Fat-soluble vitamin calibrator, freeze-drying preparation thereof and kit

Abstract

The invention relates to the technical field of analysis and detection, in particular to a fat-soluble vitamin calibrator, a freeze-drying preparation thereof and a kit. The fat-soluble vitamin calibrator comprises fat-soluble vitamins, an excipient, a stabilizer, a dispersing agent and a diluent, wherein the dispersing agent comprises Tween 80 and sodium dodecyl sulfate; the diluent comprises a mixed solution of isopropanol and water. According to the fat-soluble vitamin calibrator, the freeze-drying preparation thereof and the kit provided by the invention, the dispersing agent and the diluent are optimally introduced, so that the stability and the uniformity of the fat-soluble vitamin calibrator can be greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, in particular to a fat-soluble vitamin calibrator, a freeze-dried preparation thereof and a kit. Background Art

[0002] Vitamins are essential trace elements for maintaining human health. Acting as enzymes or coenzymes, they participate in human metabolism and regulate growth and development. Vitamin A, 25-hydroxyvitamin D2 (25-OHD2), 25-hydroxyvitamin D3 (25-OHD3), vitamin E (VE), and vitamin K1 (VK1) are fat-soluble vitamins. Because the human body cannot synthesize these vitamins or only synthesizes them in extremely small quantities, they can only be obtained from food. Inadequate intake or deficiency of fat-soluble vitamins can easily lead to disorders of physiological function, metabolism, and cellular regulation, and can induce a variety of diseases. Generally speaking, adults with a balanced diet do not require additional fat-soluble vitamin supplementation. However, vitamin deficiencies are common in special populations, such as pregnant women, the elderly, those with osteoporosis, and children. Blindly supplementing with large amounts of these vitamins can easily lead to accumulation and toxicity.

[0003] Fat-soluble vitamins are highly susceptible to oxidation and are sensitive to light, heat, acidity, alkali, and metal ions. Therefore, special measures are needed to improve the stability of vitamins in calibrators, thereby extending their shelf life and stability. Patent CN103126992A improves the stability of fat-soluble vitamins through freeze-drying, but this method produces a powder injection for the freeze-dried preparation. Patent CN117517019A discloses a freeze-dried preparation and freeze-drying method for a 25-hydroxyvitamin calibrator, and evaluates the product's reconstitution stability and accelerated stability. However, its stability and homogeneity still leave room for improvement.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention discovered that fat-soluble vitamins undergo nonspecific adsorption to the inner wall of glass bottles during processing, which can affect their stability and uniformity to a certain extent. After extensive research, the present invention discovered that the synergistic effect of Tween 80 and sodium lauryl sulfate helps reduce sample adsorption by the inner wall of glass bottles and enhances the solubility of vitamins. Furthermore, there is room for improvement in the precision of existing freeze-dried preparations of fat-soluble vitamins. The present invention discovered that introducing a mixed solution of isopropyl alcohol and water into fat-soluble vitamin calibrants can help improve detection precision, enhance preparation stability, and enhance quality control.

[0006] Based on this, the present invention has the following technical solutions: In a first aspect, the present invention provides a fat-soluble vitamin calibrator, comprising a fat-soluble vitamin, an excipient, a stabilizer, a dispersant, and a diluent; Wherein, the dispersant includes Tween 80 and sodium lauryl sulfate; and the diluent is a mixed solution of isopropyl alcohol and water.

[0007] Preferably, the diluent is isopropyl alcohol and water in a mass ratio of (1-2):(1-2).

[0008] Preferably, the dispersant is Tween 80 and sodium dodecyl sulfate in a mass ratio of (1-2): (1-2).

[0009] Preferably, the excipient comprises bovine serum albumin.

[0010] Preferably, the stabilizer comprises one or more of tea polyphenols, chlorogenic acid and vitamin C; more preferably, the stabilizer comprises tea polyphenols, chlorogenic acid and vitamin C in a mass ratio of 1:(1-3):(4-6).

[0011] Preferably, the fat-soluble vitamins include one or more of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1.

[0012] In a second aspect, the present invention provides a method for preparing the fat-soluble vitamin calibrator, comprising: S1: Dissolve the excipient in PBS and then mix with the stabilizer to obtain diluent A; S2: mixing the dispersant and the diluent to obtain a diluent B; S3: Dissolve the fat-soluble vitamins in diluent B respectively, and then mix with the diluent A to obtain fat-soluble vitamin calibrators.

