A quality detection method for bivalirudin raw material and its preparation

Abstract

The invention discloses a quality detection method for a bivalirudin raw material and a preparation thereof. The method uses a specific buffer to prepare a test solution and a standard solution, respectively uses the natural logarithm of the concentration of the test solution and the standard solution as the abscissa, and uses the thrombin inhibition percentage (%TI) as the ordinate to obtain a standard curve equation and a regression equation of a test product. The concentration of the standard product when %TI=45% is calculated according to the standard curve, and the concentration is substituted into the regression equation of the test product to calculate the thrombin inhibition percentage (%TI) of bivalirudin. The method has high specificity, high accuracy, high detection efficiency, and good reproducibility, and is suitable for the quality control of the bivalirudin raw material and a preparation thereof.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis and detection, and in particular to a quality detection method for a bivalirudin raw material drug and a preparation thereof. Background Art

[0002] Bivalirudin (CAS: 128270-60-0, molecular formula: C 98 H 138 N 24 O 33 ) is a synthetic anticoagulant drug, mainly used for elective percutaneous coronary intervention (PCI) in adults. Bivalirudin is a 20-peptide analog of hirudin, a direct, specific, and reversible inhibitor of thrombin. Whether thrombin is in the blood circulation or bound to a thrombus, this product can specifically bind to its catalytic site and anion binding site, thereby directly inhibiting the activity of thrombin.

[0003] At present, there are two main methods reported for determining the biological activity of bivalirudin and its preparations. The first method is to use the coagulation method, which determines the coagulation time of fibrinogen based on the visual observation of the tester, and is subject to significant subjective interference by the tester. The second method is to use ultraviolet spectrophotometry to determine the biological activity of bivalirudin and its preparations. This method is greatly affected by room temperature, has no self-shaking function, has a small intermolecular contact area, is insufficient in reaction, has low reproducibility, and has poor linearity of the obtained standard curve.

[0004] For example, the method for determining biological activity in the quality standard of bivalirudin for injection with the currently disclosed drug standard number YBH00142019 adopts ultraviolet spectrophotometry, and uses potassium hydrogen phosphate trihydrate to prepare 0.1 mol / L potassium hydrogen phosphate buffer (pH 7.0). It is necessary to use blank excipient solution to prepare three quality control solutions of high, medium and low concentrations, and to carry out enzymatic reaction to obtain system suitability solution and sample solution. The preparation process is complicated and easy to mix. The ultraviolet spectrophotometer cannot achieve temperature control, resulting in low accuracy of results, poor reproducibility, and low detection efficiency.

[0005] In addition, the quality standard of standard number JX20160176 discloses a method for determining the activity of bivalirudin for injection: namely, using ultraviolet spectrophotometry, which uses bovine serum albumin as an enzyme stabilizer to prevent enzyme decomposition and nonspecific adsorption, but requires the determination of the absorbance to determine the substrate volume, thrombin volume and the accurate concentration of the test sample stock solution, which increases uncontrollable factors, and the ultraviolet spectrophotometer has poor reproducibility due to the inability to achieve temperature control, resulting in low accuracy and poor reproducibility of the results. In addition, after adding the buffer, substrate, reference substance or test sample, it is necessary to water bath for 10±5 minutes, and adding thrombin requires another 20 minutes of water bath, which has a long reaction time and cumbersome operation.

[0006] In view of the limitations of the above methods, this patent discloses a method for determining the biological activity of bivalirudin and its preparations with exclusive, accurate, rapid and good reproducibility, which can effectively control the quality of bivalirudin and its preparations. Summary of the invention

[0007] In view of this, the present invention provides a quality detection method for bivalirudin raw materials and preparations thereof. The method has high specificity, high accuracy, high detection efficiency, and good reproducibility, and is suitable for quality control of bivalirudin raw materials and preparations thereof.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A quality detection method for a bivalirudin raw material drug and a preparation thereof comprises the following steps:

[0010] Step 1: Mix the bivalirudin raw material or its preparation to be tested with Tris-HCl buffer A to obtain a test solution; mix the bivalirudin standard with Tris-HCl buffer A to obtain a standard solution;

[0011] The Tris-HCl buffer A is a Tris-HCl buffer with a pH value of 7.60-9.20, containing 0.03-0.07 mol / L of tris(hydroxymethyl)aminomethane, 10.20 g / L of sodium chloride, 2.80 g / L of disodium ethylenediaminetetraacetic acid, and 1.0 g / L of polyethylene glycol 6000.

