Method for detecting content of fat-soluble vitamins a, d, e in blood

Abstract

The application discloses a detection method of fat-soluble vitamin A, D and E content in blood, which comprises the following steps: adding 250 muL of BHT internal standard solution into every 50 muL of blood sample; adding 20 muL of zinc sulfate heptahydrate solution into the blood sample after adding the BHT internal standard solution, and sequentially performing first mixing and centrifugal treatment; taking 100 muL of supernatant after the centrifugal treatment to perform concentration and drying; adding 50 muL of PTAD (4-phenyl-1,2,4-triazoline-3,5-diketone) acetonitrile solution into the supernatant after the concentration and drying to perform derivatization reaction at normal temperature, then adding a quenching agent to terminate the derivatization reaction, and obtaining a derivative sample; and taking 100 muL of supernatant after the centrifugal treatment to add into the derivative sample to perform mixing again and then detection. Through the screening of the quenching agent, the response of the vitamin can be obviously enhanced, and the detection of the fat-soluble vitamin A, D and E content can be quickly and accurately completed.

Description

Technical Field

[0001] This invention belongs to the field of vitamin detection technology, specifically relating to a method for detecting the content of fat-soluble vitamins A, D, and E in blood. Background Technology

[0002] Fat-soluble vitamins are a class of vitamins that are soluble in organic solvents but insoluble in water. Examples include vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E. Currently, methods for determining fat-soluble vitamins mainly include fluorescence analysis, enzyme immunoassay, liquid chromatography (LC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Among these, LC-MS / MS offers advantages such as high precision and sensitivity, enabling the detection of trace amounts of substances at low concentrations, and it also boasts high performance and reliability. Common pretreatment methods used in LC-MS / MS for analytes include liquid-liquid extraction, solid-liquid extraction, protein precipitation, and derivatization.

[0003] However, both liquid-liquid extraction (LiL-PE) and solid-liquid extraction (SLE) require multiple extraction, drying, and purification steps in their pretreatment, necessitating significant manual operation. Furthermore, LiL-PE is difficult to automate and achieve high throughput. SLE primarily relies on 96-well solid-phase supported liquid-liquid extraction plates (SLE plates), resulting in high costs and hindering cost reduction and efficiency improvement. Existing protein precipitation methods, after sample dilution, cannot meet the sensitivity requirements of low-performance instruments for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. Additionally, derivatization methods for determining fat-soluble vitamins exist, but these require cumbersome and time-consuming steps such as extraction, drying, derivatization, rehydration, and reprocessing before instrumentation. The derivatization process is lengthy (e.g., one hour) and requires stringent conditions (e.g., 70°C), hindering widespread adoption. Therefore, there is an urgent need to develop a convenient and rapid method for detecting fat-soluble vitamin content. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by proposing a method for detecting the content of fat-soluble vitamins A, D, and E in blood, which can quickly and accurately detect the content of fat-soluble vitamins A, D, and E.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The present invention proposes a method for detecting the content of fat-soluble vitamins A, D, and E in blood, comprising the following steps:

[0007] S1. Add 250 μL of BHT internal standard solution to every 50 μL of blood sample;

[0008] S2. Add 20 μL of zinc sulfate heptahydrate solution to the blood sample after adding BHT internal standard solution, and perform initial mixing and centrifugation in sequence.

[0009] S3. Take 100 μL of the supernatant after centrifugation and concentrate and dry it.

[0010] S4. At room temperature, add 50 μL of PTAD (4-phenyl-1,2,4-triazolline-3,5-dione) acetonitrile solution to the concentrated and dried supernatant to carry out the derivatization reaction, and then add a quencher to terminate the derivatization reaction to obtain the derivatized sample.

[0011] S5. Take 100 μL of the supernatant after centrifugation and add it to the derivatized sample for remixing before detection.

[0012] Preferably, the volume ratio of the BHT internal standard solution is methanol:acetonitrile:isopropanol = (5-20):(45-60):(30-35).

[0013] Preferably, the concentration of the BHT internal standard solution is 1 mg / mL to 5 mg / mL, and the concentration of the zinc sulfate heptahydrate solution is 0.1 mol / L to 0.5 mol / L.

