CN108508129B - Method for measuring biological potency of heparin drugs - Google Patents
Method for measuring biological potency of heparin drugs Download PDFInfo
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- CN108508129B CN108508129B CN201810298534.5A CN201810298534A CN108508129B CN 108508129 B CN108508129 B CN 108508129B CN 201810298534 A CN201810298534 A CN 201810298534A CN 108508129 B CN108508129 B CN 108508129B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides a method for measuring the biological potency of heparin drugs, belonging to the field of biological analysis. The determination method comprises the following steps: mixing the blood coagulation factor with a to-be-detected drug, antithrombin III and a specific substrate corresponding to the blood coagulation factor to construct a potency system of the in vitro anti-coagulation factor; measuring the content of a reaction product in a titer system by a high performance liquid chromatography-mass spectrometry combined analysis method; and (3) calculating the biological potency of the drug to be detected according to a quantity reaction parallel line method by using heparin drugs with known potency as standard substances. The determination method can effectively reduce interference, has strong specificity, is simple and convenient to operate, saves time and can effectively improve the determination efficiency.
Description
Technical Field
The invention relates to the field of biological analysis, in particular to a method for measuring the biological potency of heparin drugs.
Background
Blood coagulation consists of a series of complex chemical reactions linked to form a coagulation cascade. There are 12 coagulation factors currently recognized, and they are marked by the roman numerals I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, in the order of discovery, internationally and uniformly. Each chain reaction is a proteolytic reaction that converts a proenzyme to the corresponding serine protease and ultimately to thrombin (FIIa, where a represents the active form of the coagulation factor). Anticoagulants are inhibitors of most of the key enzymes in the blood coagulation cascade, and are clinically used for preventing and treating thromboembolic diseases.
The most widely used anticoagulants at present are the heparins, including heparin normal (UFH) and Low Molecular Weight Heparin (LMWH). The anticoagulant effect of heparins is achieved by antithrombin III (ATIII). The binding of heparin drugs and ATIII accelerates the inhibition of several key proteases in the coagulation cascade, such as coagulation factor Ea (E ═ II, VII, IX, X, XI, XII), where factor IIa (FIIa) and factor Xa (FXa) are most sensitive to their inhibition. The heparin medicine with high negative charge density can be combined with the lysine with positive charge on the ATIII, so that the molecular structure of the ATIII is changed, and the arginine at the active site is exposed, thereby greatly increasing the contact probability of the ATIII and the serine in the blood coagulation factor, and enhancing the anticoagulant activity of the ATIII by about 1000 times.
The measurement of the biological potency is 1 important biological index for the quality control of the heparin drugs, and can reflect the anticoagulation activity of the heparin drugs. The traditional biological potency measurement of heparin drugs comprises: coagulation methods (activated partial thromboplastin time (APTT) and Activated Clotting Time (ACT)), immunoblotting, and chromogenic methods (ultraviolet and fluorescent measurements). The blood coagulation method is easily influenced by the change of conditions such as laboratories, reagents, instruments and equipment, has poor reproducibility and low sensitivity, and can only obtain semi-quantitative results generally; immunoblotting is a method established based on reactions with several unique anti-heparin antibodies, and has limited application. For the color development method: the uv spectrophotometry used in the current chinese pharmacopoeia (ChP2015) requires a large and expensive amount of reagents, has a high test cost, and is interfered by the sample matrix. The fluorescence method has the advantages of strong specificity and high sensitivity, but the background fluorescence of the fluorescent substrate can generate obvious interference on detection. In addition, the fluorescence method uses human plasma as an enzyme source, so that a reaction system of the enzyme is established, the interference of individual differences of the plasma is easy to occur, and the supply and the storage of the human plasma are also limited by harsh conditions.
In view of the above, the present invention provides a novel method for determining the biological potency of heparin drugs.
