A preservation reagent, preservation method and detection method for fat-soluble vitamins D2, D3, A and E in peripheral blood

Abstract

The present invention discloses a preservation reagent, preservation method and detection method for fat-soluble vitamins D2, D3, A and E in peripheral blood. The preservation reagent includes the following components: 1-15% PBS buffer (v / v), 80%-95% sodium chloride solution (v / v), 0.5-2.5% Tris-HCl (v / v) and 0.5-2.5% stabilizer (w / v). The preservation reagent and preservation method of the present invention can stably preserve fat-soluble vitamins D2, D3, A and E in human whole blood samples under 2-8°C light-proof conditions for up to 28 days, which is convenient for the preservation and transportation of human whole blood samples and the detection of fat-soluble vitamins. It can improve the precision and accuracy of the detection, and can also avoid the interference caused by triglycerides and bilirubin on the detection of fat-soluble vitamins.

Description

Technical Field

[0001] The present invention relates to the technical field of storage and detection of fat-soluble vitamins D2, D3, A and E in blood, and in particular to a storage reagent, storage method and detection method of fat-soluble vitamins D2, D3, A and E in peripheral blood. Background Art

[0002] The existing samples for testing fat-soluble vitamins on the market are usually collected from veins and stored in blood collection tubes. The advantage of venous blood collection is that the results are accurate, but the disadvantage is that patients have to go to hospitals or professional medical institutions for blood collection, which takes up a certain amount of commuting time; secondly, hospitals and other institutions need professional blood collection personnel, and blood collection consumables are relatively expensive. In addition, infants and the elderly have their own vascular problems, sampling is more difficult, and venous blood collection patients are more painful and traumatic. Therefore, certain requirements are put forward for the training of medical professionals. Commuting time, consumables costs, sampling difficulties, and patient pain also impose some burdens on patients. Everyone is also looking forward to new and more convenient testing methods. Afterwards, peripheral blood sampling and blood collection tube storage methods came into being. Peripheral blood sampling greatly improves the convenience of blood collection for infants and the elderly. It belongs to non-venous blood collection, and the trauma to patients is also greatly reduced, but the commuting cost and consumables cost of going to the hospital have not improved. Later, the method of preserving samples by peripheral blood sampling dry blood spots was also invented. The preservation of dried blood spots only requires 1 to 3 drops of blood, and the amount of blood collected is greatly reduced compared to previous methods, which also further reduces the trauma to the patient during the sampling process.

[0003] As of December 2024, there is only one registered and marketed Class II dried blood spot 25-hydroxyvitamin D assay kit (liquid chromatography-tandem mass spectrometry), which is Hunan Haosi Biotechnology Co., Ltd. registered with Xiangji No. 20222400574, which can detect the content of 25-hydroxyvitamin D in human whole blood dried blood spots. Its kit uses protein precipitation method to process samples, which is simple to operate, but correspondingly, the preservation of dried blood spots also brings new problems. The preservation of blood spots requires a drying time of about 2 hours, so a large floor space is required for drying, which is easy to cause sample contamination due to improper preservation, the operation time is long, the punching operation is difficult, and it is easy to cause biological contamination to the operator. At the same time, for unstable compounds such as vitamins A and E, the preservation effect of dried blood spots is not good. In order to solve the pain points of the above products on the market, the present invention provides a mode of peripheral blood sampling and special preservation solution preservation. It not only maintains the advantages of convenient blood sampling and less trauma of dried blood spots, but also avoids the disadvantages of complex operation, easy pollution of the environment or easy contamination of samples. It also has the advantages of small sample requirements, rapid blood collection and good sample stability.

