CN111707512A - Ready-to-use vitamin standard substance, preparation method, use method and storage stabilizer thereof - Google Patents

Ready-to-use vitamin standard substance, preparation method, use method and storage stabilizer thereof Download PDF

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CN111707512A
CN111707512A CN202010546255.3A CN202010546255A CN111707512A CN 111707512 A CN111707512 A CN 111707512A CN 202010546255 A CN202010546255 A CN 202010546255A CN 111707512 A CN111707512 A CN 111707512A
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standard
vitamin
ready
concentration
vitamin standard
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王舒乐
齐延林
张恒
胡云平
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Realassay Biotechnology Co ltd
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Realassay Biotechnology Co ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N2001/2893—Preparing calibration standards

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Abstract

The invention discloses a high-stability ready-to-use vitamin standard substance, a preparation method, a using method and a storage stabilizer thereof. The preparation method comprises the following steps: (1) diluting the vitamin standard stock solution by using a storage stabilizer to obtain a vitamin standard working solution; (2) sterilizing the vitamin standard working solution and verifying the consistency of the concentration of standard substances; (3) fixing the vitamin standard mixed solution with the required concentration in a container by adopting a vacuum freeze drying technology; the vitamin standard mixed solution with the required concentration is a series of standard solutions with gradient concentration, which are obtained by diluting the vitamin standard working solution in the step (2) by using a storage stabilizer; (4) and (5) packaging and storing. The preparation method of the ready-to-use vitamin standard substance not only improves the long-term storage stability of the low-concentration standard substance, but also enables the prepared vitamin standard substance to be dissolved immediately after being rehydrated by operation, and omits the complicated preparation process of the standard solution.

Description

Ready-to-use vitamin standard substance, preparation method, use method and storage stabilizer thereof
Technical Field
The invention belongs to the technical field of preparation of vitamin standard products, and particularly relates to a ready-to-use vitamin standard product, a preparation method, a using method and a storage stabilizer thereof.
Background
The standard is used to calibrate the measurement device, evaluate the measurement method, or assign a material or substance. Most high-purity standard products on the market are small in specification and expensive, powdery standard substances are easily dispersed on the bottle wall and the cover, the bottle is opened for multiple times, moisture absorption and agglomeration are easily caused, and the liquid standard substances can be adsorbed on the bottle wall to form a liquid layer which is difficult to distinguish by naked eyes, so that the loss of the weighing process is caused, and the waste is caused. The vitamin standard solution has low preparation concentration, needs to be weighed by a high-precision balance, and has small weighing amount and large loss amount.
When the standard substance is used for the calibration of a chemical analyzer, the detection principle of the chemical analyzer is a relative measurement method, and a standard curve is required to be prepared for analysis and quantification during detection, so the standard substance is also called as a 'measuring tool' in the analytical measurement industry. Most vitamin items are detected to be ng/mL magnitude, a low-concentration vitamin standard solution is unstable to store, and the vitamin standard working solution is required to be prepared and used on site in national standards. At present, the vacuum freeze drying technology is widely applied to the production of medicines and foods such as serum, plasma, vaccines, enzymes, antibiotics, hormones and the like, so that the dried products can be stored for a long time without deterioration. But most of the freeze-dried products are easy to absorb moisture and deteriorate during the storage period, the appearance of the products is not good, the rehydration performance is poor and the like.
The preparation methods of the standard products provided in the chinese patents CN201910188685, CN201710001500 and the US patent US8357504B2 solve the tedious preparation process of the vitamin standard curve to a certain extent, but still have the following obvious problems: 1) the dried standard substance is non-sterile and needs to be filtered before use; 2) the concentration of the dry standard substance is higher, and the dry standard substance needs to be further diluted one by one for later use; 3) the shelf life of the dried standard is short.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a high-stability ready-to-use vitamin standard substance, a preparation method, a use method and a storage stabilizer thereof. The preparation method provided by the invention overcomes the technical problems that the existing powdery and liquid vitamin standard substances are complex in preparation process, easy to lose in weighing process, easy to degrade in storage process and the like, the vitamin standard substance provided by the invention comprises a series of gradient concentration vitamin standard substances and a storage stabilizer which are required by standard curve preparation, can be stably stored for a long time (less than or equal to 8 ℃ for 1 year) at a low temperature, and is suitable for vitamins such as vitamin B12, biotin, folic acid, pantothenic acid, vitamin B1, vitamin B2, vitamin B6, nicotinic acid and the like.