[0013] Preferably, in S1, the order of adding the stabilizers is: first add tea polyphenols, then add vitamin C, and finally add chlorogenic acid.

[0014] In a third aspect, the present invention provides a lyophilized preparation of a fat-soluble vitamin calibrator, characterized in that it comprises: a fat-soluble vitamin, an excipient, a stabilizer and a dispersant; wherein the dispersant comprises Tween 80 and sodium lauryl sulfate.

[0015] Preferably, the dispersant is Tween 80 and sodium dodecyl sulfate in a mass ratio of (1-2): (1-2).

[0016] Preferably, the excipient comprises bovine serum albumin.

[0017] Preferably, the stabilizer comprises one or more of tea polyphenols, chlorogenic acid and vitamin C; more preferably, the stabilizer comprises tea polyphenols, chlorogenic acid and vitamin C in a mass ratio of 1:(1-3):(4-6).

[0018] And / or, the fat-soluble vitamins include one or more of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1.

[0019] In a fourth aspect, the present invention provides a method for preparing the lyophilized preparation of the fat-soluble vitamin calibrator, comprising: freeze-drying the fat-soluble vitamin calibrator; the freeze-drying comprising: S41: Pre-freeze: keep warm at -60~-40℃ for 120~180min; S42: Sublimation drying: Control the air entrainment vacuum degree to 0.05~0.15mbar, the sublimation temperature to -35~-25℃, and the sublimation time to 15~30 hours; S43: Drying: drying temperature 20-30°C, drying time 6-12 hours.

[0020] Preferably, the preparation method of the lyophilized preparation of the fat-soluble vitamin calibrator comprises: S1: bovine serum albumin is dissolved in PBS to obtain a 3-7% BSA PBS solution, and then tea polyphenols, vitamin C and chlorogenic acid are added in sequence to obtain a diluent A; the stabilizer is tea polyphenols, chlorogenic acid and vitamin C in a mass ratio of 1: (1-3): (4-6); S2: mixing a dispersant and a diluent to obtain a dilution solution B; the dispersant is Tween 80 and sodium lauryl sulfate in a mass ratio of (1-2): (1-2); the diluent is isopropyl alcohol and water in a mass ratio of (1-2): (1-2); S3: dissolving the fat-soluble vitamins in diluent B respectively, and then mixing with the diluent A to obtain fat-soluble vitamin calibrators; S4: freeze-drying the fat-soluble vitamin calibrator; the freeze-drying comprises: S41: Pre-freeze: keep warm at -60~-40℃ for 120~180min; S42: Sublimation drying: Control the air entrainment vacuum degree to 0.05~0.15mbar, the sublimation temperature to -35~-25℃, and the sublimation time to 15~30 hours; S43: Drying: drying temperature 20-30°C, drying time 6-12 hours.

[0021] In a fifth aspect, the present invention provides a fat-soluble vitamin kit, which contains the fat-soluble vitamin calibrator or the lyophilized preparation of the fat-soluble vitamin calibrator.

[0022] The fat-soluble vitamin calibrators, their lyophilized preparations, and kits provided by the present invention can greatly improve the stability and homogeneity of the fat-soluble vitamin calibrators by optimizing the introduction of dispersants and diluents. The calibrators after lyophilization have good homogeneity and can be stably stored for at least 12 months at 2-8°C. After reconstitution, they can be stably stored at -20°C for at least 1 month. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the various raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0025] Example 1 This embodiment provides a fat-soluble vitamin calibrator, the preparation method of which comprises the following steps: 1. Preparation of diluent A 1) Measure 100 mL of 1× PBS into a suitable container; 2) Weigh 5 g of BSA and slowly add it to step 1) while stirring to prepare a 5% BSA solution. 3) Weigh 10 mg of tea polyphenols and slowly add it to step 2) while stirring; 4) Weigh 50 mg of vitamin C and slowly add it to step 3) while stirring. 5) Weigh 20 mg of chlorogenic acid and slowly add it to 4) under stirring to prepare diluent A.

