Extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography

Abstract

The application relates to an extraction and detection method for detecting antioxidant vitamins in blood plasma based on liquid chromatography. 300 uL of serum or blood plasma is taken and added into a centrifugal tube, and 300 uL of a first reagent is added to obtain a first sample liquid; 800 uL of a second reagent is added into the centrifugal tube, mixed and centrifuged to obtain 600 uL of supernatant; 600 uL of the second reagent is added into the centrifugal tube, mixed and centrifuged to obtain 600 uL of supernatant, and the first supernatant and the second supernatant are combined to obtain a second sample liquid; the second sample liquid is blown dry to obtain a solid, and a third reagent is added to obtain a to-be-detected sample; gradient elution is adopted, the to-be-detected sample is injected into an HPLC-UV detector. The application can be used for simultaneously detecting multiple indexes of antioxidant vitamins in blood plasma, i.e. vitamin A, alpha-vitamin E, beta-vitamin E, beta-carotene and coenzyme Q10, the antioxidant vitamins are reasonably separated and extracted, the extract is effectively separated by using a liquid chromatograph, and the content is accurately detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of vitamin clinical detection, in particular to an extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography. BACKGROUND

[0002] There is a set of efficient antioxidant defense system in human body, including antioxidant enzyme system (such as SOD, CAT, GSH-Px, GR which can be synthesized in vivo), and non-enzyme antioxidant defense system (including endogenous and exogenous), among which the endogenous antioxidant substances such as uric acid and glutathione are supplemented by taking various natural antioxidants from food to make up for the deficiency of antioxidant system, so as to enhance the antioxidant capacity of human body, thereby reducing and delaying the occurrence and development of diseases, and the antioxidant vitamins are the main components of exogenous antioxidants, such as vitamin A, vitamin E, beta-carotene and coenzyme Q10 which have antioxidant properties.

[0003] The advantages of various antioxidant substances mentioned above are summarized, among which vitamin A participates in the physiological functions of vision, immunity, hematopoiesis and other physiological functions, has the advantages of promoting growth and development, increasing resistance to infectious diseases, preventing and treating dry eye disease, etc., it is not an antioxidant, but it can regulate a series of genes through the function of its transcription regulator, thereby indirectly playing the role of antioxidant; beta-carotene is the precursor of vitamin A, but vitamin A precursor carotene is not vitamin A, and vitamin A precursor carotene is only a part of the large family of carotenoids, beta-carotene has direct antioxidant effect; vitamin E can maintain normal reproductive capacity and normal muscle metabolism, maintain the integrity of central nervous system and vascular system, has direct antioxidant effect, and is the main component of body lipid oxidation, vitamin E includes α, β, γ and δ four kinds, among which α-vitamin E has the highest content in human body and the highest physiological activity; finally, coenzyme Q10 has direct antioxidant effect and is also a substance for energy production (ATP) in mitochondria, and coenzyme Q10 and vitamin E have complete synergistic effect in body antioxidant.

[0004] Obviously, the antioxidant system of human body can be fully evaluated only by comprehensive detection of antioxidant vitamins.

[0005] However, antioxidant vitamins are easily oxidized, and most of them are fat-soluble, and the water phase commonly used in chromatographic separation will produce solvent effect, resulting in poor linear correlation of part of the target, affecting the accuracy of quantitative.

[0006] Furthermore, clinical testing demands high throughput, but recent clinical testing methods for antioxidant vitamins in the human body offer limited detection capabilities for these vitamins, failing to meet the high-throughput requirements of clinical practice. For example, Chinese Patent Publication No. CN 115524419 A discloses a "Method and Kit for Detecting Fat-Soluble Vitamins in Milk Samples," and Chinese Patent Publication No. CN 115236254 A discloses a "Detection Kit and Method for Vitamin A and Vitamin E," but these methods can only detect two vitamins, A and E. Similarly, Chinese Patent Publication No. CN110231425 A discloses a "Method for Extracting and Detecting Coenzyme Q10 in Blood," but this method can only detect one coenzyme Q10. It is certain that existing similar technologies are significantly insufficient in terms of detection range and are limited by the inherent limitations of their technical solutions.

