Method for detecting contents of fat-soluble vitamins A, D and E in blood

Abstract

The invention discloses a method for detecting the content of fat-soluble vitamins A, D and E in blood. The method comprises the following steps: adding 250 mu L of BHT (butylated hydroxytoluene) internal standard solution into every 50 mu L of a blood sample; adding 20 [mu] L of a zinc sulfate heptahydrate solution into the blood sample added with the BHT internal standard solution, and sequentially carrying out primary uniform mixing and centrifugal treatment; taking 100 microliters of centrifuged supernate, and concentrating and drying; adding 50 [mu] L of a PTAD (4-phenyl-1, 2, 4-triazoline-3, 5-diketone) acetonitrile solution into the concentrated and dried supernatant at normal temperature, carrying out a derivatization reaction, and then adding a quenching agent to terminate the derivatization reaction so as to obtain a derivative sample; and adding 100 microliters of centrifuged supernate into the derivative sample, uniformly mixing again, and detecting. Through screening of the quenching agent, the response of the vitamins can be obviously enhanced, and the detection of the contents of the fat-soluble vitamins A, D and E can be rapidly and accurately completed.

Description

Technical Field

[0001] The invention belongs to the technical field of vitamin detection, and specifically relates to a method for detecting the content of fat-soluble vitamins A, D and E in blood. Background Art

[0002] Fat-soluble vitamins are a class of vitamins that are soluble in organic solvents but insoluble in water, such as vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E. Currently, the methods used to determine fat-soluble vitamins mainly include fluorescence analysis, enzyme immunoassay, liquid chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS / MS), among which the advantages of liquid chromatography-tandem mass spectrometry are its high precision and high sensitivity, which can detect trace substances with low concentrations, and has high performance and reliability. When using liquid chromatography-tandem mass spectrometry technology to detect the analyte, the commonly used pre-treatment methods mainly include liquid-liquid extraction, solid-liquid extraction, protein precipitation method, and derivatization method.

[0003] However, the pretreatment of liquid-liquid extraction and solid-liquid extraction requires multiple extraction, drying, purification and other steps, which requires a lot of manual operation, and liquid-liquid extraction is difficult to achieve high throughput and automation. The solid-liquid extraction method mainly relies on 96-well solid-phase supported liquid-liquid extraction plates (SLE plates), which is costly and not conducive to cost reduction and efficiency improvement. However, the existing protein precipitation method cannot meet the sensitivity requirements of low-performance instruments after sample dilution for 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3. In addition, the market also uses derivatization methods for fat-soluble vitamin determination, but there are operations such as extraction, drying, derivatization, blowing and re-dissolving by liquid-liquid extraction methods, and then on the machine. The steps are cumbersome and time-consuming, and the derivatization time is long (such as 1 hour for derivatization reaction) and the derivatization conditions are harsh (such as 70°C derivatization reaction), which is not conducive to promotion and use. Therefore, it is urgent to develop a convenient and rapid method for detecting fat-soluble vitamin content. Summary of the invention

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

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

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

[0007] S1. Take a blood sample and add 250 μL of BHT internal standard solution to every 50 μL;

[0008] S2, adding 20 μL of zinc sulfate heptahydrate solution to the blood sample after adding BHT internal standard solution, and performing first mixing and centrifugation in sequence;

[0009] S3, taking 100 μL of the supernatant after centrifugation and concentrating and drying;

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

[0011] S5. Take 100 μL of the supernatant after centrifugation and add it to the derivative sample for remixing and 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-triazolin-3,5-dione) acetonitrile solution is 0.05 mg / mL to 2 mg / mL.

[0015] Preferably, the quencher is an ammonium acetate solution and has a concentration of 0.1 mol / L-0.5 mol / L.

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

[0017] Preferably, the derivatization reaction time is 10 min-40 min.

