Method for detecting the content of various vitamins in blood

Abstract

The application describes a method for detecting the content of multiple vitamins in blood, the multiple vitamins being vitamin A, vitamin D2, vitamin D3 and vitamin E, the detection method comprising: respectively providing a to-be-detected serum sample and a standard sample; adding an internal standard solution to the to-be-detected serum sample and the standard sample; adding a first precipitant, and mixing, wherein the first precipitant is a zinc sulfate solution with a concentration of 7.8 g / mL to 10 g / mL; adding a second precipitant, and mixing, wherein the second precipitant comprises acetonitrile and methanol; adding an extraction agent, and extracting; adding a re-dissolving solution, and re-dissolving; detecting the to-be-detected solution and the standard solution by using liquid chromatography tandem mass spectrometry; obtaining a standard curve according to the detection result of the standard solution, and obtaining the content of the multiple vitamins in the to-be-detected serum sample according to the detection result of the to-be-detected solution. According to the application, a method for detecting the content of multiple vitamins in blood with high accuracy can be provided.

Description

Technical Field

[0001] This invention generally relates to the field of chemical detection, and specifically to a method for detecting the content of multiple vitamins in blood. Background Technology

[0002] Vitamins can be divided into fat-soluble vitamins and water-soluble vitamins. Vitamins A, D, and E are common fat-soluble vitamins and are essential micronutrients for the human body, playing a regulatory role in metabolism. Vitamin A (also known as retinol) can cause birth defects in the fetus, such as cleft lip and palate, and congenital heart disease, if a pregnant woman is deficient or has an excess. Supplementing with vitamin A during pregnancy can increase hemoglobin levels and reduce the incidence of anemia. Vitamin D deficiency may lead to rickets, decreased bone density, fractures, and postpartum osteoporosis. The hydrolysis product of vitamin E is tocopherol, which can promote the secretion of sex hormones, improve fertility, and prevent miscarriage. Vitamin E deficiency may lead to placental aging and premature rupture of membranes. Therefore, detecting the levels of various vitamins in the human body has important clinical significance.

[0003] Currently, the detection of vitamin content in human blood is commonly performed using methods such as liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Existing methods typically involve shaking and mixing plasma and organic reagents during sample pretreatment to precipitate proteins, followed by extraction with an extractant, reconstitution with a reconstituted solution, and analysis using LC-MS / MS. However, the vigorous shaking and thorough mixing required during sample pretreatment and extraction to extract fat-soluble vitamins from the aqueous phase into the organic solvent can easily lead to emulsification. Simultaneously, the organic and aqueous solvents may undergo trace amounts of mutual dissolution, resulting in indistinct layering, difficulty in transferring the organic phase, and loss of the target vitamins. This leads to inter-experimental bias and affects the accuracy of the results. In particular, the levels of vitamins A and E in human blood are low, typically in the microgram range, while vitamin D levels are extremely low, in the nanogram range; therefore, vitamin loss should be minimized during detection.

[0004] Therefore, there is a need to provide a highly accurate method for detecting the content of multiple vitamins in human blood. Summary of the Invention

[0005] The present invention is proposed in view of the above-mentioned state of the prior art, and its purpose is to provide a method for detecting the content of multiple vitamins in blood with high accuracy.

[0006] Therefore, the present invention provides a method for detecting the content of multiple vitamins in blood, comprising providing a serum sample to be tested and a standard sample; adding an internal standard solution to the serum sample to be tested and the standard sample; adding a first precipitant and mixing, wherein the first precipitant is a zinc sulfate solution with a concentration of 7.8 g / mL to 10 g / mL; adding a second precipitant and mixing, wherein the second precipitant includes acetonitrile and methanol; adding an extractant and extracting; adding a reconstitution solution and reconstituteing to obtain a test solution and a standard solution; detecting the test solution and the standard solution using liquid chromatography-tandem mass spectrometry; obtaining a standard curve based on the detection results of the standard solution; and obtaining the content of the multiple vitamins in the serum sample to be tested based on the detection results of the test solution and the standard curve; wherein the multiple vitamins are vitamin A, vitamin D2, vitamin D3 and vitamin E, the standard sample is a solution of vitamin A, vitamin D2, vitamin D3 and vitamin E with a specific concentration, and the internal standard solution is a solution of vitamin A, vitamin D2, vitamin D3 and vitamin E with a specific concentration and labeled with isotopes.

