Methods for separating fat-soluble vitamins from human serum and detecting the content of each component

Abstract

This invention belongs to the field of biochemical analysis technology, specifically relating to a method for separating fat-soluble vitamins from human serum and detecting the content of each component. The method involves: pretreatment of the serum sample using normal-phase solid-phase extraction, including elution with a solid-phase column packed with diatomaceous earth, Florisil, silica gel, alumina, or amino-bonded silica gel; then, separation and determination of vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in human serum using LC-MS / MS; the chromatographic column is packed with C18, C8, or pentafluorophenyl as the packing material, and gradient elution is performed using a mixed solution of formic acid and ammonium acetate aqueous solution as mobile phase A and a mixed solution of formic acid and ammonium acetate methanol solution as mobile phase B; finally, detection is performed using tandem quadrupole mass spectrometry. This detection method is simple to operate, quick, efficient, and easier to perform in high-throughput operations, facilitating clinical promotion and application.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, specifically relating to a method for separating fat-soluble vitamins from human serum and detecting the content of each component. Background Technology

[0002] Vitamins are a class of organic chemicals essential for maintaining human life processes. Common vitamins include vitamins A, D, E, and K. Vitamin A, in particular, is a compound with retinol bioactivity, closely related to physiological functions such as vision, cell proliferation and differentiation, reproduction, immunity, bone metabolism, and iron-deficiency anemia. Vitamin A deficiency can lead to a range of eye symptoms, anemia, and weakened resistance to infection, increasing the severity of infectious diseases and the risk of death, and causing tissue damage; excessive vitamin A can cause poisoning. Vitamin D is an essential nutrient for maintaining life in higher animals, related to normal bone mineralization, muscle contraction, nerve conduction, and the function of all cells in the body. Vitamin E, also known as tocopherol, is an antioxidant. In animal tissue cell membranes, it protects polyunsaturated fatty acids, the sulfhydryl groups of the cytoskeleton and other proteins, and intracellular nucleic acids from free radical attack. Vitamin E has anti-inflammatory properties and may help prevent atherosclerosis. Compared to other cells in the body, immune cells have a higher concentration of vitamin E, thus playing an important role in the immune system. Vitamin K is a group of compounds containing a 2-methyl-1,4-naphthoquinone group. Vitamin K1 (phylloquinone) is mainly derived from plants and is the primary source of vitamin K in the human diet. Vitamin K deficiency can lead to blood clotting disorders and slow bone development; vitamin K deficiency in newborns is a significant cause of intracranial hemorrhage in children; vitamin K excess can easily lead to hyperbilirubinemia and hemolytic anemia. Therefore, the detection of fat-soluble vitamins can assess the nutritional status of fat-soluble vitamins in patients and has auxiliary diagnostic significance in the clinical judgment, treatment management, and physiological assessment of fat-soluble vitamin deficiency or excess.

[0003] Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are currently the most commonly used techniques for purifying fat-soluble vitamins from biological matrices. They achieve effective analyte recovery by reducing ion inhibition and enhancing analyte signals. LLE has a wide range of applications, is technically mature, and can handle large sample volumes. However, post-extraction pipetting is cumbersome, may not completely remove all analytes, is time-consuming, and is not easily automated. Emulsification can occur in the intermediate layer of the sample. Because the solvents used are usually nonpolar organic compounds, hydrophobic analytes are extracted into the organic layer, but other nonpolar interfering substances (e.g., serum lipids) are often co-extracted, which can easily cause matrix effects. This not only leads to lower-than-expected purity of the extract, but also the co-extract may accumulate on the analytical column (usually a reversed-phase column). These factors result in high automation costs for LLE, hindering its widespread clinical application.

[0004] The invention patent with publication number CN110967424A discloses a liquid chromatography-mass spectrometry method for detecting fat-soluble vitamins in serum, which can simultaneously determine vitamin A, vitamin E, vitamin K1, 25OHD2 and 25OHD3 in serum. However, this invention uses liquid-liquid extraction for sample pretreatment, resulting in poor peak shapes and more impurities for vitamin K and 25-hydroxyvitamin D2, leading to inaccurate integrated quantitative results.