[0012] Step 2: Incubate Tris-HCl buffer A, the test solution and the standard solution with the thrombin solution respectively; then add the bivalirudin chromogenic substrate solution and incubate again; measure the absorbance at 405 nm, respectively, and use the natural logarithm of the concentration of the bivalirudin standard solution and the test solution as the abscissa and %TI as the ordinate to obtain the standard curve and the regression equation of the bivalirudin sample to be tested;

[0013] According to the standard curve, the concentration of the standard solution when %TI=45% is calculated, and the concentration is substituted into the regression equation of the bivalirudin sample to calculate the thrombin inhibition percentage of the bivalirudin bulk drug or its preparation to be tested.

[0014] In the detection process of bivalirudin and its preparation in the present invention, an ELISA instrument with a constant temperature and mixing function is used. One end of the chromogenic substrate is a chromogenic group (PNA). Thrombin can catalyze the dissociation of PNA, causing a color change in the sample to be tested. The free PNA is yellow and produces an absorption peak at a wavelength of 405nm. Within a certain period of time, the absorbance of the reaction product is linearly correlated with the enzyme activity. The change in light absorption of the sample to be tested at 405nm caused by the release of PNA is detected, thereby determining the activity of bivalirudin.

[0015] In some specific embodiments of the present invention, the Tris-HCl buffer A is a Tris-HCl buffer with a pH of 8.40, containing 0.05 mol / L tris(hydroxymethyl)aminomethane, 10.20 g / L sodium chloride, 2.80 g / L disodium ethylenediaminetetraacetic acid, and 1.0 g / L polyethylene glycol 6000.

[0016] In the present invention, the concentrations of the standard solution and the test solution are 1.0 μg / ml-20 μg / ml, more preferably 2.5 μg / ml-10 μg / ml.

[0017] In the present invention, the thrombin solution is prepared by the following method: 100 NIH thrombin lyophilized powder is added with 1000 μl of purified water for re-dissolution, and then 9000 μl of Tris-HCl buffer A is added and mixed.

[0018] In the present invention, there is no special requirement for the type of bivalirudin chromogenic substrate, and any commonly used one in the art can be used. The concentration of the bivalirudin chromogenic substrate solution is preferably 0.1-1.0 mg / ml.

[0019] In the present invention, the bivalirudin preparation includes bivalirudin powder for injection or bivalirudin injection.

[0020] In the present invention, the detected ambient temperature in step 2 is 18°C ​​to 22°C, specifically 18°C, 20°C or 22°C.

[0021] In the present invention, the incubation in step 2 is shaking incubation at 35-39° C. for 50-150 s, preferably shaking incubation at 37° C. for 120 s; the further incubation is shaking incubation at 35-39° C. for 5-15 s, preferably shaking incubation at 37° C. for 10 s.

[0022] In the present invention, the incubation after adding thrombin in step 2 is shaking incubation at 35-39°C for 50-150s, preferably shaking incubation at 37°C for 120s; the further incubation is shaking incubation at 35-39°C for 5-15s, preferably shaking incubation at 37°C for 10s after adding the chromogenic substrate.

[0023] In the present invention, the absorbance values ​​are measured at 405 nm, wherein the absorbance values ​​are measured 50s to 70s after the end of the re-incubation, and the measurement time can be specifically 50s, 60s or 70s.

