the segmented standard curve, the first concentration range is 30-650 ng / mL, and the second concentration range is 650-1500 ng / mL; for calibrators or samples in the second concentration range, a 1:2 dilution is performed using sample diluent.
8. The determination method according to claim 6, characterized in that, In the sample pretreatment and detection process, the reaction conditions for the detection process are as follows: reaction temperature 37℃, volume ratio of sample, reagent R1, and reagent R2 10:50:50; after adding reagent R1, incubate for 180±20 seconds, then add reagent R2; after adding reagent R2, continuously monitor absorbance within a time interval of 60 to 90 seconds.
9. The determination method according to claim 6, characterized in that, In the sample preprocessing and detection process, the estimated concentration range of the sample is determined based on clinical medication information, sample source, and rapid screening through preliminary experiments.
10. The determination method according to claim 6, characterized in that, It also includes step S4, quality control: In each batch test or within a predetermined time interval, a quality control sample of known concentration is used for synchronous testing. When the test result of the quality control sample falls within the preset range, the test result of that batch and that time period is determined to be valid.