[0026] 2. Preparation of calibrators 1) Prepare diluent B by adding 0.05 g of Tween 80 and 0.05 g of sodium dodecyl sulfate to 5 mL of 50% isopropyl alcohol solution. 2) Add vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1 standard substances to 1), vortex mix, and prepare concentrated stock solutions with a series of concentration gradients of calibrators 1-6; 3) Under stirring, add the concentrated stock solutions of the calibrators 1-6 prepared in 2) to diluent A to prepare calibrators 1-6; wherein the concentration range of vitamin A for calibrators 1-6 is 60-2400 ng / mL, the concentration range of 25-hydroxyvitamin D2 is 2-80 ng / mL, the concentration range of 25-hydroxyvitamin D3 is 4-160 ng / mL, the concentration range of vitamin E is 800-32000 ng / mL, and the concentration range of vitamin K1 is 0.2-8 ng / mL.

[0027] This embodiment further provides a lyophilized preparation of a fat-soluble vitamin calibrator, the preparation method of which comprises the following steps: The calibrants prepared above were divided into brown glass bottles and half-stoppered; the half-stoppered samples were placed in a freeze-drying chamber, the circulating pump shelf refrigeration was turned on, the shelf temperature was set to -50°C for 90 minutes, and the temperature was kept warm for 120-180 minutes, the cold trap was refrigerated, the vacuum was turned on, and the product was sublimated and dried. At the same time, the aeration control was performed to control the aeration vacuum degree to 0.1 mbar, the sublimation temperature to -30°C, and the sublimation time to 15-30 hours. After the sublimation stage, the product was subjected to analytical drying at a drying temperature of 20-30°C and a drying time of 6-12 hours.

[0028] Test example 1. Bottle-to-bottle uniformity test data 1.1 Verification Method: Reconstitute 10 vials of lyophilized calibrator and determine the amounts of vitamin A, 25-OHD2, 25-OHD3, vitamin E, and vitamin K1 in the lyophilized powder by HPLC-MS / MS. The coefficient of variation (CV) for repeatability can be calculated using the following formula; CV should be ≤15%.

[0029] CV=S / ×100% CV: coefficient of variation of repeatability; : The average value of 10 measurement results; S: standard deviation of 10 measurement results.

[0030] 1.2. Acceptance criteria: Coefficient of variation of repeatability CV≤15%.

[0031] 1.3 Experimental Results Table 1 Coefficient of variation of five vitamin calibrators freeze-dried products

[0032] 1.4 Conclusion: The CV of homogeneity between bottles 1 to 6 of the five fat-soluble vitamin calibrators was ≤ 15%, which met the acceptance criteria.

[0033] 2. Real-time stability evaluation 2.1 Experimental Process The freeze-dried powder samples were stored in a refrigerator at 2-8°C, and samples were taken out at 0, 1, 3, 6, 12, and 13 months to test the accuracy of the calibrators.

[0034] 2.2 Acceptance Criteria Accuracy (%) = C test / C, accuracy is within the range of 85% to 115%; C test : measured value; C: Theoretical value.

[0035] 2.3 Experimental Results Table 2. Sample accuracy of real-time stability samples in month 0

[0036] Table 3 Real-time stability sample accuracy for the first month

[0037] Table 4. Accuracy of real-time stability samples in the third month

[0038] Table 5. Accuracy of real-time stability samples in the sixth month

[0039] Table 6 Real-time stability sample accuracy for the 12th month

[0040] Table 7 Real-time stability sample accuracy for the 13th month

[0041] 2.4 Experimental Summary When the freeze-dried product is stored at 2-8°C for up to 13 months, the accuracy of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1 is within the range of 85% to 115%, meeting the 12-month storage requirement.

[0042] 3. Redissolution stability 3.1 Experimental process After reconstitution, the lyophilized product was stored in a -20°C refrigerator to simulate the stability of the calibrator during use; samples were taken out at 0 days, 7 days, 14 days, 28 days, 30 days, and 31 days to determine the accuracy of the calibrator.

[0043] 3.2 Acceptance Criteria Accuracy (%) = Ctest / C, the accuracy is in the range of 85%~115%; Ctest: measured value; C: Theoretical value.

[0044] 3.3 Experimental Results Table 8 Accuracy of reconstituted stability samples on day 0

[0045] Table 9 Accuracy of reconstituted stability samples on day 7

[0046] Table 10 Accuracy of samples on day 14 of reconstitution stability

[0047] Table 11 Accuracy of reconstituted stability samples on day 28

[0048] Table 12 Accuracy of reconstituted stability samples on day 30

[0049] Table 13 Accuracy of reconstituted stability samples on day 31

[0050] 3.4 Experimental Summary When the freeze-dried product is opened and stored at -20°C for 31 days, the accuracy of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E, and vitamin K1 is within the range of 85% to 115%, meeting the storage requirement of 1 month.

