Protective agent for fat-soluble vitamins and carotenoids in serum and application of protective agent
By using a combination of tea polyphenols, ascorbic acid and L-ascorbyl palmitate antioxidants in serum samples, the stability problem of fat-soluble vitamins and carotenoids in serum was solved, and long-term preservation and efficient extraction were achieved.
Patent Information
- Application Number
- CN202510739921.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-04
- Publication Date
- 2025-09-16
AI Technical Summary
In the prior art, fat-soluble vitamins and carotenoids in serum have poor stability and are easily oxidized, which affects the preservation effect of the sample.
A specific ratio of water-soluble and oil-soluble antioxidants, including tea polyphenols, ascorbic acid and L-ascorbyl palmitate, is used to protect fat-soluble vitamins and carotenoids in serum samples, and the extraction effect and efficiency are improved by optimizing the pretreatment process.
The stability of fat-soluble vitamins and carotenoids in serum samples was significantly improved, ensuring long-term storage while improving the extraction effect and detection response value.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of protective agents, in particular to protective agents for fat-soluble vitamins and carotenoids in serum and applications thereof. Background Art
[0002] Fat-soluble nutrients in serum primarily include fat-soluble vitamins and carotenoids. Fat-soluble vitamins primarily include vitamins A, D, E, and K, while carotenoids primarily include α-carotene, β-carotene, and lycopene. Fat-soluble vitamins all contain ring structures and long aliphatic hydrocarbon chains. They are insoluble in water or glycerol but readily soluble in anhydrous ethanol, methanol, chloroform, ether, and oil. Vitamin A is susceptible to oxidation, and ultraviolet light accelerates its oxidative damage. Vitamins A and E are common antioxidants that prevent oxidative damage to easily oxidized substances such as unsaturated fatty acids within cells, thereby protecting cell membranes. Vitamin D, a sterol derivative, has anti-rickets effects and is also known as the anti-rickets vitamin. Vitamin K is a general term for a class of menadione derivatives that have physiological functions, including promoting normal blood coagulation. Carotenoids are lipophilic isoprenoid plant pigments found in red, yellow, orange, and dark green fruits and vegetables. Of the more than 750 carotenoids found in nature, approximately 15 are found in human serum. Among the higher concentrations in serum are lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, and lycopene. Carotenoids are effective antioxidants due to the unsaturated double bonds in their chemical structure.
[0003] A β-carotene molecule is essentially two retinol molecules linked at the tails. Through central or eccentric cleavage, it can be converted into two or one vitamin A molecules. β-carotene is further divided into all-trans and cis isomers. Central cleavage of all-trans β-carotene produces two molecules of all-trans retinol (vitamin A), while the yield of cis β-carotene converted to vitamin A is lower.
[0004] α-carotene and β-carotene have similar molecular structures and are isomers. The difference lies in the alteration of the 5', 6' double bond in the β-ionone ring at one end, which is essential for vitamin A activity. Therefore, the yield of vitamin A converted from α-carotene is only half that of β-carotene. Aside from its vitamin A activity, the properties and effects of α-carotene are similar to those of β-carotene.
[0005] Lycopene, α-carotene, and β-carotene have the same molecular weight and are isomers. Lycopene lacks the β-ionone ring structure of β-carotene and, therefore, cannot be converted into vitamin A in the body and is not considered provitamin A. Lycopene molecules contain 11 conjugated double bonds and 2 unconjugated double bonds, making them very unstable and prone to cis-trans isomerization and oxidative degradation. Factors that affect lycopene's stability include oxygen, light, heat, acid, metal ions, oxidants, and antioxidants. Fat-soluble vitamins and carotenoids are sensitive to light and heat, easily oxidized, and exhibit poor stability in blood samples.
[0006] Although some antioxidants targeting fat-soluble vitamins and carotenoids have been disclosed in the prior art, their effects still need further improvement. How to increase the stability of fat-soluble vitamins and carotenoids in serum samples remains a difficult problem in this field.
[0007] In view of this, the present invention is proposed. Summary of the Invention
[0008] In order to solve the above problems in the prior art, the present invention provides a protective agent for fat-soluble vitamins and carotenoids in serum and its application, which is used to protect the stability of fat-soluble vitamins and carotenoids in serum samples, and the serum samples can be placed in sample tubes for long-term storage.
