A preparation method and stabilized composition of freeze-dried standard product of folic acid

Abstract

The present invention discloses a method for preparing a freeze-dried standard of a folate substance, a freeze-dried standard of a folate substance, a stabilized composition and its use. The preparation method comprises the following steps: (1) diluting the basic working solution of a folate substance to a series of gradient concentration standard solutions using a stabilized composition; (2) using a vacuum freeze drying technique to prepare a freeze-dried powder from the standard solution obtained in step (1), to obtain a freeze-dried standard of a folate substance; wherein the stabilized composition is composed of the following components in parts by weight: 0.5-5 parts of an antioxidant, 0.5-5 parts of a synergist dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride, 1-10 parts of trehalose and a buffer, and the buffer is supplemented to 100 parts. The freeze-dried standard of a folate substance obtained by the preparation method has good uniformity, high storage stability, can be stably stored for a long time under low temperature conditions, and the re-dissolution step is simple, and the liquid is clear after re-dissolution.

Description

Technical Field

[0001] The invention belongs to the technical field of standard product preparation, and specifically relates to a method for preparing a freeze-dried standard product of a folic acid substance, the prepared freeze-dried standard product of a folic acid substance, a stabilized composition and uses thereof. Background Art

[0002] Folic acid (FA) is a water-soluble B vitamin that provides one-carbon units in the process of nucleotide synthesis and DNA methylation. It has the functions of promoting cell division, differentiation and repair, and is an essential substance for embryonic development. Folic acid deficiency not only easily leads to hyperhomocysteinemia, neural tube defects, and megaloblastic anemia, but also increases the risk of cardiovascular disease and tumors. At the same time, synthetic folic acid may interfere with the metabolism of naturally occurring reduced folic acid in food. Long-term excessive intake may lead to the appearance of unmetabolized folic acid in the circulation, masking vitamin B 12 Symptoms of deficiency, and promotion of colorectal tumor formation, etc.

[0003] The existing folic acid detection kits on the market mainly include fluorescent immunochromatography, chemiluminescence, and liquid chromatography-tandem mass spectrometry. Due to different detection principles, different detection methods have certain differences in detection sensitivity, specificity, linear range, etc. Among them, liquid chromatography-tandem mass spectrometry can simultaneously determine folic acid and its metabolites, with good reproducibility and online analysis, and has gradually become the development trend of clinical folic acid detection. At present, the total folic acid (tFOL) of the LC-MS method mainly includes folic acid (FA), 5-formyltetrahydrofolate (5FoTHF), and 5-methyltetrahydrofolate (5MeTHF).

[0004] A major factor affecting the accurate quantification of folic acid is the instability of the folic acid structure. The parent compound of folic acid is composed of three components: pteridine, p-aminobenzoic acid and glutamic acid. The chemical structure is as follows Figure 1 As shown. Folic acid has poor stability and can be degraded by light (long-wave ultraviolet rays, medium-wave ultraviolet rays UVA, UVB), pH value, temperature (heat), concentration, oxygen and oxidative degradation, singlet oxygen, electron beam, etc. Studies have shown that under acidic conditions, folic acid and its metabolites can be mutually converted.

[0005] Therefore, accurate determination of folic acid requires a stable quality control material to calibrate and monitor the instrument status and the effectiveness of the reagents. It is known that freeze-drying technology has the advantages of protecting the activity of heat-sensitive substances, preventing substances from being oxidized, and easy recovery by adding water, but most of the existing freeze-dried product patents on folic acid are for folic acid preparations. The present invention is aimed at screening prescriptions for folic acid standard substances required in the liquid chromatography-mass spectrometry detection process. Select a suitable stabilizing composition to improve the stability of folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate standards during long-term storage, so as to achieve the purpose of extending the validity period of the standard. Summary of the invention

[0006] The present invention aims at the deficiencies of the prior art and provides a method for preparing a freeze-dried standard product of a folate substance, a freeze-dried standard product of a folate substance obtained, a stabilized composition and uses thereof. The freeze-dried standard product of a folate substance obtained by the preparation method has good uniformity, high storage stability, can be stably stored for a long time under low temperature conditions, has a simple reconstitution step, and the liquid is clear after reconstitution.