[0005] Therefore, there is an urgent need for a pretreatment method for serum fat-soluble vitamins that is simple to operate, has high processing efficiency, and can meet the requirements of high-throughput automation, in order to overcome the shortcomings of existing technologies. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a method for separating fat-soluble vitamins in serum based on LC-MS / MS. This method utilizes normal-phase solid-phase extraction for sample pretreatment, enabling rapid detection of fat-soluble vitamins in human serum.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for separating fat-soluble vitamins in serum based on LC-MS / MS, wherein the separated components include vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2 (25OHD2), and 25-hydroxyvitamin D3 (25OHD3); the method comprises: pretreatment of serum samples using normal-phase solid-phase extraction, followed by separation using high-performance liquid chromatography (HPLC); the HPLC column is a column packed with octadecylsilane-bonded silica gel, C8, and / or pentafluorophenyl; the mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A is a mixture of 0.01-0.2% (v / v) formic acid and 0.5-5.0 mM ammonium acetate aqueous solution, and mobile phase B is a mixture of 0.01-0.2% (v / v) formic acid and 0.5-5.0 mM ammonium acetate methanol solution; and 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, vitamin A, vitamin E, and vitamin K1 are separated sequentially by gradient elution.

[0009] Furthermore, the components can be qualitatively identified based on the rate of separation.

[0010] Furthermore, the separation time was 9 minutes.

[0011] Preferably, the formic acid has a volume fraction of 0.1%; the ammonium acetate aqueous solution has a concentration of 2 mM; and the ammonium acetate methanol solution has a concentration of 2 mM.

[0012] Furthermore, the preprocessing includes the following steps:

[0013] (1) A solid column packed with polar packing material was used for normal phase solid-phase extraction.

[0014] (2) The serum sample was mixed with fat-soluble vitamin deuterated internal standard and antioxidant, then loaded onto the sample and allowed to stand.

[0015] (3) Elution and separation, collection of eluent;

[0016] (4) Nitrogen blowing, reconstitution, and obtain the sample to be tested.

[0017] This pretreatment method involves first passing an aqueous layer containing the target analyte through a solid-phase column. The analyte then adsorbs onto the packing material of the solid-phase column. Since this material is polar, polar molecules adsorb more strongly onto the surface. An organic solvent is then added to the column. The solvent interacts with and carries away the weakly bound molecules of interest. This also strengthens the adsorption of water molecules embedded in the polar packing material. This is advantageous because most impurities are polar or charged. Compared to liquid-liquid extraction, this method is simpler to operate, faster, more efficient, and easier to perform at high throughput, making it suitable for clinical application and promotion.

[0018] Furthermore, the polar material is any one or more of diatomaceous earth, Florisil, silica gel, alumina, and / or amino-bonded silica gel.

[0019] Furthermore, the polar material is preferably Florisil.

[0020] Furthermore, in step (1), the amount of filler is 100-500 mg; preferably 300 mg.

[0021] Further, in step (2), 10-200 μL of serum is mixed with a fat-soluble vitamin deuterated internal standard and 10-100 μL of antioxidant before loading the sample.

[0022] Furthermore, the fat-soluble vitamin deuterated internal standards are retinol (vitamin A), α-tocopherol (vitamin E), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin K1 deuterated internal standards.

[0023] Furthermore, the antioxidant includes one or more of citric acid, β-mercaptoethanol, 2,6-di-tert-butyl-p-cresol (BHT), and butylated hydroxyanisole (BHA).

[0024] Furthermore, the concentration of the antioxidant is 0.5-10 mg / mL.

[0025] Furthermore, in step (3), the extract includes one or more of methanol, ethanol, acetonitrile, ethyl acetate, n-hexane, and dichloromethane.

[0026] Further, the target analyte was eluted with 0.5-4 mL of extract.

[0027] Furthermore, in step (4), the reconstituted solution is any one or more of methanol, ethanol, and acetonitrile.

[0028] Furthermore, the gradient elution program is set as follows:

[0029] 0.00 min, the volume ratio of mobile phase A to mobile phase B is set to 20-40:60-80;

[0030] 0.50 min, the volume ratio of mobile phase A to mobile phase B is set to 20-40:60-80;

[0031] 3.50 min, set the volume ratio of mobile phase A to mobile phase B to be 0-5:95-100;

[0032] 6.50 min, the volume ratio of mobile phase A to mobile phase B is set to 0-5:95-100;

[0033] 6.51 min, the volume ratio of the mobile phase A to the mobile phase B is set to 20-40:60-80.