Disclosure of Invention
The invention aims to provide a method for measuring biological potency of heparin drugs, which constructs a potency system of in vitro anticoagulant factors, utilizes a high performance liquid chromatography-mass spectrometry combined analysis system to carry out quantitative measurement, has a tiny reaction system, high sensitivity and strong specificity, and can avoid the interference of substances in complex biological samples on measurement results.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for determining the biological potency of heparin drugs, which comprises the following steps:
mixing the blood coagulation factor with a to-be-detected drug, antithrombin III and a specific substrate corresponding to the blood coagulation factor to construct a potency system of the in vitro anti-coagulation factor;
measuring the content of a reaction product in a titer system by a high performance liquid chromatography-mass spectrometry combined analysis method;
and (3) calculating the biological potency of the drug to be detected according to a quantity reaction parallel line method by using heparin drugs with known potency as standard substances.
Compared with the prior art, the beneficial effects of the invention comprise:
according to the method for measuring the biological potency of the heparin drugs, a potency system of external anticoagulant factors is constructed, so that the direct use of biological samples for measurement is avoided, and the content of reaction products in the potency system is measured by using a high performance liquid chromatography-mass spectrometry combined analysis method, so that the reaction activity of the coagulation factors in the potency system is measured; and then comparing with a standard substance to obtain the biological potency of the drug to be detected.
The principle of the potency system of the in vitro anticoagulant factor is as follows: the drug to be tested, such as common heparin (UFH), after being mixed with Antithrombin (ATIII) and excessive blood coagulation factors, forms a UFH-ATIII-blood coagulation factor ternary complex, the blood coagulation factors forming the complex have no blood coagulation activity and can not catalyze the reaction of converting specific substrates into products, namely, the reaction activity of the part of blood coagulation factors is inhibited, and only the blood coagulation factors which do not form the complex in the system can continuously catalyze the conversion of the specific substrates into the products, thereby reducing the production amount of the products. Therefore, the anticoagulation effect of the drug to be detected can be reflected by measuring the content of the product, and the biological potency of the drug to be detected can be obtained. Compared with the prior art, the method for measuring the biological potency of the heparin drugs can replace plasma with commercial enzyme, avoid the interference of plasma specificity difference and save the complex work of plasma purchase, storage and the like; on the other hand, a reaction system established by utilizing commercial enzyme and commercial substrate is more beneficial to repetition and popularization, and the reaction system is small and low in cost. In addition, the reaction product is measured by using a high performance liquid chromatography-mass spectrometry combined analysis method, so that the interference can be effectively reduced, the measurement sensitivity is high, the reproducibility is strong, a large batch of samples to be measured can be continuously analyzed, and the method has the characteristic of high flux. Therefore, the determination method can effectively reduce interference, has strong specificity, is simple and convenient to operate, saves time and can effectively improve the determination efficiency. The method can not only test whether the heparin medicine produced by a pharmaceutical factory is qualified, but also be used for detecting the dosage of the heparin medicine in clinical medication and the in vivo blood coagulation level.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram of an online sample pretreatment system setup;
FIG. 2 is a schematic diagram of the method for determining the biological potency of heparin drugs according to the present invention;
FIG. 3 is the peak area ratio (I/I) of the product paranitroaniline in example 1 to the internal standard0) A standard curve for pNA concentration;
FIG. 4 is the peak area ratio (I/I) of the product 7-amino-4-methylcoumarin of example 1 to the internal standard0) A standard curve for the concentration of 7-amino-4-methylcoumarin;
FIG. 5 is the peak area ratio (I/I) of the product benzoylarginine of example 1 to the internal standard0) A standard curve for the concentration of benzoylarginine;
FIG. 6 is the peak area ratio (I/I) of the product 4-nitrophenol of example 1 to the internal standard0) A standard curve for 4-nitrophenol concentration;
FIG. 7 shows the results of determining the biological potency of heparin sodium against IIa in example 1;
FIG. 8 shows the results of anti-IXa activity in the biological potency of heparin sodium to be determined in example 1;
FIG. 9 shows the results of anti-Xa assay to be performed on the biological potency of heparin sodium in example 1
FIG. 10 shows the results of anti-XIa in the biological potency of heparin sodium to be measured in example 1;
FIG. 11 shows the results of determination of anti-XIIa in the biological potency of heparin sodium in example 1;
FIG. 12 shows the results of anti-VIIa assay in the biological potency of heparin sodium to be determined in example 1.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The embodiment provides a method for measuring the biological potency of heparin drugs, which comprises the following steps:
and step S1, mixing the blood coagulation factor with a to-be-detected drug, antithrombin III and a specific substrate corresponding to the blood coagulation factor to construct a titer system of the in vitro anti-coagulation factor.