The above object of the present invention is achieved by the following technical solutions:
a preparation method of a ready-to-use vitamin standard substance comprises the following steps:
(1) diluting the vitamin standard stock solution by using a storage stabilizer to obtain a vitamin standard working solution;
(2) sterilizing the vitamin standard working solution and verifying the consistency of the concentration of standard substances;
(3) fixing the vitamin standard mixed solution with the required concentration in a container by adopting a vacuum freeze drying technology; the vitamin standard mixed solution with the required concentration is a series of standard solutions with gradient concentration, which are obtained by diluting the vitamin standard working solution in the step (2) by using a storage stabilizer;
(4) and (5) packaging and storing.
As an embodiment of the preparation method of the ready-to-use vitamin standard provided by the invention, the storage stabilizer consists of the following components: 2-10 wt% of mannitol, 0.5-2 wt% of glucan, 0.2-1 wt% of polyvinylpyrrolidone, 2-4 wt% of trehalose, 2-4 wt% of polyethylene glycol, 0.02-0.05M of disodium hydrogen phosphate, 1-5 mM of sodium dihydrogen phosphate, 0.05-0.2wt% of polysorbate and the balance of water. The storage stabilizer is sterilized at high temperature for standby before use.
Preferably, the high-temperature sterilization refers to sterilization for 5-15 min at 121 +/-3 ℃ by using an autoclave.
As an embodiment of the preparation method of the ready-to-use vitamin standard substance provided by the invention, the step (1) is to dilute the vitamin standard stock solution by using the storage stabilizer to obtain the vitamin working solution, and at least 10 times of the vitamin working solution are needed3And (4) performing double dilution. Ensuring that the vitamin standard solution medium contains sufficient content of storage stabilizer and ensuring the homogeneity of the subsequent further gradient dilution standard solution medium, thereby obtaining consistent freeze-drying protection stabilization effect.
As an embodiment of the preparation method of the ready-to-use vitamin standard product, the step (2) of sterilizing the vitamin standard working solution and verifying the conformity of the concentration of the standard substance refers to filtering and sterilizing the vitamin standard working solution by using a filter membrane with the thickness of 0.22-0.45 μm, and then verifying the conformity of the vitamin standard working solution by combining a vitamin quality control sample with a known concentration.
As an embodiment of the method for preparing the ready-to-use vitamin standard product provided by the present invention, the step (3) of fixing the vitamin standard mixed solution refers to diluting the vitamin standard working solution in the step (2) to a series of gradient concentration standard solutions by using a storage stabilizer according to the concentration and volume required by the experimental method to obtain a series of gradient concentration vitamin standard mixed solutions, then filling the series of gradient concentration vitamin standard mixed solutions into a container to fully soak the bottom of the container for one week, performing freeze-drying for 20-40 h by using a vacuum freeze-drying technology, and fixing the standard solutions in the container.
In one embodiment of the method for preparing the ready-to-use vitamin standard, the liquid level of the vitamin standard mixed solution in the container is less than or equal to 10 mm.
As an embodiment of the preparation method of the ready-to-use vitamin standard substance provided by the invention, the packaging in the step (4) is to ensure that the standard substance is maintained under the conditions of sterility, shading and drying and stored at the temperature of less than or equal to 8 ℃.
A ready-to-use vitamin standard prepared by any of the above methods of preparing a ready-to-use vitamin standard.
The use method of the ready-to-use vitamin standard substance is that sterile water is added into a container in which a vitamin standard mixed solution is freeze-dried and is uniformly mixed to obtain a standard curve series gradient solution.
