Automatic detection method for heparin activity

Abstract

The invention belongs to the technical field of chemical analysis, and particularly relates to an automatic heparin activity detection method. According to the automatic detection method for the heparin activity, the heparin activity is detected by taking sheep plasma as a biological reagent, and key parameters such as adaptability, weighing precision, dissolution concentration, dilution concentration, sample adding amount, test adding amount, test tube selection specification, incubation temperature and time requirements and the like of the sheep plasma are optimized and limited. According to the automatic detection method for the heparin activity, the reagent cost is low, and compared with an existing detection technology, the accuracy and precision are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and in particular to an automated detection method for heparin activity. Background Art

[0002] Heparin is a mucopolysaccharide sulfate composed of glucosamine, L-iduronide, N-acetylglucosamine and D-glucuronic acid. It is a natural anticoagulant in animals and has anticoagulant effects both in vivo and in vitro. It is mainly used in clinical practice for thromboembolic diseases, myocardial infarction, cardiovascular surgery, cardiac catheterization, extracorporeal circulation, hemodialysis, etc. It is currently the most commonly used anticoagulant drug in clinical practice. Especially with the progress of pharmacology and clinical medicine, the application of heparin has been continuously expanded.

[0003] As a biologically active drug, heparin needs to undergo necessary biological activity tests in addition to testing its physical and chemical properties. Especially for anticoagulants, the anticoagulant effect is used as a measure of its biological activity. According to the Chinese Pharmacopoeia, heparin activity testing methods mainly include anti-factor IIa / anti-factor Xa titer determination method and coagulation time determination method (using rabbit blood or pig plasma). The European Pharmacopoeia and the United States Pharmacopoeia both record anti-factor IIa / anti-factor Xa titer determination methods, while the old version of the United States Pharmacopoeia (31st edition) records the method of using sheep plasma coagulation state for anticoagulant activity testing.

[0004] At present, there are many shortcomings in the application of mainstream pharmacopoeia detection methods in large-scale heparin production. First, the anti-IIa factor / anti-Xa factor titer determination method requires purified coagulation factors, antithrombin and other human biological reagents, and the high detection cost makes it unsuitable for large-scale sample testing; secondly, the solution environment and anti-interference requirements of the biological reagents required for the anti-IIa factor / anti-Xa factor titer determination method and the clotting time determination method are high, and are not suitable for the detection of intermediate process samples and crude heparin samples in large-scale heparin production; thirdly, the anti-IIa factor / anti-Xa factor titer determination method and the clotting time determination method both require complex biostatistical models to calculate the test results, which requires a high threshold of biostatistical knowledge and is not suitable for grassroots laboratory inspection personnel to operate and use; and the old version of the United States Pharmacopoeia sheep plasma method is cumbersome to operate, and is manually operated, which brings a lot of human operation errors, and the accuracy and precision are poor.

[0005] Therefore, in the large-scale heparin production process, it is necessary to deal with different types of heparin samples produced at various stages of the industrial chain. The experimental scale is getting larger and larger, and the number of samples is increasing. This field urgently needs a heparin activity detection method with low reagent cost, strong tolerance, automated operation and calculation, and high efficiency. Summary of the invention

[0006] To this end, the technical problem to be solved by the present invention is to provide an automated heparin activity detection method, which has the advantages of low reagent cost, strong method tolerance, automated operation and calculation, and high detection efficiency, and is more suitable for a method for detecting heparin activity in a large number of samples.

[0007] This method belongs to the biological assay method. It uses a specific experimental design to compare the estimated potency of the test sample with the specific reaction (coagulation state) produced by the equivalent standard under certain conditions, and then determines the biological activity of the test sample through calculation. Heparin potency standard is required in this method.

[0008] In order to solve the above technical problems, the present invention provides an automated heparin activity detection method, comprising the following steps:

[0009] (1) taking a heparin sample to be tested and using an automated dissolution device to prepare a heparin stock solution, and using an automated dilution device to dilute the heparin stock solution by adding a sodium chloride solution to obtain a heparin sample test solution of a suitable concentration;

[0010] (2) using an automatic sample adding device, taking selected amounts of the heparin sample test solution and the heparin standard solution, adding sodium chloride solution, sheep plasma and calcium chloride solution, mixing and incubating, and taking out the standard and sample to determine the coagulation state;

[0011] (3) Calculate the heparin activity of the sample based on the difference in coagulation state between the standard and the sample.

[0012] Specifically, in the automated heparin activity detection method, in step (1), the concentration of the heparin mother solution is 2 mg / ml-3 mg / ml;

[0013] Preferably, the concentration of sodium chloride is 0.9wt%.