[0007] Specifically, the present invention is realized through the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a freeze-dried standard of folic acid, comprising the following steps:

[0009] (1) using the stabilizing composition to dilute the basic working solution of folic acid substances to a series of gradient concentration standard solutions;

[0010] (2) using vacuum freeze drying technology to prepare freeze-dried powder from the standard solution of folic acid substances of the required concentration obtained in step (1), thereby obtaining a freeze-dried standard product of folic acid substances;

[0011] The stabilization composition is composed of the following components in parts by weight: 0.5-5 parts of antioxidant, 0.5-5 parts of synergist dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride, 1-10 parts of trehalose, and buffer, and the buffer is supplemented to 100 parts.

[0012] In the present invention, the folic acid substance includes one or more of folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate.

[0013] Preferably, the antioxidant in the above-mentioned stabilization composition is at least one of sodium thiosulfate, cysteine, sodium metabisulfite and glutathione.

[0014] More preferably, the antioxidant in the above-mentioned stabilization composition is cysteine.

[0015] Preferably, the buffer in the above-mentioned stabilization composition is sodium carbonate-sodium bicarbonate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, sodium dihydrogen phosphate-sodium hydroxide buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, PBS buffer or sodium hydroxide buffer.

[0016] Preferably, in the above step (1), the mass ratio of the folic acid substance to the stabilizing composition is (0.0005-0.1):1.

[0017] Preferably, the vacuum freeze-drying technology in step (2) above comprises the following steps:

[0018] 1) Pre-freeze-drying stage:

[0019] The temperature of the freeze drying chamber is -60℃~-40℃, and the product is pre-frozen for 2~4h;

[0020] 2) Primary drying stage:

[0021] The temperature is raised to -30℃~-35℃, and maintained for 1~3h under vacuum conditions → the temperature is raised to -20~-30℃, and maintained for 1~3h → the temperature is raised to -15~-25℃, and maintained for 4~7h → the temperature is raised to -10~0℃, and maintained for 4~7h under vacuum conditions;

[0022] 3) Analysis and drying stage:

[0023] The temperature was adjusted to 10-20°C and maintained under vacuum for 6-8 hours.

[0024] Preferably, the vacuum degree in the above step 2) primary drying stage and step 3) analytical drying stage is 1-20 Pa.

[0025] In a second aspect, the present invention provides a freeze-dried standard product of folate, wherein the freeze-dried standard product is prepared by the preparation method of the freeze-dried standard product of folate.

[0026] In a third aspect, the present invention provides a stabilizing composition, which is composed of the following components in parts by weight: 0.5-5 parts of an antioxidant, 0.5-5 parts of a synergist dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride, 1-10 parts of trehalose, and a buffer, wherein the buffer is supplemented to 100 parts;

[0027] The antioxidant is at least one of sodium thiosulfate, cysteine, sodium pyrosulfite and glutathione;

[0028] The buffer is sodium carbonate-sodium bicarbonate buffer, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer, sodium dihydrogen phosphate-sodium hydroxide buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, PBS buffer or sodium hydroxide buffer.

[0029] In a fourth aspect, the present invention provides a use of the above-mentioned stabilization composition in the preparation of a freeze-dried standard of folic acid substances.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The stabilized composition of the present invention uses trehalose as a skeleton and protective agent, which plays a role in supporting the skeleton and preventing the active ingredients from being denatured during the freeze-drying process; antioxidants such as cysteine ​​can be used as free radical scavengers during the antioxidant process to avoid the oxidation of folic acid substances; the synergist dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride is used in combination with the antioxidant to improve the antioxidant effect, better prevent or delay the oxidation of folic acid substances, and improve the storage stability of the freeze-dried standard of folic acid substances. Experiments have shown that the antioxidant, synergist dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride and trehalose in the stabilized composition of the present invention have synergistic effects, and are used in the method for preparing the freeze-dried standard of folic acid substances, which significantly improves the storage stability of the freeze-dried standard of folic acid substances and prolongs the shelf life.

