Method for simultaneously detecting multiple fat-soluble vitamins

Abstract

The invention relates to the field of detection, in particular to a method for simultaneously detecting multiple fat-soluble vitamins, which comprises the following steps: (S.1) carrying out protein precipitation and liquid-liquid extraction on a sample to be detected to obtain an extract containing fat-soluble vitamins; (S.2) carrying out derivatization reaction on the extract by using a PTAD derivatization reagent; and (S.3) analyzing the derivatized product by adopting a liquid chromatography-tandem mass spectrometry method, wherein the detected target fat-soluble vitamins comprise vitamin A, vitamin E, 25-hydroxy vitamin D2, 25-hydroxy vitamin D3, vitamin K1, vitamin K2 and vitamin K2. According to the application, PTAD is creatively adopted as a unified derivatization reagent, simultaneous and efficient derivatization of seven fat-soluble vitamins is successfully realized, the detection sensitivity of low-content targets, especially vitamin D, K2 and the like, is greatly improved, and the core problems of weak signal response and difficulty in accurate quantification are solved.

Description

Technical Field

[0001] This invention relates to the field of detection, and more particularly to a method for simultaneously detecting multiple fat-soluble vitamins. Background Technology

[0002] In clinical diagnosis and nutritional research, the accurate detection of fat-soluble vitamins is of paramount importance. Vitamins A, D, E, and K jointly participate in many core physiological processes in the human body, such as maintaining vision, calcium and phosphorus metabolism, antioxidation, and blood clotting. Abnormalities in their serum concentrations are closely related to various diseases, including rickets, osteoporosis, neurological disorders, and bleeding tendencies. Therefore, establishing a method that can accurately and efficiently assess the levels of multiple fat-soluble vitamins in the human body is of irreplaceable value for disease prevention, diagnosis, and treatment monitoring.

[0003] Currently, the industry has developed various technical approaches for the detection of fat-soluble vitamins, each with its own application scenarios, but all also have significant limitations. Early detection methods, such as radioimmunoassay and chemiluminescence, while providing feasible solutions in their early stages, typically suffer from poor specificity, are easily affected by interference from other structural analogs in the sample, leading to an increased risk of false positives or false negatives, and are difficult to use for simultaneous analysis of multiple vitamins, resulting in low efficiency. With advancements in chromatography, high-performance liquid chromatography (HPLC) has gradually gained application, significantly improving its separation capabilities. However, for trace amounts of vitamins in serum, especially vitamins D and K, its detection sensitivity often falls short of the clinical requirements for precise quantification of low-concentration samples.

[0004] To overcome sensitivity bottlenecks, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) has become the mainstream choice. This technique combines the superior separation capabilities of chromatography with the powerful qualitative and quantitative functions of mass spectrometry, offering high sensitivity, high specificity, and rapid analysis. Numerous methods for detecting single or a few fat-soluble vitamins using LC-MS / MS have been reported and validated in practice. However, when the target is expanded to simultaneously cover the vitamin A, D, E, and K families, significant challenges arise. The primary difficulty lies in the extreme complexity of serum sample matrices and the orders of magnitude differences in physiological concentrations among different fat-soluble vitamins. For example, vitamins A and E are relatively abundant, typically at the μg / mL level, while vitamin D and its metabolites, as well as vitamins K1 and K2 (such as MK-4 and MK-7), are much less abundant, at the ng / mL or even pg / mL level. This significant concentration gap places almost stringent requirements on the dynamic range of instrument detection. To accurately determine all target analytes in a single injection, the signal intensity of low-abundance vitamins D and K is often submerged in background noise, making effective detection and quantification impossible.

[0005] To address the issue of insufficient sensitivity in the detection of low-concentration compounds, chemical derivatization techniques have been introduced as an effective strategy to enhance mass spectrometry ionization efficiency. By reacting derivatizing reagents with target molecules, their mass spectrometric behavior can be altered, typically significantly improving ionization efficiency and thus amplifying the detection signal. Existing technologies do indeed include some derivatization methods for vitamins A, D, E, or K1, which have improved the detection limits of these vitamins to some extent. However, these methods often have limitations: either the derivatization target range is narrow, failing to consider the important vitamin K2 subtype (especially MK-4 and MK-7 with different side chain lengths); or the derivatization reaction conditions are incompatible with all target compounds, resulting in low derivatization efficiency or no derivatization at all for some vitamins. Furthermore, an ideal comprehensive screening scheme also faces the challenge of cumbersome pretreatment procedures. If different extraction, purification, or derivatization steps are required for vitamins of different properties or concentration ranges, and multiple processed samples are then analyzed separately, it will undoubtedly increase the sample volume, operation time, reagent costs, and human error many times over, making it difficult to promote and apply the method on a large scale in routine clinical testing laboratories and losing its practical value for high-throughput screening.