[0014] Specifically, the automated heparin activity detection method, in step (1), further comprises the step of preparing a heparin standard solution, specifically comprising the step of taking a heparin standard solution or a working standard solution and adding sodium chloride solution to prepare a desired concentration.

[0015] Specifically, the automated heparin activity detection method, as an exemplary device, the automatic dissolution device is usually composed of an automated robotic arm, an execution gripper, a precision electric piston pump, a filling needle and other components. Through computer control, the volume of liquid to be added can be automatically calculated according to the weight of the solid sample and the target concentration provided. The calculation formula is: volume to be added = sample weight / target concentration; and the automatic injection process is completed. Automatic calculation and automatic injection avoid errors that may be caused by manual calculation and pipetting errors caused by manual liquid addition operations.

[0016] Specifically, the automated heparin activity detection method, as an exemplary device, the automatic dilution device is composed of a robotic arm, a precision electric piston pump, a filling needle, a precision electric pipette and other components. It can be controlled by a computer and can automatically calculate the volume of the required initial solution (the volume to be aspirated) and the volume of 0.9% sodium chloride solution (the volume to be added) according to the provided parameters: initial concentration (mg / ml, i.e., the sample concentration of step (1)), target concentration (IU / ml), estimated activity (IU / mg), target volume (ml), etc., and return the actual concentration, and complete the automatic aspiration, pipetting, injection and other processes. Automatic calculation and automatic pipetting and injection avoid errors that may be caused by manual calculation and pipetting errors caused by manual addition operations. The specific calculation formula is as follows:

[0017] (1) Sample volume to be aspirated = target volume × target concentration ÷ initial concentration ÷ estimated titer;

[0018] (2) Volume to be added = target volume - volume to be aspirated;

[0019] (3) Actual concentration = initial concentration × estimated potency × volume to be aspirated ÷ target volume.

[0020] Specifically, in the automated heparin activity detection method, in step (2), an automatic sample loading device is used for sample loading. As an exemplary device, the automatic sample loading device is composed of a mechanical arm, 8-channel and various independent precision pipettes and other components, and is controlled by a computer. It can automatically perform operations such as liquid level detection, rinsing the pipette tip, sucking samples and reagents, adding samples and reagents, withdrawing the pipette tip, and automatically calculating the sample loading cycle. Automatic continuous liquid loading can improve operating efficiency and avoid pipetting errors caused by fatigue caused by continuous manual liquid loading operations.

[0021] Specifically, in the automated heparin activity detection method, in step (2), the temperature of the incubation step is 37±1° C., and the incubation time is 60 min±1 min.

[0022] Specifically, the automated heparin activity detection method, in step (2), further comprises the steps of taking sheep blood, adding sodium citrate to the mixture, and preparing the sheep plasma by solid-liquid separation;

[0023] Preferably, the concentration of the sodium citrate solution is 6-10wt%;

[0024] Preferably, the volume ratio of the sheep blood to the sodium citrate solution is 90-95%:5-10%.

[0025] Specifically, the automated heparin activity detection method further comprises the step of adding calcium chloride solution to the sheep plasma for pre-detection;

[0026] Preferably, the concentration of the calcium chloride solution is 0.5-1.5wt%.

[0027] Specifically, in the automated heparin activity detection method, in the step (2), in the coagulation state judgment step, the coagulation degree is divided into 5 levels, including:

[0028] The degree of solidification is 0: no solidification at all;

[0029] The degree of coagulation is 0.25: the clot formed by the contents occupies 1 / 4 of the contents after being divided into four equal parts, that is, 0.25;

[0030] The degree of coagulation is 0.5: the clot formed by the contents occupies 2 / 4 of the contents after being divided into four equal parts, i.e. 0.5;

[0031] The degree of coagulation is 0.75: the clot formed by the contents occupies 3 / 4 of the contents after being divided into four equal parts, that is, 0.75;

[0032] The degree of solidification is 1: completely solidified.

[0033] Specifically, in the automated heparin activity detection method, in step (2), the coagulation state judgment step includes: when the results of the same group of samples in the same experiment show that the number of samples whose coagulation degree is greater than 0.5 or less than 0.5 is less than 2, the experiment is invalid, and the concentration of the working standard or the test sample should be adjusted and the experiment should be repeated; otherwise, the heparin activity calculation is performed.