[0018] Preferably, the concentration temperature of the concentrated drying is 30° C. to 45° C., and the concentration time is 15 min to 60 min.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The method is mainly based on protein precipitation pretreatment combined with derivatization technology, and by optimizing the time and temperature of derivatization, the conditions of the derivatization process can achieve an effect that is easier to meet, and can meet the requirements of the accuracy of the added recovery rate, that is, it will not affect the accuracy of the sample due to incomplete derivatization due to a short derivatization time, nor will it be unfavorable for promotion due to a long overall detection time caused by a long derivatization time, and it will not cause the problem of poor derivatization effect caused by the easy hydrolysis of PTAD due to a high derivatization temperature and poor accuracy due to a derivatization temperature that is too low. At the same time, the quencher after derivatization is also optimized, that is, part of the PTAD in the derivatized sample will react with vitamins, so ammonium acetate solution (or vitamin C, ammonium formate, vitamin A acetate, etc.) is added during the derivatization process to quench PTAD, which is more conducive to the detection of vitamins, especially through the screening of quenchers, the response of vitamin A can be significantly enhanced, the detection of the content of fat-soluble vitamins A, D, and E can be completed quickly and accurately, and the detection sensitivity of 25-hydroxyvitamins in the blood can be significantly improved. At the same time, the accuracy of vitamins A and E can be guaranteed, making the detection results more accurate and stable, which helps to meet the detection needs of low-performance instruments for detecting fat-soluble vitamins A, D, and E. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 2 This is the MRM chromatogram of the internal and external standards of the quantification lower limit sample (3ng / mL) of 25-hydroxyvitamin D2 in the existing technology;

[0023] Figure 3 This is the MRM chromatogram of the internal and external standards of the existing 25-hydroxyvitamin D3 quantitative lower limit sample (3ng / mL);

[0024] Figure 4 This is the MRM chromatogram of the internal and external standards after PTAD derivatization of the quantitative lower limit sample (3 ng / mL) of 25-hydroxyvitamin D2 of the present invention;

[0025] Figure 5 This is the MRM chromatogram of the internal and external standards after PTAD derivatization of the quantitative lower limit sample (3 ng / mL) of 25-hydroxyvitamin D3 of the present invention;

[0026] Figure 6 This is the internal and external standard MRM chromatogram of the vitamin A quantitative lower limit sample (45 ng / mL) of the present invention;

[0027] Figure 7 The internal and external standard MRM chromatograms of the vitamin A quantitative lower limit sample (45 ng / mL) added with quencher;

[0028] Figure 8This is the internal and external standard MRM chromatogram of the vitamin E quantitative lower limit sample (750 ng / mL) of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work 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 those commonly understood by those skilled in the art in the technical field of this application. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

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

[0032] S1. Take a blood sample and add 250 μL of BHT internal standard solution to every 50 μL;

[0033] S2, adding 20 μL of zinc sulfate heptahydrate solution to the blood sample after adding BHT internal standard solution, and performing first mixing and centrifugation in sequence;

[0034] S3, taking 100 μL of the supernatant after centrifugation and concentrating and drying;

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

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

[0037] Among them, the blood sample can be serum, plasma or peripheral blood, etc. This method is mainly based on protein precipitation pretreatment combined with derivatization technology, and optimizes the pretreatment method, reasonably selects the derivatization time, temperature and post-derivation quenching agent as the optimal conditions such as simple operation and short derivatization time, and achieves the standard requirements of the added recovery rate by adopting the optimal conditions, which can enhance the vitamin response 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-triazolin-3,5-dione) acetonitrile solution is 0.05 mg / mL to 2 mg / mL.

[0041] In one embodiment, the quencher is an ammonium acetate solution with a concentration of 0.1 mol / L-0.5 mol / L, or it may be vitamin C, ammonium formate, vitamin A acetate, etc., and those skilled in the art may adjust it according to actual needs.

[0042] In one embodiment, the first mixing is vortex mixing with a rotation speed of 1000rpm-2500rpm and a time of 3min-10min; the second mixing is vortex mixing with a rotation speed of 300rpm-600rpm and a time of 3min-10min; the speed of centrifugal treatment is 3000rpm-5000rpm and the time is 5min-30min.

[0043] In one embodiment, the derivatization reaction time is 10 min-40 min.

[0044] In one embodiment, the concentration temperature of the concentrated 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 invention is described in detail below through specific embodiments.