[0004] The preservation solution in the product of the present invention is used to preserve fat-soluble vitamins D2, D3, A, and E in whole blood, and can keep the concentration of the target compound in the whole blood unchanged. At present, the commonly used solutions for preserving blood on the market are ACD solution (citric acid, citrate and glucose) or CPD solution (phosphate is added to the ACD solution), which can effectively preserve blood for 28 days at 2 to 8°C, during which the red blood cells remain active, but this solution does not meet the requirements. Because clinical sample testing hopes that the content of various compounds in the sample will no longer change after blood collection, it is necessary to separate cells such as red blood cells as soon as possible after blood collection, because the metabolism of cells will cause the content of various compounds in the sample to change. The purpose of ACD solution or CPD solution is to provide sufficient energy and a suitable environment to ensure cell activity, which is inevitably accompanied by the metabolism of compounds. Therefore, the present invention abandons ACD solution or CPD solution and does not serve as a solution of the present invention. The present invention uses a preservation reagent prepared from 0.6%-0.9% sodium chloride solution and phosphate buffer solution, Tris-HCl and a stabilizer to preserve whole blood. On the one hand, the preservation solution can maintain the osmotic pressure of red blood cells in the whole blood, so that the red blood cell membrane is not damaged due to osmotic pressure imbalance, thereby releasing a large amount of hemoglobin and other substances to interfere with the determination of the target compound. On the other hand, it does not provide energy, reduces the metabolism of red blood cells, and can also solve the problem in the prior art that the test compound cannot be stably preserved for 28 days under the light-proof condition of 2-8°C in the whole blood sample. Summary of the invention

[0005] The present invention aims to provide a novel preservation reagent, preservation method and detection method for preserving fat-soluble vitamins D2, D3, A and E in peripheral blood. The preservation reagent and the preservation method can stably preserve the fat-soluble vitamins D2, D3, A and E in human whole blood samples under light-proof conditions at 2-8°C, and the preservation time is up to 28 days, which is convenient for the preservation and transportation of human whole blood samples.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a reagent for preserving fat-soluble vitamins D2, D3, A, and E in peripheral blood, comprising the following components in percentages:

[0008] PBS buffer 1-15% (v / v)

[0009] 80-95% (v / v) sodium chloride solution with a mass volume fraction of 0.6%-0.9%

[0010] Tris-HCl 0.5-2.5% (v / v)

[0011] Stabilizer 0.5-2.5% (w / v).

[0012] As some specific embodiments of the present invention, the stabilizer includes a combination of one or more of ascorbic acid, isoascorbic acid, tea polyphenols, chlorogenic acid, 2,6-di-tert-butyl-p-cresol, and dithiothreitol.

[0013] As some specific embodiments of the present invention, the stabilizer is a combination of ascorbic acid, isoascorbic acid, tea polyphenols and chlorogenic acid.

[0014] As some specific embodiments of the present invention, the preservation reagent includes the following stabilizers in the following mass volume fractions (w / v): ascorbic acid 0.15-0.5%, isoascorbic acid 0.15-0.5%, tea polyphenols 0.1-0.5%, and chlorogenic acid 0.1-0.5%.

[0015] As some specific embodiments of the present invention, the preservation reagent includes the following stabilizers in the following mass volume fractions (w / v): ascorbic acid 0.35%, isoascorbic acid 0.35%, tea polyphenols 0.20%, and chlorogenic acid 0.10%.

[0016] In a second aspect, the present invention provides a method for preserving fat-soluble vitamins D2, D3, A, and E in peripheral blood, using any of the above-mentioned preservation reagents, comprising the following steps:

[0017] S1. Add peripheral blood to the preservation reagent and mix well;

[0018] S2. After sealing, store stably at 2~8℃ in the dark.

[0019] As some specific embodiments of the present invention, in step S1, the ratio of the sampling amount of the peripheral blood to the sampling amount of the preservation reagent is 1:2-1:20.

[0020] As some specific embodiments of the present invention, in step S1, the sampling volume of the peripheral blood is 10-50 μL, and / or the sampling volume of the preservation reagent is 100-200 μL.

[0021] In a third aspect, the present invention provides a method for detecting fat-soluble vitamins in peripheral blood, wherein the peripheral blood is stored in any one of the storage reagents described above, and the fat-soluble vitamins include vitamin D2 (VD2), vitamin D3 (VD3), vitamin A (VA), and / or vitamin E (VE), and specifically comprises the following steps:

[0022] The internal standard solution is added to the preservation solution of the calibrator, quality control product and the sample to be tested, wherein the preservation solution contains the preservation reagent, and after mixing, a precipitant is added for precipitation, centrifugation, nitrogen blowing, derivatization, termination and then centrifugation again, and the supernatant is taken for liquid chromatography tandem mass spectrometry detection.