[0034] Specifically, in the automated heparin activity detection method, in step (3), the heparin activity calculation step includes the step of automatically calculating the coagulation state value of each experimental sample according to the following formula, specifically including:

[0035] Calculate the average of the logarithmic values ​​of the volume of sodium heparin in the three adjacent tubes of the standard and the test sample and the average of the coagulation degree of the three consecutive tubes; if the average level of coagulation degree of the three consecutive tubes is 0.5, then the logarithmic value of the volume of sodium heparin in the middle corresponding to the standard or sample is X S or X U ; If the average degree of coagulation of any three consecutive samples is not 0.5, it shall be calculated according to the following formula:

[0036] X S =X Si +〔(y Si -0.5)(X Si+1 -X Si ) / (y Si -y Si+1 )〕;

[0037] X U =X Ui+〔(y Ui -0.5)(X Ui+1 -X Ui ) / (y Ui -y Ui+1 )〕;

[0038] Where:

[0039] y Si : The average value of the coagulation degree of the working standard of three consecutive samples is less than and close to the average value of the coagulation degree of 0.5;

[0040] X Si : With yS i The logarithmic mean of the volume of the working standard added in the corresponding three consecutive samples;

[0041] y Ui : The average value of the coagulation degree of the test sample in three consecutive samples is less than and close to the average value of the coagulation degree of 0.5;

[0042] X Ui : With y Ui The logarithmic mean of the volume of the test sample added in the corresponding three consecutive samples;

[0043] y Si+1 : The average coagulation value of the working standard in three consecutive samples is greater than and close to 0.5;

[0044] X Si+1 : With y Si+1 The logarithmic mean of the volume of the working standard added in the corresponding three consecutive samples;

[0045] y Ui+1 : The average value of the coagulation degree of the test sample in three consecutive test tubes is greater than and close to the average value of the coagulation degree of 0.5;

[0046] X Ui+1 : With y Ui+1 The logarithmic mean of the volume of the test sample added to three consecutive test tubes;

[0047] Logarithmic activity of the test article: M = X S -X U +㏒R; among them, In the formula,

[0048] V S : Heparin sodium activity units contained in 1 ml of working standard;

[0049] V U : The mass of heparin sodium contained in 1 ml of the test sample, mg;

[0050] If the difference in M ​​values ​​between any two experiments is greater than 0.05, continue to increase the number of experiments and calculate the confidence interval (L) of the logarithmic potency M of the test product according to the following formula, which generally does not exceed 0.2;

[0051] In the formula,

[0052] S: standard deviation of the log titer M;

[0053] k: the number of estimated valences corresponding to the logarithmic valence M;

[0054] N: the number of M values;

[0055] n: degree of freedom, n = N-1;

[0056] t: lookup table value: t value table;

[0057] The sample activity was calculated as follows:

[0058] Specifically, the automated heparin activity detection method, in step (3), further comprises a step of calculating the credibility distance of the heparin test sample;

[0059] The confidence limit range of the sample logarithmic activity is calculated as follows:

[0060]

[0061] The automated heparin activity detection method of the present invention uses sheep plasma as a biological reagent to detect heparin activity, which is easier to obtain and has lower cost than human blood purification reagents; and optimizes and limits key parameters such as the adaptability, weighing precision, dissolution concentration, dilution concentration, sample addition amount, reagent addition amount, test tube selection specifications, incubation temperature and time requirements of sheep plasma. The automated heparin activity detection method of the present invention not only has low reagent cost, but also improves accuracy and precision compared to existing detection technologies.

[0062] The automated heparin activity detection method of the present invention adopts automated operation modes such as automatic calculation, automatic pipetting, and automatic injection, which avoids errors that may be caused by human calculation to the greatest extent and avoids error factors caused by manual operation. The operation and calculation are automated, the detection efficiency is high, the accuracy and precision of the detection are improved, the detection efficiency is improved, and it is more suitable for the detection of a large number of samples.

[0063] The automated heparin activity detection method of the present invention designs a biological detection repetition strategy suitable for the method, which is more conducive to eliminating biological variability in the experiment, improving detection consistency, enhancing accuracy and precision, and the method has higher tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0065] Figure 1 A schematic diagram of the structure of the automated dissolution equipment selected for use in the present invention;

[0066] Figure 2 This is a schematic diagram of the structure of the automated dilution device selected for use in the present invention;

[0067] Figure 3 A schematic diagram of the structure of the automated sample adding device selected for use in the present invention;

[0068] The reference numerals in the figure are as follows: 1-automatic dissolution equipment cabinet, 2-four-axis robotic arm, 3-parallel clamp, 4-rotating clamp, 5-automatic dilution equipment cabinet, 6-four-axis robotic arm, 7-CNC pipette, 8-automatic sampling equipment cabinet, 9-multi-channel linkage CNC pipette, 10-six-axis robotic arm, 11-display stand. DETAILED DESCRIPTION

[0069] In the following embodiments of the present invention, as an exemplary device, the automatic dissolution device is generally composed of an automated mechanical arm, an execution gripper, a precision electric piston pump, a filling needle and other components. Through computer control, the volume of liquid to be added can be automatically calculated according to the weight of the solid sample provided and the target concentration. The calculation formula is: volume to be added = sample weight / target concentration; and the automatic injection process is completed. Automatic calculation and automatic injection avoid errors that may be caused by manual calculation and pipetting errors caused by manual liquid addition operations.

