CN115032164A - Method for detecting whether heparin sodium contains coagulation factor Xa direct inhibitor - Google Patents
Method for detecting whether heparin sodium contains coagulation factor Xa direct inhibitor Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, relates to an analysis and detection method of medicine components, and particularly provides a detection method for whether heparin sodium contains a blood coagulation factor Xa direct inhibitor. The method specifically comprises the following steps: adding heparin sodium solution, adding blood coagulation factor Xa, adding buffer solution to make the volume of the solution reach 150 mu L, adding thrombin chromogenic substrate S-2238 after incubation, adding buffer solution to make the volume reach 200 mu L, and measuring the absorbance at the wavelength of 405 nm after incubation; the inhibition was then calculated, wherein when the inhibition > 9.0%, it is indicated that the sample contains a factor Xa direct inhibitor. The detection method is convenient to operate, fast and efficient, high in sensitivity, low in detection limit and good in stability, and can be used for detecting and screening various blood coagulation factor Xa direct inhibitors.
Description
Technical Field
The invention belongs to the field of biological medicines, relates to an analysis and detection method of medicine components, and particularly provides a detection method for whether heparin sodium contains a blood coagulation factor Xa direct inhibitor.
Background
Heparin is a highly sulfated glycosaminoglycan extracted from the porcine small intestine and is the most widely used anticoagulant in the clinic. The anticoagulant mechanism of heparin sodium mainly forms a heparin-AT-III compound by combining with antithrombin III (AT-III), and the compound inhibits the activity of blood coagulation factors II and Xa so as to play an anticoagulant role, thereby being an indirect inhibitor of the blood coagulation factors II and Xa. Direct inhibitors of factor Xa include known inhibitors of apixaban, rivaroxaban, edoxaban and the like and unknown direct inhibitors of factor Xa. The production process of extracting heparin from small intestines of pigs is long, early extraction is not generally performed by medicine enterprises, in order to increase activity, the risk of adding a chemical coagulation factor Xa direct inhibitor into heparin exists, the activity of heparin sodium is mainly determined by determining the inhibitory activity of the coagulation factor Xa in Chinese pharmacopoeia, the method cannot distinguish the direct inhibitors of the heparin sodium and the chemical coagulation factor Xa, and the medicine enterprises can not be detected and are finally transmitted to a terminal product.
The pharmacological properties of heparin sodium, such as drug efficacy, pharmacokinetics and the like, are significantly different from those of the direct coagulation factor Xa inhibitor, for example, after the heparin sodium anticoagulant drug is used excessively, protamine sulfate is often used to inactivate the heparin sodium when spontaneous bleeding is caused, but the protamine sulfate is ineffective for the direct coagulation factor Xa inhibitor, and if the direct coagulation factor Xa inhibitor is added into the heparin sodium injection, a huge clinical risk exists when patients suffer from spontaneous bleeding. Because the unit activity price of heparin sodium is higher than that of a coagulation factor Xa direct inhibitor, and the possibility of adding the coagulation factor Xa direct inhibitor into heparin sodium exists, the conventional detection method can adopt HPLC (high performance liquid chromatography), liquid chromatography and mass spectrometry and the like for adding a known coagulation factor Xa direct inhibitor into heparin sodium, but the method is complex to operate and has high requirements on equipment, and the coagulation factor Xa direct inhibitor added in the heparin extraction process is unknown at many times, so that the conventional detection method cannot effectively prevent the illegal feeding problem of extracting heparin from small intestines, and the prior art has no document for monitoring and inspecting the behaviors.
Disclosure of Invention
Aiming at the problem that a mature and effective method for screening a blood coagulation factor Xa direct inhibitor from heparin sodium is not established in the prior art, the invention provides the detection method of the blood coagulation factor Xa direct inhibitor from heparin sodium, the detection method is convenient to operate, is rapid and efficient, has the detection limit as low as 1 nM, has good stability, and can be used for detecting and screening various blood coagulation factor Xa direct inhibitors.