[0023] As some specific embodiments of the present invention, the precipitant is acetonitrile.

[0024] As some specific embodiments of the present invention, the internal standard solution is obtained by preparing the isotope internal standard VD2-d3 of vitamin D2, the isotope internal standard VD3-d6 of vitamin D3, the isotope internal standard VA-d4 of vitamin A, and / or the isotope internal standard VE-d6 of vitamin E using a methanol solution containing 2,6-di-tert-butyl-p-cresol.

[0025] As some specific embodiments of the present invention, the mass volume content of 2,6-di-tert-butyl-p-cresol in the methanol solution is 0.1-0.5%.

[0026] As some specific embodiments of the present invention, the volume ratio of the internal standard solution to the preservation solution containing the preservation reagent and the sample to be tested is 1:0.4-1:4.

[0027] As some specific embodiments of the present invention, the derivatization liquid used for the derivatization is PTAD (4-phenyl-3H-1,2,4-triazine-3,5-dione) acetonitrile solution.

[0028] As some specific embodiments of the present invention, the derivatization temperature is 40-70°C; the rotation speed is 300-1000 rpm; and the time is 10-40 min.

[0029] As some specific embodiments of the present invention, the concentration of the PTAD acetonitrile solution is 0.1-0.8 mg / mL.

[0030] As some specific embodiments of the present invention, the stopping solution used for the stopping is a formic acid methanol aqueous solution;

[0031] And / or, the termination rotation speed is 300-1000 rpm; the time is 5-20 min.

[0032] As some specific embodiments of the present invention, in the formic acid-methanol aqueous solution, the volume content of formic acid is 0.05-0.2%, and the volume content of methanol is 40-60%.

[0033] As some specific embodiments of the present invention, the nitrogen blowing time is 30-60 min.

[0034] As some specific embodiments of the present invention, the centrifugal speed is 10000-14000 rpm, the time is 5-20 min, and the temperature is 0-8°C;

[0035] And / or, the re-centrifugation has a rotation speed of 3000-5000 rpm, a time of 4-8 min, and a temperature of 0-8°C.

[0036] As some specific embodiments of the present invention, the detection method specifically comprises the following steps:

[0037] (1) Sample pretreatment

[0038] a. Sample addition: Take 20 μL of calibration and quality control products, add 100 μL of sodium chloride solution, take out the collector containing 120 μL of the sample to be tested, add 30 μL of internal standard solution, and shake at 2000 rpm for 1 min; add 300 μL of acetonitrile, and shake at 2000 rpm for 10 min;

[0039] b. Centrifugation: Centrifuge at 4°C, 12,000 rpm for 10 min;

[0040] c. Nitrogen blowing: Take 200 μL of the supernatant into a 96-well plate and blow with nitrogen for 30 min until the solvent evaporates completely;

[0041] d. Derivatization: Add 50 μL of derivatization solution, seal the membrane, and derivatize at 50°C, 500 rpm for 30 min;

[0042] e. Stop: Add 100 μL of stop solution, seal the membrane and shake at 500 rpm for 10 min at room temperature;

[0043] f. Detection: After centrifugation at 4°C, 4000 rpm for 5 min, the sample was injected into the instrument for liquid chromatography-tandem mass spectrometry detection.

[0044] * For testing VA and VE, skip steps c, d, and e and directly test the supernatant after centrifugation.

[0045] (2) On-machine testing

[0046] Mass spectrometry analysis was performed using an ultra-high performance liquid chromatography tandem mass spectrometry system (ACQUITY UPLC I-Class IVD / Xevo TQ-S IVDSystem mass spectrometer) in positive ion mode.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] 1) By adopting a preservation method containing a solution of a preservation reagent formula, the problem of large blood collection volume and great trauma to patients in the process of blood collection and sample preservation by other methods is solved; at the same time, the problem that vitamins A and E in peripheral blood cannot be stably preserved when peripheral blood samples are preserved in the form of dried blood spots, and the sample needs about 2 hours to dry, requiring a large area for drying, and is prone to sample contamination due to improper preservation is solved.

[0049] 2) Sample testing does not require drilling and can be directly pre-processed, which shortens the operation time and reduces the difficulty of operation. It is also less likely to cause biological contamination to operators, and the precision of the test results is significantly improved.