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

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

[0049] 2) adding 20 μL of a precipitant (such as a 0.2 mol / L zinc sulfate heptahydrate solution) to the solution obtained in step 1), vortexing at 2000 rpm for 5 min, and centrifuging 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-well plate;

[0051] 4) Add 50 μL of 0.5 mg / mL PTAD (4-phenyl-1,2,4-triazolin-3,5-dione) acetonitrile solution to the dried sample, react at room temperature for 30 minutes, then add 0.5 mol / L ammonium acetate solution to terminate the reaction (because vitamin A easily reacts with PTAD (4-phenyl-1,2,4-triazolin-3,5-dione) acetonitrile solution, adding a portion of ammonium acetate solution during the quenching process can quickly quench the PTAD derivatization reagent (PTAD (4-phenyl-1,2,4-triazolin-3,5-dione) acetonitrile solution), thereby increasing the response of vitamin A);

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

[0053] In this embodiment, PTAD derivatization reagent is used to carry out derivatization reaction with vitamin D (25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, respectively) to form a high molecular weight derivative that is easily ionized, which significantly improves the sensitivity of liquid phase mass spectrometry determination of the compound, wherein:

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

[0055]

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

[0057]

[0058] The existing technology uses liquid chromatography tandem mass spectrometry in accordance with the national standard method for detection. For blood samples with low content, the sensitivity of mass spectrometry analysis cannot meet the detection requirements and cannot be quantified accurately. Figure 2 and Figure 3 As shown in the figure, the external standard peak area response of the lower limit of quantification of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 before derivatization was low, which could not meet the quantitative requirements, affecting the stability of the test results and unable to guarantee the accuracy of the samples. Figure 2-Figure 8 The upper figure in the figure represents the external standard mass spectrum extracted ion current diagram (external standard MRM chromatogram), and the lower figure represents the internal standard mass spectrum extracted ion current diagram (internal standard MRM chromatogram).

[0059] There are two configurations of 25(OH)D derivatization products (i.e., the result on the right side of the equal sign in the above chemical formula), 6S and 6R. For the convenience of quantification, the main peak 6S configuration is selected for quantitative analysis. The 25(OH)D derivatization product easily loses a molecule of water in the mass spectrometry environment, and the quasi-molecular ion dehydration peak formed is the main peak of the derivative ESI (electrospray ionization) primary mass spectrum. Therefore, [M+H-H2O]+ is selected as the parent ion for quantitative analysis using multiple reaction monitoring (MRM). The samples after derivatization reaction are shown in Figure 2. Figure 4 and Figure 5 It can be seen that the chromatographic peak responses of the internal and external standards of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 after derivatization can meet the detection requirements. That is, when this method is used for analysis, there is no interfering peak at the peak time of the target compound in the MRM chromatogram, the selectivity is strong, and it can greatly increase the sensitivity of the compound and improve the accuracy of quantification.

[0060] Because the PTAD derivatization reagent easily reacts with the conjugated double bonds in vitamin A, the response of vitamin A is reduced, such as Figure 6 As shown in the figure, it can be clearly seen that the vitamin A external standard has been obviously derivatized before the quencher is added. Therefore, after the derivatization reaction is completed, an appropriate concentration of ammonium acetate solution is added to quench the reaction, that is, to terminate the derivatization reaction and improve the accuracy of vitamin A, such as Figure 7 As shown, after quenching with ammonium acetate solution, the response of vitamin A was significantly improved, and accurate quantification could be performed. Figure 8 This indicates that PTAD derivatization reagent has no effect on the detection of vitamin E.

[0061] In addition, the following tests were performed in this embodiment: the test results of linearity and quality control products are shown in Table 1, the derivatization time optimization results are shown in Table 2, the derivatization temperature optimization results are shown in Table 3, and the post-derivation quenching agent optimization results are shown in Table 4.

[0062] Table 1 Linearity and quality control results of 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 within the linear range of 45-1200 ng / mL, 25-hydroxyvitamin D2 or 25-hydroxyvitamin D3 within the linear range of 3-80 ng / mL, and vitamin E within the linear range of 750-25000 ng / mL. Therefore, the protein precipitation method plus the derivatization method was selected for subsequent parameter optimization, which proved the feasibility of the method of the present application.

