CN114814129A

CN114814129A - Calibration product for rivaroxaban detection and preparation method thereof

Info

Publication number: CN114814129A
Application number: CN202210759935.2A
Authority: CN
Inventors: 胡彦勇; 曹佳强; 蔡晓霞; 赵伟
Original assignee: Shenzhen Dymind Biotechnology Co Ltd
Current assignee: Shenzhen Dymind Biotechnology Co Ltd
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2022-07-29

Abstract

The invention discloses a calibration product for rivaroxaban detection and a preparation method thereof, wherein the calibration product is freeze-dried powder, the calibration product comprises serum and a freeze-drying protective agent, the calibration product also comprises rivaroxaban, and the content of the rivaroxaban in the calibration product is 0-600 ng/mL; the preparation method comprises the following steps: carrying out biological safety detection on the serum, and reserving the serum after the serum is qualified; preparing rivaroxaban into mother liquor for later use; adding a freeze-drying protective agent into the serum and uniformly mixing to obtain a mixed solution; dividing the mixed solution into a plurality of parts, and respectively adding rivaroxaban mother solutions with different amounts to obtain a plurality of calibrator solutions; subpackaging the calibrator solution and performing vacuum freeze drying to obtain a calibrator; the calibrator of the invention takes serum as a substrate, and a freeze-drying protective agent is added to prepare the calibrator with good stability through a freeze-drying process; the calibrator is matched with an anti-Xa kit for use, so that the rivaroxaban content in a patient sample can be accurately detected, the sensitivity is high, the specificity is strong, the time consumption is short, the raw materials are convenient to obtain, and the preparation method is simple.

Description

Calibration product for rivaroxaban detection and preparation method thereof

Technical Field

The invention relates to the technical field of immunoassay, in particular to a calibration material for rivaroxaban detection and a preparation method thereof.

Background

Rivaroxaban is a novel oral anticoagulant, and belongs to the Xa factor competitive inhibitor class. Rivaroxaban inhibits the formation of thrombin by inhibiting the activity of Xa (blood coagulation factor) and blocks the intrinsic and extrinsic coagulation pathways, thereby effectively preventing thrombosis, and belongs to a Novel Oral Anticoagulant (NOAC). In some special cases, such as elderly patients (> 75 years), liver and kidney insufficiency, trauma, acute surgery, higher bleeding risk score, multiple medications, etc., it is necessary to evaluate the anticoagulation effect of rivaroxaban.

Attempts are currently made to evaluate rivaroxaban for anticoagulant activity using the results of PT and APTT tests. Rivaroxaban prolongs PT concentration-dependently, but different PT agents are different in sensitivity to rivaroxaban and PT is not sensitive to small doses of oral anticoagulants. In addition, PT is not a specific detection method, and its characteristic of relying on reagent sensitivity makes PT unsuitable as a method for quantitatively detecting rivaroxaban concentration. The APTT value and the rivaroxaban blood concentration have no unified metrological correlation, and for a patient who is known to take rivaroxaban anticoagulation treatment, APTT prolonging can indicate that the rivaroxaban blood concentration is higher, but when APTT is normal, rivaroxaban with low treatment dose cannot be excluded. And compared with PT, the sensitivity of evaluating rivaroxaban anticoagulant activity by APTT is lower, the concentration of a detection reagent is difficult to calculate, the difference of laboratory detection results is large, and the method is not suitable for qualitative and quantitative detection of rivaroxaban. In addition, the sensitivity of the dRVVT detection for evaluating the rivaroxaban blood concentration is high but the specificity is poor, the TEG may have higher sensitivity and specificity for qualitative detection of rivaroxaban, but the quantitative detection of the rivaroxaban blood concentration is lack of related research; generally, it is generally accepted that the rivaroxaban blood concentration can be more accurately detected by a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) determination method. However, the HPLC-MS/MS method is generally used in a few laboratories, and has the disadvantages of long running time, high cost, limited application range and is not suitable for being used as a clinical routine detection mode.

Therefore, a method with high sensitivity, strong specificity and high testing speed is needed for detecting the blood concentration of rivaroxaban so as to carry out clinical monitoring, and the simple and quick anti-Xa factor color development method can meet the requirements. In order to obtain accurate rivaroxaban blood concentration, a calibration curve needs to be established, so that a rivaroxaban calibrator with accurate concentration needs to be prepared. The calibration product is mainly a reference substance used as an independent variable value in a calibration function and used for calibrating detection items during quantitative detection, so that the detection results of all detection systems have higher accuracy and consistency. Most of the current preparation of the calibrator needs to use human plasma to be closer to the real sample condition, but the human plasma is difficult to obtain in large batch, which has great influence on the development and subsequent production of the calibrator.

Disclosure of Invention

The invention aims to solve the technical problem of providing a calibration product for rivaroxaban detection and a preparation method thereof aiming at the defects of the prior art.

The technical scheme adopted by the invention for solving the technical problems is as follows: the utility model provides a calibrator for rivaroxaban detects, the calibrator is the freeze-dried powder, the calibrator includes serum, freeze-drying protective agent, the calibrator still includes rivaroxaban, rivaroxaban is in the content of calibrator is 0~600 ng/mL.