Wherein the blood coagulation factor includes any one of blood coagulation factor IIa, blood coagulation factor VIIa, blood coagulation factor IXa, blood coagulation factor Xa, blood coagulation factor XIa and blood coagulation factor XIIa.
The anticoagulation activity of the above coagulation factors, the desired substrates and the resulting reaction products were measured, as shown in Table 1:
TABLE 1 establishment of anticoagulant factor potency System
Further, the construction method of the potency system of the in vitro anticoagulant factor comprises the following steps:
mixing the drug to be tested with antithrombin III, adding the blood coagulation factor and the specific substrate, mixing and reacting for 3-7 minutes, and adding a quencher to prepare the test sample.
Preferably, the quenching agent is an organic solvent, such as methanol, acetonitrile, and the like.
The preparation of the test article comprises: and centrifuging the reaction solution after the quenching agent is added, and taking supernate, namely the test sample.
Further, in order to facilitate subsequent quantitative determination, the supernatant is mixed with an internal standard with the same volume to obtain a test sample to be detected.
Step S2, measuring the content of the reaction product in the titer system by a high performance liquid chromatography-mass spectrometry combined analysis method;
the principle that the corresponding products, namely p-nitroaniline (pNA), 7-amino-4-methylcoumarin, benzoylarginine and 4-nitrophenol, are respectively generated by a substrate under the action of corresponding coagulation factors is utilized, and the products, namely the p-nitroaniline, 7-amino-4-methylcoumarin, the benzoylarginine and the 4-nitrophenol, are used as quantitative standards.
Further, the content of the reaction product was measured by an internal standard method.
The step adopts a common high performance liquid chromatography-mass spectrometry (HPLC-MS) for determination. In the measuring process, in order to further reduce the interference of substances such as enzyme, protein and the like in a titer system on an analysis result, the inventor self develops a device and a method capable of carrying out online sample pretreatment in the HPLC-MS analysis process, thereby realizing the online treatment of the sample to be measured.
The HPLC-MS analysis comprises a high-efficiency online sample pretreatment system and a high-performance liquid chromatography-mass spectrometry combined system, the structure of which is shown in figure 1, wherein the online sample pretreatment system comprises a first pump body (namely a pump A), an automatic sample injector, a multi-way valve, a pretreatment chromatographic column, the multi-way valve and a waste liquid collector which are connected in sequence; the high performance liquid chromatography-mass spectrometry system is connected or disconnected with the pretreatment chromatographic column through the multi-way valve.
In the detection process, an online sample pretreatment system is used as a first-dimension sample pretreatment system, a sample pretreatment command is executed, and a reaction system is purified, so that a reaction product with low polarity is retained on a pretreatment chromatographic column; meanwhile, the liquid phase solution is communicated with the analytical chromatographic column through a second pump body (namely a pump B) and a multi-way valve, and the analytical chromatographic column is subjected to equilibrium treatment. And then, switching the multi-way valve to enable the liquid phase solution, the pump B, the multi-way valve, the pretreatment chromatographic column, the multi-way valve, the analysis chromatographic column and the mass spectrum detector to be sequentially connected to serve as a second-dimensional sample elution and analysis system to execute an elution and analysis command. In the process, the reaction product with lower polarity is eluted to an analytical column and then is analyzed; at the moment, the liquid phase solution, the pump A, the automatic sample introduction system and the waste liquid collection system are connected to prepare for next sample introduction; and after the second-dimension analysis is finished, switching the multi-way valve to the initial connection position again, entering the sample pre-processing state after the first-dimension sampling again, and circulating the process until the analysis of the part of the sample to be analyzed is finished.