[0007] In this invention, during sample pretreatment, a first precipitant (zinc sulfate solution with a concentration of 7.8 g / mL to 10 g / mL) is added. The high concentration of zinc sulfate solution can compete with proteins for water molecules, disrupt the water film on the surface of protein colloidal particles, and neutralize the charge on the protein particles, causing them to accumulate and precipitate in the water. This alters the solubility of proteins in the aqueous solution, promoting protein precipitation and separation, thus reducing the impact on subsequent detection. Furthermore, the high concentration of zinc sulfate solution can also change the polarity of the aqueous solvent, reducing the mutual dissolution or emulsification between organic solvents and aqueous solutions, which helps reduce vitamin loss during subsequent sample processing. Therefore, it can improve the accuracy of detecting vitamin A, vitamin D2, vitamin D3, and vitamin E in blood. The addition of a second precipitant (acetonitrile and methanol) increases the polarity of water, promoting phase separation and further promoting protein precipitation in the sample. In addition, adding the first precipitant (an aqueous solution of zinc sulfate) can dilute the sample, thereby reducing the concentration of proteins in the sample and reducing the interaction and interference between proteins. Adding the second precipitant can further promote the precipitation of proteins in the sample.

[0008] Optionally, in the method of this invention, the second precipitant further includes a stabilizer, wherein the stabilizer is butylated hydroxyanisole (BHA), which is dissolved in methanol at a mass fraction of 0.1%. In this case, since both vitamin A and vitamin E are easily oxidized in air, adding BHA to the precipitant helps maintain the stability of vitamin A and vitamin E during the precipitation step, reduces the loss of vitamin A and vitamin E, and thus improves the accuracy of detection.

[0009] In the method of this invention, optionally, the volume ratio of acetonitrile and methanol with a mass fraction of 0.1% butylated hydroxyanisole in the second precipitant is 1:1. This can help improve the accuracy of the detection results.

[0010] In the method of this invention, the extractant may optionally be n-hexane. This facilitates vitamin recovery and improves detection accuracy.

[0011] In the method of this invention, optionally, the reconstitution solution comprises water and methanol containing the stabilizer. In this case, adding the stabilizer during reconstitution helps maintain the stability of vitamin A and vitamin E in the reconstitution step, reduces the loss of vitamin A and vitamin E, and thus improves detection accuracy.

[0012] In the method of this invention, optionally, the volume ratio of water to methanol containing the stabilizer in the reconstituted solution is 3:7. This facilitates vitamin recovery and improves detection accuracy.

[0013] In the method of this invention, optionally, when performing liquid chromatography, a C18 column is used, the column temperature is 40°C, and the injection volume is 10 μL. In this case, using a C18 column allows for selective separation based on the polarity of the analyte molecules, is more resistant to aqueous phases, and thus extends the column's lifespan. Furthermore, by selecting appropriate column temperature and mobile phase, it is beneficial to obtain good separation results and improve detection accuracy.

[0014] In the method of this invention, optionally, during liquid chromatography, mobile phase A is a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B is a 0.1% (v / v) methanol solution of formic acid. In this case, by selecting a suitable composition of the mobile phase, it is beneficial to obtain good separation effect and improve detection accuracy.

[0015] In the method of this invention, optionally, gradient elution is used during liquid chromatography, and the elution procedure is as follows:

[0016]

[0017]

[0018] In the method of this invention, optionally, the mass-to-charge ratio of the parent ion and daughter ion used to detect the multiple vitamins and the multiple vitamins labeled with isotopes is:

[0019] Analytes Mother ion daughter ions Vitamin A 269.300 93.100 Vitamin E 431.300 165.300 Vitamin A with isotope labeling 275.200 171.300 Vitamin E with isotope labeling 437.300 171.300 Vitamin D2 413.300 355.300 Vitamin D3 401.300 365.300 Vitamin D3 with isotope labeling 404.300 386.300 Vitamin D2 with isotope labeling 419.300 401.300

[0020] According to the present invention, a method for detecting the content of multiple vitamins in blood with high accuracy can be provided. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating a method for detecting the content of multiple vitamins in blood according to an embodiment of the present invention.