Further, in the calibrator, it is preferable that the serum is human serum.

Further, in the calibrator, the calibrator preferably comprises a plurality of calibrators with at least 5 different rivaroxaban contents, and the rivaroxaban contents in the 5 calibrators are respectively 0ng/mL, 40-60 ng/mL, 90-120 ng/mL, 280-320 ng/mL and 480-520 ng/mL.

Further, in the calibrator, it is preferable that the lyoprotectant includes at least one of an amino acid lyoprotectant, a saccharide lyoprotectant, an alcohol lyoprotectant, a protein lyoprotectant, and a salt lyoprotectant.

Further, in the calibrator, it is preferable that the amino acid-based lyoprotectant includes at least one of alanine, glycine, and arginine;

further, in the calibrator, it is preferable that the saccharide lyoprotectant includes at least one of trehalose, sucrose, and glucose;

further, in the calibrator, it is preferable that the alcohol lyoprotectant includes at least one of glycerol, mannitol, xylitol, inositol, sorbitol, and polyethylene glycol;

further, in the calibrator, preferably, the salt-type lyoprotectant includes at least one of sodium chloride, potassium chloride and sodium glutamate;

further, in the calibrator, it is preferable that the protein lyoprotectant includes at least one of bovine serum albumin and HSA.

Further, in the calibrator, it is preferable that the lyoprotectant includes the components: mannitol, trehalose, bovine serum albumin and potassium chloride.

Further, in the calibrator, it is preferable that the mass-to-volume ratio of the lyoprotectant to the serum is: 3-8% of mannitol, 1-5% of trehalose, 3-8% of bovine serum albumin and 0.5-1.5% of potassium chloride.

Further, in the calibrator, it is preferable that the mass-to-volume ratio of the lyoprotectant to the serum is: 5% of mannitol, 3% of trehalose, 5% of bovine serum albumin and 1% of potassium chloride.

A preparation method of the calibration product for rivaroxaban detection comprises the following steps:

s1, performing biological safety detection on the serum, and detecting the serum to be qualified for later use;

s2, preparing rivaroxaban into mother liquor for later use;

s3, adding a freeze-drying protective agent into the serum and uniformly mixing to obtain a mixed solution;

s4, dividing the mixed solution into multiple parts, and respectively adding rivaroxaban mother solutions with different amounts according to preset target values to obtain multiple calibrator solutions;

and S5, subpackaging the plurality of calibrator solutions and performing vacuum freeze drying to obtain a plurality of calibrators with different rivaroxaban contents.

Further, in the method for preparing the calibrator, preferably, a plurality of calibrator solutions are separately dispensed into 3 mL vials, each vial containing 1 mL of penicillin.

Further, in the method for preparing the calibrator, it is preferable that the vacuum freeze-drying includes a prefreezing stage, a sublimation drying stage, and a desorption drying stage;

in the pre-freezing stage, the pre-freezing temperature is-35 to-50 ℃, the temperature reduction time is 180 to 260min, and the time is maintained for 300 to 400min after temperature reduction;

the temperature of the sublimation drying stage is-25 to-40 ℃, the temperature reduction time is 50 to 100min, the maintenance time after temperature reduction is 1200 to 2000min, and the vacuum degree is 0.05 to 0.15 bar;

and (3) at the resolving and drying stage, the temperature is 25-32 ℃, the temperature rise time is 200-300 min, the maintaining time after temperature rise is 450-540 min, the vacuum degree is 0.05-0.15 bar, and the calibrator is obtained after freeze-drying.

Further, in the preparation method of the calibrator, the prefreezing temperature in the prefreezing stage is preferably-40 ℃, the temperature reduction time is preferably 240min, and the time is preferably 360min after temperature reduction;

the temperature of sublimation drying stage is-30 deg.C, cooling time is 60min, maintaining time is 1500min after cooling, and vacuum degree is 0.1 bar;

the temperature of the desorption drying stage is 30 ℃, the temperature rise time is 240min, the maintaining time is 480min after temperature rise, and the vacuum degree is 0.1 bar.

The invention has the beneficial effects that: according to the calibration product for rivaroxaban detection and the preparation method thereof, the calibration product takes serum as a matrix to prepare calibration products with different rivaroxaban levels, so that the limitation on raw materials caused by taking plasma as a matrix is overcome, and the cost is reduced; the calibration product has good accuracy, uniformity and stability, and is beneficial to production and use of clients; according to the preparation method of the calibration product, the rivaroxaban calibration product with low concentration and high concentration level is prepared by a stable process and adding a proper protective agent and a proper freeze-drying process, so that the problems of unstable process, short storage period and the like in the preparation process of the conventional calibration product are solved, and the batch production is facilitated; the detection system formed by the calibrator matched with the matched reagent and the instrument has good correlation with the imported brand analysis system for testing real samples, has high consistency, can realize the replacement of the imported system, reduces the detection and maintenance cost of hospitals, and can reduce the treatment cost of patients and the burden.

