Stable rivaroxaban content detection kit with strong anti-interference capability

Abstract

The invention relates to a stable rivaroxaban content detection kit with strong anti-interference capability. The kit comprises a chromogenic substrate reagent and an Xa factor reagent, the chromophoric substrate reagent comprises an Xa factor sensitive substrate, a buffer solution, a surfactant and a preservative; the Xa factor reagent comprises an Xa factor, a buffering agent, inorganic salt, a complexing agent and a preservative. The kit is high in accuracy and good in repeatability, and the stability and the anti-interference capability of the detection reagent can be guaranteed at the same time. The kit disclosed by the invention can be suitable for different types of blood coagulation analyzers, and provides a reliable detection means for quantitatively detecting the content of rivaroxaban in vitro.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine and in vitro diagnosis; specifically, it relates to a kit for detecting the content of rivaroxaban in in vitro samples, and its preparation method and application. Background Art

[0002] According to different mechanisms of action, anticoagulant drugs can be divided into four categories: vitamin K antagonists, indirect thrombin inhibitors, direct thrombin inhibitors and factor Xa inhibitors. Vitamin K antagonists are easily interfered by a variety of substances during use, while factor Xa is at the center of the intrinsic coagulation pathway and the extrinsic coagulation pathway and is the first step in the common coagulation pathway. Each activation of factor Xa can produce about 1000 times of thrombin, so inhibiting factor Xa has a better ability to inhibit fibrin formation than inactivating thrombin.

[0003] Rivaroxaban is a new oral anticoagulant that can directly act on activated factor X. Compared with heparin, rivaroxaban can specifically inhibit free or bound factor Xa without any cofactors and has a stronger anticoagulant effect.

[0004] Rivaroxaban has relatively stable pharmacokinetics and generally does not require routine monitoring in clinical practice. However, in some special circumstances, such as when the patient is suspected to have a high risk of bleeding, suspected drug overdose, or a serious bleeding event, it is very necessary to test the rivaroxaban level in the patient's blood.

[0005] It is currently known that the gold standard for evaluating direct oral anticoagulants is liquid chromatography tandem mass spectrometry. Although this method can accurately detect the content of rivaroxaban, the detection cost is expensive and it is difficult to be widely used in clinical practice.

[0006] CN107576799A discloses a test kit for the content of rivaroxaban, which is based on the chromogenic substrate method and is detected by the principle that the inhibitory effect of rivaroxaban on factor Xa is inversely proportional to the absorbance of the reaction. Although this method can detect the content of rivaroxaban in plasma, the anti-interference ability of the reagent is not explained, and the occurrence of hemolysis, lipemia or hyperbilirubin in clinical samples may affect the accuracy of the test results. The reagent in CN107576799A is a lyophilized powder reagent, which is more cumbersome to use than liquid reagents.

[0007] CN116426510B discloses a rivaroxaban detection kit containing modified factor Xa, which reduces the sensitivity of heparin-ATIII conjugate to modified factor Xa by modifying factor Xa, thereby improving the accuracy of detection. However, CN116426510B does not describe the reagent's ability to resist hemoglobin, fat emulsion and bilirubin.

[0008] Hemolysis, lipemia and high bilirubin samples in clinical practice may still affect the accuracy of the test. Therefore, a rivaroxaban content detection kit with high accuracy, strong anti-interference ability and good stability is currently needed in clinical practice. Summary of the invention

[0009] In order to take into account the repeatability, accuracy, stability and anti-interference ability of the detection kit, the present application provides the following aspects:

[0010] According to some embodiments of the present application, a rivaroxaban content detection kit is provided, comprising:

[0011] 1) A chromogenic substrate reagent comprising:

[0012] 50mM to 200mM first buffer,

[0013] 1.2mM to 2.0mM Factor Xa sensitive substrate,

[0014] 0.2% w / v to 1% w / v surfactant,

[0015] 0.05% w / v to 0.1% w / v of a first preservative;

[0016] 2) A factor Xa reagent comprising:

[0017] 50mM to 200mM second buffer,

[0018] 0.5 IU / mL to 1.0 IU / mL factor Xa,

[0019] 100mM to 200mM inorganic salts,

[0020] 3mM to 10mM complexing agent,

[0021] 0.05% w / v to 0.1% w / v secondary preservative.

[0022] In some embodiments, the first buffer and the second buffer are each independently selected from: 2-morpholineethanesulfonic acid-sodium hydroxide buffer, tris-hydrochloric acid buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, or a combination thereof.