Further, the number of the ports of the multi-way valve is larger than 6 and is an even number, for example, the multi-way valve can be a six-way valve, an eight-way valve, a ten-way valve, and the like.
Taking a two-position ten-way valve as an example, as shown in fig. 1, in the sample pretreatment stage, a liquid phase solution, a pump a, an automatic sample feeding system, the ten-way valve (9 → 10), a pretreatment chromatographic column, the ten-way valve (7 → 8) and a waste liquid collector are sequentially communicated to serve as a sample pretreatment channel; meanwhile, the liquid phase solution, the pump B, the ten-way valve (1 → 2), the analytical column, the ten-way valve (5 → 6 → 3 → 4), and the mass spectrometer are communicated as an analytical column equilibrium channel.
In the sample elution and analysis stage, the liquid phase solution, the pump B, the ten-way valve (1 → 10), the sample pretreatment column, the ten-way valve (7 → 6 → 3 → 2), the analysis chromatographic column, the ten-way valve (5 → 4) and the mass spectrum detector are communicated to be used as a sample elution and analysis channel; at this time, the liquid phase solution, the pump a, the ten-way valve (9 → 8), and the waste liquid collector are communicated in order.
Further, the method for detecting the reaction product by the HPLC-MS analysis method comprises the following steps:
a. injecting a sample by using an automatic sample injector, eluting the pretreatment chromatographic column by using an organic solvent-water system, and allowing the eluent to enter the waste liquid collector;
preferably, in the on-line sample pretreatment system, the pretreatment chromatographic column is a reverse phase chromatographic column, the mobile phase is methanol-water, and the flow rate is 0.3-0.8 mL/min-1(ii) a The sample amount is 1-20 mu L; the rinsing time is 0.5-1.5 minutes.
b. And switching the multi-way valve to communicate the second pump body of the liquid chromatogram with the pretreatment chromatographic column, eluting the analysis chromatographic column by a methanol-water solvent system, and allowing the eluent to enter a mass spectrum detector for analysis.
Preferably, the conditions of the liquid chromatography in the HPLC-MS include: adopting reverse chromatographic column as analysis chromatographic column, and making flow rate of mobile phase be 0.1-0.5 mL/min-1(ii) a The analysis time is 3-10 minutes.
Preferably, the conditions of mass spectrometry in the HPLC-MS comprise: adopting electrospray mass spectrometry positive ion mode, drying gas temperature of 250-350 deg.C, atomizer voltage of 20-60psi, capillary voltage of 3000-4500V, capillary outlet voltage of 50-150V, and drying gas flow rate of 4-12 L.min-1。
c. After the analysis is finished, the multi-way valve is switched to balance the pretreatment chromatographic column and the analysis chromatographic column, so that the next sample introduction analysis is facilitated.
And step S3, calculating the biological potency of the drug to be detected according to a quantitative reaction parallel line method by taking heparin drugs with known potency as standard substances.
Further, the biological potency P of the drug to be testedTCalculated according to the following formula:
PT＝PS×(ST/SS)
in the formula, PSThe biological value of the standard heparin medicine; sTAnd SSThe slope of the drug to be detected and the slope of the standard are respectively.