[0032] (2) The freeze-dried standard product of folic acid prepared by the present invention has a cake-like appearance and does not stick to the bottle stopper. The freeze-dried standard product has good uniformity, low water content, high storage stability, and can be stably stored for a long time under low temperature conditions. The reconstitution step is simple, and the liquid is clear after reconstitution. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0034] Figure 1 is the chemical structural formula of folic acid. DETAILED DESCRIPTION

[0035] The embodiments of the present invention are described in detail below. The embodiments are given to better illustrate the contents of the present invention and are only used to explain the present invention, but should not be understood as limiting the present invention.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0038] Example 1

[0039] A stabilizing composition, the stabilizing composition is composed of the following components in parts by weight:

[0040]

[0041] A freeze-dried folic acid standard

[0042] The preparation method of the freeze-dried folic acid standard product comprises the following steps:

[0043] (1) using the stabilizing composition of Example 1 to dilute the folic acid basic working solution to a series of gradient concentration standard solutions, the mass ratio of folic acid to the stabilizing composition being 0.005:1;

[0044] (2) The folic acid standard solution of the required concentration obtained in step (1) is made into a freeze-dried powder by using vacuum freeze-drying technology to obtain a freeze-dried folic acid standard product.

[0045] The folic acid basic working solution in step (1) is obtained by weighing a folic acid standard and fixing the volume with a 0.02 mol / L sodium hydroxide ethanol solution.

[0046] Wherein, the vacuum freeze-drying technology described in step (2) specifically comprises the following steps:

[0047] 1) Pre-freeze-drying stage:

[0048] The temperature of the freeze drying chamber is -40°C, and the product is pre-frozen for 4 hours;

[0049] 2) Primary drying stage:

[0050] The temperature was raised to -30°C, and kept under vacuum for 1 hour → the temperature was raised to -25°C, and kept under vacuum for 1 hour → the temperature was raised to -15°C, and kept under vacuum for 4 hours → the temperature was raised to 0°C, and kept under vacuum for 4 hours, and the vacuum was maintained at 20 Pa;

[0051] 3) Analysis and drying stage:

[0052] The temperature was adjusted to 20°C and maintained under vacuum conditions for 6 h at a vacuum of 20 Pa.

[0053] The 5-methyltetrahydrofolate lyophilized standard product can be obtained by replacing the folic acid basic working solution in the above step (1) with a 5-methyltetrahydrofolate basic working solution using the same preparation method as described above.

[0054] The 5-formyltetrahydrofolate lyophilized standard product can be obtained by replacing the folic acid basic working solution in the above step (1) with a 5-formyltetrahydrofolate basic working solution using the same preparation method as described above.

[0055] Example 2

[0056] A stabilizing composition, the stabilizing composition is composed of the following components in parts by weight:

[0057]

[0058] A freeze-dried folic acid standard

[0059] The preparation method of the freeze-dried folic acid standard product comprises the following steps:

[0060] (1) using the stabilizing composition of Example 2 to dilute the folic acid basic working solution to a series of gradient concentration standard solutions, the mass ratio of folic acid to the stabilizing composition being 0.01:1;

[0061] (2) The folic acid standard solution of the required concentration obtained in step (1) is made into a freeze-dried powder by using vacuum freeze-drying technology to obtain a freeze-dried folic acid standard product.

[0062] The folic acid basic working solution in step (1) is obtained by weighing a folic acid standard and fixing the volume with a 0.02 mol / L sodium hydroxide ethanol solution.

[0063] Wherein, the vacuum freeze-drying technology described in step (2) specifically comprises the following steps:

[0064] 1) Pre-freeze-drying stage:

[0065] The temperature of the freeze drying chamber is -50°C, and the product is pre-frozen for 2 hours;

[0066] 2) Primary drying stage:

[0067] The temperature was raised to -35°C, and kept under vacuum for 1 hour → raised to -30°C, and kept under vacuum for 3 hours → raised to -20°C, and kept under vacuum for 4 hours → raised to -5°C, and kept under vacuum for 5 hours, with the vacuum maintained at 10 Pa;

[0068] 3) Analysis and drying stage:

[0069] The temperature was adjusted to 15°C and maintained under vacuum conditions for 8 h at a vacuum of 10 Pa.

[0070] The 5-methyltetrahydrofolate lyophilized standard product can be obtained by replacing the folic acid basic working solution in the above step (1) with a 5-methyltetrahydrofolate basic working solution using the same preparation method as described above.

[0071] The 5-formyltetrahydrofolate lyophilized standard product can be obtained by replacing the folic acid basic working solution in the above step (1) with a 5-formyltetrahydrofolate basic working solution using the same preparation method as described above.