[0009] Based on this, the present invention has the following technical solutions: In a first aspect, the present invention provides a protective agent for fat-soluble vitamins and carotenoids in serum, comprising a water-soluble antioxidant and an oil-soluble antioxidant in a mass ratio of (0.2-4):(0.1-5); the water-soluble antioxidant is a mixture of tea polyphenols and ascorbic acid in a mass ratio of (0.1-2):(0.1-2); and the oil-soluble antioxidant is L-ascorbyl palmitate; The fat-soluble vitamins include vitamin A (VA), 25-hydroxyvitamin D2 (25-OH-VD2), 25-hydroxyvitamin D3 (25-OH-VD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25-OH-VD3), vitamin E (VE), vitamin K1 (VK1) and vitamin K2 (MK4); the carotenoids include α-carotene (α-carotene), β-carotene (β-carotene) and lycopene (Lycopene).
[0010] The present invention finds that, among numerous antioxidants, by optimizing the specific types and dosage relationships of water-soluble antioxidants and oil-soluble antioxidants, the synergistic effect of these three antioxidants can be effectively exerted, effectively improving their antioxidant activity against fat-soluble vitamins and carotenoids in serum samples.
[0011] Preferably, the mass ratio of the water-soluble antioxidant to the oil-soluble antioxidant is (3-4):(2-3).
[0012] Preferably, the fat-soluble vitamins and carotenoid protective agents in the serum include the following components in parts by mass: 1~2 parts of tea polyphenols; 1-2 parts of ascorbic acid; 1-5 parts of L-ascorbyl palmitate.
[0013] Preferably, in the serum sample, the amount of the fat-soluble vitamins and carotenoid protective agent is 3-9 mg / mL.
[0014] Preferably, the fat-soluble vitamins and carotenoid protectants in the serum are composed of the following components: 1~2 mg / mL tea polyphenols; 1-2 mg / mL ascorbic acid; 1~5 mg / mL ascorbyl palmitate.
[0015] In a second aspect, the present invention provides an application of the fat-soluble vitamin and carotenoid protective agent in serum in detecting fat-soluble vitamins and / or carotenoids in serum samples, comprising pre-treating the serum sample containing the protective agent, wherein the pre-treatment method comprises: mixing the serum sample containing the protective agent with a protein precipitant and vortexing the sample, and then mixing the serum sample with an extractant for liquid-liquid extraction, centrifuging the supernatant, and re-dissolving the sample by nitrogen blowing.
[0016] The present invention found that the serum sample after adding the protective agent had poor extraction effect and efficiency in the subsequent liquid-liquid extraction. That is, although the addition of the protective agent can significantly improve the stability of fat-soluble vitamins and carotenoids, it also affects the extraction effect of the serum sample.
[0017] After extensive research, the present invention has found that performing the above-mentioned pretreatment on the serum sample after adding the protective agent can improve its extraction effect and extraction efficiency, and affect the detection response value of the target object to be detected.
[0018] Preferably, the protein precipitant is a mixture of methanol, acetonitrile and isopropanol, and the volume ratio of methanol, acetonitrile and isopropanol is (5-10): (1-3): (0.5-2).
[0019] Preferably, the volume of the protein precipitant is more than twice the volume of the serum sample containing the protective agent; more preferably, the volume ratio of the serum sample containing the protective agent to the protein precipitant is 1:(2-5).
[0020] Preferably, the protein precipitant, the extractant, and the reconstitution solution are frozen before use to eliminate interference from dissolved oxygen. More preferably, they are placed at -25 to -10°C for more than 30 minutes.
[0021] Preferably, the extractant is n-hexane containing an antioxidant.
[0022] In the present invention, the antioxidant can be any one or a combination of several conventional antioxidants containing phenolic hydroxyl groups in their chemical structure, for example, it can be a water-soluble antioxidant, such as any one or a combination of several of tea polyphenols, chlorogenic acid, rosmarinic acid, 3-hydroxytyrosol, β-cyclodextrin and ascorbic acid; it can also be an oil-soluble antioxidant, such as any one or a combination of at least two of butylated hydroxyanisole, L-ascorbyl palmitate, 2,6-di-tert-butyl-4-methylphenol (hereinafter also referred to as "BHT") and 2,5-di-tert-butylhydroquinone, or it can be the protective agent described in the present invention, and its selection is not specifically limited here.
[0023] Preferably, in g / L, the mass volume ratio of the antioxidant to the extractant is (0.3-0.6):1.