[0034] At 9:00 min, the volume ratio of mobile phase A to mobile phase B is set to 20-40:60-80.

[0035] Furthermore, the flow rate of the high-performance liquid chromatography is 0.2-0.6 mL / min, the column temperature is 35-40℃, and the injection volume is 1-20 μL.

[0036] The second objective of this invention is to provide a method for simultaneously detecting vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in serum. This method can determine the content of vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in serum within 9 minutes or more.

[0037] To achieve the above objectives, the present invention adopts the following technical solution:

[0038] The method for simultaneously detecting vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in serum, based on the method described in Objective 1, involves separating vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in serum using the method described in Objective 1, and then detecting them using a detector; the detector is a tandem quadrupole mass spectrometer.

[0039] Furthermore, the ion pair for vitamin A was determined to be 269.15 m / z → 93.2, for vitamin E 431.35 m / z → 137.1, for vitamin K1 451.15 m / z → 187.1, for 25-hydroxyvitamin D2 413.4 m / z → 355.5, and for 25-hydroxyvitamin D3 401.3 m / z → 257.2; vitamins The ion pair for internal standard A is 275.05 m / z→96.15, the ion pair for internal standard E is 437.45 m / z→171.2, the ion pair for internal standard K1 is 451.15 m / z→187.1, the ion pair for internal standard 25-hydroxyvitamin D2 is 416.2 m / z→358.4, and the ion pair for internal standard 25-hydroxyvitamin D3 is 389.4 m / z→371.45.

[0040] Furthermore, the atomizing gas flow rate (N) is 3 L / min; the heating gas flow rate (F) is 15 L / min; the interface temperature (I) is 300℃; the DL temperature (D) is 150℃; the heating block temperature (H) is 480℃; and the drying gas flow rate (G) is 15 L / min.

[0041] Preferably, the retention times of the five components, from longest to shortest, are: 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, vitamin A, vitamin E, and vitamin K1.

[0042] As a more preferred embodiment, mobile phase A is a mixed solution of 0.1% formic acid (v / v) and 2mM ammonium acetate aqueous solution; mobile phase B is a mixed solution of 0.1% formic acid (v / v) and 2mM ammonium acetate methanol solution; the chromatographic column has dimensions of 1.7μm × 2.1mm × 50mm; the high-performance liquid chromatography (HPLC) flow rate is 0.4mL / min; the column temperature is 40℃; the injection volume is 5μL; the detector is a tandem quadrupole mass spectrometer, the ion source is an ESI source, the scanning mode is positive ion mode; and the determination method is multiple reaction monitoring (MRM).

[0043] Gradient elution was performed using a gradient elution program to obtain a chromatogram; the gradient elution program was set as follows:

[0044] 0.00 min, the volume ratio of mobile phase A to mobile phase B is set to 30:70;

[0045] 0.50 min, the volume ratio of mobile phase A to mobile phase B is set to 30:70;

[0046] 3.50 min, set the volume ratio of mobile phase A to mobile phase B to 0:100;

[0047] 6.50 min, set the volume ratio of mobile phase A to mobile phase B to 0:100;

[0048] 6.51 min, the volume ratio of mobile phase A to mobile phase B is set to 30:70;

[0049] At 9:00 min, the volume ratio of mobile phase A to mobile phase B was set to 30:70.

[0050] Furthermore, the vitamin D3 was retained for 3.00±0.5 min; the vitamin D2 was retained for 3.08±0.5 min; the vitamin A was retained for 3.22±0.5 min; the vitamin E was retained for 4.69±0.5 min; and the vitamin K1 was retained for 5.33±0.5 min.

[0051] The aforementioned retention time can be used for qualitative detection of components.