[0007] For example, in current clinical testing, the simultaneous detection of multiple vitamins typically uses liquid chromatography-tandem mass spectrometry (LC-MS / MS). While this method offers high sensitivity, the expensive instruments and deuterated internal standards (i.e., the optimal internal standard for the internal standard method is a deuterated derivative with a structure very similar to the analyte) result in high testing costs and demanding operational requirements. Furthermore, most vitamin E tested in the market is α-vitamin E. Although α-vitamin E has the highest content and physiological activity, β-vitamin E and γ-vitamin E are also present in significant amounts in the body and still retain 10%-40% of the physiological activity of α-vitamin E. Clearly, existing techniques can only detect a limited range of vitamin E types, thus limiting the overall effectiveness of the detection methods.

[0008] This invention, based on existing metal rust-preventive grease formulation technology, aims to provide a method for the simultaneous detection of multiple indicators of antioxidant vitamins (including vitamin A, α-vitamin E, β-vitamin E, β-carotene, and coenzyme Q10) in plasma. This method effectively separates and extracts antioxidant vitamins from plasma, and the extracts are efficiently separated and accurately detected using liquid chromatography with ultraviolet detectors (including fixed wavelength and full wavelength). Summary of the Invention

[0009] To address the problems existing in existing detection methods, this invention provides an extraction and detection method for antioxidant vitamins in plasma based on liquid chromatography. This method rationally separates and extracts antioxidant vitamins from plasma, and the extracts are effectively separated and accurately detected using a liquid chromatograph with ultraviolet detectors (including fixed wavelength and full wavelength).

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] An extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography, which is used for separating and extracting antioxidant vitamins in plasma and accurately detecting multiple indicators at the same time, comprising the following steps:

[0012] Step one: first take 300uL serum or plasma into a centrifuge tube, and add 300uL of the first reagent and mix for 1min to obtain the first sample solution, the first reagent is an ethanol solution containing 2,6-di-tert-butyl-4-methyl phenol 30mg / mL and vitamin E acetate 30ug / mL;

[0013] Step two: add 800uL of the second reagent to the centrifuge tube and mix for 5min, and centrifuge to take 600uL of supernatant;

[0014] Step three: add 600uL of the second reagent to the centrifuge tube and mix for 5min, and centrifuge to take 600uL of supernatant and combine with the first supernatant to obtain the second sample solution;

[0015] Step four: dry the obtained second sample solution to obtain a solid, then add 100uL of the third reagent to obtain the sample to be tested;

[0016] Step five: use gradient elution or isocratic elution, and inject 0.1-15uL of the sample to be tested into HPLC-UV for detection;

[0017] Step six: according to the detection results of chromatographic analysis, vitamin A, alpha-vitamin E, beta-vitamin E, beta-carotene, coenzyme Q10 and the internal standard are completely separated and there is no interference, and the peak shape is symmetrical;

[0018] Step seven: finally, take six samples of two different plasma samples each, and perform the same sample preparation and detection, and the coefficient of variation CV of each index is less than 10%.

[0019] In order to promote the significant improvement of technical effect, the technical personnel can also use corresponding technical means to further implement the above technical solutions, including:

[0020] For step five, the detection conditions further include: a C18 chromatographic column or a F5 chromatographic column can be selected;

[0021] For step five, the detection conditions further include: the column temperature can be 25-50℃;

[0022] For step five: the detection conditions further include the selection of mobile phase, i.e. mobile phase A is an acetonitrile solution containing 2% isopropyl alcohol and 0.5% triethylamine, and mobile phase B is a water solution containing 2% isopropyl alcohol and 0.5% triethylamine.