[0024] In the present invention, the calculation formula for the percentage of thrombin inhibition (%TI) in the reference solution series and the sample solution series is:

[0025] %TIstandard=(Absblank-Absstandard)*100% / Absblank;

[0026] %TIsample=(Absblank-Abssample)*100% / Absblank;

[0027] Absblank = the average value of blank absorbance readings corresponding to each series of solutions;

[0028] Absstandard = the average value of the absorbance readings at each concentration of the reference substance;

[0029] Abssample = the average of the absorbance readings at each sample concentration.

[0030] The invention adopts a specific buffer to prepare a test solution and a standard solution, respectively uses the natural logarithm of the concentration of the test solution and the standard solution as the abscissa, and uses the thrombin inhibition percentage (%TI) as the ordinate, to obtain a standard curve equation and a regression equation of the test product. The concentration of the standard product when %TI=45% is calculated according to the standard curve, and the concentration is substituted into the regression equation of the test product to calculate the thrombin inhibition percentage (%TI) of bivalirudin. The method has high specificity, high accuracy, high detection efficiency, good reproducibility, and is suitable for the quality control of bivalirudin raw materials and preparations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 1. The selected analysis diagram of the linear concentration range in Example 1 is shown;

[0032] Figure 2 The linear curve of the reference substance in Example 1 is shown;

[0033] Figure 3 The linear curve of the sample to be tested in Example 1 is shown;

[0034] Figure 4 The linear curve of the reference substance in Example 3 is shown;

[0035] Figure 5 The linear curve of reference preparation-1 in Example 3 is shown;

[0036] Figure 6The linear curve of reference preparation-2 of Example 3 is shown. DETAILED DESCRIPTION

[0037] The present invention provides a quality detection method for bivalirudin raw materials and preparations thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0038] The present invention provides a quality detection method for bivalirudin raw material and its preparation, and the specific process is as follows:

[0039] 1. Instruments and reagents

[0040] Instrument: Microplate reader (BioTek EPOCH2NS)

[0041] Reagents: chromogenic substrate S-2238 (HD-Phe-Pip-Arg-pNa·2HCl) (HYPHEN BioMed, specification: 25 mg, batch number: F2000197), thrombin (HYPHEN BioMed, specification: 100 NIH, batch number: F2000915), bivalirudin reference substance (Chengdu Shengnuo Bio-Pharmaceutical Co., Ltd., content ≥88.9%, batch number: C1-006 / D1901F04), bivalirudin reference preparation (Sandoz Inc. batch number: KF5245).

[0042] Tris(hydroxymethyl)aminomethane (Sinopharm Chemical Reagent Co., Ltd., 20210118), sodium chloride (Xilong Chemical Co., Ltd., 2009081), disodium ethylenediaminetetraacetic acid (Sichuan Xilong Scientific Co., Ltd., 2012251), polyethylene glycol 6000 (Sinopharm Chemical Reagent Co., Ltd., 20201215), and water was purified water.

[0043] 2. Experimental Methods

[0044] (1) Preparation of Tris-HCl buffer A

[0045] Weigh 0.03-0.07 mol of tris(hydroxymethyl)aminomethane, 10.20 g of sodium chloride, 2.80 g of disodium ethylenediaminetetraacetic acid, and 1.0 g of polyethylene glycol 6000, add 800 ml of purified water to dissolve, adjust the pH value to 7.60-9.20 with hydrochloric acid, add water to make the volume to 1 L, and obtain Tris-HCl buffer A. The specific concentration and pH value settings are shown in the specific embodiment.

[0046] (2) Preparation of sample test series solutions

[0047] Take a bottle of bivalirudine preparation (specification: 250 mg), add an appropriate amount of purified water to dissolve and dilute to 100 μg / ml as the sample stock solution, then measure an appropriate amount of the sample stock solution, add Tris-HCl buffer to dilute to a solution with a concentration of 1.0-20.0 μg / ml. The specific concentration setting is shown in the specific embodiment.