[0072] Example 3

[0073] A stabilizing composition, the stabilizing composition is composed of the following components in parts by weight:

[0074]

[0075] A freeze-dried folic acid standard

[0076] The preparation method of the freeze-dried folic acid standard product comprises the following steps:

[0077] (1) using the stabilizing composition of Example 3 to dilute the folic acid basic working solution to a series of gradient concentration standard solutions, the mass ratio of folic acid to the stabilizing composition being 0.005:1;

[0078] (2) The folic acid standard solution of the required concentration obtained in step (1) is made into a freeze-dried powder by using vacuum freeze-drying technology to obtain a freeze-dried folic acid standard product.

[0079] The folic acid basic working solution in step (1) is obtained by weighing a folic acid standard and fixing the volume with a 0.02 mol / L sodium hydroxide ethanol solution.

[0080] Wherein, the vacuum freeze-drying technology described in step (2) specifically comprises the following steps:

[0081] 1) Pre-freeze-drying stage:

[0082] The temperature of the freeze drying chamber is -40°C, and the product is pre-frozen for 4 hours;

[0083] 2) Primary drying stage:

[0084] The temperature was raised to -30°C, and kept under vacuum for 1 hour → the temperature was raised to -25°C, and kept under vacuum for 1 hour → the temperature was raised to -15°C, and kept under vacuum for 4 hours → the temperature was raised to 0°C, and kept under vacuum for 4 hours, and the vacuum was maintained at 20 Pa;

[0085] 3) Analysis and drying stage:

[0086] The temperature was adjusted to 20°C and maintained under vacuum conditions for 6 h at a vacuum of 20 Pa.

[0087] The 5-methyltetrahydrofolate lyophilized standard product can be obtained by replacing the folic acid basic working solution in the above step (1) with a 5-methyltetrahydrofolate basic working solution using the same preparation method as described above.

[0088] The 5-formyltetrahydrofolate lyophilized standard product can be obtained by replacing the folic acid basic working solution in the above step (1) with a 5-formyltetrahydrofolate basic working solution using the same preparation method as described above.

[0089] Comparative Example 1

[0090] A stabilizing composition, the stabilizing composition is composed of the following components in parts by weight:

[0091] 1 part cysteine

[0092] 1 part trehalose

[0093] 98 parts of 0.01 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

[0094] A freeze-dried folic acid standard

[0095] The preparation method of the freeze-dried folic acid standard product comprises the following steps:

[0096] (1) using the stabilizing composition of comparative example 1 to dilute the folic acid basic working solution to a series of gradient concentration standard solutions, the mass ratio of folic acid to the stabilizing composition being 0.005:1;

[0097] (2) The folic acid standard solution of the required concentration obtained in step (1) is made into a freeze-dried powder by using vacuum freeze-drying technology to obtain a freeze-dried folic acid standard product.

[0098] The folic acid basic working solution in step (1) is obtained by weighing a folic acid standard and fixing the volume with a 0.02 mol / L sodium hydroxide ethanol solution.

[0099] Wherein, the vacuum freeze-drying technology described in step (2) specifically comprises the following steps:

[0100] 1) Pre-freeze-drying stage:

[0101] The temperature of the freeze drying chamber is -40°C, and the product is pre-frozen for 4 hours;

[0102] 2) Primary drying stage:

[0103] The temperature was raised to -30°C, and kept under vacuum for 1 hour → the temperature was raised to -25°C, and kept under vacuum for 1 hour → the temperature was raised to -15°C, and kept under vacuum for 4 hours → the temperature was raised to 0°C, and kept under vacuum for 4 hours, and the vacuum was maintained at 20 Pa;

[0104] 3) Analysis and drying stage:

[0105] The temperature was adjusted to 20°C and maintained under vacuum conditions for 6 h at a vacuum of 20 Pa.

[0106] Comparative Example 2

[0107] A stabilizing composition, the stabilizing composition is composed of the following components in parts by weight:

[0108] 1 part dithiothreitol

[0109] 1 part trehalose

[0110] 98 parts of 0.01 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

[0111] A freeze-dried folic acid standard

[0112] The preparation method of the freeze-dried folic acid standard product comprises the following steps:

[0113] (1) using the stabilizing composition of comparative example 2 to dilute the folic acid basic working solution to a series of gradient concentration standard solutions, the mass ratio of folic acid to the stabilizing composition being 0.005:1;

[0114] (2) The folic acid standard solution of the required concentration obtained in step (1) is made into a freeze-dried powder by using vacuum freeze-drying technology to obtain a freeze-dried folic acid standard product.