[0052] Furthermore, for vitamin A in the range of 25 ng / mL to 1600 ng / mL, y = 0.00780649x - 0.0309512; for vitamin E in the range of 625 ng / mL to 40000 ng / mL, y = 8.40836e^ -5 x-0.00554548; for vitamin K1 in the range of 0.1 ng / mL to 6.4 ng / mL, y = 0.0261429x-0.000913363; for 25-hydroxyvitamin D2 in the range of 1 ng / mL to 64 ng / mL, y = 0.00664607x+0.0101028; for 25-hydroxyvitamin D3 in the range of 2 g / mL to 128 g / mL, y = 0.00764757x+0.00170462, where y is the y-axis, representing the peak area ratio of the standard to the internal standard, and x is the x-axis, representing the concentration.

[0053] Based on the peak areas detected by the detector, the levels of vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in serum were calculated using the isotope internal standard method. Specifically, a standard curve was established with the concentration of the standard as the x-axis and the peak area ratio of the standard to the internal standard as the y-axis. The levels of each fat-soluble vitamin in the serum sample were calculated by substituting the measured area ratio of the human serum sample into the standard curve.

[0054] The beneficial effects of this invention are as follows:

[0055] 1. The method for separating fat-soluble vitamins from serum provided by this invention employs normal-phase solid-phase extraction, which can rapidly and effectively extract fat-soluble vitamins from serum. This method is very simple and, compared with the traditional liquid-liquid extraction method, can achieve high-throughput detection simultaneously. Furthermore, the recovery rate of fat-soluble vitamins, especially vitamins A, E, K1, 25OHD2, and 25OHD3, is high, and the matrix effect is small. After pretreatment, the sensitivity of high-performance liquid chromatography-tandem mass spectrometry in detecting fat-soluble vitamins in human serum is also improved. The detection process is simpler and more efficient, and the detection results are more accurate and stable. It is particularly suitable for clinical application, reducing manual operation in the detection process.

[0056] 2. The pretreatment operation of the present invention is extremely simple. After adding the internal standard and antioxidant to human serum, it can be directly mixed by pipetting, which provides convenience for 96-well plate operation. In addition, the solid-phase column does not need to be activated in advance, saving time and cost.

[0057] 3. In the method of the present invention, after extraction with the extractant, the resulting extractant can be directly dried with nitrogen gas without the need to transfer the liquid, and the nitrogen blowing time is also very fast, which greatly saves the operation time.

[0058] 4. The entire pretreatment process of the method of the present invention uses low-adsorption pipette tips and low-adsorption 96-well plates, which can reduce the loss of low target analytes during the pretreatment process and ensure the precision and accuracy of the detection results.

[0059] 5. The method for separating fat-soluble vitamins in serum provided by this invention simultaneously detects the peak time and ion pairs of the target analyte, exhibiting extremely high specificity and greatly avoiding interference from cross-reactions, thereby improving accuracy. Furthermore, the method employs an isotope internal standard method for quantification, which can greatly eliminate matrix effects, resulting in accurate and reliable detection results.

[0060] 6. The method provided by this invention can simultaneously separate vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human serum in a very short time, and complete qualitative and quantitative detection, with higher detection efficiency. Attached Figure Description

[0061] Figure 1 MRM spectra of calibrators for vitamins A, E, K1, 25OHD2, and 25OHD3;

[0062] Figure 2 MRM chromatograms of internal standard calibrators for vitamins A, E, K1, 25OHD2, and 25OHD3;

[0063] Figure 3 MRM spectra of vitamins A, E, K1, 25OHD2, and 25OHD3 in human serum after treatment with Florisil;

[0064] Figure 4 Calibration curve for vitamin A;

[0065] Figure 5 Calibration curve for Vitamin E;

[0066] Figure 6 Calibration curve for vitamin K1;

[0067] Figure 7 Calibration curve for 25-hydroxyvitamin D2;

[0068] Figure 8 Calibration curve for 25-hydroxyvitamin D3;

[0069] Figure 9 The elution results are for five types of packing materials; among them, ① is diatomaceous earth, ② is Florisil, ③ is silica gel, ④ is alumina, and ⑤ is amino-bonded silica gel.