[0006] Therefore, there is an urgent need in this field for a new technical solution that can overcome the above-mentioned multiple obstacles and achieve true "one-stop" testing. Summary of the Invention

[0007] This application aims to overcome the shortcomings of existing technologies that cannot simultaneously and with high sensitivity analyze serum fat-soluble vitamins A, E, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin K1, vitamin K2 (MK-4), and vitamin K2 (MK-7) in a single detection using a unified derivatization method. Therefore, it provides a method for simultaneously detecting multiple fat-soluble vitamins to overcome the above-mentioned deficiencies.

[0008] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a method for simultaneously detecting multiple fat-soluble vitamins, comprising the following steps: (S.1) Protein precipitation and liquid-liquid extraction were performed on the sample to be tested to obtain an extract containing fat-soluble vitamins; (S.2) The extract was derivatized using the PTAD derivatization reagent; (S.3) The derivatized products were analyzed by liquid chromatography-tandem mass spectrometry. The target fat-soluble vitamins to be detected included vitamin A, vitamin E, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin K1, vitamin K2 (MK-4) and vitamin K2 (MK-7).

[0009] As described in the background section, while the detection of fat-soluble vitamins using LC-MS / MS technology is well-established, existing methods exhibit significant limitations when the goal shifts to simultaneously covering all key members of vitamins A, D, E, and K, particularly when including vitamin K2 (MK-4 and MK-7), which have extremely low physiological concentrations and structural differences. A particularly challenging aspect is that existing derivatization strategies were mostly designed for specific vitamin classes, lacking a universal method capable of encompassing all seven target vitamins and significantly enhancing their mass spectrometry response. This limitation forces laboratories to either perform multiple separate assays, resulting in low efficiency and high sample consumption, or abandon precise monitoring of some vitamins (such as K2), failing to provide a complete nutritional status assessment. Therefore, the industry urgently needs an innovative solution that integrates sample pretreatment, unified derivatization, and achieves highly sensitive simultaneous detection.

[0010] To address this complex challenge, the core inventiveness of this invention lies in the unexpected selection of PTAD (4-phenyl-1,2,4-triazolline-3,5-dione) as a unified derivatization reagent, and its systematic integration with a meticulously designed sample pretreatment process and LC-MS / MS detection conditions. Extensive experimental verification revealed that PTAD can effectively derivatize these seven structurally diverse target compounds, laying the foundation for a unified processing flow. Building upon this, the scheme further designs a coherent operational sequence: first, proteins are precipitated using a methanol-acetonitrile mixed solvent to remove major matrix interferences; then, liquid-liquid extraction with n-hexane is performed to efficiently enrich lipid-soluble target compounds; finally, PTAD derivatization is directly performed in the concentrated and dried extract. The ingenuity of this process lies in its seamless integration of extraction, purification, and derivatization steps, which might otherwise require separate steps, forming a standardized and repeatable operating procedure that significantly reduces operational complexity and the risk of human error.

[0011] Ultimately, this complete technical solution achieves true "one-time injection, comprehensive analysis." Through PTAD derivatization, the ionization efficiency of vitamin D and all forms of vitamin K (including K1, MK-4, and MK-7), which previously exhibited weak responses in mass spectrometry, was significantly improved. This allows for clear and stable detection and quantification of these substances using the same method, effectively solving the core challenge of insufficient sensitivity in detecting low-abundance target analytes. Simultaneously, the standardized sample pretreatment process greatly simplifies the operation, significantly reducing analysis time, reagent costs, and required sample volume. This makes the method practically valuable for large-scale clinical sample screening and possesses high-throughput potential. The method demonstrates good precision and accuracy, indicating strong resistance to matrix interference and stable and reliable results.

[0012] In summary, by selecting a key universal derivatization reagent (PTAD) and constructing a novel and optimized systematic detection process around it, this invention successfully solves multiple challenges in the simultaneous detection of multiple fat-soluble vitamins, including sensitivity, throughput, and ease of operation, providing an unprecedented, efficient, comprehensive, and reliable solution for clinical use.