[0023] In some embodiments, the factor Xa sensitive substrate is selected from: ZD-Arg-Gly-Ar g-pNA, NaZD-Arg-Gly-Arg-pNA.2HCl, Suc-Ile-Glu(gamma-pip)-Gly-Arg-pNA.HCl, Suc-Ile-Glu(gamma-pip)-Gly-Arg-pNA.HCl, or a combination thereof.

[0024] In some embodiments, the surfactant is selected from: fatty alcohol polyoxyethylene ether, fatty alcohol polyoxyethylene ether sodium sulfate, Tween-20, polyoxyethylene lauryl ether, or a combination thereof.

[0025] In some embodiments, the inorganic salt is selected from the group consisting of sodium nitrite, potassium nitrite, sodium chloride, or a combination thereof.

[0026] In some embodiments, the complexing agent is selected from: ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, citric acid, or a combination thereof.

[0027] In some embodiments, the first preservative and the second preservative are each independently selected from: sodium azide, PC300, PC950, thimerosal, or a combination thereof.

[0028] In some embodiments, the pH of the chromogenic substrate reagent is between 6.6 and 7.0.

[0029] In some embodiments, the pH value of the Factor Xa reagent is not less than 7.2, preferably 7.3 to 7.6; more preferably 7.6.

[0030] In some embodiments, the Factor Xa sensitive substrate is 1.4 mM ZD-Arg-Gly-Arg-pNA.

[0031] In some embodiments, the first buffer is 50 mM 2-morpholineethanesulfonic acid-sodium hydroxide buffer.

[0032] In some embodiments, the second buffer is 50 mM Tris-HCl buffer.

[0033] In some embodiments, the surfactant is a combination of fatty alcohol polyoxyethylene ether and fatty alcohol polyoxyethylene ether sodium sulfate at a concentration ratio of 4:1.

[0034] In some embodiments, the concentration of the fatty alcohol polyoxyethylene ether is 0.8% w / v, and the concentration of the fatty alcohol polyoxyethylene ether sodium sulfate is 0.2% w / v.

[0035] In some embodiments, the first preservative and the second preservative are 0.1% w / v sodium azide.

[0036] In some embodiments, the Factor Xa is 0.6 IU / mL.

[0037] In some embodiments, the inorganic salt is 100 mM sodium chloride.

[0038] In some embodiments, the complexing agent is 5 mM disodium ethylenediaminetetraacetate.

[0039] In some embodiments, the first preservative and the second preservative are 0.1% w / v sodium azide.

[0040] In some embodiments, the chromogenic substrate reagent and the Factor Xa reagent are liquid reagents.

[0041] In some specific embodiments, the rivaroxaban content detection kit of the present application comprises:

[0042] 1) A chromogenic substrate reagent comprising:

[0043] 2-Morpholineethanesulfonic acid-NaOH buffer 50 mM

[0044] Fatty alcohol polyoxyethylene ether 0.8% w / v

[0045] Sodium fatty alcohol polyoxyethylene ether sulfate 0.2% w / v

[0046] Sodium azide 0.1% w / v

[0047] ZD-Arg-Gly-Arg-pNA 1.4mM

[0048] pH 6.6;

[0049] 2) A factor Xa reagent comprising:

[0050] Tris-HCl buffer 50 mM

[0051] Sodium chloride 100mM

[0052] EDTA disodium 5mM

[0053] Sodium azide 0.1% w / v

[0054] Factor Xa 0.6 IU / mL

[0055] pH 7.6.

[0056] The present application also provides a method for preparing a rivaroxaban detection kit, which comprises the steps of:

[0057] 1) Add in the following order:

[0058] 50 mM to 200 mM 2-morpholineethanesulfonic acid;

[0059] 1.2mM to 2.0mM Factor Xa sensitive substrate;

[0060] 0.2% w / v to 1% w / v of a first surfactant, preferably a fatty alcohol polyoxyethylene ether;

[0061] 0.2% w / v to 1% w / v of a second surfactant, preferably sodium fatty alcohol polyoxyethylene ether sulfate;

[0062] 0.05% w / v to 0.1% w / v preservative;

[0063] 2) adjusting the pH to 6.6 to 7.0, preferably 6.6;

[0064] 3) obtaining a chromogenic substrate reagent;

[0065] The Xa factor sensitive substrate is selected from: ZD-Arg-Gly-Arg-pNA, NaZD-Ar g-Gly-Arg-pNA.2HCl, Suc-Ile-Glu(gamma-pip)-Gly-Arg-pNA.HCl, Suc-Ile-Glu(gamma-pip)-Gly-Arg-pNA.HCl, or a combination thereof; preferably ZD-Arg-Gly-Arg-pNA; the preservative is selected from: sodium azide, PC300, PC950, thimerosal, or a combination thereof; preferably sodium azide.