[0022] Figure 2 This is a flowchart illustrating step S300 involved in an embodiment of the present invention.

[0023] Figure 3 This is a standard curve diagram of vitamin A according to Embodiment 1 of the present invention.

[0024] Figure 4 This is a standard curve diagram of vitamin E involved in Example 1 of the present invention.

[0025] Figure 5 This is a standard curve diagram of vitamin D2 according to Embodiment 1 of the present invention.

[0026] Figure 6 This is a standard curve diagram of vitamin D3 according to Embodiment 1 of the present invention.

[0027] Figure 7 This is a graph showing the detection results of vitamin A according to Example 2 of the present invention.

[0028] Figure 8 This is a graph showing the detection results of vitamin E according to Example 2 of the present invention.

[0029] Figure 9 This is a graph showing the detection results of vitamin D2 according to Example 2 of the present invention.

[0030] Figure 10 This is a graph showing the detection results of vitamin D3 according to Example 2 of the present invention. Detailed Implementation

[0031] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals are used for the same parts, and repeated descriptions are omitted. Furthermore, the drawings are merely schematic diagrams, and the proportions of the parts or the shapes of the parts may differ from the actual figures.

[0032] It should be noted that the terms "comprising" and "having" and any variations thereof in this invention, such as a process, method, system, product, or device that includes or has a series of steps or units, are not necessarily limited to those steps or units that are explicitly listed, but may include or have other steps or units that are not explicitly listed or that are inherent to such processes, methods, products, or devices.

[0033] Furthermore, the subheadings and similar terms used in the following description of this invention are not intended to limit the content or scope of this invention; they are merely for reading guidance. Such subheadings should not be construed as dividing the content of the article, nor should the content under a subheading be limited to the scope of that subheading.

[0034] The method for detecting the content of multiple vitamins in blood according to this invention is liquid chromatography-tandem mass spectrometry. The multiple vitamins are fat-soluble vitamins and may include vitamin A, vitamin D2, vitamin D3, and vitamin E. This method for detecting the content of multiple vitamins in blood can be simply referred to as the detection method. The detection method of this invention can be applied to the detection of vitamin content in human blood and has high accuracy.

[0035] The present invention will now be described in detail with reference to the accompanying drawings. Figure 1 This is a flowchart illustrating the method for detecting the content of multiple vitamins in blood according to this embodiment.

[0036] In some examples, such as Figure 1 As shown, a method for detecting the content of multiple vitamins in blood may include: providing a serum sample to be tested and a standard sample (step S100); adding an internal standard solution (step S200); performing sample pretreatment to obtain a test solution and a standard solution (step S300); detecting the test solution and the standard solution (step S400); and obtaining the content of multiple vitamins in the serum sample to be tested based on the detection results of the standard solution and the test solution (step S500).

[0037] In this embodiment, the method for detecting the content of multiple vitamins in blood may include providing a serum sample to be tested and a standard sample, respectively (step S100).

[0038] In some examples, in step S100, the serum sample to be tested can be obtained by drawing peripheral blood from the subject. In some examples, the serum sample can be prepared by: collecting blood using a blood collection tube without anticoagulant, allowing it to stand at 4°C for 2 hours (avoiding vibration to prevent hemolysis), centrifuging to collect the supernatant, which can be done at 4°C, at a speed of 3000g, for 20 minutes, and can be done twice. In this case, using a serum sample can reduce interference from blood cells, fibrinogen, etc.

[0039] In some examples, the standard sample can be a solution of multiple vitamins at specific concentrations. In other words, the standard sample contains specific concentrations of vitamin A, vitamin D2, vitamin D3, and vitamin E.

[0040] In some examples, multiple standard samples can be used, with the concentrations of various vitamins set in a gradient. This allows for the generation of different detection signals based on the different vitamin concentrations in the standard samples, thus enabling the creation of a standard curve.

[0041] In some examples, in the standard samples, vitamin A can be gradient-set from 0.1 μg / mL to 2 μg / mL, with a total of 4 to 8 gradients. Vitamin E in the standard samples can be gradient-set from 1 μg / mL to 100 μg / mL, with a total of 4 to 8 gradients. Vitamin D2 in multiple standard samples can be gradient-set from 1 ng / mL to 20 ng / mL, with a total of 4 to 8 gradients. Vitamin D3 in multiple standard samples can be gradient-set from 5 ng / mL to 100 ng / mL, with a total of 4 to 8 gradients. This provides a suitable linear range.