[0070] As an exemplary operable device, the present application performs heparin dissolution and selects an automated detection and dissolution device model TNAT-01DS, and its structure is as shown in the attached Figure 1 As shown, a four-axis robot arm 2, a parallel gripper 3 and a rotating gripper 4 are arranged in the cabinet 1 of the automatic dissolution device, and the volume of liquid required to be added can be automatically calculated according to the weight of the solid sample provided and the target concentration. Figure 1 The automated detection and dissolution equipment shown comprehensively considers and solves the consumables, arrangement methods, repetition strategies, test throughput, pipetting accuracy, etc. in the dissolution, dilution, and sample addition steps. At the same time, the calculation method and parameters that match the detection of this method are set in the control software of the equipment. Since the entire set of equipment matches the various operating steps of the test method, it can replace manual calculations, setting complex parameters, etc., and can further improve efficiency. In this regard, it is more efficient than conventional equipment.

[0071] In the following embodiments of the present invention, as an exemplary device, the automatic dilution device is composed of a mechanical arm, a precision electric piston pump, a filling needle, a precision electric pipette and other components. It can be controlled by a computer and can automatically calculate the volume of the required initial solution (the volume to be aspirated) and the volume of 0.9% sodium chloride solution (the volume to be added) according to the provided parameters: initial concentration (mg / ml, i.e., the sample concentration in step (1)), target concentration (IU / ml), estimated activity (IU / mg), target volume (ml), etc., and return the actual concentration, and complete the automatic aspiration, pipetting, injection and other processes. Automatic calculation and automatic pipetting and injection avoid errors that may be caused by manual calculation and pipetting errors caused by manual addition operations. The specific calculation formula is as follows:

[0072] (1) Sample volume to be aspirated = target volume × target concentration ÷ initial concentration ÷ estimated titer;

[0073] (2) Volume to be added = target volume - volume to be aspirated;

[0074] (3) Actual concentration = initial concentration × estimated potency × volume to be aspirated ÷ target volume.

[0075] As an exemplary operable device, the automatic dilution device model TNAT-01AN is selected for heparin dilution in this application, and its structure is as shown in the attached Figure 2 As shown, a four-axis robotic arm 6 and a numerically controlled pipette 7 are arranged in the automatic dilution equipment cabinet 5, which can complete the processes of automatic sample suction, pipetting, injection, etc.

[0076] In the following embodiments of the present invention, as an exemplary device, the automatic sample loading device is composed of a mechanical arm, 8-channel and various independent precision pipettes and other components, and is controlled by a computer. It can automatically perform operations such as liquid level detection, rinsing the pipette tip, sucking samples and reagents, adding samples and reagents, withdrawing the pipette tip, and automatically calculating the sample loading cycle. Automatic continuous liquid loading can improve operating efficiency and avoid pipetting errors caused by fatigue caused by continuous manual liquid loading operations.

[0077] As an exemplary operable device, the automatic loading device model TNAT-01PT is selected for loading heparin samples in this application, and its structure is as shown in the attached Figure 3 As shown, a multi-channel linkage CNC pipette 9, a six-axis robotic arm 10 and a display stand 11 are arranged in the automatic loading device cabinet 8, which can automatically perform operations such as liquid level detection, rinsing the pipette tip, sucking samples and reagents, adding samples and reagents, withdrawing the pipette tip, and automatically calculating the loading cycle.

[0078] Example 1

[0079] Test preparation

[0080] Preparation of 0.9% sodium chloride solution: Weigh 9.00 g of sodium chloride, place it in a reagent bottle, add 800 ml of test water to dissolve, and make up to 1000 ml.

[0081] Preparation of 1% calcium chloride solution: Weigh 10.00 g of anhydrous calcium chloride, place it in a reagent bottle, add 800 ml of test water to dissolve, and make up to 1000 ml.

[0082] Preparation of Sheep Plasma: Collect fresh sheep blood directly into a container containing 8% sodium citrate solution at a ratio of 1 volume per 19 volumes of blood. Immediately mix by gently stirring and inverting the container. Immediately centrifuge the sheep blood and pool the separated plasma. Add 1 mL of plasma and 0.2 mL of calcium chloride solution (1 / 100) to a clean test tube, mix, and if a solid clot forms within 5 minutes, it is considered to be suitable for testing. To store plasma for future use, aliquot the sheep plasma into volumes not exceeding 100 mL and store in a frozen state. When storing, prevent partial thawing before use. Before using the plasma, thaw the frozen plasma in a water bath at a temperature not exceeding 37°C. Filter the thawed plasma through a coarse filter to remove particulate matter.