[0066] In some embodiments, the method of the present application further comprises the steps of:

[0067] A Factor Xa reagent is provided, comprising:

[0068] 50mM to 200mM buffer,

[0069] 0.5 IU / mL to 1.0 IU / mL factor Xa,

[0070] 100mM to 200mM inorganic salts,

[0071] 3mM to 10mM complexing agent,

[0072] 0.05% w / v to 0.1% w / v preservative;

[0073] Wherein, the buffer is selected from: 2-morpholineethanesulfonic acid-sodium hydroxide buffer, tris(hydroxymethylaminomethane)-hydrochloric acid buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, or a combination thereof; the inorganic salt is selected from: sodium nitrite, potassium nitrite, sodium chloride, or a combination thereof; the complexing agent is selected from: ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, citric acid, or a combination thereof; the preservative is selected from: sodium azide, PC300, PC950, thimerosal, or a combination thereof; the pH value of the factor Xa reagent is not less than 7.2, preferably 7.3 to 7.6; more preferably 7.6.

[0074] The present application also provides a rivaroxaban content detection kit, which is prepared by the aforementioned method. DETAILED DESCRIPTION

[0075] Example 1. Confirmation of the composition of the kit

[0076] In order to verify the advantages of the kit of the present invention in terms of anti-interference, stability and repeatability, the kit of the present invention was prepared with kits using different ratios of surfactants and different complexing agents. The specific preparation composition is as follows:

[0077] Table 1. Kits of this application and control kits

[0078]

[0079]

[0080] Example 2. Confirmation of the preparation order of the chromogenic substrate reagent

[0081] The present application found that different addition orders of raw materials in the chromogenic substrate reagent will lead to different performance of the reagent. In order to verify the optimal addition order of the reagent during preparation, the effects of several different addition orders on the performance of the reagent were compared:

[0082] Table 2. Different loading orders of chromogenic substrate reagents

[0083]

[0084] After adding the above reagents in sequence, stir at the same stirring speed for 1 hour and observe the appearance of the reagents.

[0085] Table 3. Appearance of chromogenic substrate reagents prepared in different sample addition orders

[0086]

[0087] Combining Table 2 and Table 3, it can be seen that the sample addition order of Methods 2-3 will cause the chromogenic substrate reagent to have visible suspended matter or precipitation, while the sample addition order of Method 1 can obtain a chromogenic substrate reagent with an ideal appearance.

[0088] Test Example 1. Test of the Anti-interference Ability of the Kit of the Application and the Control Kit

[0089] The kit of the present application prepared according to Example 1 and different control kits were simultaneously tested for anti-interference ability.

[0090] Contains different interferents at different concentrations, including:

[0091] 100mg / dL, 200mg / dL, 300mg / dL, 400mg / dL human hemoglobin Hb,

[0092] 10mg / dL, 15mg / dL, 20mg / dL of free bilirubin and 10mg / dL, 15mg / dL, 20mg / dL of conjugated bilirubin,

[0093] Intralipid at 100mg / dL, 200mg / dL, 300mg / dL, and 400mg / dL;

[0094] The recovery rate was calculated by comparing with the sample spiked with pure water in the same proportion.

[0095] Different types of coagulation analyzers can be selected as the detection instruments, such as the STA RMax coagulation analyzer from Stago in France, the CS-5100 coagulation analyzer from Sysmex in Japan, the ACL TOP750 coagulation analyzer from Wolfen in the United States, etc. The specific instrument usage parameters need to be adjusted accordingly according to different instruments. The anti-interference test results of the test kit taking the STA R Max coagulation analyzer as an example are shown in the following table.

[0096] Table 4. Anti-interference ability test (the data unit in the table is ng / mL)

[0097]

[0098]

[0099]

[0100] The results show that the kit of the present application has outstanding ability in resisting interference from hemoglobin, bilirubin and fat emulsion. By comparison, it is found that replacing the concentration ratio of the surfactant in the chromogenic substrate reagent will unexpectedly affect the anti-interference ability of the reagent.

[0101] Test Example 2. Long-term stability test of the present kit and the control kit

[0102] The long-term stability monitoring of the kit of the present application and different control kits prepared according to Example 1 was carried out simultaneously for a total of 18 months. The kits were stored at 2-8° C. and the stability of the reagents was monitored every two months.