In order to achieve the above object, the technical solution of the present invention includes the following contents:
the invention provides a method for detecting a coagulation factor Xa direct inhibitor in heparin sodium, which specifically comprises the following steps:
step 1: adding a proper amount of heparin sodium solution into the ELISA plate, adding a proper amount of blood coagulation factor Xa, adding a proper amount of buffer solution to ensure that the volume of the mixed solution is 150 mu L, and incubating;
and 2, step: adding a proper amount of thrombin chromogenic substrate S-2238 into the mixed solution obtained in the step 1, adding a buffer solution to ensure that the volume of the mixed solution is 200 mu L, incubating to obtain a sample solution, and measuring the absorbance of the sample solution at the wavelength of 405 nm to obtain A Sample (I) ；
And step 3: the blank group replaces the heparin sodium solution in the sample solution with buffer solution with the same volume, and the absorbance of the blank group solution at the wavelength of 405 nm is measured to obtain A Blank space (ii) a Calculating the inhibition rate of the blood coagulation factor Xa direct inhibitor, wherein the inhibition rate (%) = (A) Blank space -A Sample (I) ）/A Blank space 100% percent of inhibition rate>At 9.0%, indicating that the sample contains a factor Xa direct inhibitor; when the inhibition rate is less than or equal to 9.0 percent, the sample does not contain the direct coagulation factor Xa inhibitor.
Further, the factor Xa direct inhibitor is a currently known factor Xa direct inhibitor and an unknown factor Xa direct inhibitor.
The known direct blood coagulation factor Xa inhibitor is one or more of apixaban, rivaroxaban and edoxaban.
Further, the buffer solutions described in step 1 and step 2 each contained 20 mM Tris-HCl, 100mM NaCl, and buffer pH = 7.4.
Further, the temperature of the incubation in the step 1 is 0-50 ℃, and the incubation time is 1-120 min; preferably, the incubation temperature is 37 ℃ and the incubation time is 30 min.
Further, the final concentration of the heparin sodium in the step 1 is 1-100 IU/mL; preferably 1-50 IU/mL; more preferably 50 IU/mL.
Further, the final concentration of the blood coagulation factor Xa in the step 1 is 0.2-10.0 nkat/mL; preferably 2 nkat/mL.
Further, the final concentration of the chromogenic substrate S-2238 in the step 2 is 0.05-1.00 mM; preferably 0.25 mM.
Further, the temperature of the incubation in the step 2 is 0-50 ℃, and the incubation time is 1-5 min; preferably, the incubation temperature is 37 ℃ and the incubation time is 2 min.
Compared with the prior art, the invention provides a detection method of a coagulation factor Xa direct inhibitor in heparin sodium, which has the advantages of convenient operation, rapidness, high efficiency, high sensitivity, low detection limit and good stability, can be used for detecting and screening various coagulation factor Xa direct inhibitors, and has the following beneficial effects:
the invention overcomes the defect that the existing method for measuring the activity of the heparin sodium can not distinguish the coagulation factor Xa indirect inhibitor heparin sodium from the direct inhibitor, can detect whether the known coagulation factor Xa direct inhibitor is contained or not, can realize the detection of the unknown coagulation factor Xa direct inhibitor, and can prevent the addition of the Xa direct inhibitor in the process of extracting and producing the heparin. In addition, the detection limit of the blood coagulation factor Xa direct inhibitor is 1 nM, the sensitivity is high, and the detection limit is low.
Detailed Description
The present application is described in further detail below by way of examples to enable those skilled in the art to practice the present application. It is to be understood that other embodiments may be utilized and that changes may be made without departing from the spirit or scope of the present application. To avoid detail not necessary to enable those skilled in the art to practice the application, the description may omit certain information known to those skilled in the art. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims. The following examples are presented to facilitate a better understanding of the present application and are not intended to limit the scope of the present application.
Example 1 measurement of coagulation factor Xa direct inhibitory Activity of heparin sodium solution
The pH =7.4 was prepared with a buffer containing 20 mM Tris-HCl and 100mM NaCl, 50. mu.L of a coagulation factor Xa solution (8 nkat/mL) was added to the microplate, appropriate amounts of heparin sodium solutions were added to give final concentrations of 1 IU/mL, 10 IU/mL, 50 IU/mL and 100 IU/mL, the buffer was added to give a volume of 150. mu.L, the mixture was incubated at 37 ℃ for 30 min, and 50. mu.L of thrombin chromogenic substrate S-2238(1 mM) was added. The blank group replaced the heparin sodium solution in the sample solution with an equal volume of buffer. After the reaction, the absorbance of the reaction system at the wavelength of 405 nm is measured by a microplate reader 5 min before the reaction system is started, the absorbance is measured every 1 min, and the measurement result is shown in table 1. The absorbance measured at 405 nm after 2 min of the blank solution did not increase any more, indicating that the substrate was completely reacted after 2 min. When the concentration of the heparin sodium is more than 50 IU/mL, the activity of the blood coagulation factor Xa is inhibited, and when the concentration of the heparin sodium is 1 IU/mL, 10 IU/mL or 50 IU/mL, the activity of the blood coagulation factor Xa is not inhibited, so that the concentration of the heparin sodium in the detection system is 1-50 IU/mL.