Drawings

The invention will be further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a calibration curve of a calibrator of example 2 of the present invention;

FIG. 2 is a calibration graph of a calibrator of comparative example 2 according to the present invention;

FIG. 3 is a linear regression curve of the test results of the calibrator and the reference system of example 2 of the present invention;

FIG. 4 is a linear regression curve of the test results of the calibrator of comparative example 2 of the present invention and the reference system;

FIG. 5 is a substrate interchangeability evaluation of calibrators of example 2 of the present invention;

FIG. 6 is a substrate interchangeability evaluation of a calibrator of comparative example 2 according to the present invention.

Detailed Description

In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments of the present invention will now be described in detail.

A calibrator for rivaroxaban detection is freeze-dried powder and comprises serum and a freeze-drying protective agent, and the calibrator further comprises rivaroxaban, wherein the content of rivaroxaban in the calibrator is 0-600 ng/mL.

The calibrator of the invention takes the serum as the substrate to prepare calibrators with different rivaroxaban levels, thereby overcoming the limitation on raw materials caused by taking the plasma as the substrate and simultaneously reducing the cost; the calibration product has good accuracy, uniformity and stability, and is beneficial to production and use of clients; according to the preparation method of the calibration product, the rivaroxaban calibration product with low concentration and high concentration level is prepared by a stable process and adding a proper protective agent and a proper freeze-drying process, so that the problems of unstable process, short storage period and the like in the preparation process of the conventional calibration product are solved, and the batch production is facilitated; the detection system formed by the calibrator matched with the matched reagent and the instrument has good correlation with the imported brand analysis system for testing real samples, has high consistency, can realize the replacement of the imported system, reduces the detection and maintenance cost of hospitals, and can reduce the treatment cost of patients and the burden.

Furthermore, preferably, the serum is human serum, the human serum is a basic matrix, the effect of the solvent is similar to that of the human serum, meanwhile, the human serum contains a large amount of protein, and the human serum can also be used as a freeze-drying excipient of the calibrator for maintaining the shape of the calibrator in the freeze-drying process, can bear larger temperature range change and is beneficial to maintaining the cake-shaped structure of the calibrator.

The rivaroxaban calibrator is subjected to multi-point calibration during calibration, wherein the 1 st point is a point 0 which does not contain rivaroxaban, and therefore rivaroxaban is not added, and is also one of the 1 calibration points belonging to a calibrator, and other calibrators are calibrators containing different amounts of rivaroxaban, so that part of calibrators also contain rivaroxaban, and the rivaroxaban is added because the rivaroxaban is required to be a component in the calibrator, so that a patient sample treated by clinical rivaroxaban is simulated; the specific addition amount of rivaroxaban is determined according to the target value of the calibrator, namely the addition is performed according to the fixed value range of the calibrator; further, the calibrator comprises a plurality of calibrators with at least 5 different rivaroxaban contents, and the rivaroxaban contents in the 5 calibrators are respectively 0ng/mL, 40-60 ng/mL, 90-120 ng/mL, 280-320 ng/mL and 480-520 ng/mL.

The addition of the freeze-drying protective agent can improve the stability of the calibrator, inhibit nonspecific adsorption in a system on the premise of not influencing the sensitivity of a detection reaction system, improve the solid content in the calibrator, be used for maintaining the form of the calibrator in the freeze-drying process, bear larger temperature range change, be beneficial to maintaining the cake-shaped structure and have extremely small using amount; meanwhile, the freeze-drying protective agent is also used for protecting rivaroxaban in the freeze-drying process of the calibrator, so that the loss of rivaroxaban in the freeze-drying process is reduced, and the due content and activity of rivaroxaban are maintained, so that the detection result of the freeze-dried calibrator is within an expected acceptance range and meets the requirement of the calibrator; further, the freeze-drying protective agent comprises at least one of amino acid freeze-drying protective agent, saccharide freeze-drying protective agent, alcohol freeze-drying protective agent, salt freeze-drying protective agent and protein freeze-drying protective agent; further, it is preferable that the amino acid type lyoprotectant comprises at least one of alanine, glycine and arginine, the amino acid is a basic constituent unit of the protein, and the amino acid is added to prevent the protein from being denatured and to raise the collapse temperature of the calibrator during lyophilization so as to prevent the protein in the calibrator from being denatured due to collapse; the saccharide freeze-drying protective agent comprises at least one of trehalose, sucrose and glucose, the alcohol freeze-drying protective agent comprises at least one of glycerol, mannitol, xylitol, inositol, sorbitol and polyethylene glycol, the addition of the saccharide and the alcohol freeze-drying protective agent is favorable for inhibiting the freeze denaturation of protein on one hand and can ensure that the condition of precipitation and non-specific aggregation of a calibrator due to the freezing of the calibrator can not be caused, so that the stability of the calibrator in production, storage, transportation and the like is ensured, particularly the stability in a vacuum freeze-drying stage is ensured, oligosaccharides such as sucrose, trehalose and the like can play a low-temperature protection function and a dehydration function on the calibrator, trehalose can also ensure that the calibrator has relatively high glass transition temperature, the plasticizing effect of water on a glass transition state is effectively prevented, and the protection of rivaroxaban is favorable. The salt freeze-drying protective agent comprises at least one of sodium chloride, potassium chloride and sodium glutamate, preferably potassium chloride, has a shaping effect, is used for maintaining the balance of the whole system, plays a role in protection and also plays a role in regulating the osmotic pressure of a calibrator; the protein freeze-drying protective agent comprises at least one of bovine serum albumin and HSA, has a shaping effect, can increase the protein concentration in the whole system, and has a protective effect on rivaroxaban.