The features and properties of the present invention are further described in detail below with reference to examples:
example 1
The present embodiment provides a method for measuring the biological potency of heparin sodium, the detection principle of which is shown in fig. 2, and the method comprises the following steps:
establishing an in-vitro anticoagulant factor (IIa, VIIa, IXa, Xa, XIa, XIIa) titer reaction system:
1. solution preparation:
preparing a tris-hydroxymethyl-aminomethane-polyethylene glycol 6000 buffer (pH 8.4): 50mM Tris, 7.5mM EDTA, 175mM NaCl, 0.1% PEG 6000, 800mL water, hydrochloric acid to adjust pH 8.4, water dilution to 1000mL as buffer solution; an appropriate amount of blood coagulation factor (IIa, IXa, Xa, XIa, XIIa, VIIa) powder was precisely weighed and diluted to 0.05 IU. mL with a buffer solution-1、0.25IU·mL-1、0.015IU·mL-1、0.25IU·mL-1、0.5IU·mL-1And 0.25 IU. mL-1(ii) a Precisely weighing a proper amount of substrate powder, and preparing a solution of 5 mu M, 50 mu M, 15 mu M, 50 mu M, 100 mu M and 50 mu M of S2238, D-Leu-Phg-Arg-4-nitroanilide (LPAN), S2765, Z-Gly-Gly-Arg-7-amido-4-methyloumarin (GGAM), benzyl aryl ethyl ester (BAE) and benzyl oxy-Arg-p-nitrobenzyl ester (BAN) respectively by using distilled water; accurately weighing appropriate amount of ATIII powder, and diluting to 0.00015IU & mL with buffer solution-1(ii) a Precisely weighing appropriate amount of heparin sodium powder, and diluting heparin standard product with distilled water to 0.00020 IU/mL-1、0.0001IU·mL-1、0.00005IU·mL-1、0.000025IU·mL-1A range of concentrations.
2. Constructing a titer system:
according to Table 1, 0.0002 IU. mL was measured precisely-1、0.0001IU·mL-1、0.00005IU·mL-1、0.000025IU·mL -150 μ L of heparin sodium at four concentrations is added into 50 μ L of heparin sodium, 0.00015 IU/mL-1And (3) mixing ATIII solution, adding the same volume of blood coagulation factor and corresponding specific substrate as shown in Table 1, mixing, reacting for 5 minutes, and adding 100 mu L of 0.2% acetic acid acetonitrile solution for quenching. Centrifuging at 12000 r for 10 min, mixing 100 μ L of supernatant with 100 μ L of internal standard, and performing on-line sample pretreatment and high performance liquid chromatography-mass spectrometry.
Secondly, establishing a high-efficiency online sample pretreatment-based HPLC-MS method for analyzing reaction products in a titer system:
1. establishment of high-efficiency on-line sample pretreatment HPLC-MS analysis method
An efficient online sample pretreatment HPLC-MS analysis system is set up as shown in FIG. 1, when the switching valves are communicated at positions 1-2, a sample pretreatment channel is communicated, a sample pretreatment command is executed, a reaction system is purified, and reaction products with small polarity are retained on a pretreatment chromatographic column; at this time, the mobile phase is also communicated with the analysis channel, and the analysis chromatographic column is subjected to equilibrium treatment. After the pretreatment is finished, the multi-way valve is automatically switched to enable the 1-10 positions of the multi-way valve to be communicated, at the moment, the sample pretreatment column is communicated with the analysis channel, a sample elution and analysis command is executed, and a reaction product with lower polarity is eluted onto the analysis column to be subjected to HPLC-MS analysis treatment.
The detection process comprises the following steps:
a. loading: quantitatively sucking 2 mu L of titer system reaction liquid;
b. sample online treatment: the sample pretreatment column adopts common C18(or C)8) A chromatographic column; methanol and 0.05% formic acid water solution (v: v ═ 1:9) are adopted as a mobile phase; the flow rate was 0.6 mL. min-1Washing for 1 minute, and allowing eluent to enter a waste liquid collector;
c. elution and analysis: the analytical chromatographic column adopts common C18A column; methanol and 0.01% formic acid water solution (v: v ═ 6:4) are adopted as a mobile phase; the flow rate was 0.4 mL/min-1(ii) a Elute 3 min and elute into mass spectrometer.
