Method for determining multiple fat-soluble vitamins based on mass spectrometry

Abstract

The invention discloses a method for determining multiple fat-soluble vitamins based on mass spectrometry, which comprises the following steps: S1, pretreating a sample, and centrifuging to take supernate; s2, carrying out solid-phase extraction on the supernate, collecting an eluent, and redissolving; s3, injecting the redissolved solution treated in S2 into a high performance liquid chromatography system for gradient elution; s4, components flowing out of the high performance liquid chromatography system are guided into a tandem mass spectrometer for detection; and S5, acquiring detection data in the step S4, and performing qualitative judgment and quantitative calculation to obtain the content of each fat-soluble vitamin in the sample. Compared with the prior art, the method for determining the multiple fat-soluble vitamins based on the mass spectrometry has the advantages that the efficient and accurate determination of the multiple fat-soluble vitamins in a sample is completed by optimizing sample pretreatment, chromatographic separation and mass spectrometry detection.

Description

Technical Field

[0001] This invention relates to the field of vitamin content determination technology, specifically to a method for determining multiple fat-soluble vitamins based on mass spectrometry. Background Technology

[0002] Water-soluble vitamins are a group of vitamins containing cyclic structures and long aliphatic hydrocarbon chains, mainly including vitamins A, D, E, and K.

[0003] The vitamins mentioned above are a class of low-molecular-weight organic compounds that are essential for maintaining normal metabolism and certain special physiological functions in the human body; Currently, traditional detection methods for fat-soluble vitamins, such as HPLC, suffer from insufficient sensitivity or low separation efficiency, making it difficult to accurately determine multiple vitamins simultaneously. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned technical defects and provide a method for the determination of multiple fat-soluble vitamins based on mass spectrometry, which optimizes sample pretreatment, chromatographic separation and mass spectrometry detection to achieve efficient and accurate determination of multiple fat-soluble vitamins in a sample.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is: a method for determining multiple fat-soluble vitamins based on mass spectrometry, comprising the following steps: S1: Sample pretreatment, centrifugation to obtain supernatant; S2: Solid-phase extraction of the supernatant is performed to collect the eluent and then reconstitute it; S3: Take the reconstituted solution after treatment in S2 and inject it into the high performance liquid chromatography system for gradient elution; S4: The components eluting from the high-performance liquid chromatography system are introduced into a tandem mass spectrometer for detection; S5: Obtain the detection data from S4 for qualitative and quantitative analysis to obtain the content of each fat-soluble vitamin in the sample.

[0006] Preferably, step S1 includes taking a sample and placing it in a centrifuge tube, adding an isotope internal standard mixture and a protein precipitant, and then taking the supernatant after vortexing.

[0007] Preferably, the solid-phase extraction column in S2 is activated into a hydrophilic-lipophilic balanced solid-phase extraction column, and methanol and ultrapure water are injected sequentially for activation.

[0008] Preferably, step S2 further includes: S2.1: Slowly load the supernatant from S1 onto the activated solid-phase extraction column. After the liquid has completely passed through the column, rinse the column with methanol-water solution to remove impurities. Discard the eluent and then vacuum dry the column. S2.2: Elute the target analyte with a methanol-dichloromethane mixture, collect the eluent, dry it, and then reconstitute it through an organic phase filter membrane to obtain the test solution.

[0009] Preferably, S4 includes: S4.1: The components eluting from the high-performance liquid chromatography system are split according to a preset ratio using a splitter; S4.2: Ionize the sample by introducing it into the ionization source of the tandem mass spectrometer; S4.3: The tandem mass spectrometer adopts a multi-reaction monitoring mode and sets characteristic precursor ion-daughter ion pairs; S4.4: Standards should be used periodically for mass calibration during mass spectrometry testing.

[0010] Preferably, the preset splitting ratio in S4.1 is 1~5:1.

[0011] Preferably, step S4.3 includes acquiring the primary mass spectrum of each fat-soluble vitamin through full scan mode, determining the mass number of the parent ion, performing product ion scanning on the selected parent ion, and screening out characteristic fragment ions as daughter ions. By combining the parent ion-daughter ion pairs of isotope internal standards, a multi-channel monitoring list is constructed for the simultaneous qualitative and quantitative analysis of multiple fat-soluble vitamins.

[0012] Preferably, step S5 includes establishing a standard curve using the internal standard method, and calculating the concentration by substituting the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard into the standard curve.