A storage stabilizer for a ready-to-use vitamin standard substance, which consists of the following components: 2-10 wt% of mannitol, 0.5-2 wt% of glucan, 0.2-1 wt% of polyvinylpyrrolidone, 2-4 wt% of trehalose, 2-4 wt% of polyethylene glycol, 0.02-0.05M of disodium hydrogen phosphate, 1-5 mM of sodium dihydrogen phosphate, 0.05-0.2wt% of polysorbate and the balance of water.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method of the high-stability ready-to-use vitamin standard substance provided by the invention can accurately fix the vitamin standard substance in trace amount, and can be used as a directly used standard substance for an internal standard method and an external standard method in vitamin analysis and detection. The vitamin standard solution can be fixed in various container carriers according to the requirements of a detection method, after the vitamin standard solution and the storage stabilizer are mixed and freeze-dried, the structure is firm, no flying powder exists, and the vitamin standard solution is not easy to absorb moisture and collapse, the container carrier can be a sealed container with a cover or a sealed container without a cover, the freeze-dried material is easy to dissolve after rehydration, the loss rate of the freeze-dried material after long-term low-temperature storage (less than or equal to 8 ℃ and 1 year) is less than or equal to 5 percent, and the loss rate of the freeze-dried material under short-term high-temperature storage conditions (less than or equal to 60 ℃ and 7 days) can be avoided, so.
The preparation method of the high-stability ready-to-use vitamin standard substance solves the problems of complex preparation and unstable storage of the existing standard substance in the using process, and belongs to the technical field of vitamin standard substances. The ready-to-use standard substance has the advantages of high stability and good rehydration, meets the requirement of long-term stable storage (less than or equal to 8 ℃ for 1 year) under the condition of low temperature, can realize accurate immobilization of large-batch standard curve solutions, has small difference in batches, reduces preparation before experiments, and improves the detection efficiency.
When the high-stability ready-to-use vitamin standard substance provided by the invention is used, the preparation of a standard curve solution can be completed only by 'adding water → blowing and beating/vortex mixing', and the preparation processes of weighing, constant volume, constant value, sterilization and the like of standard substances are omitted.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a schematic diagram of a lath-type ready-to-use vitamin B12 standard of the present invention, in which 11-microwell lath, 12-vitamin B12 standard;
FIG. 2 is a schematic diagram of a bottled ready-to-use biotin standard of the present invention, in which 21-bottle container, 22-biotin standard is shown.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, but the examples are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The materials and reagents used in the examples of the present invention are commercially available.
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
Example 1 Ready-to-use vitamin B12 slate standard
Vitamin B12 is present in food in very small amounts, generally in the range of 2-10. mu.g/100 g, and the national standard uses ATCC 7830 for the quantitative determination of the specificity and high sensitivity of vitamin B12, the sensitivity of the method is up to 0.001ng/ml, and the required vitamin B12 standard is also a lower concentration.
The vitamin B12 powder standard substance described in this example was purchased from Supelco 47869 and has a purity of 99.2%.
A preparation method of a high-stability ready-to-use vitamin B12 lath type standard substance is prepared by the following steps:
(10) preparing a vitamin B12 standard stock solution and a storage stabilizer;
(11) fully mixing and diluting the vitamin B12 standard stock solution prepared in the step (10) by using the storage stabilizer prepared in the step (10) to obtain a vitamin B12 standard working solution;
(12) performing sterilization and concentration conformance verification on the vitamin B12 standard working solution;
(13) fixing the vitamin B12 standard mixed solution with required concentration in a container by vacuum freeze drying technology, as shown in figure 1; the vitamin B12 standard mixed solution with the required concentration is a series of standard solutions with gradient concentration, which are obtained by diluting the vitamin B12 standard working solution in the step (12) by using a storage stabilizer;
(14) and (5) packaging and storing.
The vitamin B12 standard stock solution in the step (10) is prepared by accurately weighing a vitamin B12 powder standard substance, and adding a 25% ethanol solution to a constant volume of 10 mug/mL to obtain a vitamin B12 standard stock solution.