[0050] 3) The preservation reagent and preservation method of the present invention are used to preserve peripheral blood samples, which can ensure that the samples are stably stored at 2-8°C for 28 days, have good preservation effects on vitamins D2, D3, A, and E, and have good preservation stability.

[0051] 4) The preservation reagent is used for the preservation of vitamins D2, D3, A, and E in peripheral blood, which can improve the precision and accuracy of detection and avoid the interference of triglycerides and bilirubin on the detection of fat-soluble vitamins. DETAILED DESCRIPTION

[0052] The embodiments of the present invention are described in detail below. The embodiments are for better illustrating the content of the present invention and are only used to explain the present invention, but cannot be understood as limiting the present invention. It should be pointed out that the present invention can also make several variations and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0053] Example

[0054] 1. Storage of reagents

[0055] A reagent for preserving fat-soluble vitamins D2, D3, A, and E in peripheral blood, the formula of which is shown in Table 1 below:

[0056] Table 1 Preservative reagent formula

[0057]

[0058] *Here is mass volume fraction (w / v).

[0059] 2. Saving method

[0060] The fat-soluble vitamins in peripheral blood are preserved using the prepared preservation reagent, specifically including:

[0061] Take 100 μL of the preservation reagent of the above formula and put it into the selected plastic tube, take 20 μL of peripheral blood whole blood sample and put it into the plastic tube, mix well, and store at 2~8℃ in the dark.

[0062] 3. Detection method

[0063] 3.1 Preparation of reagents

[0064] (1) Preparation of standard products:

[0065] The standards of vitamin D2 (VD2), vitamin D3 (VD3), vitamin A (VA), and vitamin E (VE) were diluted with a methanol solution containing 0.3% 2,6-di-tert-butyl-p-cresol to the concentrations shown in Table 2:

[0066] Table 2 Calibrators of different concentrations

[0067]

[0068] (2) Preparation of pretreatment reagents

[0069] Internal standard solution: VD2-d3, VD3-d6, VA-d4, and VE-d6 were diluted with 0.3% 2,6-di-tert-butyl-p-cresol methanol solution to prepare the internal standard solutions. The initial concentrations of the internal standards were 10 ng / mL for VD2-d3, 30 ng / mL for VD3-d6, 600 ng / mL for VA-d4, and 5000 ng / mL for VE-d6.

[0070] Derivatization solution: 0.5 mg / mL PTAD acetonitrile solution;

[0071] Stop solution: 0.1% formic acid in 50% methanol solution.

[0072] 3.2 Collecting samples to be tested

[0073] Use a disposable distal blood collection device, remove the protective cap, press on the selected finger or other position, and the blood beads will flow out. Put the capillary glass tube of the disposable micro-sampling pipette in contact with the blood beads. After the capillary glass tube is filled with whole blood, insert the capillary glass tube of the disposable micro-blood collection pipette below the liquid level in the disposable sample collector, press the air bag of the disposable micro-blood collection pipette to discharge the whole blood into the disposable sample collector, press repeatedly three times to ensure that all samples are discharged. Close the cap of the disposable quantitative storage tube.

[0074] 3.3 Detection steps

[0075] (1) Sample pretreatment

[0076] a. Sample addition: Take 20 μL of calibration product and quality control product, add 100 μL of preservation reagent, take out the collector containing 20 μL of peripheral blood and 100 μL of preservation reagent, add 30 μL of internal standard solution, and shake at 2000 rpm for 1 min; add 300 μL of acetonitrile, and shake at 2000 rpm for 10 min;

[0077] b. Centrifugation: Centrifuge at 4°C, 12,000 rpm for 10 min;

[0078] c. Nitrogen blowing: Take 200 μL of the supernatant into a 96-well plate and blow with nitrogen for 30 minutes until the solvent evaporates completely;

[0079] d. Derivatization: Add 50 μL of derivatization solution, seal the membrane, and derivatize at 50°C, 500 rpm for 30 min;

[0080] e. Stop: Add 100 μL of stop solution, seal the membrane, and shake at 500 rpm for 10 min at room temperature;

[0081] f. Detection: After centrifugation at 4°C, 4000 rpm for 5 min, the sample was injected into the instrument for liquid chromatography-tandem mass spectrometry detection.