[0014] Preferably, the concentration of the PTAD (4-phenyl-1,2,4-triazolline-3,5-dione) acetonitrile solution is 0.05 mg / mL to 2 mg / mL.

[0015] Preferably, the quenching agent is an ammonium acetate solution with a concentration of 0.1 mol / L to 0.5 mol / L.

[0016] Preferably, the first mixing is vortex mixing at a speed of 1000rpm-2500rpm for 3min-10min; the second mixing is vortex mixing at a speed of 300rpm-600rpm for 3min-10min; and the centrifugation is centrifuged at a speed of 3000rpm-5000rpm for 5min-30min.

[0017] Preferably, the derivatization reaction takes 10-40 minutes.

[0018] Preferably, the concentration temperature for concentration and drying is 30℃~45℃, and the concentration time is 15min~60min.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This method primarily utilizes protein precipitation pretreatment combined with derivatization technology. By optimizing the derivatization time and temperature, the derivatization process conditions are made easily achievable, meeting the requirements for recovery accuracy. It avoids the issues of incomplete derivatization due to short derivatization times affecting sample accuracy, and excessively long derivatization times hindering widespread adoption. It also avoids the problems of PTAD hydrolysis and poor derivatization at high temperatures, and inaccurate results at low temperatures. Furthermore, the post-derivatization quencher has been optimized. Since some PTAD in the derivatized sample reacts with vitamins, adding ammonium acetate solution (or alternatively, vitamin C, ammonium formate, vitamin A acetate, etc.) during derivatization quenches PTAD, improving vitamin detection. In particular, the quencher selection significantly enhances the response to vitamin A, enabling rapid and accurate detection of fat-soluble vitamins A, D, and E. It also significantly improves the detection sensitivity of 25-hydroxyvitamin in blood while maintaining the accuracy of vitamins A and E, resulting in more accurate and stable detection results. This approach helps meet the needs of detecting fat-soluble vitamins A, D, and E using low-performance instruments. Attached Figure Description

[0021] Figure 1 This is a flowchart of the method for detecting the content of fat-soluble vitamins A, D, and E in blood according to the present invention;

[0022] Figure 2 The MRM chromatogram of a sample (3 ng / mL) representing the limit of quantification of 25-hydroxyvitamin D2 using existing techniques with internal and external standards;

[0023] Figure 3 The MRM chromatogram of a sample (3 ng / mL) representing the limit of quantification of 25-hydroxyvitamin D3 using existing techniques with internal and external standards;

[0024] Figure 4 This is the chromatogram of internal and external standard MRM after PTAD derivatization of the 25-hydroxyvitamin D2 sample (3 ng / mL) according to the limit of quantification of this invention;

[0025] Figure 5 This is the chromatogram of internal and external standard MRM after PTAD derivatization of the 25-hydroxyvitamin D3 sample (3 ng / mL) for the limit of quantification of this invention;

[0026] Figure 6 This is the MRM chromatogram of the vitamin A lower limit of quantification sample (45 ng / mL) of the present invention, including both internal and external standards.

[0027] Figure 7 The internal and external standard MRM chromatograms of the vitamin A quantification limit sample (45 ng / mL) of the present invention were added to the sample.

[0028] Figure 8This is the MRM chromatogram of the vitamin E quantification limit sample (750 ng / mL) of the present invention, which is an internal and external standard. Detailed Implementation

[0029] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0030] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the application.

[0031] like Figure 1 As shown, a method for detecting the levels of fat-soluble vitamins A, D, and E in blood includes the following steps:

[0032] S1. Add 250 μL of BHT internal standard solution to every 50 μL of blood sample;

[0033] S2. Add 20 μL of zinc sulfate heptahydrate solution to the blood sample after adding BHT internal standard solution, and perform initial mixing and centrifugation in sequence.

[0034] S3. Take 100 μL of the supernatant after centrifugation and concentrate and dry it.