Wherein, the storage stabilizer in the step (10) consists of the following components: 2wt% mannitol, 0.5wt% dextran, 0.5wt% polyvinylpyrrolidone, 2wt% trehalose, 2wt% polyethylene glycol, 0.02M disodium hydrogen phosphate, 1mM sodium dihydrogen phosphate, 0.1wt% polysorbate, and the balance water, and the storage stabilizer is sterilized by autoclave at 121 ℃ for 15min for use.
Wherein the vitamin B12 standard working solution in the step (11) is prepared by diluting a vitamin B12 standard stock solution by using a storage stabilizer to reach the concentration of 0.6ng/mL of the working solution;
wherein the sterilization and concentration conformance verification of the vitamin standard working solution in the step (12) refers to performing filtration sterilization on 0.6ng/mL vitamin B12 standard working solution by using a 0.22-micron filter membrane, and performing concentration conformance verification on the vitamin standard working solution by combining a vitamin quality control sample (NO: 4044755861, quality control range: 2.12-2.88 μ g/100 g) with known concentration.
Wherein, in the step (13), according to the vitamin B12 detection kit experimental method, the volume of the required standard solution is 150 muL, the concentration of the required biotin standard curve is 0, 0.0075, 0.015, 0.03, 0.045, 0.06 and 0.09 ng/mL, the standard working solution in the step (12) is further diluted by a series of gradient concentration standard solutions by a storage stabilizer for 0, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.6ng/mL, 48 muL of standard working solution is transferred and orderly loaded into each micropore of the lath for one week with the bottom of the micropore being fully soaked and the liquid level height being less than or equal to 10mm, and the standard mixed solution of a series of vitamin B12 is freeze-dried and immobilized in the micropore in vacuum freeze-drying technology for 20 hours.
Wherein, the packaging and storing in the step (14) means that the micropores fixed with the vitamin B12 standard substances are placed in aluminum foil bags with corresponding customized specifications, vacuumized, sealed and placed in an environment with the temperature of less than or equal to 8 ℃ for storage.
When the vitamin B12 standard substance prepared in the embodiment is used, 320 mu L of sterile water is added into each micropore, the mixture is uniformly blown and beaten, 150 mu L of standard solution is transferred into a pore of the micropore, and the preparation of the vitamin B12 standard curve solution can be completed.
Example 2 Ready-to-use Biotin bottled standards
The biotin powder standard described in this example was purchased from Supelco 47868 with a purity of 98%.
A preparation method of a high-stability ready-to-use biotin bottled standard substance is prepared by the following steps:
(20) preparing a biotin standard stock solution and a storage stabilizer;
(21) fully mixing and diluting the biotin standard stock solution prepared in the step (20) by using the storage stabilizer prepared in the step (20) to obtain biotin standard working solution;
(22) performing sterilization and concentration conformance verification on the biotin standard working solution;
(23) fixing the biotin standard mixed solution with the required concentration in a container by adopting a vacuum freeze-drying technology, as shown in figure 2; the biotin standard mixed solution with the required concentration is a serial gradient concentration standard solution obtained by diluting the biotin standard working solution in the step (2) by using a storage stabilizer;
(24) and (5) packaging and storing.
The biotin standard stock solution in the step (20) is prepared by accurately weighing a biotin powder standard substance, and fixing the volume to 100 mu g/mL by using a 50% ethanol solution.
Wherein the storage stabilizer in the step (20) consists of the following components: 2wt% mannitol, 1wt% dextran, 0.4wt% polyvinylpyrrolidone, 4wt% trehalose, 1wt% polyethylene glycol, 0.04M disodium hydrogen phosphate, 1mM sodium dihydrogen phosphate, 0.1wt% polysorbate, and the balance water, and the storage stabilizer is sterilized by an autoclave at 121 ℃ for 15min for use.
Wherein, the biotin standard working solution in the step (21) is prepared by diluting the biotin standard stock solution with a storage stabilizer until the concentration of the working solution is 5 ng/mL.