[0013] The advantages of this invention compared with the prior art are as follows: the invention employs specific solid-phase extraction technology and high-performance liquid chromatography gradient elution conditions, which can effectively separate multiple fat-soluble vitamins with similar structures in the sample, reduce interference, and improve the accuracy of analysis. Simultaneously, by utilizing the multi-reaction monitoring mode of the tandem mass spectrometer and setting characteristic precursor ion-daughter ion pairs, the selectivity and sensitivity of the detection are greatly improved. In this invention, an internal standard method is used to establish a standard curve, and the concentration is calculated by substituting the standard curve into the standard curve, which improves the detection efficiency and reduces the detection cost. Attached Figure Description

[0014] Figure 1 This is a flowchart illustrating a method for determining multiple fat-soluble vitamins based on mass spectrometry. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to the accompanying drawings.

[0016] Combined with appendix Figure 1As shown, a method for determining multiple fat-soluble vitamins based on mass spectrometry includes the following steps: S1: sample pretreatment, centrifugation to obtain the supernatant; S2: solid-phase extraction of the supernatant to collect the eluent and redissolve it; S3: injection of the redissolved solution from S2 into a high-performance liquid chromatography system for gradient elution; S4: introduction of the components eluted from the high-performance liquid chromatography system into a tandem mass spectrometer for detection; S5: acquisition of the detection data from S4 for qualitative and quantitative calculation to obtain the content of each fat-soluble vitamin in the sample.

[0017] In specific implementation of the present invention, S1 involves placing the sample into a centrifuge tube, adding an isotope internal standard mixture and a protein precipitant, and then taking the supernatant after vortexing. In S2, the solid-phase extraction column is activated into a hydrophilic-lipophilic balanced solid-phase extraction column, and methanol and ultrapure water are injected sequentially for activation.

[0018] In one embodiment: S2 also includes: S2.1: Slowly load the supernatant from S1 onto the activated solid-phase extraction column. After the liquid has completely passed through the column, rinse the column with methanol-water solution to remove impurities. Discard the eluent and then vacuum dry the column. S2.2: Elute the target analyte with a methanol-dichloromethane mixture, collect the eluent, dry it, and then reconstitute it through an organic phase filter membrane to obtain the test solution.

[0019] S4 includes: S4.1: The components eluting from the high-performance liquid chromatography system are split according to a preset ratio using a splitter; S4.2: Ionize the sample by introducing it into the ionization source of the tandem mass spectrometer; S4.3: The tandem mass spectrometer adopts a multi-reaction monitoring mode and sets characteristic precursor ion-daughter ion pairs; S4.4: During the mass spectrometry detection process, standard products are used regularly for mass calibration. In S4.1, the preset split ratio is 1~5:1, while S4.3 includes obtaining the primary mass spectrum of each fat-soluble vitamin through full scan mode, determining the mass number of the precursor ion, performing product ion scanning on the selected precursor ion, and screening out characteristic fragment ions as daughter ions. By combining the parent ion-daughter ion pairs of isotope internal standards, a multi-channel monitoring list is constructed for the simultaneous qualitative and quantitative analysis of multiple fat-soluble vitamins.

[0020] In one embodiment: S5 includes using the internal standard method to establish a standard curve, and calculating the concentration by substituting the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard into the standard curve.

[0021] In practical use: Place an appropriate amount of sample into a centrifuge tube, and accurately add a certain volume of isotope internal standard mixture and protein precipitant. Place the centrifuge tube on a vortex mixer and vortex at an appropriate speed for a certain period of time to ensure thorough mixing of the sample and reagents. Then place the centrifuge tube in a centrifuge and centrifuge at a suitable speed for a certain period of time, then collect the supernatant for later use. The hydrophilic-lipophilic balanced solid-phase extraction column was then installed on the solid-phase extraction device. The solid-phase extraction column was activated sequentially with appropriate amounts of methanol and ultrapure water. Methanol activation removes impurities from the column and wets the packing material, while ultrapure water activation brings the column to a suitable hydration state. During activation, the flow rate was controlled to allow the liquid to slowly pass through the column. The supernatant obtained was then slowly loaded onto the activated solid-phase extraction column. The flow rate was controlled to avoid liquid impact on the column and loss of the target substance. After the liquid had completely passed through the column, the column was rinsed with an appropriate amount of methanol-water solution to remove impurities. After rinsing, the rinsing solution was discarded, and the column was then vacuum-dried to remove residual water. After eluting the target analyte with a methanol-dichloromethane mixture and collecting the eluent, the eluent was dried using a nitrogen blow-dryer. After drying, a reconstitution solvent was added, and the solution was filtered through an organic phase filter membrane to obtain the test solution. This solution was then injected into a high-performance liquid chromatography (HPLC) system, and a gradient elution program was set. The components eluted from the HPLC system were split according to a preset ratio using a splitter. A portion of the split solution was then introduced into a tandem mass spectrometer (MS / MS) for detection. The solution was then introduced into the ionization source of the MS / MS for ionization treatment, including setting ionization parameters such as spray voltage and capillary temperature, to ionize the target analyte. The tandem mass spectrometer employs multiple reaction monitoring (MRM) mode, acquiring primary mass spectra of each fat-soluble vitamin through full scan mode to determine the precursor ion mass number. Product ion scanning is then performed on selected precursor ions to identify characteristic fragment ions as daughter ions. A multi-channel monitoring list is constructed by combining precursor ion-daughter ion pairs with isotopic internal standards.