[0048] (3) Preparation of reference test series solutions

[0049] Accurately weigh 10 mg of the bivalirudin reference substance, add Tris-HCl buffer to dissolve and dilute to prepare a reference substance stock solution containing about 100 μg of bivalirudin per 1 ml, then measure an appropriate amount of the reference substance stock solution, add Tris-HCl buffer to dilute to prepare a solution with a concentration of 1.0-20.0 μg / ml. The specific concentration setting is shown in the specific embodiment.

[0050] (4) Using Tris-HCl buffer as a blank, add the reference test solution to the microplate reader and the sample test solution to the microplate reader according to the following procedure and sequence (parallel determination twice).

[0051] Table 1 Test procedures

[0052]

[0053] Table 2 Test sequence

[0054]

[0055]

[0056] Note: Blank (B), reference solution concentration (S1, S2, S3, S4), first sample solution concentration (T1, T2, T3, T4), second sample solution concentration (U1, U2, U3, U4).

[0057] With the natural logarithm of the concentration of the reference series solution and the sample series solution as the horizontal axis and %TI as the vertical axis, the linear regression graphs of the reference and samples were drawn respectively.

[0058] According to the reference substance regression equation, the reference substance concentration when %TI=45% was calculated, and the concentration was substituted into the sample regression equation to calculate the percentage of thrombin inhibition (%TI) of bivalirudin.

[0059] The present invention will be fully described below in conjunction with the technical solutions of the embodiments of the present invention, but the technical solutions of the present invention are not limited to the specific implementation modes listed below.

[0060] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0061] The present invention will be further described below in conjunction with embodiments:

[0062] Example 1

[0063] The thrombin solution, the bivalirudin reference solution and the bivalirudin sample solution were prepared respectively with Tris-HCl buffer A (0.05 mol / L, pH=8.40).

[0064] 10.47 mg of bivalirudin reference substance was accurately weighed, and reference substance stock solution was prepared with Tris-HCl buffer A (0.05 mol / L, pH=8.40), and then diluted to concentrations of 1.16 μg / ml, 2.33 μg / ml, 4.65 μg / ml, 9.31 μg / ml, 18.62 μg / ml, 37.23 μg / ml, and 55.85 μg / ml as bivalirudin reference substance solution. The microplate reader was preheated to 37°C, and Tris-HCl buffer A (0.05 mol / L, pH=8.40) was used as blank. The absorbance of the above different series of solutions at 60 s was measured at 405 nm according to the procedures and sequences in Tables 1 and 2 above. The results are shown in Table 3 below.

[0065] Table 3 Linear concentration selection determination results

[0066]

[0067]

[0068] A linear regression graph was drawn with the natural logarithm of the concentration of the reference substance series solution as the abscissa and %TI as the ordinate. It can be seen that the linearity is significant within the concentration range of 1.16μg / ml-9.31μg / ml. The curve equation is: y=16.746x+27.405, r=0.9862, see Figure 1 .

[0069] Accurately weigh 10.41 mg of bivalirudin reference substance, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) to prepare reference substance stock solution, and then dilute to concentrations of 1.16 μg / ml, 2.31 μg / ml, 4.63 μg / ml, 9.25 μg / ml as bivalirudin reference substance series solutions, and then take bivalirudin preparation to prepare bivalirudin sample series solutions with concentrations of 1.36 μg / ml, 2.71 μg / ml, 5.43 μg / ml, and 10.85 μg / ml. Preheat the microplate reader to 37°C, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) as blank, and measure the absorbance of the above different series of solutions at 405 nm at 60s according to the procedures and sequences in Tables 1 and 2 above. The results are shown in Table 4 below. Figure 2 and Figure 3 .

[0070] Table 4 Linearity determination results

[0071] Linear equations r Reference y=17.228x+24.045 0.9916 sample y=16.598x+26.412 0.9949

[0072] Example 2

[0073] A series of reference solutions were prepared using Tris-HCl buffer. Tris-HCl buffer was prepared according to the method described under "Specific Implementation Methods". The pH of the buffer (7.60, 8.00, 8.40, 8.80, 9.20) and the concentration of tris(hydroxymethyl)aminomethane (0.03, 0.04, 0.05, 0.06, 0.07 mol / L) were changed to determine the biological activity.