[0042] In this embodiment, the method for detecting the content of multiple vitamins in blood may include adding an internal standard solution (step S200).

[0043] In some examples, in step S200, internal standard solutions can be added to both the serum sample to be tested and the standard sample, respectively. In some examples, the same volume of internal standard solution can be added to both the serum sample to be tested and the standard sample. In some examples, the internal standard solution can be a solution of isotopically labeled vitamin A, vitamin D2, vitamin D3, and vitamin E with specific concentrations. For example, the internal standard solution can have specific concentrations of deuterated vitamin A, deuterated vitamin D2, deuterated vitamin D3, and deuterated vitamin E. Therefore, using multiple isotopically labeled vitamins as internal standards facilitates the quantitative detection of vitamin A, vitamin D2, vitamin D3, and vitamin E in the sample.

[0044] In some examples, the concentration of isotopically labeled vitamin A in the internal standard solution can be 20 to 25 μg / mL. The concentration of isotopically labeled vitamin D2 in the internal standard solution can be 0.5 to 2 μg / mL. The concentration of isotopically labeled vitamin D3 in the internal standard solution can be 0.5 to 2 μg / mL. The concentration of isotopically labeled vitamin E in the internal standard solution can be 60 to 65 μg / mL.

[0045] In some examples, the samples can be mixed after the internal standard solution is added, for example, by shaking.

[0046] In this embodiment, the method for detecting the content of multiple vitamins in blood may include sample pretreatment to obtain a test solution and a standard solution (step S300). Figure 2 This is a flowchart illustrating step S300 as described in an embodiment of the present invention. Figure 2 As shown, step S300 may include: adding a first precipitant (step S310), adding a second precipitant (step S320), adding an extractant (step S330), and adding a complex solution to obtain the test solution and the standard solution (step S340).

[0047] In some examples, in step S310, a first precipitant may be added to the serum sample to be tested containing the internal standard solution and the standard sample containing the internal standard solution, respectively.

[0048] In some examples, the first precipitant can be a high-concentration zinc sulfate solution. In some examples, the concentration of the zinc sulfate solution can be from 7.8 g / mL to 10 g / mL. For example, the concentration of the zinc sulfate solution can be 7.8 g / mL, 8 g / mL, 8.5 g / mL, 9 g / mL, 9.5 g / mL, or 10 g / mL. In this case, the zinc sulfate solution can compete with proteins for water molecules, disrupt the water film on the surface of protein colloidal particles, and neutralize the charge on the protein particles, thereby causing the protein particles to accumulate and precipitate in the water. This can alter the solubility of proteins in the sample in aqueous solution, promoting protein precipitation and separation. Additionally, it can alter the polarity of the aqueous solvent, reducing the mutual dissolution or emulsification between organic solvents and aqueous solutions, which helps reduce vitamin loss during subsequent sample processing. Therefore, it is beneficial for detecting vitamin A, vitamin D2, vitamin D3, and vitamin E in the blood.

[0049] In some examples, mixing can be performed after the first precipitant is added, for example, by shaking the sample. This facilitates the precipitation of proteins in the sample.

[0050] In some examples, in step S320, the second precipitant may include acetonitrile and methanol. This further promotes the precipitation of proteins in the sample.

[0051] In some examples, a second precipitant can be added after the first precipitant. In this case, the second precipitant increases the polarity of water, promoting phase separation and thus further promoting protein precipitation in the sample. Furthermore, adding the first precipitant dilutes the sample, reducing the protein concentration and minimizing protein interactions and interference; adding the second precipitant then further promotes protein precipitation.

[0052] In some examples, the second precipitant may also include a stabilizer. In some examples, the stabilizer may be an antioxidant. In some examples, the stabilizer may be butylated hydroxyanisole (BHA). The stabilizer may be soluble in methanol at a mass fraction of 0.1%. In other words, the second precipitant may include a methanol solution of acetonitrile and butylated hydroxyanisole at a mass fraction of 0.1%. Vitamins A and E are readily oxidized in air. In this case, adding BHA to the precipitant can help maintain the stability of vitamins A and E during the precipitation step, reduce the loss of vitamins A and E, and thus improve detection accuracy.