[0070] Figure 10 Results of serum treatment with Florisil columns of different packing volumes;

[0071] Figure 11 This is a chromatogram of serum after liquid-liquid extraction. Detailed Implementation

[0072] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0073] Example 1. Investigation of the packing material

[0074] This embodiment investigated five different fillers: diatomaceous earth, Florisil, silica gel, alumina, and amino-bonded silica gel. The results are as follows: Figure 9 As shown, all five packing materials achieved normal-phase elution. This invention patent further investigates the effect of different packing material amounts on the pretreatment results, using Florisil as an example. The Florisil packing material amounts were 60 mg, 100 mg, 200 mg, 300 mg, and 500 mg. The test results are as follows: Figure 10 As shown, when 100 μL of serum is added, serum flow-through does not occur with filler amounts of 300 mg and 500 mg. Considering cost, this invention ultimately selected a filler amount of 300 mg.

[0075] Example 2. Method for separating and determining fat-soluble vitamins in human serum

[0076] This embodiment provides a method for separating and detecting vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 based on LC-MS / MS, including the following steps:

[0077] (1) Column packing: The normal phase solid phase extraction method is adopted, and polar packing is used to pack the solid phase column into a small column;

[0078] (2) Sample loading: Take serum, add fat-soluble vitamin deuterated internal standard and antioxidant, mix well and add directly to solid phase extraction column, squeeze all liquid into packing material, and let stand for 5-10 min;

[0079] (3) Elution and separation;

[0080] (4) The extracted solution was dried under nitrogen at room temperature, reconstituted, vortexed for 2 min, and detected by LC-MS / MS.

[0081] 1. Chromatographic conditions

[0082] Column type: Waters ACQUITY BEH C18 1.7μm 2.1×50mm Column; flow rate: 0.4ml / min; column temperature: 40℃; injection volume: 5μL; mobile phase A: a mixture of formic acid and ammonium acetate aqueous solution, wherein the volume / mass fraction of formic acid is 0.1% and the concentration of ammonium acetate aqueous solution is 2mM; mobile phase B: a mixture of formic acid and ammonium acetate methanol solution, wherein the volume / mass fraction of formic acid is 0.1% and the concentration of ammonium acetate methanol solution is 2mM; gradient elution program is shown in Table 2.

[0083] Table 2. Ultra-high performance liquid chromatography elution program

[0084] Washing time (min) Mobile phase A (%) Mobile phase B (%) 0 30 70 0.5 30 70 3.50 0 100 6.50 0 100 6.51 30 70 9.00 30 70

[0085] 2. Mass spectrometry detection conditions

[0086] The ion source was an ESI source; the scanning mode was positive ion mode; the measurement mode was multiple reaction monitoring (MRM mode); the nebulizer gas flow rate (N) was 3 L / min; the heating gas flow rate (F) was 15 L / min; the interface temperature (I) was 300 °C; the DL temperature (D) was 150 °C; the heating block temperature (H) was 480 °C; the drying gas flow rate (G) was 15 L / min; the ion pairs of each analyte and the collision energies and Q1 and Q3 voltages under MRM conditions are shown in Table 3.

[0087] Table 3. Ion pairs of each analyte and collision energies and Q1 and Q3 voltages under MRM conditions

[0088]

[0089] MRM chromatograms of various fat-soluble vitamin standards and internal standards are as follows: Figure 1 and Figure 2 As shown.

[0090] 3. Sample preparation

[0091] Sample preparation includes the preparation of standard curves, quality control samples, and internal standards. First, vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 standards are prepared into standard stock solutions, which will serve as standard working solutions and quality control stock solutions. Then, negative blank human serum is used to serially dilute the stock solutions by volume to obtain the standard curve (as shown in Table 4) and quality control samples (as shown in Table 5).

[0092] Table 4. Concentration points of the standard curves for five fat-soluble vitamins

[0093] Serial Number Dilution ratio VAng / mL VE μg / mL VK ng / mL 25OHD2 ng / mL 25OHD3 ng / mL 1 1 1600 40 6.4 64 128 2 1 / 2 800 20 3.2 32 64 3 1 / 4 400 10 1.6 16 32 4 1 / 8 200 5 0.8 8 16 5 1 / 16 100 2.5 0.4 4 8 6 1 / 32 50 1.25 0.2 2 4 7 1 / 64 25 0.625 0.1 1 2

[0094] Table 5. Concentration points of low, medium, and high quality control samples of five fat-soluble vitamins

[0095]

[0096]

[0097] Preparation of internal standards: Prepare mixed internal standard working solutions with concentrations of 500, 100, 50, 200 and 200 ng / mL for vitamin A-d6, vitamin E-d6, vitamin K1-d4, 25-hydroxyvitamin D2-d3 and 25-hydroxyvitamin D3-d6, respectively.