[0115] The folic acid basic working solution in step (1) is obtained by weighing a folic acid standard and fixing the volume with a 0.02 mol / L sodium hydroxide ethanol solution.

[0116] Wherein, the vacuum freeze-drying technology described in step (2) specifically comprises the following steps:

[0117] 1) Pre-freeze-drying stage:

[0118] The temperature of the freeze drying chamber is -40°C, and the product is pre-frozen for 4 hours;

[0119] 2) Primary drying stage:

[0120] The temperature was raised to -30°C, and kept under vacuum for 1 hour → the temperature was raised to -25°C, and kept under vacuum for 1 hour → the temperature was raised to -15°C, and kept under vacuum for 4 hours → the temperature was raised to 0°C, and kept under vacuum for 4 hours, and the vacuum was maintained at 20 Pa;

[0121] 3) Analysis and drying stage:

[0122] The temperature was adjusted to 20°C and maintained under vacuum conditions for 6 h at a vacuum of 20 Pa.

[0123] Comparative Example 3

[0124] A stabilizing composition, the stabilizing composition is composed of the following components in parts by weight:

[0125] 1 part of tri(2-carboxyethyl)phosphine hydrochloride

[0126] 1 part trehalose

[0127] 98 parts of 0.01 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer.

[0128] A freeze-dried folic acid standard

[0129] The preparation method of the freeze-dried folic acid standard product comprises the following steps:

[0130] (1) using the stabilizing composition of comparative example 3 to dilute the folic acid basic working solution to a series of gradient concentration standard solutions, the mass ratio of folic acid to the stabilizing composition being 0.005:1;

[0131] (2) The folic acid standard solution of the required concentration obtained in step (1) is made into a freeze-dried powder by using vacuum freeze-drying technology to obtain a freeze-dried folic acid standard product.

[0132] The folic acid basic working solution in step (1) is obtained by weighing a folic acid standard and fixing the volume with a 0.02 mol / L sodium hydroxide ethanol solution.

[0133] Wherein, the vacuum freeze-drying technology described in step (2) specifically comprises the following steps:

[0134] 1) Pre-freeze-drying stage:

[0135] The temperature of the freeze drying chamber is -40°C, and the product is pre-frozen for 4 hours;

[0136] 2) Primary drying stage:

[0137] The temperature was raised to -30°C, and kept under vacuum for 1 hour → the temperature was raised to -25°C, and kept under vacuum for 1 hour → the temperature was raised to -15°C, and kept under vacuum for 4 hours → the temperature was raised to 0°C, and kept under vacuum for 4 hours, and the vacuum was maintained at 20 Pa;

[0138] 3) Analysis and drying stage:

[0139] The temperature was adjusted to 20°C and maintained under vacuum conditions for 6 h at a vacuum of 20 Pa.

[0140] Example 4

[0141] Detection of reconstitution time of freeze-dried standard of folic acid substances:

[0142] The reconstitution time is one of the important indicators of whether the preparation of the freeze-dried standard product is successful or not. Six bottles of the freeze-dried folic acid standard product prepared in Example 1 were taken, purified water was added to reconstitute, and the reconstitution time was recorded. The experimental results are shown in Table 1:

[0143] Table 1: Reconstitution time of lyophilized folic acid standard

[0144]

[0145] It can be seen from Table 1 that the lyophilized product can be reconstituted by adding a fixed volume of purified water, the reconstitution steps are simple, the reconstitution time is short, and the solution is clear after reconstitution.

[0146] Example 5

[0147] Stability test of freeze-dried standard of folic acid substances:

[0148] The lyophilized folic acid standard, 5-methyltetrahydrofolate lyophilized standard, and 5-formyltetrahydrofolate lyophilized standard prepared in Example 2 were placed in a 37° C. incubator for 7 days and then taken out. 1 ml of purified water was added for reconstitution. The area ratios of folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate to the corresponding internal standard were simultaneously detected by LC-MS / MS method, and compared with the lyophilized product at time 0 (control group). The experimental results are shown in Table 2:

[0149] Table 2: Stability test of freeze-dried standard of folic acid substances (ratio of folic acid substances to corresponding internal standard areas)

[0150]

[0151] It can be seen from Table 2 that the deviation of the test results at 7 days of accelerated storage is within ±10% compared with the test results at 0 time. According to the Arrhenius formula, the freeze-dried standard of folic acid prepared by the present invention can be stored at -20°C for 1 year.