Wherein the step (22) of sterilizing the biotin standard working solution and verifying the consistency of the concentration refers to filtering and sterilizing 5 ng/mL of the biotin standard working solution by using a 0.22-micron filter membrane, and then verifying the consistency of the concentration of the biotin standard working solution by combining a vitamin quality control sample (NO: 4044755861, quality control range: 22.3-30.8-microgram/100 g) with known concentration.
According to the method for detecting biotin in GB5009.259-2016 food, the volume of a required biotin standard solution is 5mL, the concentration of a required biotin standard curve is 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.16 and 0.2ng/mL, the standard working solution is further diluted by a storage stabilizer in a series of gradient concentration standard solutions for 0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 ng/mL, 200 mu L of the standard working solution is transferred and subpackaged into a penicillin bottle, the penicillin bottle can be soaked at the bottom of the penicillin bottle for one week, the liquid level is less than or equal to 10mm, the cover is lightly covered, and freeze-drying is carried out for 24 hours by utilizing a vacuum freeze-drying technology, so that the standard substance is fixed in the penicillin bottle.
Wherein, the packaging and storing in the step (24) refers to that the penicillin bottle with the immobilized standard substance is subjected to vacuum capping and sealed and placed in an environment with the temperature less than or equal to 8 ℃ for storage.
When the biotin standard substance prepared in this embodiment is used, 5mL of sterile water is added into each penicillin bottle, and the mixture is vortexed and shaken uniformly, so that the preparation of a biotin standard curve solution can be completed.
EXAMPLE 3 Ready-to-use folate centrifuge tube standards
The folic acid powder standard described in this example was purchased from Supelco 47866 and had a purity of 98.5%.
A preparation method of a high-stability ready-to-use folic acid standard substance comprises the following steps:
(30) preparing folic acid standard stock solution and a storage stabilizer;
(31) fully mixing and diluting the folic acid standard stock solution prepared in the step (30) by using the storage stabilizer prepared in the step (30) to obtain folic acid standard working solution;
(32) sterilizing the folic acid standard working solution and verifying the consistency of the concentration;
(33) fixing the folic acid standard mixed solution with the required concentration in a container by adopting a vacuum freeze drying technology; the folic acid standard mixed solution with the required concentration is a series of standard solutions with gradient concentration, which are obtained by diluting the folic acid standard working solution in the step (32) by using a storage stabilizer;
(34) and (5) packaging and storing.
Wherein the folic acid standard stock solution in the step (30) is obtained by accurately weighing folic acid powder standard substance, and using 0.01mol/L sodium hydroxide ethanol solution to fix the volume to 20 mug/mL.
Wherein the storage stabilizer in the step (30) consists of the following components: 4wt% of mannitol, 1wt% of dextran, 1wt% of polyvinylpyrrolidone, 2wt% of trehalose, 2wt% of polyethylene glycol, 0.02M disodium hydrogen phosphate, 5mM sodium dihydrogen phosphate, 0.05 wt% of polysorbate, and the balance of water, and the storage stabilizer is sterilized by an autoclave at 121 ℃ for 15min for later use.
Wherein the folic acid standard working solution in the step (31) is prepared by diluting folic acid standard stock solution with a storage stabilizer to a working solution concentration of 3.2 ng/mL.
Wherein the sterilization and concentration conformance verification of the folic acid standard working solution in the step (32) refers to performing filtration sterilization on 3.2 ng/mL folic acid standard working solution by using a 0.22-micron filter membrane, and performing concentration conformance verification on the folic acid standard working solution by combining a vitamin quality control sample (NO: 4044755861, quality control range: 95.16-126.52 μ g/100 g) with known concentration.
Wherein, in the step (33), according to the condition that the volume of the standard solution required in the experimental method of the folic acid detection kit is 150 muL, the concentration of the standard curve of the folic acid required is 0, 0.025, 0.05, 0.1, 0.15, 0.2 and 0.3ng/mL, the standard working solution is further diluted by 0, 0.125, 0.25, 0.5, 0.75, 1 and 1.5 ng/mL through a series of gradient concentration standard solutions by a storage stabilizer, 100 muL of the standard working solution is transferred and subpackaged into a centrifuge tube which can be fully soaked at the bottom of the centrifuge tube for one week and the liquid level is less than or equal to 10mm, a cover is lightly covered, and freeze-drying is carried out for 22 h by utilizing a vacuum freeze-drying technology, and the standard substance is immobilized.