[0013] Preferably, step (S.1) specifically includes: adding a protein precipitant to the sample to be tested, vortexing and mixing, adding n-hexane for liquid-liquid extraction, centrifuging and taking the organic phase, drying it with nitrogen to obtain the extract.

[0014] Preferably, the protein precipitant is a mixed solution of methanol and acetonitrile.

[0015] Preferably, in the derivatization reaction of step (S.1), the concentration range of PTAD derivatization reagent is 100-1000 μg / mL, the derivatization reaction is carried out at room temperature, and the reaction time is 5-15 minutes.

[0016] Although PTAD is known as a Diels-Alder reaction derivatization reagent, its application to the complex multi-component systems targeted in this invention—that is, systems simultaneously containing vitamin A, E, D metabolites, K1, and different forms of K2 (MK-4, MK-7)—requires a much more complex process than simply applying existing literature or conducting routine experiments. Different target molecules exhibit variations in spatial structure, accessibility of reaction sites, and inherent reactivity. This means that derivatization conditions suitable for a particular class of vitamins (e.g., only vitamin D) may not simultaneously guarantee optimal and stable derivatization efficiency for other types of vitamins (especially vitamin K2). If the reagent concentration is too low or the reaction time is too short, vitamins with low reactivity (such as some K2 homologues) may not be completely derivatized, thus failing to fully enhance their detection sensitivity and defeating the purpose of derivatization. Conversely, if the reagent concentration is too high or the reaction time is too long, some vitamins that have already completed the reaction (such as vitamins A or E) may undergo over-derivatization, produce byproducts, or even degrade. This not only fails to enhance the signal but may also introduce additional impurities, increasing the difficulty of chromatographic separation and affecting the accuracy of quantification.

[0017] Therefore, the reagent concentration range of 100-1000 μg / mL and the mild reaction conditions of room temperature and 5-15 minutes, determined through extensive experimental optimization, ensure that the PTAD reagent provides sufficient but not excessive reaction driving force for all seven target analytes. This allows for efficient and stable derivatization reactions to be completed simultaneously and within a relatively short time without the need for harsh heating or lengthy waiting periods. This results in stable and reliable signal responses for subsequent LC-MS / MS detection, laying a solid foundation for high-precision quantitative analysis.

[0018] Preferably, the derivatization reaction is terminated by adding ethanol.

[0019] Preferably, the liquid chromatography in step (S.3) uses a reversed-phase column.

[0020] Preferably, the reversed-phase chromatographic column is a C18 column, the mobile phase A of the liquid chromatography is an aqueous solution containing formic acid, the mobile phase B is acetonitrile, and gradient elution is used.

[0021] Preferably, in step (S.3), the tandem mass spectrometry uses an electrospray ionization source and is performed in positive ion mode, with the detection mode being multiple reaction monitoring mode.

[0022] Preferably, before step (S.1), the method further includes adding an isotope internal standard solution to the sample to be tested, wherein the isotope internal standard is a deuterated product corresponding to the target fat-soluble vitamin.

[0023] As a preferred method, the internal standard method is used for quantitative analysis. A standard curve is established with the ratio of the concentration of the analyte to the concentration of the isotope internal standard as the abscissa and the ratio of the peak area of ​​the analyte to the peak area of ​​the isotope internal standard as the ordinate, and the concentration of the analyte in the sample is calculated.

[0024] PTAD was used to uniformly derivatize all seven target analytes to improve sensitivity. However, the derivatization efficiency can be affected by minor fluctuations in factors such as sample matrix, temperature, and time, directly impacting the amount of product entering the mass spectrometer. Furthermore, a standardized pretreatment process (including liquid-liquid extraction and nitrogen blowing reconstitution) inevitably introduces operational errors and losses between samples. If only the external standard method is used, these variations generated during sample preparation and derivatization will directly translate to the final detection signal, leading to decreased accuracy and repeatability of the quantitative results.

[0025] This invention precisely configures corresponding deuterated isotope internal standards for each analyte. These internal standards are chemically almost identical to their corresponding target vitamins. Therefore, throughout the entire pretreatment process (such as protein precipitation and extraction) and the crucial PTAD derivatization reaction, the internal standards and target analytes undergo the same efficiency changes and losses. As a result, the final measured peak area ratio of the target analyte to the internal standard can effectively offset most of the variations and interferences introduced throughout the entire process from sample preparation to instrumental analysis.