[0074] Accurately weigh 10.37 mg of the bivalirudin reference substance, use the above-mentioned Tris-HCl buffer A under different conditions to prepare the reference substance stock solution, and then dilute to a concentration of 1.18 μg / ml, 2.35 μg / ml, 4.69 μg / ml, 9.55 μg / ml as a series of bivalirudin reference substance solutions. Preheat the microplate reader to 37°C, use the Tris-HCl buffer as a blank, and measure the absorbance of the above-mentioned different series of solutions at 405 nm at 60 seconds according to the procedure in Table 1 above.

[0075] The linear regression graph of the reference substance was drawn with the natural logarithm of the concentration of the reference substance series solution as the horizontal axis and %TI as the vertical axis. Based on the linear regression graph of the reference substance, the linear correlation of the reference substance under different buffer conditions was compared. The specific results are shown in Tables 5 to 6 below.

[0076] Table 5 Linear equation results obtained in Example 2

[0077]

[0078] Table 6 shows the corresponding correlation coefficient (r) results measured in Table 5

[0079]

[0080] From the results, we can see that Tris-HCl buffer with a pH value of 8.40 and a concentration of 0.05 mol / L was used to prepare the Tris. The results showed that the linearity of the regression equation detected after treatment with the buffer was significant and the reproducibility was good.

[0081] Example 3

[0082] Accurately weigh 10.99 mg of the bivalirudin reference substance, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) to prepare the reference substance stock solution, and then dilute to a concentration of 1.22 μg / ml, 2.43 μg / ml, 4.86 μg / ml, 9.73 μg / ml as a series of bivalirudin reference substance solutions, and then take the bivalirudin reference preparation to prepare a series of solutions with concentrations of 1.39 μg / ml, 2.79 μg / ml, 5.57 μg / ml, and 11.14 μg / ml. Preheat the microplate reader to 37°C, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) as a blank, and measure the absorbance of the above different series of solutions at 405 nm at 60s according to the procedure in Table 1 above. Parallel determination is performed twice.

[0083] The linear regression graphs of the reference substance and the reference preparation were drawn with the natural logarithm of the concentration of the reference substance series solution and the concentration of the reference preparation series solution as the abscissa and %TI as the ordinate. The equation of the standard curve of the reference substance was: y = 15.487x + 33.901, r = 0.9891, see Figure 4 According to the regression equation of the reference substance, the natural logarithm of the concentration corresponding to 45% TI is 0.717. Substituting the natural logarithm of the concentration corresponding to 45% TI of the reference substance into the standard curve equation of the reference preparation, the thrombin inhibition percentage (% TI) is obtained. The results are shown in Table 7 and Figures 5-6 .

[0084] Table 7 Example 3 Measurement results

[0085]

[0086] Example 4

[0087] Three batches of bivalirudin preparations produced by Kunming Longjin Pharmaceutical Co., Ltd. were selected, with batch numbers 20210413, 20210420, and 20210422, and the specifications were all 250 mg.

[0088] 10.60 mg of the bivalirudin reference substance was accurately weighed, and a reference substance stock solution was prepared with Tris-HCl buffer A (0.05 mol / L, pH=8.40), and then diluted to a concentration of 1.18 μg / ml, 2.36 μg / ml, 4.71 μg / ml, and 9.42 μg / ml as a series of bivalirudin reference substance solutions, and then two bottles of bivalirudin preparations of each batch were taken to prepare a series of bivalirudin sample solutions, and the specific dilution concentrations were shown in Table 6. The microplate reader was preheated to 37°C, and Tris-HCl buffer A (0.05 mol / L, pH=8.40) was used as a blank, and the absorbance of the above different series of solutions at 60 s was measured at 405 nm according to the procedures and sequences in Tables 1 and 2 above.