Further, it is preferred that the lyoprotectant comprises the components: mannitol, trehalose, bovine serum albumin and potassium chloride, wherein the mass volume ratio of the components in serum is as follows: 3-8% of mannitol, 1-5% of trehalose, 3-8% of bovine serum albumin and 0.5-1.5% of potassium chloride; under the proportion, the freeze-drying protective agent has good protection effect, and the prepared calibrator has good stability, high accuracy, good appearance of finished products, short redissolution time and good clarity after redissolution.

In one embodiment, the lyoprotectant includes the components: trehalose, mannitol, bovine serum albumin and potassium chloride, wherein the mass volume percentage of the components in the mixed serum is as follows: the protective effect of the freeze-drying protective agent is good, the prepared calibrator has good stability, high accuracy, good appearance of finished products, short redissolution time and good clarity after redissolution.

A preparation method of a calibrator for rivaroxaban detection comprises the following steps:

and S1, performing biological safety detection on the serum, and detecting the serum to be qualified for later use. Specifically, the serum is preferably normal human serum, a special biological safety detection kit can be used for performing biological safety detection on the serum, the biological safety detection items comprise HIV, HBV, HCV, TP and the like, and if the test results of all the items are negative, no biological risk is proved, namely the serum is qualified.

S2, preparing rivaroxaban into mother liquor for later use, wherein the specific step can be dissolving rivaroxaban in ultrapure water with a certain volume, and preparing the mother liquor into 50 mug/mL for later use.

And S3, adding a freeze-drying protective agent into the serum and mixing uniformly to obtain a mixed solution.

S4, dividing the mixed solution into multiple parts, and respectively adding rivaroxaban mother solutions with different amounts according to preset target values to obtain multiple calibrator solutions; the method comprises the steps that a fixed value range of rivaroxaban in a target, namely a calibration product, is preset, the rivaroxaban calibration product is subjected to multi-point calibration during calibration, wherein the 1 st point is a point 0, namely a point without rivaroxaban, no rivaroxaban is added, the calibration product also belongs to 1 calibration point of the calibration product, other calibration products are calibration products containing different amounts of rivaroxaban, part of calibration products also comprise rivaroxaban, and the addition of rivaroxaban is used as a component needing rivaroxaban in the calibration product, so that a patient sample treated by clinical rivaroxaban is simulated; the specific addition amount of rivaroxaban is determined according to the target value of the calibrator, namely, the addition amount is determined according to the fixed value range of the calibrator.

S5, subpackaging the multiple calibrant solutions and carrying out vacuum freeze drying to obtain multiple calibrants with different rivaroxaban contents. Further, it is preferable that a plurality of calibrator solutions are separately dispensed into 3 mL vials, each containing 1 mL of penicillin. The vacuum freeze drying comprises a prefreezing stage, a sublimation drying stage and a desorption drying stage. Vacuum freeze-drying refers to a method of freezing a calibrator into a solid at a low temperature, and then raising the temperature at a low pressure to directly sublimate a solvent (water) in the calibrator into a gas phase without passing through a liquid phase, thereby removing water and preserving a blood product. The vacuum freeze-drying process typically includes a prefreezing stage, a sublimation drying stage, and a desorption drying stage. Compared with the traditional drying method such as drying or sun drying, the freeze-drying method has a plurality of unique advantages, rivaroxaban in the calibrator can well keep the activity of rivaroxaban under the low-temperature condition, and because most of water is removed, the action of microorganisms and enzymes can hardly be carried out, so that the original properties of the substance can be kept to the maximum extent, and the storage life of the substance can be prolonged. In addition, the inside of the calibrator obtained by freeze-drying is loose and porous, so that the calibrator is easy to redissolve and convenient for clients to use. The setting of the freeze-drying process is a key factor for determining whether the freeze-drying calibration product is successful, and in order to obtain the optimal freeze-drying process, the conditions of temperature, temperature rise/temperature fall time, maintenance time, vacuum degree and the like of each freeze-drying stage need to be groped; in addition, in the freeze-drying process, attention needs to be paid to several key temperature points, namely eutectic point temperature, glass transition temperature, eutectic point temperature and collapse temperature, and a freeze-drying process is set according to the key temperature, so that a good freeze-drying calibrator is obtained.