[0024] Preferably, the pretreatment method comprises: mixing the serum sample containing the protective agent with a protein precipitant and vortexing the mixture, then mixing the mixture with n-hexane containing an antioxidant to perform liquid-liquid extraction more than once, centrifuging and taking the supernatant for nitrogen blowing and re-dissolving; wherein the protein precipitant is a mixture of methanol, acetonitrile and isopropanol in a mass ratio of (5-10): (1-3): (0.5-2).
[0025] In the present invention, a mixed internal standard is added to a protein precipitant for the above-mentioned pretreatment. The pretreatment method comprises: mixing the serum sample containing a protective agent with the protein precipitant containing the mixed internal standard and vortexing the mixture, and then mixing the mixture with n-hexane containing an antioxidant for one or more liquid-liquid extractions, centrifuging and collecting the supernatant for nitrogen re-dissolution; wherein the protein precipitant is a mixture of methanol, acetonitrile and isopropanol in a mass ratio of (5-10): (1-3): (0.5-2).
[0026] Based on this, the technical solution of the present invention has the following beneficial effects: The fat-soluble vitamin and carotenoid protective agent in serum provided by the present invention and its application can effectively improve the stability of fat-soluble vitamins and carotenoids in serum samples, thereby placing the serum samples in sample tubes for long-term storage. DETAILED DESCRIPTION
[0027] 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.
[0028] 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.
[0029] In the following examples, the protein precipitant containing the internal standard, n-hexane containing BHT, and anhydrous ethanol used in the pretreatment process were all placed at -20°C for more than 30 minutes before use.
[0030] Example 1 This example provides a fat-soluble vitamin and carotenoid protective agent in serum, which consists of 1.5 mg / mL tea polyphenols, 2 mg / mL ascorbic acid, and 3 mg / mL ascorbyl palmitate.
[0031] The specific preparation method includes: 1. Experimental Procedure: Mixed human samples were divided into several 4 mL aliquots and placed in centrifuge tubes. High and low point spikes were added to the samples to achieve the following target concentrations: Vitamin A (retinol): 100 ng / mL and 500 ng / mL; 25-hydroxyvitamin D2: 10 ng / mL and 50 ng / mL; 25-hydroxyvitamin D3: 10 ng / mL and 50 ng / mL; 3-epi-25-hydroxyvitamin D3: 10 ng / mL and 50 ng / mL; Vitamin E (α-tocopherol): 1 μg / mL and 5 μg / mL; Vitamin K1: 1 ng / mL and 5 ng / mL; Vitamin K2 (MK4): 0.5 ng / mL and 2.5 ng / mL; α-carotene: 50 ng / mL and 250 ng / mL; β-carotene: 100 ng / mL and 500 ng / mL; Lycopene: 100 ng / mL and 500 ng / mL.
[0032] 2. Add a protective agent to obtain a serum sample containing the protective agent. Finally, the serum sample containing the protective agent contains 1.5 mg / mL tea polyphenols, 2 mg / mL ascorbic acid, and 3 mg / mL ascorbyl palmitate.
[0033] This embodiment further provides the use of the above-mentioned protective agent in detecting fat-soluble vitamins and carotenoids in serum samples, including pre-treating the serum sample containing the above-mentioned protective agent: The specific pretreatment process is as follows: methanol, acetonitrile and isopropanol in a mass ratio of 6.5:3:0.5 are mixed to obtain a protein precipitant, and 117.5 μL of mixed internal standards is added to 200 mL of protein precipitant, of which the volumes of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, vitamin E, vitamin K1 and vitamin K2 (MK4), α-carotene, β-carotene and lycopene isotope internal standards added are 10 μL, 20 μL, 20 μL, 20 μL, 12.5 μL, 10 μL, 10 μL and 15 μL respectively, to obtain a protein precipitant containing isotope internal standards.
[0034] Add the above-mentioned frozen protein precipitant containing isotope internal standard to 100 μL of serum containing the above-mentioned protective agent, vortex at 2000 rpm for 5 minutes, add 800 μL of frozen 0.4 g / L BHT and n-hexane (the volume ratio of BHT and n-hexane is 0.4:1), vortex at 2000 rpm for 10 minutes, and centrifuge to obtain the supernatant; add 800 μL of frozen 0.4 g / L BHT and n-hexane (the volume ratio of BHT and n-hexane is 0.4:1) to the precipitate, vortex at 2000 rpm for 10 minutes, centrifuge to obtain the supernatant, combine the supernatants, blow dry with nitrogen, and add 100 μL of frozen ethanol to reconstitute.