In this step, the mass spectrometry condition is electrospray mass spectrometry (ESI-MS) positive ion mode; capillary exit voltage: 90V; temperature of the drying gas: 350 ℃; atomizer voltage: 15 psi; capillary voltage: 4000V; the quantification mode was multiplex ion monitoring mode (MRM), and the product quantitative ion pairs are shown in table 2:
TABLE 2 product quantitation ion pairs
d. Rebalancing: at the end of step c, the multi-way valve was switched and the pre-treatment column and the analytical column were equilibrated for 1 minute.
The assay procedure took a total of 5 minutes, pretreatment (1 min) + elution and assay (3 min) + re-equilibration (1 min).
2. The method for quantifying the product of the enzyme reaction catalyzed by the blood coagulation factor is established by adopting an internal standard method:
an appropriate amount of paranitroaniline (pNA), 7-amino-4-methylcoumarin, benzoylarginine and 4-nitrophenol powder are accurately weighed, respectively dissolved in a 250mL volumetric flask, and stored in a refrigerator at 4 ℃. When used, each of the solutions was prepared to be 0.05. mu.g.L in 50% acetonitrile water (v: v)-1To 2000. mu.g.L-1The product standard solutions with series concentrations are quantified by adopting an internal standard method, wherein the internal standards are respectively N-methyl-4-nitroaniline, N-methyl-7-amino-4-methylcoumarin and benzoylarginine (Arg-N)15) And 4-nitrobenzyl ether, and carrying out on-line sample pretreatment high performance liquid chromatography-mass spectrometry combined analysis.
The ratio of the product to the internal standard mass spectrum response area I/I is taken as0For the y-axis and product concentration for the x-axis, a standard curve was established:
the standard curve of the product p-nitroaniline is shown in FIG. 3, and the linear regression equation is that y is 0.2797x + -0.003311 (R20.999), the quantitative range is 0.05 mug.l-1To 1000. mu.g.L-1；
The standard curve of the product 7-amino-4-methylcoumarin is shown in fig. 4, and the linear regression equation is 0.1309x ± 0.01878 (R)20.999), the quantitative range is 0.1 mug.l-1To 1000. mu.g.L-1；
The standard curve of the product benzoylarginine is shown in fig. 5, and the linear regression equation is that y is 0.1167x ± 0.04046 (R)20.999), the quantitative range is 0.25 mug.l-1To 1200. mu.g.L-1；
The standard curve for the product, 4-nitrophenol, is shown in figure 6, with a linear regression equation of y ═ 2.018x ± 1.261 (R)20.999), the quantitative range is 0.05 mug.l-1To 1500. mu.g.L-1。
The standard curve has good linearity, so that the result of the on-line sample pretreatment high performance liquid chromatography-mass spectrometry combined analysis is accurate and reliable.
3. And (3) verification of methodology:
the established product quantitative method is verified, and the quantitative limit, the detection limit, the precision, the accuracy and the matrix effect are examined and the requirements of the analysis method verification guiding principle in the four parts of the Chinese pharmacopoeia of 2015 edition are met.
(1) Quantitative and detection limits
Detection limit of p-nitroaniline: 0.01. mu.g.L-1(SNR 3.50 ± 1.53; n 6); lower limit of quantitation: 0.05. mu.g.L-1(SNR 11.05 ± 1.26; n 6). Detection limit of 7-amino-4-methylcoumarin: 0.05. mu.g.L-1(SNR 3.65 ± 1.31; n 6); lower limit of quantitation: 0.1. mu.g.L-1(SNR 10.75 ± 1.42; n 6). Detection limit of benzoylarginine: 0.1. mu.g.L-1(SNR 3.23 ± 1.12; n 6); lower limit of quantitation: 0.25. mu.g.L-1(SNR 10.45 ± 1.56; n 6). Detection limit of 4-nitrophenol: 0.01. mu.g.L-1(SNR 3.36 ± 1.51; n 6); lower limit of quantitation: 0.05. mu.g.L-1(SNR 11.40 ± 1.31; n 6). From the results, it can be seen that the method is high in detection sensitivity.