[0035] S4. At room temperature, add 50 μL of PTAD (4-phenyl-1,2,4-triazolline-3,5-dione) acetonitrile solution to the concentrated and dried supernatant to carry out the derivatization reaction, and then add a quencher to terminate the derivatization reaction to obtain the derivatized sample.

[0036] S5. Take 100 μL of the supernatant after centrifugation and add it to the derivatized sample for remixing before detection.

[0037] The blood sample can be serum, plasma, or capillary blood. This method mainly combines protein precipitation pretreatment with derivatization technology, and optimizes the pretreatment method. It rationally selects derivatization time, temperature, and post-derivatization quenching agents as optimal conditions for simple operation and short derivatization time. By adopting optimal conditions, the standard requirements for recovery rate can be met, and the vitamin response can be enhanced through derivatization.

[0038] In one embodiment, the volume ratio of the BHT internal standard solution is methanol:acetonitrile:isopropanol = (5-20):(45-60):(30-35).

[0039] In one embodiment, the concentration of the BHT internal standard solution is 1 mg / mL to 5 mg / mL, and the concentration of the zinc sulfate heptahydrate solution is 0.1 mol / L to 0.5 mol / L.

[0040] In one embodiment, the concentration of the PTAD (4-phenyl-1,2,4-triazolline-3,5-dione) acetonitrile solution is 0.05 mg / mL to 2 mg / mL.

[0041] In one embodiment, the quenching agent is an ammonium acetate solution with a concentration of 0.1 mol / L to 0.5 mol / L. Alternatively, it may be vitamin C, ammonium formate, vitamin A acetate, etc., which can be adjusted according to actual needs by those skilled in the art.

[0042] In one embodiment, the first mixing is vortex mixing at a speed of 1000 rpm to 2500 rpm for 3 min to 10 min; the second mixing is vortex mixing at a speed of 300 rpm to 600 rpm for 3 min to 10 min; and the centrifugation is performed at a speed of 3000 rpm to 5000 rpm for 5 min to 30 min.

[0043] In one embodiment, the derivatization reaction takes 10-40 minutes.

[0044] In one embodiment, the concentration temperature for the concentration drying is 30°C to 45°C, and the concentration time is 15 min to 60 min.

[0045] Exceeding the above parameter range will affect the accuracy of the final result.

[0046] The following detailed description is provided through specific embodiments.

[0047] The method for detecting the levels of fat-soluble vitamins A, D, and E in blood in this embodiment includes the following steps:

[0048] 1) Take 50 μL of blood sample into a 1.6 mL 96-well deep plate and add 250 μL of BHT internal standard solution with a concentration of 5 mg / mL (methanol:acetonitrile:isopropanol = 15:55:30);

[0049] 2) Add 20 μL of precipitant (such as 0.2 mol / L zinc sulfate heptahydrate solution) to the solution obtained in step 1), vortex at 2000 rpm for 5 min, and centrifuge at 4000 rpm for 15 min.

[0050] 3) Take 100 μL of the supernatant after centrifugation and concentrate and dry it in a 330 μL 96-well deep plate;

[0051] 4) Add 50 μL of PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) acetonitrile solution with a concentration of 0.5 mg / mL to the dried sample. After reacting at room temperature for 30 min, add 0.5 mol / L ammonium acetate solution to terminate the reaction (because vitamin A readily reacts with PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) acetonitrile solution, adding a portion of ammonium acetate solution beforehand during the quenching process can rapidly quench the PTAD derivatization reagent (PTAD (4-phenyl-1,2,4-triazoline-3,5-dione) acetonitrile solution), thereby improving the response of vitamin A).

[0052] 5) Take 100 μL of the supernatant after centrifugation and add it to the derivatized sample obtained in step 4). Mix well at 500 rpm for 5 min and then detect.

[0053] In this embodiment, PTAD derivatization reagent was used to derivatize vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively) to form easily ionized polymeric derivatives, which significantly improved the sensitivity of liquid chromatography-mass spectrometry determination of the compounds.