[0089] Table 8 Concentration of three batches of preparation sample solutions

[0090] Preparation batch number Concentration 1 (μg / ml) Concentration 2 (μg / ml) Concentration 3 (μg / ml) Concentration 4 (μg / ml) 20200413 1.22 2.32 4.95 9.85 20210420 1.19 2.27 4.86 9.77 20210422 1.31 2.33 4.96 10.18

[0091] The natural logarithm of the concentration of the reference substance series solution and the sample series solution concentration was used as the abscissa and %TI as the ordinate to draw the reference substance linear regression graph and the sample linear regression graph. The reference substance standard curve equation was: y = 16.241x + 25.295, r = 0.9957. According to the reference substance regression equation, the natural logarithm of the concentration corresponding to 45%TI was 1.213. Substituting it into the sample standard curve equation, the thrombin inhibition percentage (%TI) of bivalirudin was obtained. The results are shown in Table 9 below.

[0092] Table 9 Test results of three batches of preparations

[0093]

[0094] Example 5

[0095] Take about 10 mg of blank auxiliary material, weigh accurately, add Tris-HCl buffer A (0.05 mol / L, pH = 8.40) to dissolve and dilute to scale to prepare 0.01 mg / ml blank auxiliary material solution. Preheat the microplate reader to 37 ° C, measure the absorbance of the blank auxiliary material solution and the blank solution at 405 nm according to the procedure in Table 1 above, and calculate the interference of the blank auxiliary material on the blank solution.

[0096] Table 10 Example 5 Measurement results

[0097]

[0098] The absorbance of the blank excipient should be within the range of 90%-100% of the absorbance of the blank solvent, that is, after being placed at room temperature for 24 hours, the results of the reference stock solution and the sample stock solution are compared with those at 0 hours, and the relative deviation of %TI is ≤10%. It can be seen from the table above that the interference rate is -5.3%, which is small.

[0099] Example 6

[0100] The thrombin test results of the same batch of samples were examined by adjusting the detection time, thrombin incubation time, thrombin addition amount, incubation temperature, chromogenic substrate addition amount, etc.

[0101] 10.47 mg of bivalirudin reference substance was accurately weighed, and reference substance stock solution was prepared with Tris-HCl buffer A (0.05 mol / L, pH=8.40), and then diluted to concentrations of 1.16 μg / ml, 2.33 μg / ml, 4.65 μg / ml, and 9.31 μg / ml as bivalirudin reference substance series solutions, and then bivalirudin preparation was taken to prepare bivalirudin sample series solutions with concentrations of 1.36 μg / ml, 2.71 μg / ml, 5.43 μg / ml, and 10.85 μg / ml. The microplate reader was preheated to 37°C, and Tris-HCl buffer A (0.05 mol / L, pH=8.40) was used as a blank, and the absorbance of the above different series of solutions at 60s was measured at 405 nm according to a certain procedure. The determination was repeated twice, and the results are shown in Table 11 below.

[0102] Table 11 Example 6 Measurement results

[0103]

[0104] The invention obtains the optimal test parameters for determining the biological activity of the bivalirudine preparation by adjusting the detection time, the thrombin incubation time, the amount of thrombin added, the incubation temperature and the amount of the chromogenic substrate added.

[0105] Example 7

[0106] The stability of the reference substance stock solution and the sample stock solution after being placed at room temperature for 24 hours was investigated. The reference substance stock solution and the sample stock solution of bivalirudin after being placed in a volumetric flask at room temperature for 24 hours were taken to prepare a reference substance series solution and a sample series solution.

[0107] Freshly prepared reference substance series solutions were used as controls, i.e., 10.35 mg of the bivalirudin reference substance was accurately weighed, and the reference substance stock solution was prepared with Tris-HCl buffer A (0.05 mol / L, pH=8.40), and then diluted to concentrations of 1.15 μg / ml, 2.30 μg / ml, 4.60 μg / ml, and 9.20 μg / ml as the bivalirudin reference substance series solutions. The microplate reader was preheated to 37°C, and Tris-HCl buffer A (0.05 mol / L, pH=8.40) was used as a blank, and the absorbance of the above different series of solutions at 60 s was measured at 405 nm according to the procedure in Table 1 above.