(2) Precision and accuracy survey
Prepared at a low concentration (5. mu.g.L)-1) Middle concentration (50. mu.g.L)-1) High concentration (500. mu.g.L)-1) The product solutions at three concentration levels were used as quality control samples for accuracy and precision studies. The accuracy is between 85% and 115%, and the relative deviation of the precision is within +/-15%, which indicates that the precision and the accuracy of the method are good.
(3) Investigation of matrix Effect
Low concentrations (5. mu.g. L) were prepared using buffer and enzyme solution, respectively-1) Middle concentration (50. mu.g.L)-1) High concentration (500. mu.g.L)-1) The product solutions at the three concentration levels were mixed with the corresponding internal standards and analyzed for recovery and precision of product detection in the matrix (enzyme solution). The recovery rate is between 85 and 115 percentIndicating that the method is not disturbed by the substrate.
4. Establishment of biological potency measuring method represented by heparin medicine
A batch of heparin sodium samples were subjected to biological potency assay for anti-IIa activity, anti-IXa activity, anti-Xa activity, anti-XIa activity, anti-XIIa activity and anti-VIIa activity, respectively, using heparin sodium of known potency as a standard drug, and the results should be 90-110% of the indicated values based on the biological potency as an evaluation criterion, and the potency ratio of anti-Xa to anti-IIa factor should meet the specifications.
Heparin sodium standard substance, preparing a group of solutions with gradient titer concentration according to the dose-to-dose ratio of 1:0.5, the concentration is 0.0002 IU.mL respectively-1、0.0001IU·mL-1、0.00005IU·mL-1、0.000025IU·mL-1Labeled S1, S2, S3, S4, respectively. For the test heparin, the titer is estimated firstly, and then gradient solutions with the same titer concentration as the standard product are prepared according to the estimated titer, and are marked as T1, T2, T3 and T4 respectively.
During testing, according to the optimized method, the following steps are carried out: s1, S2, S3, S4, T1, T2, T3, T4, T1, T2, T3, T4, S1, S2, S3, S4, 3 replicates per sample, tested 48 times. And mixing 100 mu L of supernatant with 100 mu L of internal standard for online sample pretreatment and high performance liquid chromatography-mass spectrometry combined analysis. The peak area ratios of the product and its corresponding internal standard in each reaction were recorded. By peak area ratio (I/I)0) The ordinate is the logarithm of the titer and concentration of the standard product and the sample, and the abscissa is the logarithm of the titer and concentration of the sample, and a linear regression curve is respectively drawn. The titers and experimental errors were calculated according to the bioassay statistical method, the mean confidence limit (FL%) was not more than 15%, and the results are shown in table 3:
TABLE 3 measurement of biological potency of heparin sodium test sample
As can be seen from Table 3, the results obtained by the method are close to the results of the labeled values, FL is less than 15%, and the reliability tests are all passed. Therefore, the method for determining the biological potency of the heparin drugs by utilizing the online sample pretreatment and HPLC-MS analysis, which is established in the embodiment, is accurate and sensitive, and can be used as a method for controlling the quality in the production of the heparin drugs.
Examples 2 to 10
The drugs of fondaparinux, daparinux, aclidinarin, nadroparin, serto heparin, parnaparin, reviparin, tinzaparin and enoxaparin shown in table 4 were used to replace heparin sodium in example 1, and appropriate substrates were selected according to table 5 to establish an anticoagulant factor Ea (E ═ II, VII, IX, X, XI, XII) titer system, and an on-line sample pretreatment hplc-ms analysis method for the corresponding products was established. The bio-potency of different heparin drugs against different coagulation factors was measured using the standards as reference standards. The results are consistent with those of experiment 1, which are 90-110% of the indicated values, and the potency ratio of anti-Xa to anti-IIa factor is defined.