[0083] Pre-testing of sheep plasma: After the sheep plasma is prepared, different concentrations of heparin standards are used to screen out the concentration of heparin standards that meets the requirements for the establishment of the experiment, which is recorded as the "appropriate experimental concentration". It should be noted that the "appropriate experimental concentration" of each batch of sheep plasma is different, so the plasma needs to be pre-tested, and sheep plasma between 1IU / ml and 4IU / ml is generally selected for the experiment. The "appropriate experimental concentration" specifically set in the scheme of the present invention allows the sheep plasma to show changes in coagulation state within the set volume distribution range when used for detection, and the coagulation state span is moderate, and it shows sufficiently sensitive changes within the appropriate concentration range.

[0084] Sample preparation

[0085] An appropriate amount of heparin sample (100-120 mg) is accurately weighed with a 1 / 10,000 electronic balance and placed in a covered container. Using an automatic dissolution device, 0.9% sodium chloride solution is injected into the container to prepare a sample solution containing 2 mg / ml-3 mg / ml of heparin.

[0086] Use an automatic dilution device to dilute the above heparin stock solution to the required "experimental appropriate concentration" with 0.9% sodium chloride solution according to the estimated activity of the sample, and complete the automatic sampling, pipetting, injection and other processes.

[0087] Standard solution preparation

[0088] Prepare the heparin standard solution or working standard solution to the "appropriate experimental concentration". The "appropriate experimental concentration" of the standard and sample must be equal, accurate to two decimal places.

[0089] Adding samples and reagents

[0090] Use a clean stoppered test tube ( Round bottom, colorless and transparent, smooth inner wall) is used as a container for series concentration reaction, and each sample or standard needs to be in a group of 8 test tubes, numbered 1#-8#; when multiple samples are tested at the same time, multiple groups of test tubes need to be prepared and arranged neatly. In this embodiment, the selection of a suitable stoppered test tube helps to make the plasma clot uniform and controllable in shape and size, which is more conducive to the operator to knock and observe accurately during judgment.

[0091] Use an automatic sample adding device to add corresponding samples and reagents to each numbered test tube of each group according to the steps and volumes in Table 1. The composition of the sample adding system described herein helps to obtain a suitable data distribution.

[0092] Table 1 Sample addition parameters

[0093]

[0094] Incubation

[0095] Stopper the test tube after adding liquid, shake well to make the inner wall of the test tube completely wet and no bubbles are generated, place it in a 37±1℃ water bath (note: the longest time from adding sheep plasma to placing it in the water bath should not exceed 20 minutes) and incubate for 60 minutes±1 minute. Take it out of the water bath to judge the coagulation state.

[0096] Solidification state judgment

[0097] The same inspector knocks each group of test tubes one by one from low concentration to high concentration, knocking four times on the palm with the same force, observing the size of the clot with the naked eye, and judging the coagulation state of the contents in each tube. The coagulation degree is divided into 5 levels, namely:

[0098] Solidification degree 0: no solidification at all;

[0099] The degree of coagulation is 0.25: the clot formed by the contents occupies 1 / 4 of the contents after being divided into four equal parts, that is, 0.25;

[0100] The degree of coagulation is 0.5: the clot formed by the contents occupies 2 / 4 of the contents after being divided into four equal parts, i.e. 0.5;

[0101] The degree of coagulation is 0.75: the clot formed by the contents occupies 3 / 4 of the contents after being divided into four equal parts, that is, 0.75;

[0102] The degree of solidification is 1: completely solidified.

[0103] Judgment of experimental validity: When the results of the same group of test tubes in the same experiment show that the number of tubes with a coagulation degree greater than 0.5 or less than 0.5 is less than two, the experiment is invalid and the concentration of the working standard or test sample should be adjusted and the experiment repeated.

[0104] In this embodiment, the added volume A of each group of heparin standard solution is recorded separately. Si and the solidification coefficient Y Si ; And, record the added volume A of each group of heparin test solution Ui and the solidification coefficient Y Ui .