[0098] The standard curves and correlation coefficients for vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 are shown below. Figure 4 , 5 As shown in 6, 7, and 8.

[0099] 4. Sample pretreatment

[0100] Sample pretreatment, including human serum samples, calibrator working solutions, and quality control samples, is performed using the following methods:

[0101] (1) Take 100 μL of human serum / calibrator working solution / quality control solution into a 96-well deep plate, add 50 μL of internal standard mixture and 10 μL of LHT ethanol solution (2 mg / mL), and shake to mix.

[0102] (2) Transfer the above liquid to a 96-hole filter plate, press the positive pressure device into the packing, and let it stand for 5 minutes;

[0103] (3) Add 2.5 mL of 6% ethanol-hexane solution and collect the eluent using a 96-well plate;

[0104] (4) Place the eluent at room temperature and dry it with nitrogen gas;

[0105] (5) Add 100 μL of methanol reconstitution solution, shake to mix for 2 min, transfer the liquid to a brown vial for testing.

[0106] MRM of human serum fat-soluble vitamins after sample pretreatment, such as Figure 3 As shown, there are relatively few interference peaks.

[0107] 5. Data Processing and Analysis

[0108] The linear relationship of the standard curve is shown in Table 6.

[0109] Table 6. Linearity of Standard Curves

[0110]

[0111] 6. Spiking recovery and matrix effect

[0112] Three different concentration levels of mixed standard solution (low, medium, and high, corresponding to the three concentrations in Table 7) were added to clinical serum. The pretreatment method of Example 2 was followed. Each sample was analyzed in triplicate. The recovery rate was obtained by calculating the mean detection value and the theoretical added value. A recovery rate between 87.2% and 120% indicates a good recovery rate. The matrix effect was calculated by comparing the instrument response of the analyte in a sample with a known concentration with that of an equal volume (content × recovery rate) of pure analyte solution. A matrix effect between 87.1% and 114.7% indicates that the influence of the matrix on the analyte is negligible. The detection results are shown in Table 7.

[0113] Table 7 Spike Recovery and Matrix Effect

[0114]

[0115] 7. Accuracy and precision

[0116] Intra-batch precision requires testing three concentrations (low, medium, and high) of quality control samples in one analytical batch, with six samples for each concentration, and the measurements are performed in triplicate. Inter-batch precision requires at least three analytical batches. Results: Intra-batch precision was between 2.5% and 10.4%, ≤15%; inter-batch precision was between 2.7% and 10.5%, ≤15%; accuracy was between 94.7% and 111.9%, meeting the requirements.

[0117] Comparative Example 1

[0118] This comparative example provides a method for separating and detecting fat-soluble vitamins in human serum. The comparative example uses liquid-liquid extraction for sample pretreatment (other conditions are the same as in Example 2), and the specific method is as follows:

[0119] (1) Take 100 μL of serum into a 2 mL centrifuge tube, add 50 μL of internal standard mixture and 10 μL of LHT ethanol solution (2 mg / mL), and vortex to mix for 2 min;

[0120] (2) Add 800 μL of acetonitrile to the liquid obtained in step (1) and vortex for 10 min;

[0121] (3) Add 800 μL of n-hexane to the liquid obtained in step (2), vortex mix for 2 min, centrifuge at 10000×g at room temperature for 10 min, take the supernatant into a clean 2 mL centrifuge tube, and repeat this step once.

[0122] (4) Combine the supernatants, blow dry with nitrogen, and finally redissolve with 100 μL methanol solution. Vortex for 2 min and test for later use.

[0123] Test results as follows Figure 11As shown, the peak shapes of vitamin K and 25-hydroxyvitamin D2 in the liquid-liquid extraction method are poor and there are many impurity peaks, which makes the integral quantification results inaccurate.