[0026] Therefore, by establishing a calibration curve with the concentration ratio of the analyte to the internal standard as the x-axis and the peak area ratio as the y-axis, an "internal calibration" system relative to the entire analytical system is essentially constructed. This allows the method to still exhibit excellent precision and accuracy when faced with complex serum matrices and multi-step processing procedures, ensuring the stability and reliability of detection results for low-concentration components such as vitamin D and K2.

[0027] As a preferred method, the seven fat-soluble vitamins were separated and detected in a single injection.

[0028] The method provided in this application has the following significant beneficial effects: First, this application innovatively uses PTAD as a unified derivatization reagent, which successfully achieves simultaneous and efficient derivatization of seven fat-soluble vitamins with significant differences in physicochemical properties, namely vitamins A, E, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin K1, MK-4 and MK-7. This significantly improves the detection sensitivity of low-content target substances, especially vitamins D and K2, and solves the core problem of weak signal response and difficulty in accurate quantification. Secondly, this application designs a coherent and efficient sample pretreatment process that organically combines protein precipitation, liquid-liquid extraction and derivatization steps, which significantly simplifies the operation, reduces sample volume, reagent costs and human error, and improves analytical efficiency and throughput, making it more suitable for large-scale clinical sample screening. Finally, by combining carefully optimized chromatographic-mass spectrometry conditions with isotope internal standard quantification, this application demonstrates excellent precision and accuracy, strong resistance to matrix interference, and stable and reliable results, ensuring the accuracy of quantification across different concentration ranges. Attached Figure Description

[0029] Figure 1 This is a standard curve graph for seven fat-soluble vitamins.

[0030] Figure 2 This is a liquid chromatogram of a vitamin A standard.

[0031] Figure 3 The liquid chromatogram is for a 25-hydroxyvitamin D2 standard.

[0032] Figure 4 The liquid chromatogram is for a 25-hydroxyvitamin D3 standard.

[0033] Figure 5 This is a liquid chromatogram of a vitamin E standard.

[0034] Figure 6 This is a liquid chromatogram of a vitamin K1 standard.

[0035] Figure 7 This is a liquid chromatogram of vitamin MK4 standard.

[0036] Figure 8 This is a liquid chromatogram of vitamin MK7 standard.

[0037] Figure 9 This is a liquid chromatogram of vitamin A standard in a serum sample.

[0038] Figure 10 This is a liquid chromatogram of 25-hydroxyvitamin D2 in a serum sample.

[0039] Figure 11 This is a liquid chromatogram of 25-hydroxyvitamin D3 in a serum sample.

[0040] Figure 12 This is a liquid chromatogram of vitamin E standard in a serum sample.

[0041] Figure 13 This is a liquid chromatogram of vitamin K1 standard in a serum sample.

[0042] Figure 14 This is a liquid chromatogram of vitamin MK4 standard in a serum sample.

[0043] Figure 15 This is a liquid chromatogram of vitamin MK7 standard in a serum sample. Detailed Implementation

[0044] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0045] This embodiment provides a method for simultaneously detecting fat-soluble vitamins A, D, E, K1, and K2 in serum samples, comprising the following steps: 1. Solution preparation: (1) Preparation of standard stock solution of target analyte: Accurately weigh VA, 25-OH-D2, 25-OH-D3 and VE, and prepare standard stock solution with methanol with a concentration of 1 mg / mL. Prepare standard stock solution with anhydrous ethanol with a concentration of 1 mg / mL. Store in a refrigerator at -20℃ protected from light.

[0046] (2) Preparation of mixed standard solution: Pipette each target analyte standard into a volumetric flask, dilute to volume with methanol, and prepare a mixed standard solution with concentrations of VA: 2 μg / mL, 25-OH-D2 and 25-OH-D3: 200 ng / mL, VE: 20 μg / mL, VK1, MK-4, MK-7: 20 ng / mL.

[0047] (3) Preparation of mixed isotope internal standard solution: Take the isotope internal standard solution of each target analyte, dilute with methanol to prepare mixed isotope internal standard solutions with concentrations of VA-d6 and VE-d6: 10 μg / mL, 25-OH-D2-d3 and 25-OH-D3-d6: 1 μg / mL, and VK1-d7, MK-4-d7, and MK-7-d7: 100 ng / mL.

[0048] (4) Preparation of standard curve points: Take a pipette of the mixed standard solution and dilute it stepwise with methanol to prepare concentrations as follows: VA: 0.0156 μg / mL, 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL; 25-OH-D2 and 25-OH-D3: 1.56 ng / mL, 6.25 ng / mL, 12.5 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL; VE: 0.156 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL; VK1, MK-4 and MK-7: Standard curve points for 0.156 ng / mL, 0.625 ng / mL, 1.25 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL. The standard curves for the seven fat-soluble vitamins are shown below. Figure 1 As shown.