[0022] When using mass spectrometry, standard samples should be used regularly for mass calibration to ensure the accuracy of the test results.

[0023] Then, based on the retention times and characteristic parent-daughter ion pairs of each fat-soluble vitamin and its isotopic internal standard, the target analytes in the sample are qualitatively identified. If the retention time of the compound detected in the sample is consistent with that of the standard, and the parent-daughter ion pair matches the set characteristic pair, it can be identified as the corresponding fat-soluble vitamin.

[0024] Simultaneously, an internal standard method was used to establish a standard curve. A series of standard solutions with different concentrations were prepared, and the same amount of isotopic internal standard was added to each concentration point. The ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard in each standard solution was obtained using the above method. The standard curve was plotted with the analyte concentration on the x-axis and the peak area ratio on the y-axis. For the sample solution, the ratio of the peak area of ​​the analyte to the peak area of ​​the internal standard was measured in the same way, and the concentrations of each fat-soluble vitamin in the sample were calculated by substituting the values ​​into the standard curve.

[0025] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0026] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0027] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature being directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature being directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature. The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. A method for determining a plurality of fat-soluble vitamins based on mass spectrometry, characterized by: It comprises the following steps: S1: pretreatment of the sample, centrifugal take supernatant; S2: solid phase extraction on the supernatant to collect the eluent and redissolve; S3: take the redissolved solution after S2 processing into the high performance liquid chromatography system for gradient elution; S4: the components from the high performance liquid chromatography system are introduced into the tandem mass spectrometer for detection; S5: obtain the detection data in S4 for qualitative judgment and quantitative calculation, and obtain the content of each fat-soluble vitamin in the sample.

2. The method for determining a plurality of fat-soluble vitamins based on mass spectrometry according to claim 1, characterized by: The S1 includes taking the sample into the centrifugal tube, adding the isotopic internal standard mixed solution and the protein precipitant, taking the supernatant after vortex oscillation.

3. The method according to claim 1, wherein: the method is a method for determining a plurality of fat-soluble vitamins based on mass spectrometry. The S2 includes activating the solid phase extraction column into a hydrophilic-lipophilic balance solid phase extraction column, and sequentially injecting methanol and ultrapure water for activation.

4. The method according to claim 3, wherein the method is a method for determining a plurality of fat-soluble vitamins based on mass spectrometry. The S2 further includes: S2.1: slowly sample the supernatant in S1 into the activated solid phase extraction column, use methanol aqueous solution to wash the column body to remove impurities after the liquid completely passes through the column, discard the washing liquid, and then vacuum dry the column body; S2.2: use methanol-dichloromethane mixed solution to elute the target, collect the eluent, blow dry, redissolve through the organic phase filter membrane, and obtain the detection liquid.

5. The method for determining a plurality of fat-soluble vitamins based on mass spectrometry according to claim 1, characterized by: The S4 includes: S4.1: the components from the high performance liquid chromatography system are divided by a predetermined proportion through a shunt device; S4.2: introduce into the ionization source of the tandem mass spectrometer for ionization treatment; S4.3: the tandem mass spectrometer adopts a multiple reaction monitoring mode, and sets a characteristic parent ion-sub ion pair; S4.4: during the mass spectrometry process, periodically use standard products for quality calibration.

6. The method according to claim 5, wherein: the method is a method for determining a plurality of fat-soluble vitamins based on mass spectrometry. The predetermined shunt ratio in S4.1 is 1-5:

1.

7. The method according to claim 5, wherein: the method is a mass spectrometry-based method for determining a plurality of fat-soluble vitamins. The S4.3 includes obtaining the mass spectrum of each fat-soluble vitamin by full scan mode, determining the mass number of the parent ion, and selecting the product ion scanning of the selected parent ion to screen out the characteristic fragment ion as the daughter ion; Combine the parent ion-daughter ion pair of the isotopic internal standard to construct a multi-channel monitoring list for simultaneous qualitative and quantitative analysis of multiple fat-soluble vitamins.

8. The method according to claim 1, wherein: the method is a method for determining a plurality of fat-soluble vitamins based on mass spectrometry. The S5 includes using the internal standard method to establish a standard curve, and substituting the ratio of the peak area of the measured substance and the peak area of the internal standard into the standard curve to calculate the concentration.

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