[0105] In this embodiment, the calculation formula for the activity P of the heparin sample to be tested is as follows ①-⑥ calculation:

[0106] ①The logarithm of the volume of the heparin standard solution X Si The calculation formula is:

[0107] X Si =㏒A Si ;

[0108] ②The logarithm of the volume of the heparin test solution X Ui The calculation formula is:

[0109] X Ui =㏒ A Ui ;

[0110] ③ Logarithmic volume of heparin standard solution X S The calculation formula is:

[0111]

[0112] ④ Logarithmic volume of heparin test solution X U The calculation formula is:

[0113]

[0114] ⑤The calculation formula of the logarithmic activity M of the heparin sample to be tested is:

[0115]

[0116] ⑥ The calculation formula for the activity P of the heparin sample to be tested is:

[0117] P=anti M ;

[0118] In the above formula:

[0119] A Si is the added volume of heparin standard solution;

[0120] A ui is the added volume of heparin test solution;

[0121] V S It is the heparin sodium activity unit contained in 1 ml of heparin standard solution;

[0122] V U is the mass of heparin sodium contained in 1 ml of heparin test solution, mg;

[0123] P is the activity of the heparin sample to be tested, in IU / mg.

[0124] Result calculation

[0125] This embodiment refers to the above calculation parameters and formulas, uses the designed formula and the verified Excel spreadsheet to calculate the activity of the sample, and only needs to fill in the determined coagulation state of each test tube into the corresponding cell to automatically calculate the sample activity.

[0126] Calculate the average of the logarithmic values ​​of the volume of sodium heparin in three consecutive tubes of the standard and test sample, i.e., tubes 1, 2, 3, 2, 3, 4, 3, 4, 5, 4, 5, 6, and the average of the coagulation degree of the three consecutive tubes. If the average level of coagulation degree of the three consecutive test tubes is 0.5, then the logarithmic value of the volume of sodium heparin in the middle corresponding to the standard or sample is X S or X U If the average solidification degree of any three consecutive tubes is not 0.5, calculate according to the following formula:

[0127] X S =X Si +〔(y Si -0.5)(X Si+1 -X Si ) / (y Si -y Si+1) 〕;

[0128] X U =X Ui +〔(y Ui -0.5)(X Ui+1 -X Ui ) / (y Ui -y Ui+1) 〕;

[0129] Where:

[0130] y Si : The average value of the coagulation degree of the working standard of three consecutive test tubes is: less than and close to the average value of the coagulation degree of 0.5;

[0131] X Si : With y Si The logarithmic mean of the volume of the working standard added to the corresponding three consecutive test tubes;

[0132] y Ui : The average value of the coagulation degree of the test sample in three consecutive test tubes is: the average value of the coagulation degree that is less than and close to 0.5;

[0133] X Ui : With y Ui The logarithmic mean of the volume of the test sample added to the corresponding three consecutive test tubes;

[0134] y Si+1 : The average coagulation value of the working standard in three consecutive test tubes is: the average coagulation value greater than and close to 0.5;

[0135] X Si+1 : With y Si+1 The logarithmic mean of the volume of the working standard added to the corresponding three consecutive test tubes;

[0136] y Ui+1 : The average value of the coagulation degree of the test sample in three consecutive test tubes is: the average value of the coagulation degree greater than and close to 0.5;

[0137] X Ui+1 : With y Ui+1 The logarithmic mean of the volume of the test sample added to three consecutive test tubes.

[0138] The sample activity was calculated as follows: in,

[0139] M is the logarithmic activity of the test product: M = X S -X U +㏒R; where in,

[0140] V S : Heparin sodium activity units contained in 1 ml of working standard;

[0141] V U : The mass of heparin sodium contained in 1 ml of the test sample, mg.

[0142] If the difference in M ​​values ​​between any two experiments is greater than 0.05, continue to increase the number of experiments and calculate the confidence interval (L) of the logarithmic potency M of the test sample according to the following formula, which generally does not exceed 0.2.

[0143] Where:

[0144] S: standard deviation of the log titer M;

[0145] k: the number of estimated valences corresponding to the logarithmic valence M;

[0146] N: the number of M values;

[0147] n: degree of freedom, n = N-1;

[0148] t: table lookup value: t value table.

[0149] In this embodiment, the confidence limit range of the logarithmic activity of the sample is calculated as follows:

[0150] In this example, the activity of known heparin standards was tested according to the parameters in Table 1, and the results of each sample are shown in Table 2 below.

[0151] Table 2 Experimental results of each experimental sample

[0152]

[0153] It can be seen that the method of the present invention has good accuracy in detecting heparin activity.

[0154] Example 2 Precision Investigation

[0155] This example tests different types of heparin samples based on the scheme in Example 1, and comprehensively examines the precision under factors such as different time and different personnel.

[0156] In this example, the results of each sample are shown in Table 3 below.

[0157] Table 3 Experimental results of each experimental sample

[0158]

[0159]

[0160]

[0161] It can be seen that the accuracy of 4 parallel tests of multiple known heparin standards (covering the range of 40-250 IU / mg) using this method was within ±1%. For the same sample, the RSD% was <2% when factors such as different times and different personnel were comprehensively examined.