[0051] 4. Desorption effect The adsorption effect of glass bottles on fat-soluble vitamins was evaluated by multiple tube transfer experiments in glass bottles.

[0052] Evaluation criteria, with an adsorption rate of 15% as the acceptable standard.

[0053] Table 14

[0054] Conclusion: After 5 adsorption experiments, vitamin K1 was not significantly adsorbed.

[0055] 5. Uniformity Ten vials of freeze-dried samples were reconstituted and assayed by liquid chromatography-tandem mass spectrometry. Each vial was assayed once, and the CV of the 10 measurements was calculated to determine the inter-vial precision. The acceptance standard was CV ≤ 15%.

[0056] CV=SD / Ave.

[0057] CV---coefficient of variation; SD---test standard deviation; Ave---test mean.

[0058] Table 15

[0059] Example 2 This example provides a lyophilized preparation of a fat-soluble vitamin calibrator. The preparation method thereof differs from that of Example 1 only in that: in the fat-soluble vitamin calibrator, the mass ratio of Tween 80 to sodium lauryl sulfate is 1:2, and the concentration of the isopropanol aqueous solution is 65%.

[0060] The uniformity of the calibration product was determined using the same test method as in Example 1, step 5. The test results are as follows: Table 16

[0061] The precision CV is ≤15%, which meets the experimental requirements.

[0062] Example 3 This embodiment provides a lyophilized preparation of a fat-soluble vitamin calibrant. The preparation method thereof differs from that of Example 1 only in that the concentration of the isopropanol aqueous solution in the fat-soluble vitamin calibrant is 20%.

[0063] The following table shows the homogeneity test results of calibrators vitamin E and vitamin K1: Table 17

[0064] After reducing the isopropanol content, the less polar fat-soluble vitamin E and vitamin K1 were difficult to disperse evenly into the aqueous phase system, and the inter-bottle precision CV exceeded the acceptance standard by 15%.

[0065] Example 4 This embodiment provides a lyophilized preparation of a fat-soluble vitamin calibrator. The preparation method thereof differs from that of Example 1 only in that the mass ratio of Tween 80 to sodium lauryl sulfate in the fat-soluble vitamin calibrator is 5:1.

[0066] The corresponding values ​​of vitamin K1 are shown in the table below: Table 18

[0067] The above table shows that when the ratio of Tween 80 to sodium dodecyl sulfate is increased from 1:1 to 5:1 in the example, the mass spectrometry response of vitamin K1 is significantly inhibited, which will greatly reduce the detection sensitivity in actual tests.

[0068] Comparative Example 1 This comparative example provides a lyophilized preparation of a fat-soluble vitamin calibrator. The preparation method thereof differs from that of Example 1 only in that Tween 80 is replaced with an equal amount of sodium lauryl sulfate in the fat-soluble vitamin calibrator.

[0069] Homogeneity test results: Table 19 Coefficient of variation of five vitamin calibrants freeze-dried products

[0070] Experimental summary: Compared with Example 1, after Tween 80 was removed from the formula, the inter-bottle deviation of vitamin E and vitamin K1 increased significantly; this proved that Tween 80 and sodium dodecyl sulfate had a synergistic effect, increasing the solubility of vitamins in BSA and reducing the adsorption of samples by the inner wall of the glass bottle.

[0071] Comparative Example 2 This comparative example provides a lyophilized preparation of a fat-soluble vitamin calibrator. The preparation method thereof differs from that of Example 1 only in that isopropanol in the fat-soluble vitamin calibrator is replaced with an equal amount of methanol.

[0072] The following table shows the results of the homogeneity test of the calibrator vitamin E: Table 20

[0073] After isopropanol was replaced with methanol, the mixed solvent's ability to disperse fat-soluble vitamins was weakened due to the weaker solvent strength of methanol. Vitamin K1, which is highly hydrophobic and has the highest concentration, was difficult to disperse evenly into the aqueous phase system, and the inter-bottle precision CV exceeded the acceptance standard by 15%.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A fat-soluble vitamin calibrator, characterized in that: including fat-soluble vitamins, excipients, stabilizers, dispersants and diluents; Wherein, the dispersant includes Tween 80 and sodium lauryl sulfate; and the diluent is a mixed solution of isopropyl alcohol and water.