[0035] Example 2 This example provides a protective agent for fat-soluble vitamins and carotenoids in serum. This differs from Example 1 in that the serum sample containing the protective agent contains 2 mg / mL tea polyphenols, 2 mg / mL ascorbic acid, and 3 mg / mL ascorbyl palmitate. Results demonstrate comparable efficacy to that of Example 1.
[0036] Example 3 This example provides the use of the protective agent described in Example 1 in detecting fat-soluble vitamins and carotenoids in serum samples. This example differs from Example 1 in that the protein precipitant is replaced with an equal amount of acetonitrile during the pretreatment. The results indicate that the target detection signal is not as high as in Example 1, indicating a relatively poor response.
[0037] Example 4 This example provides the use of the protective agent of Example 1 in detecting fat-soluble vitamins and carotenoids in serum samples. This example differs from Example 1 in that the volume of the protein precipitant used in the pretreatment is 150 μL. Results demonstrate that the protein precipitation effect is slightly inferior to that of Examples 1 and 2, resulting in a relatively poor response to the target substance detected.
[0038] Example 5 This example provides the use of the protective agent of Example 1 in detecting fat-soluble vitamins and carotenoids in serum samples. This example differs from Example 1 in that the protein precipitant and extractant are not frozen and are used directly. The results show that the stability of vitamins A, D, and E, as well as carotenoids, is slightly less than that of Examples 1 and 2.
[0039] Comparative Example 1 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 3.5 mg / mL tea polyphenols and 3 mg / mL ascorbyl palmitate.
[0040] Comparative Example 2 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 3.5 mg / mL chlorogenic acid and 3 mg / mL ascorbyl palmitate.
[0041] Comparative Example 3 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 3.5 mg / mL ascorbic acid and 3 mg / mL ascorbyl palmitate.
[0042] Comparative Example 4 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 3.5 mg / mL rosmarinic acid and 3 mg / mL 2,6-di-tert-butyl-4-methylphenol.
[0043] Comparative Example 5 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 3.5 mg / mL hydroxytyrosol and 3 mg / mL 2,6-di-tert-butyl-4-methylphenol.
[0044] Comparative Example 6 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 3.5 mg / mL β-cyclodextrin and 3 mg / mL butylated hydroxyanisole.
[0045] Comparative Example 7 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 1.5 mg / mL tea polyphenols, 2 mg / mL ascorbic acid, and 3 mg / mL 2,6-di-tert-butyl-4-methylphenol.
[0046] Comparative Example 8 This comparative example provides a protective agent for fat-soluble vitamins and carotenoids in serum, which differs from Example 1 in that the serum sample containing the protective agent contains 1.5 mg / mL tea polyphenols, 2 mg / mL ascorbic acid, and 3 mg / mL chlorogenic acid.
[0047] Comparative Example 9 This comparative example provides the use of the protective agent of Example 1 in detecting fat-soluble vitamins and carotenoids in serum samples. The difference from Example 1 is that no frozen protein precipitant is added in the pretreatment, and liquid-liquid extraction is performed directly.
[0048] Test example The stability of serum samples in different examples and comparative examples was investigated when stored at room temperature for different time periods and at 2-8°C for different time periods. The samples at each time point were measured three times, and the results were compared with those at day 0 to calculate the deviation.
[0049] Fat-soluble vitamins and carotenoids using AB SCIEX Triple Quad TM 4500MD test.