[0162] Example 3

[0163] This example conducts corresponding methodological investigations based on the scheme in Example 1, and the specific experimental strategy requirements are as follows.

[0164] Single independent experiment: It is completed independently by the same inspector. It can be called an independent experiment only if all the following conditions are met at the same time:

[0165] (1) The test solution is prepared by the same inspector each time, and the coagulation state is determined by the same inspector;

[0166] (2) Each time, the standard and sample series test tubes must be tested simultaneously for comparison, and the standard series must be run twice in parallel;

[0167] (3) A control sample (also known as a known sample or system suitability sample) must be tested each time;

[0168] (4) Use only the same filtered sheep plasma / bottle each time;

[0169] (5) Place the samples in the same water bath each time and put them in and take them out at the same time.

[0170] Repeated testing of test solutions: refers to multiple dilutions and testing of the same dissolved test solution, or multiple testing of the same test solution. Multiple repeated tests can be included in a single independent experiment.

[0171] Parallel testing of samples: Repeat multiple "single independent experiments" on the same sample, and take the average of the results of multiple "single independent experiments" as the sample result. In crude product testing, at least 2 parallel test results are required to take the average of the sample result, and the two "single independent experiments" are best completed by different inspectors.

[0172] The setting of the repetition strategy specified in the present application scheme takes into account the operator factors, personnel subjective judgment factors, experimental material (plasma) variability factors, reaction condition consistency factors, etc. in this method; without these specific conditions, the test results will be affected and interfered by the above factors, and the stability of the results will be much worse, which is also higher than the requirements of biological detection methods under normal circumstances.

[0173] In this example, the heparin activity detection method was investigated in terms of accuracy, precision, and resistance to denaturation according to the requirements of ICH Q2 (R1). The results are shown in Table 4 below.

[0174] Table 4 Methodology validation results

[0175]

[0176] The above verification results show that by adopting the key parameter control and biological detection repetition strategy designed by this method, and using automated calculation and operation, the accuracy can reach within ±1% and the precision can reach <1%.

[0177] Comparative Example 1

[0178] This comparative example is based on the scheme in Example 1, but manual operation is performed and the experimental repetition strategy specified therein is not implemented. At the same time, the same batch of samples are repeatedly tested under the scheme operation in Example 1, and the comparison results are shown in Table 5.

[0179] Table 5 Comparison of Example 1 and Comparative Example 1

[0180]

[0181] It can be seen that when the method is used for automatic detection operation and the experimental repetition strategy is implemented, the precision of detecting the same batch of samples is significantly improved.

[0182] Comparative Example 2

[0183] This comparative example is based on the scheme in Example 1, and the only difference is that the dissolution step, dilution step and injection step are all manually detected, and the automatic detection working sequence and beat time are arranged as shown in Table 6. The detection efficiency of the automatic detection mode (scheme in Example 1) and the manual detection mode (scheme in this comparative example) is statistically analyzed.

[0184] Table 6 Sorting table

[0185]

[0186] As in the scheme of Example 1, under the condition of using automated equipment, 4 people are required to cooperate to test 81 batches (162 samples), and the working hours consumed are 8h*4 people=32h.

[0187] As in the comparative scheme, without the use of automated equipment, 11 people are required to collaborate to test 81 batches (162 samples) (manual testing efficiency: 15 batches / group-2 people / day), consuming 8*11 people=88 hours of work.

[0188] It can be seen that when using this method for automatic detection operation, the inspection efficiency is greatly improved.

[0189] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for automated detection of heparin activity, characterized in that: The steps include: (1) taking a heparin sample to be tested and using an automated dissolution device to prepare a heparin mother solution, and using an automated dilution device to add a sodium chloride solution to dilute the heparin mother solution to obtain heparin sample solutions of different concentrations; (2) using an automatic sampling device, taking selected amounts of the heparin sample solution, sodium chloride solution, sheep plasma, and calcium chloride solution, mixing and incubating, and taking out samples to determine the coagulation state; (3) Calculate the heparin activity of the sample based on the coagulation state of the sample.

2. The method for automated detection of heparin activity according to claim 1, characterized in that: In the step (1), the concentration of the heparin mother solution is 2 mg / ml-3 mg / ml; Preferably, the concentration of sodium chloride is 0.9wt%.

3. The method for automated detection of heparin activity according to claim 1 or 2, characterized in that: The step (1) also includes the step of preparing a heparin standard solution, specifically including the step of taking a heparin standard solution or a working standard solution and adding it to a sodium chloride solution to prepare a desired concentration.