[0049] 2. Serum sample and standard curve point processing: Take 100 μL of serum sample or standard curve point, add 10 μL of mixed isotope internal standard solution and 80 μL of ultrapure water (4% BSA for standard curve point), vortex to mix, add 200 μL of methanol-acetonitrile (50:50, v / v) to precipitate protein, vortex for 30 s, add 900 μL of n-hexane, vortex for 1 min, centrifuge at 12000 rpm for 5 min at 4℃, take 800 μL of supernatant, blow dry with nitrogen, add 100 μL of LPTAD derivatization solution with a concentration of 100-1000 μg / mL (500 μg / mL in this example), incubate at room temperature (25℃) for 5-15 min (10 min in this example), add 10 μL of anhydrous ethanol to terminate the reaction, transfer to a brown sample vial with an inner tube, and perform HPLC-MS / MS analysis.

[0050] 3. Chromatographic column: Waters ACQUITY UPLC BEH Shield RP18 (2.1×50mm, 1.7μm).

[0051] 4. Testing instrument: Shimadzu 8060LC-MS / MS.

[0052] 5. Liquid phase conditions: Mobile phase: Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: pure acetonitrile; Elution gradient: 0-2 min 40% B, 2-4 min 40-60% B, 4-5 min 60-100% B, 5-7 min 100% B, 7-7.5 min 100-40% B, 7.5-9 min 40% B; Column temperature: 40℃; Flow rate: 0.3 mL / min; Injection volume: 5 μL.

[0053] The liquid chromatograms of vitamin A standards, 25-hydroxyvitamin D2 standards, 25-hydroxyvitamin D3 standards, vitamin E standards, vitamin K1 standards, vitamin MK4 standards, vitamin MK7 standards, and serum samples of vitamin A standards, 25-hydroxyvitamin D2 standards, 25-hydroxyvitamin D3 standards, vitamin E standards, vitamin K1 standards, vitamin MK4 standards, and vitamin MK7 standards are shown below. Figures 2-15 As shown.

[0054] 6. Mass spectrometry conditions: Ion source: Electrospray ionization (ESI) source. In positive ion mode, the ion source temperature is 500℃; the atomizing gas flow rate is 3.0L / min; the drying gas flow rate is 10.0L / min; and the heating gas flow rate is 10.0L / min. The MRM (Multi-Reaction Monitoring) mode is used for detection, and the MRM parameters are shown in Table 1.

[0055] Table 1. MRM parameters of each compound compound Parent ion (m / z) Daughter ions (m / z) CE(V) VA 637.2 272.1 16 VA-d6 643.2 272.1 16 25-OH-D2 570.4 298.0 18 25-OH-D2-d3 573.4 301.0 18 25-OH-D3 558.4 298.2 16 25-OH-D2-d6 564.4 298.2 16 VE 781.4 604.4 20 VE-d6 787.4 610.4 20 VK1 626.5 449.2 15 VK1-d7 633.5 456.2 15 MK-4 620.4 443.1 14 MK-4-d7 627.4 450.1 14 MK-7 825.4 586.2 25 MK-7-d7 832.4 593.2 25

[0056] From the table above, we can determine that the detection ion pairs for derivatized VA and internal standard VA-d6 are 637.2 m / z→272.1 and 643.2 m / z→272.1, respectively; the detection ion pairs for VE and internal standard VE-d6 are 781.4 m / z→604.4 and 787.4 m / z→610.4, respectively; the detection ion pairs for 25-OH-D2 and internal standard 25-OH-D2-d3 are 570.4 m / z→298.0 and 573.4 m / z→301.0, respectively; and the detection ion pairs for 25-OH-D3 and internal standard 25-OH-D3-d6 are... The detected ion pairs were 558.4 m / z→298.2 and 564.4 m / z→298.2, respectively; the detected ion pairs of VK1 and internal standard VK1-d7 were 626.5 m / z→449.2 and 633.5 m / z→456.2, respectively; the detected ion pairs of MK-4 and internal standard MK-4-d7 were 620.2 m / z→443.4 and 627.2 m / z→450.4, respectively; and the detected ion pairs of MK-7 and internal standard MK-7-d7 were 825.4 m / z→586.2 and 832.4 m / z→593.2, respectively.