[0050] Test conditions: The chromatographic conditions for the HPLC-MS / MS detection are shown in Table 1: Table 1
[0051] The mass spectrometry conditions for the HPLC-MS / MS detection are shown in Table 2: Table 2
[0052] Test results: Table 3 Vitamin A degradation rate of serum samples in Examples and Comparative Examples stored at room temperature and 2-8°C
[0053] Table 4 Degradation rate of 25-hydroxyvitamin D2 in serum samples of Examples and Comparative Examples at room temperature and 2-8°C
[0054] Table 5 Degradation rate of 25-hydroxyvitamin D3 in serum samples of Examples and Comparative Examples at room temperature and 2-8°C
[0055] Table 6 Degradation rate of 3-epi-25-hydroxyvitamin D3 in serum samples of Examples and Comparative Examples at room temperature and 2-8°C
[0056] Table 7 Vitamin E degradation rate of serum samples in Examples and Comparative Examples stored at room temperature and 2-8°C
[0057] Table 8 Vitamin K1 degradation rate of serum samples in Examples and Comparative Examples stored at room temperature and 2-8°C
[0058] Table 9 Vitamin K2 (MK4) degradation rate of serum samples in Examples and Comparative Examples stored at room temperature and 2-8°C
[0059] Table 10 α-carotene degradation rate of serum samples in Examples and Comparative Examples stored at room temperature and 2-8°C
[0060] Table 11 β-carotene degradation rate of serum samples in Examples and Comparative Examples stored at room temperature and 2-8°C
[0061] Table 12 Lycopene degradation rate of serum samples in Examples and Comparative Examples stored at room temperature and 2-8°C
[0062] Table 13 Peak areas of 10 fat-soluble vitamins in actual serum samples from Examples and Comparative Examples under different pretreatment conditions
[0063] 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 protective agent for fat-soluble vitamins and carotenoids in serum, characterized in that: The invention comprises a water-soluble antioxidant and an oil-soluble antioxidant in a mass ratio of (0.2-4): (0.1-5); the water-soluble antioxidant is a mixture of tea polyphenols and ascorbic acid in a mass ratio of (0.1-2): (0.1-2); and the oil-soluble antioxidant is L-ascorbyl palmitate; The fat-soluble vitamins include vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, vitamin E, vitamin K1 and vitamin K2; and the carotenoids include α-carotene, β-carotene and lycopene.
2. The fat-soluble vitamin and carotenoid protective agent in serum according to claim 1, characterized in that The mass ratio of the water-soluble antioxidant to the oil-soluble antioxidant is (3-4): (2-3).
3. The protective agent for fat-soluble vitamins and carotenoids in serum according to claim 1 or 2, characterized in that The composition includes the following parts by mass: 1~2 parts of tea polyphenols; 1-2 parts ascorbic acid; 1-5 parts of L-ascorbyl palmitate.
4. The protective agent for fat-soluble vitamins and carotenoids in serum according to any one of claims 1 to 3, characterized in that In the serum sample, the amount of the fat-soluble vitamins and carotenoid protective agent is 3-9 mg / mL; Preferably, the fat-soluble vitamins and carotenoid protectants in the serum consist of the following concentrations: 1~2 mg / mL tea polyphenols; 1-2 mg / mL ascorbic acid; 1~5 mg / mL ascorbyl palmitate.
5. Use of the protective agent for fat-soluble vitamins and carotenoids in serum according to any one of claims 1 to 4 in detecting fat-soluble vitamins and / or carotenoids in serum samples, characterized in that: The serum sample containing the protective agent is pretreated, and the pretreatment method includes: mixing the serum sample containing the protective agent with a protein precipitant and vortexing, then mixing with an extractant for liquid-liquid extraction, centrifuging and taking the supernatant for nitrogen blowing and re-dissolving.
6. Use of the protective agent for fat-soluble vitamins and carotenoids in serum according to claim 5 in detecting fat-soluble vitamins and / or carotenoids in serum samples, characterized in that: The protein precipitant is a mixture of methanol, acetonitrile and isopropanol, and the volume ratio of the methanol, acetonitrile and isopropanol is (5-10): (1-3): (0.5-2).
7. Use of the protective agent for fat-soluble vitamins and carotenoids in serum according to claim 6 in detecting fat-soluble vitamins and / or carotenoids in serum samples, characterized in that: The volume of the protein precipitant is more than twice the volume of the serum sample containing the protective agent; preferably, the volume ratio of the serum sample containing the protective agent to the protein precipitant is 1:(2-5).
8. Use of the protective agent for fat-soluble vitamins and carotenoids in serum according to any one of claims 5 to 7 in detecting fat-soluble vitamins and / or carotenoids in serum samples, characterized in that: The protein precipitant, the extractant and the reconstitution solution are frozen before use; preferably, they are placed at -25 to -10°C for more than 30 minutes.
9. Use of the protective agent for fat-soluble vitamins and carotenoids in serum according to any one of claims 5 to 8 in detecting fat-soluble vitamins and / or carotenoids in serum samples, characterized in that: The extractant is n-hexane containing an antioxidant.
10. Use of the protective agent for fat-soluble vitamins and carotenoids in serum according to claim 9 in detecting fat-soluble vitamins and / or carotenoids in serum samples, characterized in that: In terms of g / L, the mass volume ratio of the antioxidant to the extractant is (0.3~0.6):1.