[0023] For step one: the first reagent is composed of a first solvent and an antioxidant, wherein the first solvent is a solvent formed by one or any combination of methanol, acetonitrile, ethanol, n-propanol, isopropanol; wherein 2,6-di-tert-butyl-4-methylphenol is the antioxidant, and the concentration thereof is 1-100 mg / mL; in addition to vitamin E acetate, the optional internal standard can also be Q9 coenzyme 0.5-50 ug / mL or vitamin A acetate 1-50 ug / mL.

[0024] In combination with the application effect, on the basis of the same concept, the skilled person can also use actual corresponding technical means to form corresponding technical solutions, including:

[0025] For step two: the second reagent is composed of a second solvent, which is a solvent formed by one or any combination of n-hexane, ethyl acetate, chloroform, carbon tetrachloride, cyclohexane, chloromethane, dichloromethane.

[0026] For step two: the second reagent is added in one batch, mixed, and then centrifuged, or added in small amounts multiple times, mixed, and then centrifuged.

[0027] In addition, for step four: the third reagent is composed of a third solvent, which is a solvent formed by one or any combination of methanol, acetonitrile, ethanol, n-propanol, isopropanol.

[0028] The above extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography includes the following preparation steps when preparing the kit:

[0029] Accurately take 10 mg of vitamin A, completely dissolve in ethanol, and then add ethanol to make up to 10 mL to obtain a 1 mg / mL vitamin A stock solution;

[0030] Completely dissolve 100 mg of α-vitamin E in ethanol, add ethanol to make up to 100 mL to obtain a 10 mg / mL α-vitamin E stock solution;

[0031] Completely dissolve 20 mg of β-vitamin E in ethanol, add ethanol to make up to 10 mL to obtain a 2 mg / mL β-vitamin E stock solution;

[0032] Completely dissolve 20 mg of coenzyme Q10 in isopropanol, add isopropanol to make up to 10 mL to obtain a 2 mg / mL coenzyme Q10 stock solution;

[0033] Completely dissolve 5 mg of β-carotene in tetrahydrofuran, add tetrahydrofuran to make up to 10 mL to obtain a 0.5 mg / mL β-carotene stock solution.

[0034] All the above stock solutions are transferred to brown glass bottles and stored at -20°C.

[0035] The present application can be used for simultaneous detection of multiple indexes of antioxidant vitamins in plasma, including vitamin A, alpha-vitamin E, beta-vitamin E, beta-carotene and coenzyme Q10, etc. The present application first reasonably separates and extracts the antioxidant vitamins in plasma, and then effectively separates and accurately detects the content of the extract by using a liquid chromatograph with an ultraviolet detector (including fixed wavelength and full wavelength). BRIEF DESCRIPTION OF DRAWINGS

[0036] The present application will be further described in detail below with reference to the accompanying drawings.

[0037] Figure 1 is the extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application, and its test chromatogram is shown;

[0038] Figure 2 is the extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application, and its linear range analysis graph of vitamin A is shown;

[0039] Figure 3 is the extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application, and its linear range analysis graph of beta-vitamin E is shown;

[0040] Figure 4 is the extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application, and its linear range analysis graph of alpha-vitamin E is shown;

[0041] Figure 5 is the extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application, and its linear range analysis graph of beta-carotene is shown;

[0042] Figure 6 is the extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application, and its linear range analysis graph of coenzyme Q10 is shown. DETAILED DESCRIPTION

[0043] The extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application aims to solve the problems of single type, low precision and high cost in the prior art.

[0044] (I) The extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography implemented by the present application can take the following steps according to actual needs during extraction:

[0045] First step: take 300uL serum / plasma into centrifuge tube, and add 300uL first reagent, mix at 2500rpm for 1min to obtain first sample liquid;

[0046] The first reagent added is an ethanol solution containing 2,6-di-tert-butyl-4-methylphenol (BHT) 30mg / mL and vitamin E acetate 30ug / mL (i.e. internal standard);

[0047] Second step: then add 800uL second reagent (i.e. n-hexane) to the centrifuge tube based on the obtained first sample liquid, mix at 2500rpm for 5min, and centrifuge to obtain 600uL supernatant;

[0048] Third step: accordingly, add 600uL second reagent (n-hexane) to the centrifuge tube, mix at 2500rpm for 5min, and centrifuge to obtain 600uL supernatant, which is combined with the first supernatant to obtain second sample liquid;

[0049] Fourth step: the obtained second sample liquid is blown dry with nitrogen to obtain solid, and then 100uL third reagent (i.e. ethanol) is added to obtain the sample to be tested.