[0054] a) The chemical formula for the 25-hydroxyvitamin D2 derivatization reaction is as follows:

[0055]

[0056] b) The chemical formula for the 25-hydroxyvitamin D3 derivatization reaction is as follows:

[0057]

[0058] Current technology uses liquid chromatography-tandem mass spectrometry (LC-MS / MS) according to national standards. However, for blood samples with low concentrations, the sensitivity of mass spectrometry analysis is insufficient to meet the detection requirements, making accurate quantification impossible. Figure 2 and Figure 3 As shown, the peak area response of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 outside the limit of quantitation is low before derivatization, which cannot meet the quantitative requirements, affects the stability of the detection results, and cannot guarantee the accuracy of the sample. Figures 2-8 The upper figure in the image represents the extracted ion chromatogram of external standard mass spectrometry (external standard MRM chromatogram), and the lower figure represents the extracted ion chromatogram of internal standard mass spectrometry (internal standard MRM chromatogram).

[0059] The 25(OH)D derivatized product (i.e., the result on the right side of the equation in the above chemical formula) has two configurations: 6S and 6R. For ease of quantification, the dominant peak of the 6S configuration was selected for quantitative analysis. The 25(OH)D derivatized product readily loses one molecule of water in the mass spectrometry environment, and the resulting quasi-molecular ion dehydration peak is the dominant peak in the ESI (electrospray ionization) primary mass spectrum of this derivative. Therefore, [M+H-H2O]+ was selected as the parent ion for quantitative analysis using multiple reaction monitoring (MRM). Samples after the derivatization reaction are shown below. Figure 4 and Figure 5 It can be seen that the chromatographic peak responses of the internal and external standards of the derivatized 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 can meet the detection requirements. That is, when the target compound is analyzed by this method, there are no interfering peaks in the MRM chromatogram, the selectivity is strong, and the sensitivity of the compound can be greatly increased, thus improving the accuracy of quantification.

[0060] Because PTAD derivatizing reagents readily react with the conjugated double bonds in vitamin A, leading to a decrease in the vitamin A response, such as... Figure 6 As shown, it is evident that the vitamin A external standard was significantly derivatized before the addition of the quencher. Therefore, after the derivatization reaction is complete, adding an appropriate concentration of ammonium acetate solution to quench the reaction, thus terminating the derivatization reaction and improving the accuracy of the vitamin A standard. Figure 7 As shown, the response of vitamin A was significantly improved after quenching with ammonium acetate solution, enabling accurate quantification. Figure 8 This indicates that the PTAD derivatization reagent has no effect on the detection of vitamin E.

[0061] In addition, the following tests were also conducted in this embodiment: the test results for linearity and quality control products are shown in Table 1, the optimization results for derivation time are shown in Table 2, the optimization results for derivation temperature are shown in Table 3, and the optimization results for the quencher after derivation are shown in Table 4.

[0062] Table 1. Results of linearity and quality control tests using the protein precipitation method + derivatization method.

[0063]

[0064] The results in Table 1 show that the protein precipitation method combined with the derivatization method can meet the detection requirements of vitamin A in the linear range of 45-1200 ng / mL, 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 in the linear range of 3-80 ng / mL, and vitamin E in the linear range of 750-25000 ng / mL. Therefore, the selection of the protein precipitation method plus the derivatization method for subsequent parameter optimization proves the feasibility of the method in this application.

[0065] Table 2

[0066]

[0067]

[0068] Table 2 shows that the derivatization time of 10-15 min had no significant effect on the concentrations of vitamin A and vitamin E. The concentration of 25-hydroxyvitamin D derivatives increased with time, but there was no significant difference in the concentration of 25-hydroxyvitamin D derivatives between 30-60 min. Therefore, the optimal derivatization time was selected as 15 min. The target values ​​were the traceability concentrations of vitamins A, D, and E, and the lower limit samples were samples with the lower limit of quantitation concentrations.

[0069] Table 3

[0070]

[0071]

[0072] Table 3 shows that the derivatization temperature had no significant effect on vitamin A and vitamin E. Within the derivatization temperature range of 20-30℃, the concentration of the 25-hydroxyvitamin D derivative compound did not differ significantly. Within the range of 30-50℃, the concentration decreased with increasing temperature. During the derivatization process, it was found that some water vapor entered the sample during temperature increases, causing premature hydrolysis of the derivatizing reagent, thus hindering the derivatization effect. Furthermore, derivatization within the 20-30℃ range was sufficient for instrument detection; therefore, room temperature was chosen as the derivatization temperature, with 25℃ being the optimal temperature.