TABLE 4 heparin class of drugs
TABLE 5 enzymes and substrates available in the anticoagulant factor Ea potency System
In conclusion, the invention utilizes the high-efficiency on-line sample pretreatment HPLC-MS analysis system to measure the biological potency of the heparin drugs, and compares the biological potency with the standard substance, and the result meets the requirement. As can be seen from the measurement results, compared with the existing method, the method has the advantages of high sensitivity, good accuracy and strong stability, and greatly saves the dosage of enzyme and substrate.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (5)
1. A method for measuring the biological potency of heparin drugs is characterized by comprising the following steps:
mixing the blood coagulation factor with a to-be-detected drug, antithrombin III and a specific substrate corresponding to the blood coagulation factor to construct a potency system of the in vitro anti-coagulation factor;
measuring the content of the reaction product in the titer system by a high performance liquid chromatography-mass spectrometry combined analysis method;
calculating the biological potency of the drug to be detected according to a quantitative reaction parallel line method by taking heparin drugs with known potency as standard substances;
the biological potency P of the drug to be testedTCalculated according to the following formula:
PT＝PS×(ST/SS)
in the formula, PSThe biological value of the standard heparin medicine; sTAnd SSThe slope of the drug to be detected and the slope of the standard product are respectively obtained;
the high performance liquid chromatography-mass spectrometry combined analysis system comprises an online sample pretreatment system and a high performance liquid chromatography-mass spectrometry combined system, wherein the online sample pretreatment system comprises a first pump body, an automatic sample injector, a multi-way valve, a pretreatment chromatographic column, a multi-way valve and a waste liquid collector which are sequentially connected, and the high performance liquid chromatography-mass spectrometry combined system is connected with or disconnected from the pretreatment chromatographic column through the multi-way valve;
in the online sample pretreatment system, the pretreatment chromatographic column is a reversed-phase chromatographic column, the mobile phase is methanol-water, and the flow rate is 0.3-0.8 mL/min-1(ii) a The sample amount is 1-20 mu L; the washing time is 0.5-1.5 minutes;
the mass spectrum in the high performance liquid chromatography-mass spectrometry system adopts an electrospray mass spectrometry positive ion mode, the temperature of the dry gas is 250-350 ℃, the pressure of the atomizer is 20-60psi, the capillary voltage is 3000-4500V, the outlet voltage of the capillary is 50-150V, and the flow rate of the dry gas is 4-12 L.min-1；
The conditions of the liquid chromatogram in the high performance liquid chromatogram-mass spectrum combined system comprise: the analytical chromatographic column is a reverse chromatographic column, and the flow rate of the mobile phase is 0.1-0.5 mL/min-1(ii) a The analysis time is 3-10 minutes.
2. The method for determining the biological potency of heparin-like drugs according to claim 1, wherein the potency system of in vitro anticoagulant factor is constructed by the following steps:
mixing the drug to be tested with antithrombin III, adding the blood coagulation factor and the specific substrate, mixing and reacting for 3-7 minutes, and adding a quencher to prepare the test sample.
3. The method for determining the biological potency of heparin-like drugs according to claim 1, wherein the blood coagulation factor comprises any one of blood coagulation factors IIa, VIIa, IXa, Xa, XIa and XIIa.
4. The method for determining the biological potency of heparin-like drugs according to claim 1, wherein the method for detecting reaction products by using the HPLC-MS method comprises:
injecting a sample by using an automatic sample injector, eluting the pretreatment chromatographic column by using an organic solvent-water system, and allowing the eluent to enter the waste liquid collector; and then, switching the multi-way valve to communicate the second pump body of the liquid chromatogram with the pretreatment chromatographic column, sequentially eluting the pretreatment chromatographic column and the analysis chromatographic column by using a methanol-water solvent system, and allowing the eluent to enter a mass spectrum detector for analysis.
5. The method for determining the biological potency of heparin-like drugs according to claim 1, wherein the number of the multi-way valve ports is larger than 6 and is an even number.
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