Further, the preferred vacuum freeze-drying comprises a prefreezing stage, a sublimation drying stage and a desorption drying stage; in the pre-freezing stage, the pre-freezing temperature is-35 to-50 ℃ so as to completely freeze the calibrator solution, and the pre-freezing temperature can be-35 ℃, 42 ℃ or 50 ℃, preferably 40 ℃ below zero; the cooling time is 180-260 min, and it can be understood that the cooling time can be 180min, 220min or 260min, and preferably 240 min; the maintaining time after cooling is 300-400 min, and it can be understood that the maintaining time after cooling can be 300min, also can be 350min, also can be 400min, and the maintaining time after cooling is 360min is preferred. Further, most of the water in the calibrator is removed in the sublimation drying stage, wherein the temperature in the sublimation drying stage is-25 to-40 ℃, namely the temperature in the sublimation drying stage can be-25 ℃, can also be-32 ℃, can also be-40 ℃, and preferably the temperature in the sublimation drying stage is-30 ℃; the cooling time is 50-100 min, namely the cooling time can be 50min, 75min or 100min, and preferably 60 min; the maintaining time after cooling is 1200-2000 min, namely the maintaining time after cooling can be 1200min, 1600min, 2000min, preferably 1500 min; the vacuum degree is 0.05-0.15 bar, namely the vacuum degree can be 0.05 bar, also can be 0.1bar, also can be 0.15bar, the preferred vacuum degree is 0.1 bar; further, removing residual small amount of water in an analysis drying stage, wherein the temperature in the analysis drying stage is 25-32 ℃, namely the temperature in the analysis drying stage can be 25 ℃, 32 ℃ or 28 ℃, and preferably the temperature in the sublimation drying stage is 30 ℃; the temperature rise time is 200-300 min, namely the temperature rise time can be 200min, 250min or 300min, and preferably the temperature rise time is 240 min; maintaining for 450-540 min after temperature rise, namely maintaining for 450min, 500min, 540min after temperature rise, preferably maintaining for 480min after temperature rise; the vacuum degree is 0.05-0.15 bar, namely the vacuum degree can be 0.05 bar, also can be 0.1bar, also can be 0.15bar, the preferred vacuum degree is 0.1 bar; and (5) obtaining a calibrator after freeze-drying. The calibrator prepared under the freeze-drying parameters has the advantages of good stability, high accuracy, good appearance of a finished product, short redissolution time and good clarity after redissolution.

In one embodiment, the pre-freezing temperature in the pre-freezing stage is-40 ℃, the cooling time is 240min, and the holding time is 360min after cooling; the temperature of sublimation drying stage is-30 deg.C, cooling time is 60min, maintaining time is 1500min after cooling, and vacuum degree is 0.1 bar; the temperature of the desorption drying stage is 30 ℃, the temperature rise time is 240min, the maintaining time is 480min after temperature rise, and the vacuum degree is 0.1 bar. The calibrator prepared under the freeze-drying parameters has the advantages of good stability, high accuracy, good appearance of a finished product, short redissolution time and good clarity after redissolution.

According to the preparation method of the calibration product, the rivaroxaban-containing calibration product is prepared by a stable process and adding a proper protective agent and a proper freeze-drying process, the problems of unstable process, short storage period and the like in the preparation process of the conventional calibration product are solved, and the preparation method is favorable for batch production.

The invention is further illustrated by the following specific embodiments, which should not be construed as limiting the invention, except that the reagents used in the invention are all commercially available reagents that are chemically pure, unless otherwise indicated.

Example 1

A calibrator for rivaroxaban detection comprises serum and a freeze-drying protective agent, wherein the freeze-drying protective agent comprises trehalose, mannitol, Bovine Serum Albumin (BSA) and potassium chloride. Note: the trehalose can be at least one of sucrose, glucose, glycine, alanine and arginine; mannitol can be replaced by at least one of glycerol, xylitol, inositol, sorbitol, polyethylene glycol, sucrose, and glucose; bovine serum albumin can be replaced by HSA; the potassium chloride can be replaced by at least one of sodium chloride and sodium glutamate.

The preparation method comprises the following steps:

and S1, performing biological safety detection on the serum, and detecting the serum to be qualified for later use.

S2, dividing the serum into 11 parts, adding a freeze-drying protective agent into each serum matrix according to the proportion (mass volume percentage) in the table 1, and mixing uniformly to obtain the mixed liquid of the examples 1-9 and the comparative examples 1-2.

S3, subpackaging 3 mL penicillin bottles with 1 mL each to obtain a calibrator solution, freeze-drying, carrying out freeze-drying according to the freeze-drying process table 2 in examples 1-3 and comparative examples 1-2, carrying out freeze-drying according to the freeze-drying process table 3 in examples 1-4-1-6, carrying out freeze-drying according to the freeze-drying process table 4 in examples 1-7-1-9, and carrying out freeze-drying to obtain the calibrator after the freeze-drying is finished. And observing the appearance, the redissolution time and the redissolution state after freeze-drying.

Table 1 shows the addition ratio and the freeze-dried state of the freeze-drying protective agent in examples 1-1 to 1-9 and comparative examples 1-1 to 1-2

TABLE 2 Freeze-drying Process for calibrator in examples 1-1 to 1-3 and comparative examples 1-1 to 1-2

TABLE 3 Freeze drying Process for calibrator in examples 1-4 to 1-6

TABLE 4 Freeze drying Process for calibrator in examples 1-7 to 1-9

According to the appearance, the redissolution time and the clarity after redissolution of the freeze-dried product, the freeze-dried finished products of the embodiments 1-1 to 1-9 have good appearance, short redissolution time and good redissolution effect, and particularly the freeze-drying protective agent which is prepared by adding 3% of trehalose, 5% of mannitol, 5% of BSA and 1% of potassium chloride into serum has better effect; the dosage of the freeze-drying protective agents trehalose, mannitol, bovine serum albumin and potassium chloride in the comparative examples 1-2 exceeds the upper limit and the lower limit, and the appearance, the redissolution time and the redissolution effect of the freeze-dried finished product are not the same as those of the calibrator in the examples 1-9 of the invention.