[0057] 7. Establish standard curves for seven vitamins using the internal standard method: A calibration curve was established using the ratio of the analyte concentration to the internal isotope standard concentration as the X-axis and the ratio of the analyte peak area to the internal isotope standard peak area as the Y-axis, thereby calculating the analyte concentration in the sample. The linear regression equation and linear correlation coefficient are shown in Table 2.

[0058] Table 2. Linear regression equations and correlation coefficients for the standard curves of seven vitamins. .

[0059] 8. Precision: Three replicate samples at low, medium, and high concentration levels were tested on the same day to assess intra-day precision, and tests were conducted for three consecutive days to assess inter-day precision. Method precision is expressed as relative standard deviation (RSD). The precision results are shown in Table 3. The precision (RSD) for each level of the seven vitamins was less than 15%, meeting the detection requirements.

[0060] Table 3. Intra-day and inter-day precision of methods for determining the content of seven vitamins in human serum. .

[0061] 9. Spike recovery rate: Clinical serum samples were selected and mixed, then divided into four equal volumes of 100 μL each. One sample was treated with 10 μL of methanol as a baseline sample, and analyzed using this method. The other three samples were treated with 10 μL of low, medium, and high concentrations of the analyte standard solution, respectively, and analyzed using this method. The recoveries at the low, medium, and high concentration levels were statistically analyzed. The recovery rate was calculated as (sample value after spiking - baseline sample value) / theoretical value × 100%. The results are shown in Table 4. The recoveries of the seven vitamins at all three concentration levels met the requirements.

[0062] Table 4. Spiked recovery rates of methods for determining the content of seven vitamins in human serum.

[0063] The test results above demonstrate that this invention establishes a method for the simultaneous detection of fat-soluble vitamins A, D (25-OH-D2 and 25-OH-D3), E, ​​K1, and K2 (MK-4 and MK-7) in serum samples. This method enables comprehensive detection of seven fat-soluble vitamins in a single injection, improving sample detection efficiency. Furthermore, this invention enhances the ionization efficiency of fat-soluble vitamins through derivatization after sample extraction, effectively reducing matrix interference and significantly improving detection sensitivity.

[0064] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for simultaneously detecting multiple fat-soluble vitamins, characterized in that, Includes the following steps: (S.1) Protein precipitation and liquid-liquid extraction were performed on the sample to be tested to obtain an extract containing fat-soluble vitamins; (S.2) The extract was derivatized using the PTAD derivatization reagent; (S.3) The derivatized products were analyzed by liquid chromatography-tandem mass spectrometry. The target fat-soluble vitamins to be detected included vitamin A, vitamin E, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, vitamin K1, vitamin K2 (MK-4) and vitamin K2 (MK-7).

2. The method according to claim 1, characterized in that, The specific steps (S.1) include: adding a protein precipitant to the sample to be tested, vortexing and mixing, adding n-hexane for liquid-liquid extraction, centrifuging and taking the organic phase, drying it with nitrogen to obtain the extract.

3. The method according to claim 2, characterized in that, The protein precipitant is a mixed solution of methanol and acetonitrile.

4. The method according to claim 1, characterized in that, In the derivatization reaction of step (S.1), the concentration range of PTAD derivatization reagent is 100-1000 μg / mL, the derivatization reaction is carried out at room temperature, and the reaction time is 5-15 minutes.

5. The method according to claim 4, characterized in that, The derivatization reaction was terminated by the addition of ethanol.

6. The method according to claim 1, characterized in that, In step (S.3), the liquid chromatography uses a reversed-phase column.

7. The method according to claim 6, characterized in that, The reversed-phase chromatographic column is a C18 column. The mobile phase A of the liquid chromatography is an aqueous solution containing formic acid, and the mobile phase B is acetonitrile, using gradient elution.

8. The method according to claim 1, characterized in that, In step (S.3), the tandem mass spectrometry uses an electrospray ionization source and is performed in positive ion mode. The detection mode is multiple reaction monitoring mode.

9. The method according to claim 1, characterized in that, Before step (S.1), the method further includes adding an isotope internal standard solution to the sample to be tested, wherein the isotope internal standard is a deuterated product corresponding to the target fat-soluble vitamin.

10. The method according to claim 9, characterized in that, Quantitative analysis was performed using the internal standard method. A standard curve was established with the ratio of the concentration of the analyte to the concentration of the isotopic internal standard as the abscissa and the ratio of the peak area of ​​the analyte to the peak area of ​​the isotopic internal standard as the ordinate, and the concentration of the analyte in the sample was calculated.

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