[0050] For the above extraction steps for detecting antioxidant vitamins in plasma based on liquid chromatography according to the present application, the technical solutions adopted by the skilled person can be refined and selected through the following specific technical means, in the first step:

[0051] The first reagent is composed of A solvent and antioxidant, wherein the A solvent is a solvent formed by one or any combination of methanol, acetonitrile, ethanol, n-propanol, and isopropanol;

[0052] The 2,6-di-tert-butyl-4-methylphenol (BHT) is an antioxidant, and its concentration is 1-100mg / mL;

[0053] The internal standard can be vitamin E acetate with a concentration of 5-100ug / mL, or coenzyme Q90.5-50ug / mL, or vitamin A acetate 1-50ug / mL.

[0054] The amount of the first reagent is 1-4 times the volume of the serum / plasma sample.

[0055] In the second step:

[0056] The second reagent is composed of C solvent, which can be a solvent formed by one or any combination of n-hexane, ethyl acetate, chloroform, carbon tetrachloride, cyclohexane, chloromethane, and dichloromethane;

[0057] The second reagent is 1-10 times the volume of the serum / plasma sample.

[0058] In addition, the second reagent can be added at one time, mixed, and then centrifuged, or added in small amounts multiple times, mixed, and then centrifuged.

[0059] In the fourth step:

[0060] The third reagent is composed of a D solvent, which can be a solvent formed by one or any combination of methanol, acetonitrile, ethanol, n-propanol, or isopropanol;

[0061] The third reagent is 0.1-1 times the volume of the plasma sample.

[0062] (II) In the detection of anti-oxidative vitamins in plasma based on the liquid chromatography method implemented by the present application, the technical solution adopted by the technical personnel can be refined and selected through the following specific technical means:

[0063] For the instruments and instrument conditions used, the instrument type can be preferably HPLC-UV; the chromatographic column can be preferably F5 chromatographic column;

[0064] For the mobile phase: mobile phase A is an acetonitrile solution containing 2% isopropanol + 0.5% triethylamine, and mobile phase B is a water solution containing 2% isopropanol + 0.5% triethylamine (v / v);

[0065] For the elution method: gradient elution; the specific time gradient and UV detection data are shown in the following table (Table 1):

[0066]

[0067] For the detection wavelength: 325 nm (vitamin A), 295 nm (α-vitamin E, β-vitamin E), 448 nm (β-carotene), and 275 nm (coenzyme Q10), 285 nm (internal standard, vitamin E acetate).

[0068] Other preferred parameters include: the column temperature can be preferably 35℃; the flow rate is 1 mL / min; the sample injection volume is 12 uL, which is injected into the HPLC-UV for detection.

[0069] Further, the detection method for detecting anti-oxidative vitamins in plasma based on the liquid chromatography method implemented by the present application can be further implemented as:

[0070] Gradient elution or isocratic elution, C18 chromatographic column or F5 chromatographic column, column temperature 25-50℃, flow rate of mobile phase 0.5-2 mL / min, sample injection volume: 0.1-15 uL;

[0071] The mobile phase A phase is a methanol solution containing isopropanol with a volume percentage of ≥1%, triethylamine content of 0.1-1%, or an acetonitrile solution, or any combination solution of methanol and acetonitrile; the mobile phase B phase is a water solution containing isopropanol with a volume percentage of ≥1% and triethylamine content of 0.1-1%.