[0082] * For testing VA and VE, skip steps c, d, and e and directly test the supernatant after centrifugation.

[0083] (2) On-machine testing

[0084] Mass spectrometry analysis was performed using an ultra-high performance liquid chromatography tandem mass spectrometry system (ACQUITY UPLC I-Class IVD / Xevo TQ-S IVDSystem mass spectrometer) in positive ion mode.

[0085] Chromatographic conditions:

[0086] Chromatographic column: Waters BEH C18 1.7μm 2.1×50mm Column;

[0087] Mobile phase A: 0.1% FA-1 mM CH3NH2-H2O;

[0088] Mobile phase B: 0.1% FA-5 mM CH3NH2-ACN.

[0089] Gradient elution conditions:

[0090] Flow rate: 0.4 mL / min; column temperature: 45°C; automatic sampler: 10°C; injection volume: 5 μL.

[0091] Table 3 Chromatographic gradient of compounds

[0092]

[0093] Mass spectrometry conditions:

[0094] The ESI source conditions are as follows: Capillary (kV): 2.00; Source Offset (V): 50.0; Source Temperature (℃): 150; Desolvation Temperature (℃): 500; Cone Gas Flow (L / Hr): 150; Desolvation Gas Flow (L / Hr): 1000; Collision Gas Flow (mL / Min): 0.15; Nebuliser Gas Flow (Bar): 7.00. The MRM mode was used to detect the ion pairs to be measured. The information of the ion pairs to be measured is shown in Table 4 below.

[0095] Table 4 Mass spectrometry parameters of compounds

[0096]

[0097] 3.4 Calculation of test results

[0098] In the samples of the calibrator series, a linear regression was performed with the ratio of the calibrator concentration to its corresponding internal standard concentration (x) as the horizontal axis and the ratio of the calibrator peak area to its corresponding internal standard peak area (y) as the vertical axis, with a weight of 1 / x. 2 , and obtain the calibration curve equation. Substitute the ratio of the peak area of ​​each compound in the sample to be tested to the peak area of ​​its internal standard into the fitted calibration curve equation to calculate the concentration of fat-soluble vitamins D2, D3, A, and E in the sample to be tested.

[0099] Table 5 Results of LC-MC / MS method for detecting the concentrations of fat-soluble vitamins D2, D3, A, and E in peripheral blood samples

[0100]

[0101] 4. Result identification

[0102] 4.1 Summary of stability and precision data

[0103] The precision and stability validation data of peripheral blood samples preserved in the preservation solution were analyzed at 2-8°C. The results are as follows:

[0104] Table 6 Coefficient of variation of sample precision of Example 1

[0105]

[0106] Table 7 Bias between sample detection concentration and detection concentration on day 0 in the short-term stability trend of samples in Example 1

[0107]

[0108] It can be clearly seen from Tables 6 and 7 that the stability and precision results of fat-soluble vitamins D2, D3, A, and E in the samples preserved using the preservation reagent of the embodiment are all less than 15% within 31 days, which can meet the clinical detection requirements. At the same time, it avoids the cost problem of blood collection tube preservation and the instability problem of dried blood spot preservation, simplifies the operation method, and facilitates detection; and can maintain good precision and stability within the 31-day storage time.

[0109] 4.2 Accuracy test

[0110] The accuracy (spike recovery) of fat-soluble vitamins D2, D3, A, and E in peripheral blood samples preserved in the preservation solution was investigated. The high-concentration sample A was added to the low-concentration basic whole blood B. The volume of the added sample A did not exceed 10% of the total volume of the mixed sample (A+B). The test was repeated 3 times and the average value was calculated. The accuracy (spike recovery) of the peripheral blood sample should be between 85% and 115%. Calculation formula:

[0111]

[0112] Where: R = recovery rate

[0113] V = volume of sample A

[0114] V0 = volume of sample B

[0115] C = average concentration of sample B after adding sample A

[0116] C0 = average detected concentration of sample B

[0117] Cs = theoretical concentration of sample A

[0118] The results are shown in Table 8.