[0053] In some examples, the volume ratio of acetonitrile and methanol with a mass fraction of 0.1% butylated hydroxyanisole in the second precipitant can be 1:1. This can help improve the accuracy of the detection results.

[0054] In some examples, mixing can be performed after adding a second precipitant, for example, by shaking the sample. This facilitates the precipitation of proteins in the sample.

[0055] In some examples, the extractant in step S330 is n-hexane.

[0056] In some examples, mixing can be performed after adding the extraction solvent, for example, by shaking the sample. This facilitates the extraction of vitamins into the organic phase. In other examples, centrifugation can be performed after mixing with the extraction solvent. This helps reduce interference from proteins and other substances.

[0057] In some examples, the organic layer can be aspirated. The vitamin to be detected is mainly extracted into the organic layer, which can be aspirated while the other layers are discarded. In this case, the influence of other impurities on the detection can be reduced.

[0058] In some examples, the obtained organic layer can be dried with nitrogen. This helps to remove organic solvents as much as possible, reducing their impact on subsequent chromatographic detection.

[0059] In some examples, a reconstitution solution may be added in step S340 for reconstitution. In some examples, the reconstitution solution may be a solution compatible with the mobile phase system used for chromatographic detection. In some examples, the reconstitution solution may include water and methanol. In some examples, the reconstitution solution may also include a stabilizer. In this case, adding a stabilizer during reconstitution helps maintain the stability of vitamin A and vitamin E in the reconstitution step, reduces the loss of vitamin A and vitamin E, and thus improves detection accuracy.

[0060] In some examples, the reconstitution solution can consist of water and a methanol solution containing 0.1% butylated hydroxyanisole (BHA). In some examples, the volume ratio of water to the 0.1% BHA methanol solution can be 3:7. This facilitates vitamin recovery and improves detection accuracy.

[0061] In some examples, after the reconstitution operation, the test solution and the standard solution can be obtained separately.

[0062] In this embodiment, the method for detecting the content of multiple vitamins in blood may include detecting the test solution and the standard solution (step S400).

[0063] In some examples, in step S400, high performance liquid chromatography-tandem mass spectrometry can be used to detect the test solution and the standard solution.

[0064] In some examples, in step S400, the chromatographic parameters may include selecting an ODS column (C18 column) as the chromatographic column. In this case, using a C18 column allows for selective separation based on the polarity of the analyte molecules, and it is more resistant to aqueous phases, thus extending the column's lifespan. In other examples, other chromatographic columns with comparable detection performance to the C18 column may also be selected.

[0065] In some examples, chromatographic parameters may include selecting a column temperature of 40°C. Chromatographic parameters may include an injection volume of 10 μL. Chromatographic parameters may include a flow rate of 0.55 mL / min. Chromatographic parameters may include using a mixture of formic acid and water as mobile phase A, and a mixture of formic acid and methanol as mobile phase B. Preferably, the volume fraction of formic acid in mobile phase A is 0.1%, and the volume fraction of formic acid in mobile phase B is also 0.1%. In this case, by selecting appropriate column temperature, injection volume, flow rate, and mobile phase, it is beneficial to obtain good separation results and improve detection accuracy.

[0066] In some examples, gradient elution can be used when performing high-performance liquid chromatography, and the elution program can be:

[0067] Time (min) Mobile phase A% Mobile phase B% Flow rate (ml / min) 0 95 5 0.55 0.5 17 83 0.55 4 1 99 0.55 4.5 1 99 0.55 4.7 95 5 0.55 5.5 95 5 0.55

[0068] In some examples, during mass spectrometry, data can be acquired using an electrospray ionization (ESI) source and a positive ion mode multiple reaction monitoring (MRM) scan mode.

[0069] In some examples, the ion source voltage and gas flow parameters can be as follows:

[0070]

[0071] In some examples, the parent ion / daughter ion mass-to-charge ratio (m / z) of the vitamins used for detection and their internal standards can be shown in the table below:

[0072]

[0073] The present invention will be described in detail below through embodiments. However, the following embodiments are merely examples provided to illustrate the present invention and do not limit or restrict the scope of the invention disclosed in this application.