Example 2

A calibrator for rivaroxaban detection comprises serum, a freeze-drying protective agent and rivaroxaban, wherein the freeze-drying protective agent comprises trehalose, mannitol, Bovine Serum Albumin (BSA) and potassium chloride. The preparation method comprises the following steps:

s1, taking human serum, carrying out biological safety detection on the human serum, and keeping the human serum for later use after the detection is qualified.

S2, adding a freeze-drying protective agent (5% mannitol, 3% trehalose, 5% BSA and 1% potassium chloride according to the mass-volume ratio) into the serum in proportion, mixing uniformly and mixing uniformly; dissolving rivaroxaban in ultrapure water with a certain volume, and preparing mother liquor with the volume of 50 mug/mL for later use; the mixed serum substrates were divided into 5 groups, the group 1 (example 2-1) was not added with rivaroxaban, the group 2 (example 2-2) was added with rivaroxaban to give a final concentration of 52 ng/mL, the group 3 (example 2-3) was added with rivaroxaban to give a final concentration of 103 ng/mL, the group 4 (example 2-4) was added with rivaroxaban to give a final concentration of 305 ng/mL, and the group 5 (example 2-5) was added with rivaroxaban to give a final concentration of 505 ng/mL.

S3, subpackaging the calibrator solution obtained in the above examples 2-1 to 2-5, subpackaging the solution into 3 mL penicillin bottles with 1 mL each, and freeze-drying according to the freeze-drying process table 2 to obtain the calibrator after the freeze-drying.

Comparative example 2

A calibrator for rivaroxaban detection comprises 50mmoL/L Tris buffer with pH of 7.4, a freeze-drying protective agent and rivaroxaban, wherein the freeze-drying protective agent comprises trehalose, mannitol, bovine serum albumin and potassium chloride.

Comparative example 2 is different from example 2 in that 50mmoL/L of Tris buffer solution with pH 7.4 is used as a base for a calibrator instead of human serum, and a lyoprotectant (5% mannitol, 3% trehalose, 5% BSA and 1% potassium chloride are added according to the mass volume ratio) is proportionally added into the serum to be mixed evenly; dissolving rivaroxaban in ultrapure water with a certain volume, and preparing a mother solution of 50 mug/mL for later use; dividing the uniformly mixed serum matrix into 5 groups, wherein the other components, the proportion and the preparation method are the same, and obtaining 5 groups to proportion 2 (comparative example 2-1 to comparative example 2-5 respectively). Wherein comparative example 2-1 was not supplemented with rivaroxaban, the final concentration of rivaroxaban of comparative example 2-2 was 52 ng/mL, the final concentration of rivaroxaban of comparative example 2-3 was 103 ng/mL, the final concentration of rivaroxaban of comparative example 2-4 was 305 ng/mL, and the final concentration of rivaroxaban of comparative example 2-5 was 505 ng/mL. And (3) subpackaging the solutions obtained in the comparative examples 2-1-2-5, subpackaging the solutions into 3 mL penicillin bottles with 1 mL of each bottle to obtain a calibrator solution, and freeze-drying the calibrator solution according to the freeze-drying process table 2 to obtain the calibrator after the freeze-drying is finished.

The calibrators obtained in the embodiments 2-1 to 2-5 and the comparative examples 2-1 to 2-5 are subjected to source tracing and assignment through a standardized process, and target values are marked. And then respectively calibrating according to the concentration values after tracing, taking the OD value of the tested absorbance as a vertical coordinate, taking the rivaroxaban content value as a horizontal coordinate, making a standard curve in a linear fitting mode, and respectively showing the tracing values of the calibrators in tables 5-6, wherein the obtained calibration curve is shown in a table 1-2.

TABLE 5 evaluation results of the calibrators of examples 2-1 to 2-5

TABLE 6 evaluation results of comparative examples 2-1 to 2-5

The results of tables 5-6 and attached figures 1-2 show that the values of the calibrators in the embodiments 2-1 to 2-5 of the invention are all within an acceptable range, that is, the result of the value assignment of the calibrators is not greater than the actual addition amount, and is not much lower than the actual addition amount (the loss is greater than 30%), so that the requirements are met. As can be seen from FIGS. 1-2, the calibration material of the present invention has a good fit of the standard curve, R ² Not less than 0.998, which proves that the calibrator can obtain a standard curve with better quality, but the final assignment result of the calibrator in comparative example 2 is lower in maximum degree due to different selected matrixes, and when the serum is replaced by a common buffer system, the assignment results of all points of the calibrator are abnormal, which indicates that the assignment results are unreliable, and indicates that the serum matrix is crucial to the successful preparation of the calibrator, and the serum is easier to obtain and has low cost.

The prepared calibrator is subjected to standardized traceability assignment (examples 2-1 to 2-5), the concentration of the calibrator is marked, and then the calibrator is tested by using a certified test system, which is specifically as follows.