[0119] Table 8 Accuracy of LC-MS / MS method for detecting fat-soluble vitamins D2, D3, A, and E

[0120]

[0121] It can be clearly seen from Table 8 that for the samples preserved using the preservation reagent of the embodiment, there is no significant difference between the added concentrations of fat-soluble vitamins D2, D3, A, and E and the detected recovery concentrations, and the accuracy (spike recovery) results are all between 85% and 115%, which can meet clinical testing needs.

[0122] 4.3 Interference Investigation

[0123] The interference of fat-soluble vitamins D2, D3, A, and E in peripheral blood samples preserved in the preservation solution was investigated. Standards were added to 6 mixed clinical samples from different sources as basic samples, and then higher concentrations of potential interfering substances (triglycerides and bilirubin) were added as experimental samples. An equal amount of blank solvent was added as a control group sample. The content of the target in the experimental samples and the control samples was detected. The deviation between the target value of the experimental sample containing the interfering substance and the control sample without the interfering substance was acceptable within ±15%. The interfering substances and their concentrations are shown in Table 9, and the interference results are shown in Tables 10 and 11.

[0124] Table 9 Types and concentrations of interferents

[0125]

[0126] Table 10 Effect of triglycerides on target compounds

[0127]

[0128] Table 11 Effect of bilirubin on target compounds

[0129]

[0130] It can be seen from Tables 10 and 11 that the deviation between the target substance measurement values ​​of the experimental samples containing interfering substances and the control samples without interfering substances in the experimental group is within ±15%, which can meet the clinical detection requirements.

[0131] Comparative Example

[0132] A method for preserving fat-soluble vitamins D2, D3, A and E in peripheral blood, wherein the preserving method is dried blood spot preservation.

[0133] 1. Storage reagent: Whatman standard blood collection card.

[0134] 2. Storage method: Take 1-3 drops of whole blood and drop them into the center of the circle marked on the blood collection card. The whole blood will spread naturally. Place it in an open area, spread the blood collection card, dry it naturally for 2 hours, and store it at 2-8℃ away from light.

[0135] 3. Detection method:

[0136] (1) Sample pretreatment

[0137] a. Sample preparation: Take out the dried blood spot and the hole puncher, select a location with uniform color and no contamination to punch a 4 mm hole, take out the blood spot, and put it into an EP tube or a 96-well plate;

[0138] b. Sample addition: Take the prepared dried blood spot calibrators, quality control products, and dried blood spot samples, add 100 μL of water, add 30 μL of internal standard solution, and shake at 2000 rpm for 10 min; add 300 μL of acetonitrile, shake at 2000 rpm for 10 min;

[0139] c. Centrifugation: Centrifuge at 4°C, 12,000 rpm for 10 min;

[0140] d. Nitrogen blowing: Take 200 μL of the supernatant into a 96-well plate and blow with nitrogen for 30-60 min until the solvent evaporates completely;

[0141] e. Derivatization: Add 50 μL of derivatization solution, seal the membrane, and derivatize at 50°C and 500 rpm for 30 min;

[0142] f. Stop: Add 100 μL of stop solution, seal the membrane, and shake at 500 rpm for 10 min at room temperature;

[0143] g. Detection: After centrifugation at 4°C and 4000 rpm for 5 min, the sample was injected into the instrument for liquid chromatography-tandem mass spectrometry detection.

[0144] * Steps c, d, and e can be skipped for detecting VA and VE alone. Other reagents used are the same as those in the example.

[0145] (2) On-machine testing

[0146] The machine test was carried out using the same method and conditions as in the embodiment.

[0147] 4. Result identification

[0148] (1) Summary of stability and precision data

[0149] The precision verification and stability verification data analysis of the whole blood samples of the comparative proportion were carried out under the condition of 2~8℃, and the results are as follows:

[0150] Table 12 Coefficient of variation of sample precision of comparative examples

[0151]

[0152] Table 13 Bias between sample detection concentration and detection concentration on day 0 in the short-term stability trend of samples in the comparative example

[0153]

[0154] *The fat-soluble vitamins A and E in the comparative samples were degraded too quickly due to the low detection peak areas, and it was speculated that the coefficient of variation and stability results could not be calculated.

[0155] It can be clearly seen from Tables 12 and 13 that when the samples are stored at 2-8°C using the dried blood spot method, the stability and precision of fat-soluble vitamins A and E cannot be guaranteed.