TABLE 1 measurement results of inhibitory Activity
| Heparin sodium concentration IU/mL | 1 min | 2 min | 3 min | 4 min | 5 min |
| Blank space | 0.451 | 0.92 | 0.902 | 0.883 | 0.93 |
| 1 | 0.449 | 0.909 | 0.884 | 0.918 | 0.927 |
| 10 | 0.456 | 0.886 | 0.918 | 0.896 | 0.929 |
| 50 | 0.446 | 0.911 | 0.925 | 0.881 | 0.917 |
| 100 | 0.307 | 0.674 | 0.682 | 0.659 | 0.678 |
Example 2 blank solution assay
The buffer solution containing 20 mM Tris-HCl and 100mM NaCl at pH =7.4 was prepared, 50. mu.L of a coagulation factor Xa solution (8 nkat/mL) and 100. mu.L of a buffer solution (pH =7.4 containing 20 mM Tris-HCl and 100mM NaCl) were added to the microplate, and the microplate was incubated at 37 ℃ for 30 min, followed by addition of 50. mu.L of thrombin chromogenic substrate S-2238(1 mM), and absorbance at a wavelength of 405 nm was measured after incubation at 37 ℃ for 2 min, as shown in Table 2, with the blank solution having an RSD of 3.4% and the blank solution having a stable absorbance at a wavelength of 405 nm. The detection limit of the inhibition rate is 9.0%.
TABLE 2 measurement results of blank solution
Example 3 detection of direct inhibitors of coagulation factor Xa
The reagent kit is prepared by preparing a buffer solution with pH =7.4 and containing 20 mM Tris-HCl and 100mM NaCl, adding 50 mu L of a coagulation factor Xa solution (8 nkat/mL) into an enzyme label plate, respectively adding a proper amount of apixaban, rivaroxaban, edoxaban and a coagulation factor Xa direct inhibitor mixture to make the final concentration of the mixture respectively 1 nM, 3 nM, 15 nM and 30 nM, adding the buffer solution to make the volume to 150 mu L, incubating the mixture at 37 ℃ for 30 min, respectively adding 50 mu L of thrombin chromogenic substrate S-2238(1 mM), and replacing the coagulation factor Xa direct inhibitor with the buffer solution with the same volume in a blank control group. After incubation for 2 min at 37 ℃, the absorbance at the wavelength of 405 nm is measured, and the inhibition rate is calculated according to the formula: inhibition (%) = (a) Blank space -A Sample (I) ）/A Blank space 100% and the results are shown in table 3, it can be seen that the inhibition rates of the apixaban, rivaroxaban, edoxaban and coagulation factor Xa direct inhibitor mixture of 1 nM are 14.7%, 24.9%, 12.5% and 18.3%, respectively, and the inhibition rate is greater than the detection limit of the blank solution and is 9.0%, which indicates that the method has high sensitivity, and can detect 1 nM of apixaban, rivaroxaban and edoxaban.
TABLE 3 detection results of coagulation factor Xa direct inhibitors
Example 4 heparin sodium sample assay with addition of known factor Xa direct inhibitors
Considering that heparin sodium may affect the factor Xa direct inhibitor, this example examines the accuracy of the method by adding the factor Xa direct inhibitor to the heparin sodium solution, measuring the inhibition rate.
Preparing a buffer solution with pH =7.4 and containing 20 mM Tris-HCl and 100mM NaCl; adding a heparin sodium solution into an enzyme label plate, adding a blood coagulation factor Xa solution, adding a blood coagulation factor Xa direct inhibitor apixaban, adding a buffer solution to make the volume reach 150 mu L, making the final concentration of the heparin sodium solution be 50 IU, making the final concentration of the blood coagulation factor Xa direct inhibitor be 8 nkat/mL, making the final concentration of the mixture of the apixaban, rivaroxaban, edoxaban and the blood coagulation factor Xa direct inhibitor be 3 nM respectively, incubating for 30 min at 37 ℃, then adding 50 mu L of thrombin chromogenic substrate S-2238(1 mM) respectively, and incubating for 2 min at 37 ℃ to obtain a solution for testing the recovery rate. The control solution replaced the sodium heparin solution in the recovery assay solution with an equal volume of buffer. After 2 min, the absorbance at 405 nm was measured, and 6 experiments were performed to calculate the recovery rate (%) = the inhibition rate of the recovery rate solution/the inhibition rate of the control solution × 100%. The results are shown in tables 4, 5, 6 and 7.