[0074] Example 1: Obtaining the standard curve

[0075] Preparation of standard samples: Prepare mixed solutions of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E standards. The four vitamins have six series concentrations (S1 to S6) in the standards, as shown in the table below:

[0076]

[0077] Prepare internal standard solutions with the concentrations shown in the table below.

[0078] Raw material name concentration VA-d6 20ug / mL VE-d6 60ug / mL 25(OH)D2-d6 0.5ug / mL 25(OH)D3-d6 0.5ug / mL

[0079] Preprocessing:

[0080] ① Transfer 100 μL of standard sample, add 10 μL of internal standard solution, mix and shake for 1 min;

[0081] ② After mixing thoroughly, add 200 μL of zinc sulfate solution (concentration of 8 g / mL), mix and shake for 1 min;

[0082] ③ Transfer 400uL of 0.1% BHA methanol:acetonitrile (1:1) solution to the sample treated in ②, mix and shake for 1 min to mix evenly;

[0083] ④ Transfer 600 μL of n-hexane to the sample treated in ③, mix and shake for 1 min, and centrifuge at 10000 g for 5 min; the sample can be seen to be divided into three layers. Take 500 μL of the upper organic layer into another centrifuge tube (be careful not to touch the middle water layer), and dry it at room temperature using a 96 microplate / deep well plate nitrogen blower.

[0084] ⑤ Take 60 μL of 0.1% BHA methanol:water (7:3) to reconstitute the sample treated in ④. After thorough mixing, transfer the solution to a 96-well V-type microplate and cover it with an aluminum foil cover for liquid chromatography-tandem mass spectrometry analysis.

[0085] Detection by liquid chromatography-tandem mass spectrometry:

[0086] Triple quadrupole mass spectrometer LC / MS / MS System (Model: API 3200MD) TM ); Column: Waters, XBridge C18, 3.5μm; 2.1×5.0mm; Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% formic acid methanol; Flow rate: 0.55mL / min; Column temperature: 40℃; Injector temperature: 15℃; Injection volume: 10μL.

[0087] High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used for detection, and data acquisition was performed using gradient elution and electrospray ionization (ESI) with positive ion mode multiple reaction monitoring (MRM).

[0088] Ion source voltage and gas flow parameters:

[0089]

[0090] The following table shows the mass-to-charge ratio (m / z) of the parent ion / daughter ion of the four fat-soluble vitamins used for detection and their internal standards:

[0091]

[0092]

[0093] The gradient elution time is 5.5 minutes, and the gradient elution program is as follows:

[0094] Time (min) Mobile phase A% Mobile phase B% Flow rate (ml / min) 0 95 5 0.55 0.5 17 83 0.55 4 1 99 0.55 4.5 1 99 0.55 4.7 95 5 0.55 5.5 95 5 0.55

[0095] Standard curves were obtained based on the detection data from liquid chromatography-tandem mass spectrometry. Specifically, the concentrations of six standard samples and the detection signal values ​​were plotted on the X and Y axes, yielding the following linear regression equations: Vitamin A: y = 9.46x - 0.0428, r = 0.9984; Vitamin E: y = 0.223x + 0.00036, r = 0.9978; Vitamin D2: y = 0.000494x + 0.00312, r = 0.9990; Vitamin D3: y = 0.0012x - 0.000429, r = 0.9986. All showed good linearity. See Figures 3 to 6 , Figure 3This shows a standard curve of vitamin A according to Embodiment 1 of the present invention. Figure 4 This is a standard curve diagram of vitamin E according to Example 1 of the present invention. Figure 5 This is a standard curve diagram of vitamin D2 according to Embodiment 1 of the present invention. Figure 6 This is a standard curve diagram of vitamin D3 according to Embodiment 1 of the present invention.

[0096] Example 2: Serum Sample Detection

[0097] Clinical serum samples were provided, and the same pretreatment and liquid chromatography-tandem mass spectrometry detection procedures as in Example 1 were used. The detection results are shown in [Figure 1]. Figures 7 to 10 . Figure 7 This is a graph showing the detection results of vitamin A according to Example 2 of the present invention. Figure 8 This is a graph showing the detection results of vitamin E according to Example 2 of the present invention. Figure 9 This is a graph showing the detection results of vitamin D2 according to Example 2 of the present invention. Figure 10 This is a graph showing the detection results of vitamin D3 according to Example 2 of the present invention. Figures 7 to 10 Based on the test results and the standard curve obtained in Example 1, it can be concluded that the content of vitamin A in this clinical serum sample is 0.356 μg / mL; the content of vitamin E is 9.39 μg / mL; the content of vitamin D2 is 1.56 ng / mL; and the content of vitamin D3 is 19.3 ng / mL.