And (4) packaging and storing, namely performing vacuum capping on the centrifuge tube with the immobilized standard substance, sealing and storing in an environment at the temperature of less than or equal to 8 ℃.
When the folic acid standard substance prepared by the embodiment is used, 500 mu L of sterile water is added into each centrifuge tube, vortex oscillation is uniform, 150 mu L of standard solution is transferred into a pore of the microporous plate, and then the preparation of the folic acid standard curve solution can be completed.
Comparative example 1 lyophilized standard of B12
In this comparative example, the preparation and application of the storage stabilizer in step (12) are not included, and a constant volume medium of a B12 standard stock solution is used instead of the storage stabilizer, namely a 25% ethanol solution, and other conditions are the same as those in example 1.
Comparative example 2 Biotin lyophilized Standard
The preparation and application of the storage stabilizer in the step (22) are not included in the comparative example, a constant volume medium of a biotin standard stock solution is used to replace the storage stabilizer, namely a 50% ethanol solution, and other conditions are the same as those in the example 2.
Comparative example 3 Folic acid lyophilized Standard
In this comparative example, the preparation and application of the storage stabilizer in step (32) are not included, and a constant volume medium of the folic acid standard stock solution is used instead of the storage stabilizer, that is, 0.01mol/L sodium hydroxide ethanol solution, and other conditions are the same as those in example 3.
Low temperature long term storage stability test
The vitamin standards prepared in examples 1 to 3 and comparative examples 1 to 3 were aseptically and hermetically packaged, and left at 4 ℃ for 12 months, and the contents of the relevant vitamins were measured at 0 month, 3 months, 6 months, 9 months, and 12 months, respectively, and the loss rates (%) of the vitamin standards in examples and comparative examples were calculated.
Loss rate of vitamin standard = (fixed concentration of vitamin standard-vitamin detection concentration in experimental example)/fixed concentration of vitamin standard = 100%.
The results of the comparison of the low-temperature long-term storage stability are shown in Table 1 below:
4℃ example 1 Example 2 Example 3 Comparative example 1 Comparative example 2 Comparative example 3
0 month 0.16 0.07 0.23 0.91 1.48 0.78
3 months old 0.41 0.30 0.62 1.48 3.70 1.95
6 months old 0.74 0.89 1.17 7.17 5.33 7.94
9 months old 1.24 1.26 1.71 13.27 7.47 12.31
12 months old 1.90 1.85 2.10 20.45 11.39 17.91
As can be seen from Table 1 above, the ready-to-use vitamin standard prepared by the invention has a vitamin loss rate of less than 5% in the low-temperature long-term storage experiment, and compared with comparative examples 1, 2 and 3, the storage stabilizer of the invention has outstanding advantages in low-temperature long-term vitamin storage stability.
High temperature short term storage stability test
After the vitamin standards prepared in examples 1 to 3 and comparative examples 1 to 3 were left at 4 ℃, 25 ℃, 37 ℃ and 60 ℃ for 7 days, the content of the vitamin standard was measured, and the loss rate (%) of the vitamin standards in examples and comparative examples was calculated.
Loss rate of vitamin standard = (fixed concentration of vitamin standard-vitamin detection concentration in experimental example)/fixed concentration of vitamin standard = 100%.
The results of the high temperature short term storage stability comparison are shown in Table 2 below:
7 days Example 1 Example 2 Example 3 Comparative example 1 Comparative example 2 Comparative example 3
4℃ 0.16 0.10 0.18 0.96 1.34 0.71
25℃ 0.40 0.57 0.53 11.80 6.21 24.02
37℃ 0.72 0.86 1.07 23.76 17.11 44.40
60℃ 1.20 1.05 1.33 100.00 100.00 100.00
As can be seen from the above Table 2, the vitamin loss rates of the ready-to-use vitamin standard prepared by the invention in the high-temperature short-term storage experiment are less than 5%, and compared with comparative examples 1, 2 and 3, the storage stabilizer of the invention has outstanding advantages in the aspect of high-temperature short-term vitamin storage stability.