[0072] (Three) The technical personnel carried out corresponding demonstration on the detection method of detecting antioxidant vitamins in plasma based on liquid chromatography, so as to prove the superiority of the technical means by objective practice:

[0073] (1) Test chromatogram: as shown in Figure 1 , using the above test method, the reference substances vitamin A, α-vitamin E, β-vitamin E, β-carotene and coenzyme Q10 and the internal standard (vitamin E acetate) are completely separated, there is basically no interference, and the peak shape is symmetrical, which shows that good separation can be obtained under this condition, and the test result is accurate.

[0074] (2) The linear range of various vitamins is as shown in Figures 2-6 , the r value of this method is greater than 0.9900, which meets the requirements of clinical test; within the concentration range of the calibration sample, the linear correlation coefficient r of the fitting curve is greater than 0.9900, and the concentration range, the fitting curve and the linear correlation coefficient of the control vitamin are shown in the following table (Table 2):

[0075] (Table II) Concentration range Fitted curve Linear correlation coefficient r Vitamin A 50-5000 ng / mL y = 1308.19x - 72.8597 0.9997 β-Vitamin E 200-8000 ng / mL y = 19707.7x - 47.1394 0.9994 α-Vitamin E 500-50000 ng / mL y = 24773.6x - 82.7915 0.9999 β-Carotene 30-3000 ng / mL y = 837.625x - 7.41894 0.9934 Coenzyme Q10 200-10000 ng / mL y = 67485.7x - 100.189 0.9963

[0076] (3) Regarding the precision, two different plasma (low, high) samples, each 6, were taken, and the same test sample preparation and detection were carried out, and the results are shown in the following table (Table 3):

[0077]

[0078]

[0079] As can be seen from the table, the coefficient of variation CV of each index is less than 10%, and the precision of the detection method shows superiority.

[0080] The above has carried out corresponding demonstration to the extraction and detection method of the application based on liquid chromatography for detecting antioxidant vitamins in plasma, and on the same basis, it can also form associated, additional, and related kit preparation method technical means, thereby proving the superiority brought by the technical scheme with objective practice, such as providing a kit preparation method based on liquid chromatography for detecting antioxidant vitamins in plasma, taking the preparation of 1 mg / mL vitamin A stock solution, 10 mg / mL α-vitamin E stock solution, 2 mg / mL β-vitamin E stock solution, 2 mg / mL coenzyme Q10 stock solution, and 0.5 mg / mL β-carotene stock solution as an example.

[0081] First, accurately weigh 10 mg of vitamin A into a 10 mL volumetric flask, add a certain amount of ethanol, cover the stopper to prevent liquid leakage, invert up and down until the vitamin A is completely dissolved, add ethanol to constant volume to 10 mL, obtain 1 mg / mL vitamin A stock solution, transfer to a brown glass bottle, and store at -20℃;

[0082] Accurately weigh 100 mg of α-vitamin E into a 10 mL volumetric flask, add a certain amount of ethanol, cover the stopper to prevent liquid leakage, invert up and down until the α-vitamin E is completely dissolved, add ethanol to constant volume to 100 mL, obtain 10 mg / mL α-vitamin E stock solution, transfer to a brown glass bottle, and store at -20℃;

[0083] Accurately weigh 20 mg of β-vitamin E into a 10 mL volumetric flask, add a certain amount of ethanol, cover the stopper to prevent liquid leakage, invert up and down until the β-vitamin E is completely dissolved, add ethanol to constant volume to 10 mL, obtain 2 mg / mL β-vitamin E stock solution, transfer to a brown glass bottle, and store at -20℃;

[0084] Accurately weigh 20 mg of coenzyme Q10 into a 10 mL volumetric flask, add a certain amount of isopropyl alcohol, cover the stopper to prevent liquid leakage, invert up and down until the coenzyme Q10 is completely dissolved, add isopropyl alcohol to constant volume to 10 mL, obtain 2 mg / mL coenzyme Q10 stock solution, transfer to a brown glass bottle, and store at -20℃.