[0152] Example 6

[0153] Data detection between different batches of freeze-dried standard folic acid substances:

[0154] Specific experimental process: The same method was used to prepare solutions at different times, and the solutions were packaged in 1 mL portions and freeze-dried to obtain three batches of freeze-dried products. 1 mL of purified water was added to each batch to reconstitute the products. The products were detected simultaneously using mass spectrometry, and the mean deviation and coefficient of variation were calculated. The experimental results are shown in Table 3:

[0155] Table 3: Data detection between different batches of lyophilized folic acid standard products (ratio of folic acid to corresponding internal standard area)

[0156] First batch Second batch The third batch Target value Mean deviation CV% Folic acid 0.950 0.947 1.047 0.983 0.981 -0.17 5.80 5-Methyltetrahydrofolate 0.185 0.184 0.184 0.182 0.184 1.28 0.31 5-Formyltetrahydrofolate 0.102 0.106 0.102 0.096 0.103 7.64 2.23

[0157] It can be seen from Table 3 that the coefficients of variation of the three batches of freeze-dried standard products of folic acid substances are all less than 10%, indicating that the preparation method of a freeze-dried standard product of folic acid substances provided by the present invention can stably prepare freeze-dried standard products of folic acid substances.

[0158] Example 7

[0159] Stability test of freeze-dried standard of folic acid substances:

[0160] The folic acid reference substances prepared in Comparative Examples 1 and 2 were tested according to the stability test method for freeze-dried standard substances of folic acid described in Example 5, and compared with the results of Example 1. The stability test results are shown in Table 4:

[0161] Table 4: Stability test of freeze-dried standard and reference substances of folic acid substances (ratio of folic acid substances to corresponding internal standard areas)

[0162]

[0163] The folic acid reference substances prepared in Comparative Example 1, Comparative Example 3 and Example 3 were tested according to the stability test method for the lyophilized standard substance of folic acid described in Example 5, and compared. The stability test results are shown in Table 5:

[0164] Table 5: Stability test of freeze-dried standard and reference substances of folic acid substances (area ratio of folic acid substances to their corresponding internal standards)

[0165]

[0166] It can be seen from Tables 4 and 5 that the antioxidant cysteine, the synergist dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride and trehalose in the stabilized composition of the present invention have synergistic effects and are applied to the method for preparing a freeze-dried standard product of folic acid substances, which significantly improves the storage stability of the freeze-dried standard product of folic acid substances and prolongs the shelf life.

[0167] Obviously, the above embodiments are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. Those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a freeze-dried standard product of folic acid, characterized in that: The following steps are involved: (1) diluting the basic working solution of folic acid substances into a series of gradient concentration standard solutions using the stabilizing composition; (2) using vacuum freeze-drying technology to prepare freeze-dried powder from the standard solution of folic acid substances of the required concentration obtained in step (1), thereby obtaining a freeze-dried standard product of folic acid substances; The stabilization composition is composed of the following components in parts by weight: 0.5 parts of antioxidant cysteine, 0.5 parts of synergist dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride, 1 part of trehalose, and 0.01 mol / L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer, wherein the buffer is supplemented to 100 parts; The folic acid substance is one or more of folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate. The mass ratio of the folic acid substance to the stabilizing composition is 0.005:

1.

2. The method for preparing the freeze-dried standard product of folic acid according to claim 1, characterized in that: The vacuum freeze drying technology of step (2) comprises the following steps: 1) Pre-freeze-drying stage: The temperature of the freeze drying chamber is -60℃~-40℃, and the product is pre-frozen for 2~4h; 2) Primary drying stage: The temperature rises to -30℃~-35℃, and keeps for 1~3h under vacuum conditions→rises to -20~-30℃, and keeps for 1~3h→rises to -15~-25℃, and keeps for 4~7h→rises to -10~0℃, and keeps for 4~7h under vacuum conditions; 3) Analysis and drying stage: Adjust the temperature to 10~20℃ and maintain it under vacuum for 6~8h.

3. A freeze-dried standard product of folic acid, characterized in that: The lyophilized standard product of folic acid is prepared by the method for preparing the lyophilized standard product of folic acid as claimed in claim 1 or claim 2.

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