1. Correlation

The calibrator of the invention of examples 2-1 to 2-5 and comparative examples 2-1 to 2-5 was used in combination with a reference instrument and a reagent (the reference instrument was a blood coagulation analyzer of model CS 5100 from the company of schirmen corporation, japan, and the reagent was a reagent of HYPHEN BioMed, both the company producing the reagent and the company producing the instrument belong to the flag of schirmen), and the calibrator of the reference system was used in combination with the reference instrument and the reagent to perform calibration (i.e., except for the calibrator, other conditions were consistent), and 2 sets of calibration lines were used to test the same set of clinical samples, respectively, and perform linear fitting comparison on 2 sets of test values, where the linear slope required to satisfy k =1.00 ± 0.10, and the correlation coefficient required to satisfy r ≥ 0.975. The clinical alignment fit is shown in table 7 below, and in fig. 3-4.

TABLE 7 correlation test results of example 2, comparative example 2 and reference system

As can be seen from the results in Table 7 and the accompanying FIGS. 3-4, the results of testing clinical samples using the reference instrument and the reagent in combination with the standard and the reference calibrator in example 2 (examples 2-1 to 2-5) were analyzed by linear regression, and the slope k and the correlation R of the linear regression equation were determined ² The standards in example 2 and the reference standard are proved to have good correlation, and the low-concentration-level and high-concentration-level calibrators prepared in example 2 can meet the correlation requirement; and the clinical comparison slope of the comparative example 2 is low, and the relative deviation is very high, so that the clinical test requirement is not met.

2. Matrix interchange assessment

Substrate interchangeability was tested and calculated for the calibrators of example 2 of the present invention and comparative example 2, respectively, according to the guidelines for evaluation of WST356-2011 for substrate effect and interoperability. The results of substrate interchangeability evaluation are shown in FIGS. 5 and 6 below.

As can be seen from fig. 5-6, the matrix interchangeability assessment results show that the calibrators of the present invention have no matrix effect (i.e., all the test points of the calibrators fall within the 95% confidence interval), but comparative example 2 has a matrix effect (some of the test points fall outside the 95% confidence interval), indicating that the matrix of the present invention is not different from the clinical sample matrix, whereas comparative example 2 is different from the clinical sample matrix.

3. Accuracy of

The calibration product of the embodiment 2-1 to 2-5 is prepared by testing the calibrated reference system, the test is repeated for 10 times, the relative deviation between the test mean value and the indication value of 10 times is calculated according to the formula (1), and each test result meets the requirement of accuracy: the relative deviation between the measured concentration and the indicated value is within. + -. 10%.

（1）

In the formula:

b-relative deviation;

-a calibrator test mean;

t-calibrator index value.

TABLE 8 accuracy test results of examples 2-1 to 2-5

As can be seen from the results in Table 8, the standards for each concentration level (examples 2-1 to 2-5) prepared in example 2 satisfy the accuracy requirements.

4. Uniformity of

Taking 10 bottles of calibrator with the same standard value in the same batch, testing each bottle of calibrator 1 time, and calculating the average value of detection results according to the formulas (2) and (3) ((

) And standard deviation S1; randomly selecting 1 bottle from the calibration sample with the same index value for 10 times, and calculating the average value of the detection results by using the same formula (

) And standard deviation S2. Then, the uniformity (CV bottle inside) of the calibrator in the bottle is calculated according to the formula (4), the uniformity (CV bottle inside) of the calibrator between the bottles is calculated according to the formulas (5) and (6), and the obtained result meets the uniformity requirement: the inner part of the CV bottles is less than or equal to 10 percent, and the space between the CV bottles is less than or equal to 10 percent. The results are shown in tables 9-13.

（2）

S =

（3）

CV =

× 100% （4）

S bottle interval =

（5）

CV bottle space (%) = S bottle space-

×100% （6）

When S1< S2, let CV bottle-to-bottle =0

In the formula:

-an average value;

s-standard deviation;

CV — coefficient of variation;

n-number of measurements;

xi-ith measurement.

TABLE 9 homogeneity test results for the calibrator of example 2-1

TABLE 10 homogeneity test results for calibrators from examples 2-2

TABLE 11 homogeneity test results for calibrators from examples 2-3

TABLE 12 homogeneity test results for calibrators from examples 2-4

TABLE 13 homogeneity test results for calibrators from examples 2-5

From the results in tables 9-13, it can be seen that the standards prepared in example 2 all meet the requirement of uniformity.

5. Stability of

Placing the prepared calibrator in a constant temperature and humidity box at 37 ℃, respectively taking out a set of calibrator on days 0, 8, 12, 14 and 16 for testing, testing each value taking point for 5 times, calculating the mean value and the relative deviation, placing for 16 days under the acceleration condition of 37 ℃, and obtaining the relative deviation between the test result and the indication value within the range of +/-10%.

TABLE 14 stability (37 ℃) test results (ng/mL) of the calibrator of example 2-1

TABLE 15 stability (37 ℃) test results (ng/mL) of the calibrator of example 2-2

TABLE 16 stability (37 ℃) test results (ng/mL) of the calibrators of examples 2-3

TABLE 17 stability (37 ℃) test results (ng/mL) of the calibrators of examples 2-4

TABLE 18 stability (37 ℃) test results (ng/mL) for the calibrators of examples 2-5

As can be seen from the test results in tables 14 to 18, the calibrators of examples 2-1 to 2-5 were left for 16 days at 37 ℃ under accelerated conditions, and the relative deviation between the test results and the indicated values was within + -10%, and the relative deviation was small, and was in accordance with the stability requirements, indicating that the calibrators of the present invention have excellent stability.