[0103] The stability experiment mainly evaluates the appearance of the reagents and detects the relative deviation of the rivaroxaban quality control product. The deviation of each test from the quality control test at month 0 should not exceed 10%. Each kit is tested three times and the average value is taken. The rivaroxaban quality control product selected for this test is the rivaroxaban quality control product (2 levels) produced by Jiuqiang Biological Company.

[0104] Table 5. Long-term stability test (data in the table are in ng / mL)

[0105]

[0106]

[0107] During the monitoring period, the appearance of the present application kit and the different control kits did not change, and they were all clear liquids without visible precipitation or suspended matter. By comparison, it was found that changing the disodium ethylenediaminetetraacetic acid in the factor Xa reagent would significantly affect the long-term stability of the kit.

[0108] Test Example 3. Repeatability test of the present kit and the control kit

[0109] The rivaroxaban quality control product (2 levels) was simultaneously detected using the kit of the present application prepared according to Example 1 and different control kits. The measurement was repeated 10 times, and the coefficient of variation (CV) was calculated. The kit was considered to have good repeatability when the CV was within ±10%.

[0110] Table 6. Repeatability test (data in the table are in ng / mL)

[0111]

[0112]

[0113] By comparison, it was found that when the pH value of the Factor Xa reagent was 7.6, the kit had better repeatability and a coefficient of variation of <3%.

[0114] Example 4. Applicability of the kit of this application to different types of coagulation analyzers

[0115] In order to evaluate the applicability of the kit of this application on different models of coagulation analyzers that have been marketed, rivaroxaban quality control products (2 levels) and samples prepared with different amounts of rivaroxaban were tested on 4 different models (A to D) of coagulation analyzers, and the model difference was calculated based on A as the benchmark. The relative deviation of the measured values ​​of different models and the host model was required to be no more than ±10%, and the model difference was determined to be controllable. The parameters of the kit on different models of coagulation analyzers are different.

[0116] Table 7. Test results of the difference between the models of the kits in this application

[0117]

[0118]

[0119] The data show that the relative deviations of the test results of the kit of the present application on coagulation analyzers B to D and the test results on coagulation analyzer A are all less than 10%, indicating that the test deviation of the kit on the four different models of coagulation analyzers is small, and the kit can be applied to the above four different models of coagulation analyzers.

[0120] Comparative Example 1

[0121] Compared with the rivaroxaban content detection kit prepared in CN 107576799A, the kit of the present application has statistically significant improvements in anti-interference ability, reagent repeatability and applicability of coagulation analyzers (data not shown).

[0122] The kit of the present application is a liquid reagent, which is more convenient to use than the lyophilized powder reagent disclosed in CN 107576799A, and its stability is not inferior to that of the lyophilized powder reagent.

[0123] Comparative Example 2

[0124] Compared with the rivaroxaban detection kit prepared in CN 116426510 B, the kit of the present application has statistically significant improvements in anti-interference ability and applicability to coagulation analyzers (data not shown).

[0125] In summary:

[0126] 1) The preparation method and kit of the present application have good anti-interference ability: human hemoglobin is not higher than 400 mg / mL, free bilirubin and conjugated bilirubin are not higher than 20 mg / mL, and fat emulsion is not higher than 400 mg / mL, which has no statistically significant effect on the detection of rivaroxaban content.

[0127] 2) The kit of this application can be stored at 2-8°C for at least 1 year with good reproducibility;

[0128] 3) The kit of the present application is compatible with different models of coagulation analyzers.

Claims

1. A rivaroxaban content detection kit, comprising: 1) A chromogenic substrate reagent comprising: 50mM to 200mM first buffer, 1.2mM to 2.0mM Factor Xa sensitive substrate, 0.2% w / v to 1% w / v surfactant, 0.05% w / v to 0.1% w / v of a first preservative; 2) A factor Xa reagent comprising: 50mM to 200mM second buffer, 0.5 IU / mL to 1.0 IU / mL factor Xa, 100mM to 200mM inorganic salts, 3mM to 10mM complexing agent, 0.05% w / v to 0.1% w / v secondary preservative; in: The first buffer and the second buffer are each independently selected from: 2-morpholineethanesulfonic acid-sodium hydroxide buffer, tris-hydrochloric acid buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, or a combination thereof; The factor Xa sensitive substrate is selected from: ZD-Arg-Gly-Arg-pNA, NaZD-Ar g-Gly-Arg-pNA.2HCl, Suc-Ile-Glu (gamma-pip)-Gly-Arg-pNA.HCl, Suc-Ile-Glu (gamma-pip)-Gly-Arg-pNA.HCl, or a combination thereof; The surfactant is selected from: fatty alcohol polyoxyethylene ether, fatty alcohol polyoxyethylene ether sodium sulfate, Tween-20, polyoxyethylene lauryl ether, or a combination thereof; The inorganic salt is selected from: sodium nitrite, potassium nitrite, sodium chloride, or a combination thereof; The complexing agent is selected from: ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, citric acid, or a combination thereof; The first preservative and the second preservative are each independently selected from: sodium azide, PC300, PC950, thimerosal, or a combination thereof; The pH value of the chromogenic substrate reagent is 6.6 to 7.0; The pH value of the Factor Xa reagent is not less than 7.2, preferably 7.3 to 7.6; more preferably 7.