4. The method for automated detection of heparin activity according to any one of claims 1 to 3, characterized in that: In step (2), the temperature of the incubation step is 37±1° C., and the incubation time is 60 min±1 min.

5. The method for automated detection of heparin activity according to any one of claims 1 to 4, characterized in that: The step (2) further comprises the step of taking sheep blood, adding sodium citrate and mixing, and preparing the sheep plasma by solid-liquid separation; Preferably, the concentration of the sodium citrate solution is 6-10wt%; Preferably, the volume ratio of the sheep blood to the sodium citrate solution is 90-95%:5-10%.

6. The method for automated detection of heparin activity according to claim 4, characterized in that: It also includes the step of adding calcium chloride solution to the sheep plasma for pre-testing; Preferably, the concentration of the calcium chloride solution is 0.5-1.5wt%.

7. The method for automated detection of heparin activity according to any one of claims 1 to 6, characterized in that: In the step (2), in the step of determining the coagulation state, the coagulation degree is divided into 5 levels, including: The degree of solidification is 0: no solidification at all; The degree of coagulation is 0.25: the clot formed by the contents occupies 1 / 4 of the contents after being divided into four equal parts, i.e. 0.25; The degree of coagulation is 0.5: the clot formed by the contents occupies 2 / 4 of the contents after being divided into four equal parts, i.e. 0.5; The degree of coagulation is 0.75: the clot formed by the contents occupies 3 / 4 of the contents after being divided into four equal parts, that is, 0.75; The degree of solidification is 1: completely solidified.

8. The method for automated detection of heparin activity according to claim 7, characterized in that: In the step (2), the coagulation state judgment step includes: when the results of the same group of samples in the same experiment show that the number of samples with a coagulation degree greater than 0.5 or less than 0.5 is less than 2, the experiment is invalid, and the concentration of the working standard or the test sample should be adjusted and the experiment should be repeated; otherwise, the heparin activity calculation is performed.

9. The method for automated detection of heparin activity according to any one of claims 1 to 8, characterized in that: In the step (3), the heparin activity calculation step includes the step of automatically calculating the coagulation state value of each experimental sample according to the following formula, specifically including: Calculate the average of the logarithmic values ​​of the volume of heparin sodium in the standard and the three adjacent test samples and the average of the coagulation degree of the three consecutive tubes; if the average level of coagulation degree of the three consecutive tubes is 0.5, then the logarithmic value of the volume of heparin sodium in the corresponding standard or sample in the middle is X S or X U ; If the average solidification degree of any three consecutive tubes is not 0.5, calculate according to the following formula: X S =X Si +〔(and Si (0.5)(X) Si+1 -X Si ) / (and Si -and Si+1 )〕; X U =X Ui +〔(and Ui (0.5)(X) Ui+1 -X Ui ) / (and Ui -and Ui+1 )〕; Where: y Si : The average value of the coagulation degree of three consecutive tubes of the working standard is less than and close to the average value of the coagulation degree of 0.5; X Si : With y Si The logarithmic mean of the volume of the working standard added in the corresponding three consecutive samples; y Ui : The average value of the coagulation degree of the test sample in three consecutive tubes is less than and close to the average value of the coagulation degree of 0.5; X Ui : With y Ui The logarithmic mean of the volume of the test sample added to the corresponding three consecutive tubes; y Si+1 : The average coagulation value of the working standard in three consecutive tubes is greater than and close to 0.5; X Si+1 : With y Si+1 The logarithmic mean of the volume of the working standard added in three consecutive tubes; y Ui+1 : The average value of the coagulation degree of the test sample in three consecutive test tubes is greater than and close to the average value of the coagulation degree of 0.5; X Ui+1 : With y Ui+1 The logarithmic mean of the volume of the test sample added to three consecutive test tubes; Logarithmic activity of the test article: M = X S -X U +㏒R; among them, In the formula, V S : Heparin sodium activity units contained in 1 ml of working standard; V U : The mass of heparin sodium contained in 1 ml of the test sample, mg; If the difference in M ​​values ​​between any two experiments is greater than 0.05, continue to increase the number of experiments and calculate the confidence interval (L) of the logarithmic potency M of the test article according to the following formula, which generally does not exceed 0.2; In the formula, S: standard deviation of the log titer M; k: the number of estimated valences corresponding to the logarithmic valence M; N: the number of M values; n: degree of freedom, n = N-1; t: table lookup value: t value table; The sample activity is calculated according to the following formula: P = anti㏒M × working standard correction factor.

10. The method for automated detection of heparin activity according to claim 9, characterized in that: The step (3) further includes a step of calculating the credibility distance of the heparin test sample; The confidence limit range of the sample logarithmic activity is calculated as follows: The confidence limit of sample logarithmic potency = M ± (L / 2).

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