The applicant states that the preparation method and application of the high-stability ready-to-use vitamin standard substance of the present invention are illustrated by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (10)

1. The preparation method of the ready-to-use vitamin standard substance is characterized by comprising the following steps:
(1) diluting the vitamin standard stock solution by using a storage stabilizer to obtain a vitamin standard working solution;
(2) sterilizing the vitamin standard working solution and verifying the consistency of the concentration of standard substances;
(3) fixing the vitamin standard mixed solution with the required concentration in a container by adopting a vacuum freeze drying technology; the vitamin standard mixed solution with the required concentration is a series of standard solutions with gradient concentration, which are obtained by diluting the vitamin standard working solution in the step (2) by using a storage stabilizer;
(4) and (5) packaging and storing.
2. The method of preparing a ready-to-use vitamin standard according to claim 1, wherein the storage stabilizer comprises the following components: 2-10 wt% of mannitol, 0.5-2 wt% of glucan, 0.2-1 wt% of polyvinylpyrrolidone, 2-4 wt% of trehalose, 2-4 wt% of polyethylene glycol, 0.02-0.05M of disodium hydrogen phosphate, 1-5 mM of sodium dihydrogen phosphate, 0.05-0.2wt% of polysorbate and the balance of water.
3. The method for preparing a ready-to-use vitamin standard according to claim 1, wherein the step (1) is to dilute the vitamin standard stock solution with a storage stabilizer to obtain a vitamin working solution, wherein the vitamin working solution is at least 10 times diluted with the storage stabilizer3And (4) performing double dilution.
4. The method for preparing the ready-to-use vitamin standard substance as claimed in claim 1, wherein the step (2) of sterilizing the vitamin standard working solution and verifying the compliance of the concentration of the vitamin standard working solution comprises filtering the vitamin standard working solution through a 0.22-0.45 μm filter membrane for sterilization, and then verifying the compliance of the vitamin standard working solution by combining a vitamin quality control sample with a known concentration.
5. The method for preparing a ready-to-use vitamin standard according to claim 1, wherein the step (3) of fixing the vitamin standard mixed solution is to dilute the vitamin standard working solution of step (2) to a series of gradient concentration standard solutions by using a storage stabilizer according to the concentration and volume required by the experimental method to obtain a series of gradient concentration vitamin standard mixed solutions, then separately pack the series of gradient concentration vitamin standard mixed solutions into a container to fully soak the bottom of the container for one week, freeze-dry the mixture for 20-40 h by using a vacuum freeze-drying technology, and fix the standard solutions into the container.
6. The method for preparing a ready-to-use vitamin standard according to claim 5, wherein the liquid level of the vitamin standard mixture in the container is less than or equal to 10 mm.
7. The method for preparing a ready-to-use vitamin standard substance according to claim 1, wherein the packaging in step (4) is to ensure that the standard substance is maintained in a sterile, light-shielding and dry condition and stored at a temperature of less than or equal to 8 ℃.
8. A ready-to-use vitamin standard prepared by the method of any one of claims 1 to 7.
9. The use method of the ready-to-use vitamin standard according to claim 8, wherein sterile water is added into a container in which the vitamin standard mixture is lyophilized, and the mixture is uniformly mixed to obtain a gradient solution of a standard curve series.
10. A storage stabilizer for a ready-to-use vitamin standard is characterized by comprising the following components: 2-10 wt% of mannitol, 0.5-2 wt% of glucan, 0.2-1 wt% of polyvinylpyrrolidone, 2-4 wt% of trehalose, 2-4 wt% of polyethylene glycol, 0.02-0.05M of disodium hydrogen phosphate, 1-5 mM of sodium dihydrogen phosphate, 0.05-0.2wt% of polysorbate and the balance of water.
CN202010546255.3A 2020-06-15 2020-06-15 Ready-to-use vitamin standard substance, preparation method, use method and storage stabilizer thereof Pending CN111707512A (en)

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