6.

2. The rivaroxaban content detection kit according to claim 1, wherein: The factor Xa sensitive substrate is 1.4 mM ZD-Arg-Gly-Arg-pNA.

3. The rivaroxaban content detection kit according to claim 1, wherein: The first buffer is 50 mM 2-morpholineethanesulfonic acid-sodium hydroxide buffer; The second buffer is 50 mM Tris-hydrochloric acid buffer.

4. The rivaroxaban content detection kit according to claim 1, wherein: The surfactant is a combination of fatty alcohol polyoxyethylene ether and fatty alcohol polyoxyethylene ether sodium sulfate at a concentration ratio of 4:1; Preferably, the concentration of the fatty alcohol polyoxyethylene ether is 0.8% w / v; Preferably, the concentration of the sodium fatty alcohol polyoxyethylene ether sulfate is 0.2% w / v.

5. The rivaroxaban content detection kit according to claim 1, wherein: The first preservative and the second preservative are 0.1% w / v sodium azide; The factor Xa is 0.6 IU / mL; The inorganic salt is 100mM sodium chloride; The complexing agent is 5mM disodium ethylenediaminetetraacetate; The first preservative and the second preservative were 0.1% w / v sodium azide.

6. The rivaroxaban content detection kit according to claim 1, wherein: The chromogenic substrate reagent and the Factor Xa reagent are liquid reagents.

7. The rivaroxaban content detection kit according to claim 1, comprising: 1) A chromogenic substrate reagent comprising: 2) A factor Xa reagent comprising:

8. A method for preparing a rivaroxaban detection kit, comprising the steps of: 1) Add in the following order: 50 mM to 200 mM 2-morpholineethanesulfonic acid; 1.2mM to 2.0mM Factor Xa sensitive substrate; 0.2% w / v to 1% w / v of a first surfactant, preferably a fatty alcohol polyoxyethylene ether; 0.2% w / v to 1% w / v of a second surfactant, preferably sodium fatty alcohol polyoxyethylene ether sulfate; 0.05% w / v to 0.1% w / v preservative; 2) adjusting the pH to 6.6 to 7.0, preferably 6.6; 3) obtaining a chromogenic substrate reagent; The factor Xa sensitive substrate is selected from: ZD-Arg-Gly-Arg-pNA, NaZD-Ar g-Gly-Arg-pNA.2HCl, Suc-Ile-Glu (gamma-pip)-Gly-Arg-pNA.HCl, Suc-Ile-Glu (gamma-pip)-Gly-Arg-pNA.HCl, or a combination thereof; preferably ZD-Arg-Gly-Arg-pNA; The preservative is selected from: sodium azide, PC300, PC950, thimerosal, or a combination thereof; preferably sodium azide.

9. The method according to claim 8, further comprising the steps of: A Factor Xa reagent is provided, comprising: 50mM to 200mM buffer, 0.5 IU / mL to 1.0 IU / mL factor Xa, 100mM to 200mM inorganic salts, 3mM to 10mM complexing agent, 0.05% w / v to 0.1% w / v preservative; in: The buffer is selected from: 2-morpholineethanesulfonic acid-sodium hydroxide buffer, tris(hydroxymethylaminomethane)-hydrochloric acid buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer, or a combination thereof; The inorganic salt is selected from: sodium nitrite, potassium nitrite, sodium chloride, or a combination thereof; The complexing agent is selected from: ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, citric acid, or a combination thereof; The preservative is selected from: sodium azide, PC300, PC950, thimerosal, or a combination thereof; The pH value of the Factor Xa reagent is not less than 7.2, preferably 7.3 to 7.6; more preferably 7.

6.

10. A rivaroxaban content detection kit, which is prepared by the method according to claim 8 or 9.

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