TABLE 4 test results of control solutions and recovery test solutions
As can be seen from the above experiments, the inhibition ratio is 47.0% and is more than 9.0% when the concentration of heparin sodium is 50 IU/mL and the concentration of apixaban is 3 nM; the recovery rate of the inhibition rate is 97.0%, which indicates that the heparin sodium has no significant interference on the detection of the coagulation factor Xa Apixaban direct inhibitor.
TABLE 5 rivaroxaban control solution and recovery test solution test results
TABLE 6 Edixaban control solution and solution test results for recovery test
TABLE 7 test results for coagulation factor Xa direct inhibitor mixture control solution and recovery test solution
As can be seen from the above experiments, the inhibition ratio is 47.0% and is more than 9.0% when the concentration of heparin sodium is 50 IU/mL and the concentration of apixaban is 3 nM; the recovery rate of the inhibition rate is 97.0 percent; the inhibition rate is 39.0% and is more than 9.0% when the rivaroxaban concentration is 3 nM; the recovery rate of the inhibition rate is 96.4%; the inhibition rate of Idoxaban is 31.8 percent and is more than 9.0 percent when the concentration of Idoxaban is 3 nM; the recovery rate of the inhibition rate is 101.6%; the inhibition rate of the mixture of the direct blood coagulation factor Xa inhibitors is 42.7 percent and is more than 9.0 percent when the concentration of the mixture is 3 nM; the recovery rate of the inhibition rate is 101.9%, which indicates that the heparin sodium has no significant interference on the detection of the factor Xa direct inhibitor.
Sample assay
Selecting 3 parts of heparin sodium solution of 10 batches, respectively adding 1 nM apixaban, 1 nM rivaroxaban or 1 nM edoxaban, then randomly numbering, detecting the samples by adopting the detection method disclosed by the invention, respectively detecting the samples with the inhibition rate of more than 9.0% by adopting high performance liquid chromatography, and determining that the samples with the inhibition rate of more than 9.0% respectively contain apixaban, rivaroxaban and edoxaban. Therefore, the method established by the invention is accurate and reliable, and plays a great role in the aspect of standardizing whether the coagulation factor Xa direct inhibitor is added into the heparin sodium injection.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited by the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, combinations, substitutions and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A method for detecting whether heparin sodium contains a coagulation factor Xa direct inhibitor is characterized by comprising the following steps:
step 1: adding blood coagulation factor Xa into a heparin sodium solution, adding a buffer solution to obtain a mixed solution, and incubating;
and 2, step: adding a thrombin chromogenic substrate S-2238 into the mixed solution obtained in the step 1, adding a buffer solution, incubating to obtain a sample solution, and measuring the absorbance of the sample solution at a wavelength of 405 nm to obtain A Sample(s) ；
And step 3: the blank group replaces the heparin sodium solution in the sample solution with buffer solution with the same volume, and the absorbance of the blank group solution at the wavelength of 405 nm is measured to obtain A Blank space (ii) a According to the formula: inhibition (%) = (a) Blank space -A Sample (I) ）/A Blank space Calculating the inhibition rate of a direct inhibitor of coagulation factor Xa by 100%, wherein the inhibition rate is calculated as>9.0%, indicating that the sample contains a factor Xa direct inhibitor.
2. The assay of claim 1, wherein the factor Xa direct inhibitor is one or more of apixaban, rivaroxaban, and edoxaban.
3. The assay of claim 1, wherein the buffer solutions of steps 1 and 2 each comprise 20 mM Tris-HCl, 100mM NaCl, and the buffer pH = 7.4.
4. The detection method according to claim 1, wherein the temperature of the incubation in step 1 is 0 to 50 ℃ and the incubation time is 1 to 120 min.
5. The detection method according to claim 1, wherein the concentration of heparin sodium in the mixed solution in the step 1 is 1 to 50 IU/m.
6. The method according to claim 1, wherein the final concentration of the coagulation factor Xa in the mixed solution of step 1 is 0.2 to 10.0 nkat/mL.
7. The detection method according to claim 1, wherein the final concentration of the chromogenic substrate S-2238 in the sample solution in step 2 is 0.05 to 1.00 mM.
8. The detection method according to claim 1, wherein the temperature of the incubation in step 2 is 0 to 50 ℃ and the incubation time is 1 to 5 min.
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