[0108] The linear regression graph of the reference substance was drawn with the natural logarithm of the concentration of the freshly prepared reference substance series solution as the abscissa and %TI as the ordinate. According to the reference substance regression equation, the reference substance concentration when %TI = 45% was calculated. The natural logarithm of the concentration corresponding to 45%TI was substituted into the standard curve equation of the sample and reference substance after 24 hours of storage to obtain the respective thrombin inhibition percentage (%TI).

[0109] Table 12 Example 7 Measurement results

[0110]

[0111] The relative deviation of %TI of the reference stock solution after being placed at room temperature for 24 hours and the sample stock solution measured at 0 hours was 0.11%, and the solution had good stability.

[0112] Example 8

[0113] Accurately weigh 10.47 mg of bivalirudin reference substance, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) to prepare reference substance stock solution, and then dilute to concentrations of 1.16 μg / ml, 2.33 μg / ml, 4.65 μg / ml, 9.31 μg / ml as bivalirudin reference substance series solutions, and then take bivalirudin preparation to prepare bivalirudin sample series solutions with concentrations of 1.36 μg / ml, 2.71 μg / ml, 5.43 μg / ml, and 10.85 μg / ml. Preheat the microplate reader to 37°C, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) as blank, and measure the absorbance of the above different series solutions at 405 nm at 60 s according to the procedure in Table 1 above. Parallel determination three times.

[0114] Calculation of %TI for reference substance at 50%, 100%, and 150% concentration points: Calculate the reference substance concentration corresponding to 45% TI as the 100% concentration point based on the reference substance linear regression equation, and use this concentration ±50% as the 50% and 150% concentration points. Substitute 50% and 150% concentrations into the regression equation to obtain their respective corresponding %TI.

[0115] Calculation of %TI at 50%, 100%, and 150% concentration points of the sample: Substitute the 50%, 100%, and 150% concentration points of the reference substance into the regression equation of the three samples to obtain their respective %TI.

[0116] Table 13 Example 8 Measurement Results

[0117]

[0118] As can be seen from the table above, the recoveries of %TI at the sample concentration points of 50%, 100%, and 150% were all between 90% and 110%, with an RSD of 2.0%.

[0119] Example 9

[0120] Different experimenters measured the same batch of samples on different days to examine the results of biological activity determination.

[0121] Accurately weigh 10.41 mg of bivalirudin reference substance, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) to prepare reference substance stock solution, and then dilute to concentrations of 1.16 μg / ml, 2.31 μg / ml, 4.63 μg / ml, 9.25 μg / ml as bivalirudin reference substance series solutions, and then take bivalirudin preparation to prepare bivalirudin sample series solutions with concentrations of 1.36 μg / ml, 2.71 μg / ml, 5.43 μg / ml, and 10.85 μg / ml. Preheat the microplate reader to 37°C, use Tris-HCl buffer A (0.05 mol / L, pH = 8.40) as blank, and measure the absorbance of the above different series solutions at 405 nm at 60 s according to the procedure in Table 1 above. Parallel determination six times.

[0122] The linear regression graph of the reference substance was drawn with the natural logarithm of the concentration of the reference substance series solution as the abscissa and %TI as the ordinate. The equation of the reference substance standard curve was: y=16.536x+25.502, r=0.9901. According to the reference substance regression equation, the natural logarithm of the concentration corresponding to 45%TI was 1.179. Substituting it into the sample standard curve equation, the thrombin inhibition percentage (%TI) of bivalirudin was obtained. The results are shown in Table 14.

[0123] Table 14 Example 9 measurement results

[0124]

[0125] The repeatability of 6 samples of thrombin inhibition percentage (%TI) of bivalirudin has an RSD ≤ 5.0%; the intermediate precision of 6 samples of thrombin inhibition percentage (%TI) of bivalirudin has an RSD ≤ 5.0%; the precision of 12 samples of thrombin inhibition percentage (%TI) of bivalirudin has an RSD ≤ 5.0%. As can be seen from the above table, the precision is good.