Claims

1. A method for separating fat-soluble vitamins from serum based on LC-MS / MS, characterized in that, The separated components include vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3. The method involves pretreatment of serum samples using normal-phase solid-phase extraction, followed by separation using high-performance liquid chromatography (HPLC). The HPLC column is packed with octadecylsilane-bonded silica gel. The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixture of 0.01-0.2% (v / v) formic acid and 0.5-5.0 mM ammonium acetate aqueous solution, while mobile phase B is a mixture of 0.01-0.2% (v / v) formic acid and 0.5-5.0 mM ammonium acetate methanol solution. Gradient elution sequentially separates 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, vitamin A, vitamin E, and vitamin K1. The preprocessing includes the following steps: (1) The normal phase solid-phase extraction method is adopted, and a solid column packed with polar material is used; (2) The serum sample was mixed with a fat-soluble vitamin deuterated internal standard and an antioxidant, then loaded onto the sample and allowed to stand. (3) Elution and separation, and collection of the eluent; (4) Nitrogen blowing, reconstitution, and obtaining the sample to be tested; The polar material is any one or more of diatomaceous earth, Florisil, silica gel, alumina and / or amino-bonded silica gel. The gradient elution includes the following settings: The elution time is 0.00 min, and the volume ratio of mobile phase A to mobile phase B is set to 30:

70. The elution time is 0.50 min, and the volume ratio of mobile phase A to mobile phase B is set to 30:

70. The elution time was 3.50 min, and the volume ratio of mobile phase A to mobile phase B was set to 0:

100. The elution time was 6.50 min, and the volume ratio of mobile phase A to mobile phase B was set to 0:

100. The elution time was 6.51 min, and the volume ratio of mobile phase A to mobile phase B was set to 30:

70. The elution time was 9.00 min, and the volume ratio of mobile phase A to mobile phase B was set to 30:

70.

2. The method according to claim 1, characterized in that, In step (1), the amount of filler is 100-500mg.

3. The method according to claim 1, characterized in that, In step (2), the antioxidant includes one or more of citric acid, β-mercaptoethanol, 2,6-di-tert-butyl-p-cresol, and butylated hydroxyanisole.

4. The method according to claim 1, characterized in that, The ion pairs for vitamin A were 269.15 m / z→93.2, vitamin E was 431.35 m / z→137.1, vitamin K1 was 451.15 m / z→187.1, 25-hydroxyvitamin D2 was 413.4 m / z→355.5, and 25-hydroxyvitamin D3 was 401.3 m / z→257.

2. The ion pairs for the internal standard vitamin A were 275.05 m / z→96.15, vitamin E was 437.45 m / z→171.2, vitamin K1 was 451.15 m / z→187.1, 25-hydroxyvitamin D2 was 416.2 m / z→358.4, and 25-hydroxyvitamin D3 was 389.

4. m / z→371.

45.

5. The method according to claim 4, characterized in that, The vitamin D3 was retained for 3.00±0.5 min; the vitamin D2 was retained for 3.08±0.5 min; the vitamin A was retained for 3.22±0.5 min; the vitamin E was retained for 4.69±0.5 min; and the vitamin K1 was retained for 5.33±0.5 min.

6. The method according to claim 4, characterized in that, The vitamin A concentration is in the range of 25 ng / mL to 1600 ng / mL, and y = 0.00780649x − 0.0309512; the vitamin E concentration is in the range of 625 ng / mL to 40000 ng / mL, and y = 8.40836e^(-1 / 2). −5 x−0.00554548; for vitamin K1 in the range of 0.1 ng / mL to 6.4 ng / mL, y=0.0261429x−0.000913363; for 25-hydroxyvitamin D2 in the range of 1 ng / mL to 64 ng / mL, y=0.00664607x+0.0101028; for 25-hydroxyvitamin D3 in the range of 2 g / mL to 128 g / mL, y=0.00764757x+0.00170462, where y is the y-axis, representing the peak area ratio of the standard to the internal standard, and x is the x-axis, representing the concentration.

7. The method according to any one of claims 1-6 for simultaneously detecting vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2, and 25-hydroxyvitamin D3 in serum, characterized in that, Vitamin A, vitamin E, vitamin K1, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum are separated using the method described in any one of claims 1-6, and the separated vitamins are then detected by a detector; the detector is a tandem quadrupole mass spectrometer.

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