[0156] Obviously, the above embodiments are examples for clearly explaining the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which does not affect the essence 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 reagent for preserving fat-soluble vitamins D2, D3, A, and E in peripheral blood, characterized in that: The following percentages are included: PBS buffer 1-15% 80-95% sodium chloride solution with a mass volume fraction of 0.6%-0.9% Tris-HCl 0.5-2.5% Stabilizer 0.5-2.5%; The amounts of the PBS buffer, sodium chloride solution, and Tris-HCl are expressed as volume fractions, and the amount of the stabilizer is expressed as mass volume fraction; The stabilizer includes one or more combinations of ascorbic acid, isoascorbic acid, tea polyphenols, chlorogenic acid, 2,6-di-tert-butyl-p-cresol, and dithiothreitol.

2. A method for preserving fat-soluble vitamins D2, D3, A, and E in peripheral blood, characterized in that: Using the preservation reagent as claimed in claim 1, the method comprises the following steps: S1. Add peripheral blood to the preservation reagent and mix well; S2. After sealing, store stably at 2~8℃ in the dark.

3. The storage method according to claim 2, characterized in that: In step S1, the volume ratio of the peripheral blood to the preservation reagent is 1:2-1:

20.

4. A method for detecting fat-soluble vitamins in peripheral blood, characterized in that: The peripheral blood is stored in the storage reagent as claimed in claim 1, and the fat-soluble vitamins include vitamin D2, vitamin D3, vitamin A, and vitamin E, specifically comprising the following steps: Add internal standard solution to the preservation solution of calibration products, quality control products and samples to be tested, wherein the preservation solution contains preservation reagent, mix well, add precipitant to precipitate, centrifuge, nitrogen blow, derivatize, terminate and centrifuge again, and take the supernatant for liquid chromatography tandem mass spectrometry detection; The derivatization liquid used in the derivatization is PTAD acetonitrile solution; The chromatographic conditions of the liquid chromatography tandem mass spectrometry detection include: Chromatographic column: Waters BEH C18 1.7 μm 2.1 × 50 mm Column; Mobile phase A: 0.1% FA-1 mM CH3NH2-H2O; Mobile phase B: 0.1% FA-5 mM CH3NH2-ACN; Gradient elution conditions: Flow rate: 0.4 mL / min; column temperature: 45 °C; autosampler: 10 °C; injection volume: 5 μL; Chromatographic gradient: ； The mass spectrometry conditions of the liquid chromatography tandem mass spectrometry detection include: The ESI source conditions were as follows: Capillary: 2.00 kV; Source Offset: 50.0 V; Source Temperature: 150 ℃; Desolvation Temperature: 500 ℃; Cone Gas Flow: 150 L / Hr; Desolvation Gas Flow: 1000 L / Hr; Collision Gas Flow: 0.15 mL / Min; Nebuliser Gas Flow: 7.00 Bar; and the MRM mode was used to detect the ion pairs to be measured.

5. The detection method according to claim 4, characterized in that: The internal standard solution is prepared by using a methanol solution containing 2,6-di-tert-butyl-p-cresol to prepare isotope internal standard VD2-d3 of vitamin D2, isotope internal standard VD3-d6 of vitamin D3, isotope internal standard VA-d4 of vitamin A, and isotope internal standard VE-d6 of vitamin E.

6. The detection method according to claim 4, characterized in that: The concentration of the PTAD acetonitrile solution is 0.1-0.8 mg / mL.

7. The detection method according to claim 4, characterized in that: The termination solution used for the termination is a formic acid-methanol aqueous solution; in the formic acid-methanol aqueous solution, the volume content of formic acid is 0.05-0.2%, and the volume content of methanol is 40-60%.

8. The detection method according to claim 4, characterized in that: The derivatization temperature is 40-70°C, the rotation speed is 300-1000 rpm, and the time is 10-40 min; The termination speed is 300-1000 rpm and the time is 5-20 min.

9. The detection method according to claim 4, characterized in that The centrifugal speed is 10000-14000 rpm, the time is 5-20 min, and the temperature is 0-8°C; The re-centrifugation has a rotation speed of 3000-5000 rpm, a time of 4-8 min, and a temperature of 0-8°C.

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