[0085] Accurately weigh 5 mg of β-carotene into a 10 mL volumetric flask, add a certain amount of tetrahydrofuran, cover the stopper to prevent liquid leakage, invert up and down until the β-carotene is completely dissolved, add tetrahydrofuran to constant volume to 10 mL, obtain 0.5 mg / mL β-carotene stock solution, transfer to a brown glass bottle, and store at -20℃.

[0086] For the preparation of the above calibrators (constant volume dilution method), taking the preparation of 10 mL calibrator as an example, first prepare the intermediate solution (Table Four) from the stock solutions:

[0087]

[0088] As shown in Table 4, the above-mentioned each stock solution is used as a sampling solution, and each intermediate solution is prepared by sampling under the conditions of a reasonable concentration, volume and solvent.

[0089] Accordingly, as shown in Table 5, the final volume and final concentration of each calibrator, low-value quality control and high-value quality control are obtained.

[0090]

[0091]

[0092]

[0093] In addition, since the amount of each raw material used in the raw material ratio is within a certain range, the formed technical solution can select different solutions according to the extraction requirements, the role played by different raw material ratios cannot be considered as completely the same effect, and the technical solution of the present application focuses on the subsequent steps formed after the extraction by using an orderly and reasonable method and then detected under specific conditions. The technical solution of the present application does not discuss the influence of the function of the equipment used. Therefore, the technical solution implemented by the present application is actually an extraction and detection method for detecting antioxidant vitamins in blood plasma based on liquid chromatography, which can be referred to and implemented by a person skilled in the art combined with conventional technical means. The technical personnel can obtain a series of advantages according to different application conditions and requirements.

[0094] In the description of the present specification, if the terms "embodiment one", "the present embodiment", "specific implementation" and the like are described, it means that the specific features, structures, materials or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present application or invention. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example; moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in an appropriate manner.

[0095] The above description of the embodiments is to facilitate the ordinary skilled in the art to understand and apply, those skilled in the art can learn from the content of this paper, and appropriately improve the process parameters to realize; It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. Therefore, the case is not limited to the above embodiments, and the following modifications should be within the scope of protection of the case: ① based on the technical scheme of the present application and combined with the known common knowledge, the new technical scheme is implemented, the technical effect produced by the new technical scheme does not exceed the technical effect of the present application, such as using centrifugation to obtain supernatant to obtain the measured material and then forming a new technical scheme by gradient elution step, and the expected effect does not exceed the present application; ② equivalent replacement of part of the features of the technical scheme of the present application by using known technology, the technical effect produced is the same as the technical effect of the present application, for example, equivalent replacement of reagents with the same effect; ③ expand the technical scheme based on the present application, the substantial content of the expanded technical scheme does not exceed the technical scheme of the present application; ④ equivalent transformation using the content recorded in the present application, and applying the obtained technical means to other related technical fields.

Claims

1. An extraction and detection method for detecting antioxidant vitamins in blood plasma based on liquid chromatography, which is used for separating and extracting antioxidant vitamins in blood plasma and accurately detecting a plurality of indexes at the same time, characterized in that, The method comprises the following steps: Step 1: 300 uL of serum or plasma is taken into a centrifuge tube, and 300 uL of a first reagent is added and mixed for 1 min to obtain a first sample solution, wherein the first reagent is an ethanol solution containing 2,6-di-tert-butyl-4-methylphenol 30 mg / mL and vitamin E acetate 30 ug / mL; Step 2: 800 uL of a second reagent is added into the centrifuge tube and mixed for 5 min, and 600 uL of supernatant is taken after centrifugation; Step 3: 600 uL of the second reagent is added into the centrifuge tube and mixed for 5 min, and 600 uL of supernatant is taken after centrifugation to obtain a second sample solution combined with the first supernatant; Step 4: The obtained second sample solution is blown dry with nitrogen to obtain a solid, and 100 uL of a third reagent is added to finally obtain a sample to be tested; Step 5: The sample to be tested is injected into HPLC-UV for detection in a gradient elution or isocratic elution mode, with a sample injection volume of 0.1-15 uL; Step 6: According to the detection results of chromatographic analysis, vitamin A, α-vitamin E, β-vitamin E, β-carotene, coenzyme Q10 and the internal standard are completely separated and there is no interference, and the peak shape is symmetrical; Step 7: Finally, six samples of two different plasma samples are taken, and the same sample preparation and detection are performed, and it is found that the coefficient of variation CV of each index is less than 10%.