[0126] Example 10

[0127] The reference solution and the sample solution to be tested were prepared using Tris-HCl buffer A (0.05 mol / L, pH=8.40), and the biological activity of the same batch of bivalirudine preparations was determined using an ultraviolet spectrophotometer under the same conditions as the microplate reader. 100 μl of each of the blank, reference test series solution, and sample test series solution was placed in a 2 ml centrifuge tube, incubated in a 37°C water bath for 30 s, 40 μl of thrombin was added, shaken, and incubated in a water bath for 120 s, and then 30 μl of the chromogenic substrate was added, mixed, and reacted in a 37°C water bath for 70 s, and glacial acetic acid was added to terminate the reaction. The parallel determination was performed twice for comparison, and linear equations 1 and 2 respectively represent the results of the two parallel determinations, and the results are shown in Table 15 below.

[0128] Table 15 Example 10 Measurement results

[0129]

[0130]

[0131] It can be seen from the above results that, compared with the ultraviolet spectrophotometer, the present invention can obtain a good linear curve by measuring the biological activity of bivalirudin by using the microplate reader with constant temperature and shaking functions, and the operation is simple and the result reproducibility is good.

[0132] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A quality detection method for bivalirudin raw materials and preparations thereof, characterized in that, The following steps are involved: Step 1: Mix the bivalirudin raw material or its preparation to be tested with Tris-HCl buffer A to obtain a test solution; mix the bivalirudin standard with Tris-HCl buffer A to obtain a standard solution; The Tris-HCl buffer A is a Tris-HCl buffer with a pH of 7.60-9.20, containing 0.03-0.07 mol / L of tris(hydroxymethyl)aminomethane, 10.20 g / L of sodium chloride, 2.80 g / L of disodium ethylenediaminetetraacetic acid, and 1.0 g / L of polyethylene glycol 6000; Step 2: Incubate Tris-HCl buffer A, the test solution and the standard solution with the thrombin solution respectively; then add the bivalirudin chromogenic substrate solution and incubate again; measure the absorbance at 405 nm, respectively, and use the natural logarithm of the concentration of the bivalirudin standard solution and the test solution as the abscissa and %TI as the ordinate to obtain the standard curve and the regression equation of the bivalirudin sample to be tested; According to the standard curve, the concentration of the standard solution when %TI=45% is calculated, and the concentration is substituted into the regression equation of the bivalirudin sample to calculate the thrombin inhibition percentage of the bivalirudin bulk drug or its preparation to be tested.

2. The detection method according to claim 1, characterized in that: The Tris-HCl buffer A is a Tris-HCl buffer with a pH of 8.40 and containing 0.05 mol / L tris(hydroxymethyl)aminomethane, 10.20 g / L sodium chloride, 2.80 g / L disodium ethylenediaminetetraacetic acid, and 1.0 g / L polyethylene glycol 6000.

3. The detection method according to claim 1, characterized in that: The concentrations of the standard solution and the test solution are both 1.0 μg / ml-60 μg / ml.

4. The detection method according to claim 1, characterized in that: The thrombin solution was prepared by the following method: 100 NIH thrombin lyophilized powder was added with 1000 μl of purified water for re-dissolution, and then 9000 μl of Tris-HCl buffer A was added and mixed.

5. The detection method according to claim 1, characterized in that: The concentration of the bivalirudin chromogenic substrate solution is 0.1-1.0 mg / ml.

6. The detection method according to claim 1, characterized in that: The bivalirudin preparation includes bivalirudin powder for injection or bivalirudin injection.

7. The detection method according to claim 1, characterized in that: The ambient temperature detected in step 2 is 18°C ​​to 22°C.

8. The detection method according to claim 1, characterized in that: The incubation in step 2 is shaking incubation at 35-39° C. for 50-150 s; the further incubation is shaking incubation at 35-39° C. for 5-15 s.

9. The detection method according to claim 1, characterized in that: The absorbance value in step 2 is measured 50s to 70s after the end of the re-incubation.

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