[0073] Table 4

[0074]

[0075]

[0076] The results in Table 4 show that using ammonium acid substances as quenchers can significantly improve the response of vitamin A. Among the two quenchers mentioned above, ammonium acetate is more effective, and the effect is best when the addition amount is 0.5M.

[0077] In summary, the method in this embodiment achieves the best results when the derivatization time is 15 min, the derivatization temperature is 25 °C, and the quenching agent after derivatization is a 0.5 mol / L ammonium acetate solution.

[0078] The accuracy (addition recovery rate) was determined using the method described in this application, as shown in Table 5.

[0079] Table 5

[0080]

[0081] Based on the optimized derivatization times (5, 10, 15, 30, 60 min), derivatization temperatures (20, 25, 30, 40, 50 °C), and quencher selection results, it can be seen that this method, by combining protein precipitation pretreatment with derivatization, and optimizing the concentration of derivatization reagents, derivatization temperature, derivatization time, quencher selection, and quencher concentration, significantly improves the sensitivity of fat-soluble vitamins in serum, enabling low-performance instruments to meet the detection requirements of fat-soluble vitamins A, D, and E.

[0082] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The embodiments described above are merely specific and detailed examples of the embodiments described in this application, and should not be construed as limiting the scope of the application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these modifications and improvements all fall within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the appended claims.

Claims

1. A method for detecting the content of fat-soluble vitamins A, D, and E in blood, characterized in that: The method for detecting the levels of fat-soluble vitamins A, D, and E in blood includes the following steps: S1. Take a blood sample and add 250 μL of BHT internal standard solution to every 50 μL. The volume ratio of the BHT internal standard solution is methanol:acetonitrile:isopropanol = (5-20):(45-60):(30-35), and the concentration of the BHT internal standard solution is 1 mg / mL to 5 mg / mL. S2. Add 20 μL of zinc sulfate heptahydrate solution to the blood sample after adding BHT internal standard solution, and perform initial mixing and centrifugation in sequence. S3. Take 100 μL of the supernatant after centrifugation and concentrate and dry it. S4. At room temperature, add 50 μL of 4-phenyl-1,2,4-triazolline-3,5-dione acetonitrile solution to the concentrated and dried supernatant to carry out the derivatization reaction. Then, add a quencher to terminate the derivatization reaction and obtain a derivatized sample. The quencher is ammonium acetate solution with a concentration of 0.1 mol / L-0.5 mol / L. The derivatization reaction takes 10 min-15 min and the temperature is 20℃-30℃. S5. Take 100 μL of the supernatant after centrifugation and add it to the derivatized sample. Mix well again and then perform liquid chromatography-tandem mass spectrometry detection.

2. The method for detecting the content of fat-soluble vitamins A, D, and E in blood as described in claim 1, characterized in that: The concentration of the zinc sulfate heptahydrate solution is 0.1 mol / L to 0.5 mol / L.

3. The method for detecting the content of fat-soluble vitamins A, D, and E in blood as described in claim 1, characterized in that: The concentration of the 4-phenyl-1,2,4-triazolline-3,5-dione acetonitrile solution is 0.05 mg / mL to 2 mg / mL.

4. The method for detecting the content of fat-soluble vitamins A, D, and E in blood as described in claim 1, characterized in that: The first mixing is vortex mixing at a speed of 1000rpm-2500rpm for 3min-10min; the second mixing is vortex mixing at a speed of 300rpm-600rpm for 3min-10min; the centrifugation is centrifuged at a speed of 3000rpm-5000rpm for 5min-30min.

5. The method for detecting the content of fat-soluble vitamins A, D, and E in blood as described in claim 1, characterized in that: The concentration temperature for the concentration and drying process is 30℃~45℃, and the concentration time is 15min~60min.

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