2. The extraction and detection method for detecting antioxidant vitamins in blood plasma based on liquid chromatography according to claim 1, characterized by, For step 5, the detection conditions further include: selecting a C18 chromatographic column or a F5 chromatographic column.

3. The extraction and detection method for detecting antioxidant vitamins in blood plasma based on liquid chromatography according to claim 2, characterized by, For step 5, the detection conditions further include: the column temperature is 25-50℃.

4. The extraction and detection method for detecting the antioxidant vitamins in plasma based on liquid chromatography according to any one of claims 1-3, characterized in that, For step 5: the detection conditions further include the selection of the mobile phase, i.e. the mobile phase A is an acetonitrile solution containing 2% isopropyl alcohol and 0.5% triethylamine, and the mobile phase B is a water solution containing 2% isopropyl alcohol and 0.5% triethylamine.

5. The extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography according to claim 4, characterized by, For step 1: the first reagent is composed of a first solvent and an antioxidant, wherein the first solvent is a solvent formed by one or any combination of methanol, acetonitrile, ethanol, n-propanol, isopropyl alcohol.

6. The method for detecting the antioxidant vitamins in the plasma based on liquid chromatography according to claim 4, characterized in that: The 2,6-di-tert-butyl-4-methylphenol is an antioxidant, and the concentration is 1-100 mg / mL.

7. The method for the extraction and detection of antioxidant vitamins in plasma based on liquid chromatography according to claim 4, characterized by the fact that: In addition to vitamin E acetate, the optional internal standard can also be Q9 coenzyme 0.5-50 ug / mL or vitamin A acetate 1-50 ug / mL.

8. The extraction and detection method for detecting the antioxidant vitamins in the plasma based on liquid chromatography according to any one of claims 1-3, characterized in that, For step 2: the second reagent is composed of a second solvent, and the second solvent is a solvent formed by one or any combination of n-hexane, ethyl acetate, chloroform, carbon tetrachloride, cyclohexane, chloromethane, dichloromethane.

9. The extraction and detection method for detecting the antioxidant vitamins in the plasma based on liquid chromatography according to any one of claims 1-3, characterized in that, For step 4: the third reagent is composed of a third solvent, and the third solvent is a solvent formed by one or any combination of methanol, acetonitrile, ethanol, n-propanol, isopropyl alcohol.

10. The extraction and detection method for detecting antioxidant vitamins in plasma based on liquid chromatography according to claim 1, characterized by, When the reagent kit is prepared, the extraction and detection method further comprises the following preparation steps: 10 mg of vitamin A is completely dissolved in ethanol, and then ethanol is added to make up to 10 mL to obtain a 1 mg / mL vitamin A stock solution; 100 mg of α-vitamin E is completely dissolved in ethanol, and then ethanol is added to make up to 100 mL to obtain a 10 mg / mL α-vitamin E stock solution; 20 mg of β-vitamin E was completely dissolved in ethanol, and then ethanol was added to make up to 10 mL, to obtain a 2 mg / mL β-vitamin E stock solution; 20 mg of coenzyme Q10 was completely dissolved in isopropyl alcohol, and then isopropyl alcohol was added to make up to 10 mL, to obtain a 2 mg / mL coenzyme Q10 stock solution; 5 mg of β-carotene was completely dissolved in tetrahydrofuran, and then tetrahydrofuran was added to make up to 10 mL, to obtain a 0.5 mg / mL β-carotene stock solution.

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