2. The fat-soluble vitamin calibrator according to claim 1, characterized in that The diluent is isopropyl alcohol and water in a mass ratio of (1-2): (1-2).

3. The fat-soluble vitamin calibrator according to claim 1 or 2, characterized in that The dispersant is Tween 80 and sodium dodecyl sulfate in a mass ratio of (1-2): (1-2).

4. The fat-soluble vitamin calibrator according to any one of claims 1 to 3, characterized in that The excipient includes bovine serum albumin; And / or, the stabilizer comprises one or more of tea polyphenols, chlorogenic acid and vitamin C; preferably, the stabilizer comprises tea polyphenols, chlorogenic acid and vitamin C in a mass ratio of 1:(1-3):(4-6); And / or, the fat-soluble vitamins include one or more of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1.

5. The method for preparing the fat-soluble vitamin calibrator according to any one of claims 1 to 4, characterized in that: include: S1: Dissolve the excipient in PBS and then mix with the stabilizer to obtain diluent A; S2: mixing the dispersant and the diluent to obtain a diluent B; S3: Dissolve the fat-soluble vitamins in diluent B respectively, and then mix with the diluent A to obtain fat-soluble vitamin calibrators.

6. The method for preparing a fat-soluble vitamin calibrator according to claim 5, wherein: In S1, the order of adding stabilizers is: first add tea polyphenols, then add vitamin C, and finally add chlorogenic acid.

7. A lyophilized preparation of a fat-soluble vitamin calibrator, characterized in that: include: Fat-soluble vitamins, excipients, stabilizers and dispersants; wherein the dispersants include Tween 80 and sodium lauryl sulfate; Preferably, the dispersant is Tween 80 and sodium dodecyl sulfate in a mass ratio of (1-2): (1-2); and / or, the excipient comprises bovine serum albumin; And / or, the stabilizer comprises one or more of tea polyphenols, chlorogenic acid and vitamin C; preferably, the stabilizer comprises tea polyphenols, chlorogenic acid and vitamin C in a mass ratio of 1:(1-3):(4-6); And / or, the fat-soluble vitamins include one or more of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin E and vitamin K1.

8. The method for preparing the lyophilized preparation of the fat-soluble vitamin calibrator according to claim 7, characterized in that: include: freeze-drying the fat-soluble vitamin calibrator according to any one of claims 1 to 4; The freeze-drying comprises: S41: Pre-freeze: keep warm at -60~-40℃ for 120~180min; S42: Sublimation drying: Control the air entrainment vacuum degree to 0.05~0.15mbar, the sublimation temperature to -35~-25℃, and the sublimation time to 15~30 hours; S43: Drying: drying temperature 20-30°C, drying time 6-12 hours.

9. The method for preparing the lyophilized preparation of fat-soluble vitamin calibrator according to claim 8, characterized in that: include: S1: bovine serum albumin is dissolved in PBS to obtain a 3-7% BSA PBS solution, and then tea polyphenols, vitamin C and chlorogenic acid are added in sequence to obtain a diluent A; the stabilizer is tea polyphenols, chlorogenic acid and vitamin C in a mass ratio of 1: (1-3): (4-6); S2: mixing a dispersant and a diluent to obtain a dilution solution B; the dispersant is Tween 80 and sodium lauryl sulfate in a mass ratio of (1-2): (1-2); the diluent is isopropyl alcohol and water in a mass ratio of (1-2): (1-2); S3: dissolving the fat-soluble vitamins in diluent B respectively, and then mixing with the diluent A to obtain fat-soluble vitamin calibrators; S4: freeze-drying the fat-soluble vitamin calibrator; the freeze-drying comprises: S41: Pre-freeze: keep warm at -60~-40℃ for 120~180min; S42: Sublimation drying: Control the air entrainment vacuum degree to 0.05~0.15mbar, the sublimation temperature to -35~-25℃, and the sublimation time to 15~30 hours; S43: Drying: drying temperature 20-30°C, drying time 6-12 hours.

10. A fat-soluble vitamin kit, characterized in that: Containing the fat-soluble vitamin calibrator according to any one of claims 1 to 4, the fat-soluble vitamin calibrator prepared by the preparation method according to claim 5 or 6, the fat-soluble vitamin calibrator lyophilized preparation according to claim 7, or the fat-soluble vitamin calibrator lyophilized preparation prepared by the preparation method according to claim 8 or 9.

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