[0098] Example 3: Analysis of the Influence of Precipitating Agent on Detection Results

[0099] A mixed solution was prepared using vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E standards as the intermediate solution for the quality control working solution, and the quality control samples were prepared accordingly. The four vitamins exhibited two series of concentrations in the quality control samples: low (L) and high (H), as shown in the table below.

[0100]

[0101] Different second precipitants were used in the pretreatment process, and the experimental setup is shown in the table below:

[0102]

[0103] The remaining pretreatment steps and liquid chromatography-tandem mass spectrometry detection procedures were the same as in the examples, and the results are shown in the following tables (where the concentrations of vitamin A and vitamin E are in μg / mL; and the concentrations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 are in ng / mL):

[0104] Experiment 1 test results:

[0105]

[0106]

[0107] Experiment 2 test results:

[0108]

[0109] Experiment 3 test results:

[0110]

[0111] Experiment 4 test results:

[0112]

[0113] Based on the detection results of Experiments 1 to 2 above, the recovery rate and precision were analyzed, and the results are shown in the table below:

[0114]

[0115] The experimental results showed that the precipitant combination in Experiment 1 had the highest accuracy (highest recovery rate, smallest relative deviation) and best precision (lowest CV value) in the detection of multiple vitamins compared to other precipitant combinations. Therefore, zinc sulfate + 0.1% BHA methanol:acetonitrile (1:1) was selected as the best protein precipitant in the detection of fat-soluble vitamins.

[0116] While the present invention has been specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the invention in any way. Those skilled in the art can make modifications and variations to the present invention as needed without departing from its essential spirit and scope, and all such modifications and variations fall within the scope of the present invention.

Claims

1. A method for detecting the content of multiple vitamins in blood, characterized in that, include: Provide both the serum sample to be tested and the standard sample; Add internal standard solution to the serum sample to be tested and the standard sample; Add a first precipitant and mix, wherein the first precipitant is a zinc sulfate solution with a concentration of 7.8 g / mL to 10 g / mL; A second precipitant is added and mixed; wherein the second precipitant is composed of acetonitrile and methanol with a stabilizer in a volume ratio of 1:1, and the stabilizer is butylated hydroxyanisole, and the methanol with the stabilizer refers to methanol with a butylated hydroxyanisole mass fraction of 0.1%; An extractant is added to perform extraction; wherein the extractant is n-hexane. Add the redissolution solution to redissolve the sample, thus obtaining the test solution and the standard solution. The test solution and the standard solution were detected by liquid chromatography-tandem mass spectrometry. A standard curve is obtained based on the detection results of the standard solution. The content of the multiple vitamins in the serum sample to be tested is obtained based on the detection results of the test solution and the standard curve. The multivitamins are vitamin A, vitamin D2, vitamin D3, and vitamin E; the standard sample is a solution of vitamin A, vitamin D2, vitamin D3, and vitamin E with a specific concentration; and the internal standard solution is a solution of vitamin A, vitamin D2, vitamin D3, and vitamin E with a specific concentration and isotope labeling.

2. The method according to claim 1, characterized in that, The complex solution comprises water and methanol containing the stabilizer.

3. The method according to claim 2, characterized in that, In the complex solution, the volume ratio of water to the methanol containing the stabilizer is 3:

7.

4. The method according to claim 1, characterized in that, For liquid chromatography, a C18 column was used at 40℃, and the injection volume was 10 μL. Mobile phase A was a 0.1% (v / v) aqueous solution of formic acid, and mobile phase B was a 0.1% (v / v) methanolic solution of formic acid. Gradient elution was used, and the elution program was as follows:

5. The method according to claim 1, characterized in that, When performing mass spectrometry, the mass-to-charge ratio of the parent ion and daughter ion used to detect the various vitamins and the various vitamins labeled with isotopes is:

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