And (3) redissolving the prepared calibrator, placing the calibrator in a refrigerator at 2-8 ℃, taking out the calibrator for testing for 0, 3, 5, 7 and 8 hours respectively, testing each value taking point for 5 times, calculating the mean value and the relative deviation, placing the calibrator for 8 hours after bottle opening redissolution at 2-8 ℃, and keeping the relative deviation of the obtained test result and the standard value within the range of +/-10%.

TABLE 19 test results of the open bottle stability (2. about.8 ℃ C.) after reconstitution of the calibrator for example 2-1 (ng/mL)

TABLE 20 test results of the open bottle stability (2. about.8 ℃ C.) after reconstitution of the calibrator for example 2-2 (ng/mL)

TABLE 21 test results of the open bottle stability (2. about.8 ℃ C.) after reconstitution of the calibrator for examples 2 to 3 (ng/mL)

TABLE 22 test results of the open bottle stability (2. about.8 ℃ C.) after reconstitution of the calibrator for examples 2 to 4 (ng/mL)

TABLE 23 test results of the open bottle stability (2-8 ℃ C.) after reconstitution of the calibrator for examples 2-5 (ng/mL)

From the test results in tables 19 to 23, it can be seen that the calibrators of examples 2-1 to 2-5 were placed at 2 to 8 ℃ for 8 hours after being reconstituted by decapping, and the relative deviation between the test results and the indicated values was within ± 10%, which meets the stability requirement, indicating that the calibrators of the present invention have excellent stability after being reconstituted.

The data and the figures show that the test result of the calibrator test data and the calibrated analysis system meets the acceptance standard. And the above-mentioned specific embodiments are chosen to verify the feasibility and rationality of the present invention, and are only part of the explanation and illustration of the present invention, but not limited thereto. All changes, modifications and substitutions that may be made by one skilled in the art without departing from the scope of the invention as defined by the appended claims are to be embraced within the scope of the invention.

Claims

1. The calibration material for rivaroxaban detection is characterized by being freeze-dried powder, comprising serum and a freeze-drying protective agent, and further comprising rivaroxaban, wherein the content of the rivaroxaban in the calibration material is 0-600 ng/mL.

2. The calibrator of claim 1, wherein the serum is human serum.

3. The calibrator according to claim 1, wherein the calibrator comprises a plurality of calibrators with at least 5 different rivaroxaban contents, and the rivaroxaban contents in the 5 calibrators are 0ng/mL, 40-60 ng/mL, 90-120 ng/mL, 280-320 ng/mL and 480-520 ng/mL respectively.

4. The calibrator according to claim 1, wherein the lyoprotectant comprises at least one of an amino acid lyoprotectant, a saccharide lyoprotectant, an alcohol lyoprotectant, a protein lyoprotectant, and a salt lyoprotectant.

5. The calibrator of claim 4, wherein the amino acid-based lyoprotectant includes at least one of alanine, glycine, and arginine;

the saccharide freeze-drying protective agent comprises at least one of trehalose, sucrose and glucose;

the alcohol freeze-drying protective agent comprises at least one of glycerol, mannitol, xylitol, inositol, sorbitol and polyethylene glycol;

the salt freeze-drying protective agent comprises at least one of sodium chloride, potassium chloride and sodium glutamate;

the protein freeze-drying protective agent comprises at least one of bovine serum albumin and HSA.

6. The calibrator according to claim 5, wherein the lyoprotectant comprises the following components: mannitol, trehalose, bovine serum albumin and potassium chloride.

7. The calibrator according to claim 6, wherein the mass-to-volume ratio of the lyoprotectant to the serum is: 3-8% of mannitol, 1-5% of trehalose, 3-8% of bovine serum albumin and 0.5-1.5% of potassium chloride.

8. The calibrator according to claim 7, wherein the mass-to-volume ratio of the lyoprotectant to the serum is: 5% of mannitol, 3% of trehalose, 5% of bovine serum albumin and 1% of potassium chloride.

9. A method for preparing a calibrator for rivaroxaban testing according to any one of claims 1 to 8, comprising the steps of:

s2, preparing rivaroxaban into mother liquor for later use;

10. The method for preparing a calibrator according to claim 9, wherein the plurality of calibrator solutions are dispensed in 3 mL vials, 1 mL per vial, respectively, at S5.

11. The method for preparing a calibration material according to claim 10, wherein the vacuum freeze-drying comprises a prefreezing stage, a sublimation drying stage, and a desorption drying stage;

in the pre-freezing stage, the pre-freezing temperature is-35 to-50 ℃, the cooling time is 180 to 260min, and the holding time is 300 to 400min after cooling;

12. The method for preparing the calibrator according to claim 11, wherein the pre-freezing temperature in the pre-freezing stage is-40 ℃, the cooling time is 240min, and the holding time is 360min after cooling;