[0065] Table 2

[0066]

[0067]

[0068] The results in Table 2 show that the derivatization time has no significant effect on the concentrations of vitamin A and vitamin E in the 10-15 minute period, and the concentration of 25-hydroxyvitamin D derivative compounds increases with time. In the 30-60 minute period, there is no significant difference in the concentration of 25-hydroxyvitamin D derivative compounds, so the optimal derivatization time is preferably 15 minutes. Among them, the target value is the traceable concentration of vitamin A, D, and E, and the lower limit sample is the sample with the quantitative lower limit concentration.

[0069] Table 3

[0070]

[0071]

[0072] The results in Table 3 show that the derivatization temperature has no significant effect on vitamin A and vitamin E. When the derivatization temperature is in the range of 20-30°C, there is no significant difference in the concentration of 25-hydroxyvitamin D derivative compounds. In the range of 30-50°C, the concentration decreases with the increase of temperature after derivatization. During the derivatization process, it was found that some water vapor entered the sample during the temperature increase, causing the derivatization reagent to hydrolyze in advance, so the derivatization effect could not be produced well. Derivatization in the range of 20-30°C can already meet the instrument detection requirements. Therefore, room temperature is selected as the derivatization temperature, and 25°C is preferably selected as the optimal temperature for derivatization.

[0073] Table 4

[0074]

[0075]

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

[0077] In summary, the method of this embodiment has the best effect when the derivatization time is 15 minutes, the derivatization temperature is 25°C, and the quencher after derivatization is 0.5 mol / L ammonium acetate solution.

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

[0079] Table 5

[0080]

[0081] From the results of the optimized derivatization time (5, 10, 15, 30, 60 min), derivatization temperature (20, 25, 30, 40, 50 °C) and quencher screening, it can be seen that this method greatly improves the sensitivity of fat-soluble vitamins in serum by combining the protein precipitation pretreatment method with the derivatization method, and optimizing the concentration of the derivatization reagent, derivatization temperature, derivatization time, quencher screening and quencher concentration, thereby achieving the detection needs of low-performance instruments for fat-soluble vitamins A, D, and E.

[0082] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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 above-described embodiments only express the more specific and detailed embodiments described in this application, but they cannot be understood as limiting the scope of the application. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of this application, which all belong to the protection scope of this application. Therefore, the protection scope of this application shall be based on the attached 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 content of fat-soluble vitamins A, D and E in the blood comprises the following steps: S1. Take a blood sample and add 250 μL of BHT internal standard solution to every 50 μL; S2, adding 20 μL of zinc sulfate heptahydrate solution to the blood sample after adding BHT internal standard solution, and performing first mixing and centrifugation in sequence; S3, taking 100 μL of the supernatant after centrifugation and concentrating and drying; S4, adding 50 μL of PTAD (4-phenyl-1,2,4-triazolidine-3,5-dione) acetonitrile solution to the concentrated and dried supernatant at room temperature to carry out a derivatization reaction, and then adding a quencher to terminate the derivatization reaction to obtain a derivatized sample; S5. Take 100 μL of the supernatant after centrifugation and add it to the derivative sample for remixing and detection.

2. The method for detecting the content of fat-soluble vitamins A, D and E in blood according to claim 1, characterized in that: The volume ratio of the BHT internal standard solution is methanol:acetonitrile:isopropanol=(5-20):(45-60):(30-35).

3. The method for detecting the content of fat-soluble vitamins A, D and E in blood according to claim 1, characterized in that: 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.

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

5. The method for detecting the content of fat-soluble vitamins A, D and E in blood according to claim 1, characterized in that: The quencher is an ammonium acetate solution and has a concentration of 0.1 mol / L-0.5 mol / L.

6. The method for detecting the content of fat-soluble vitamins A, D and E in blood according to claim 1, characterized in that: The first mixing is vortex mixing with a rotation speed of 1000rpm-2500rpm and a time of 3min-10min; the second mixing is vortex mixing with a rotation speed of 300rpm-600rpm and a time of 3min-10min; the rotation speed of the centrifugal treatment is 3000rpm-5000rpm and a time of 5min-30min.

7. The method for detecting the content of fat-soluble vitamins A, D and E in blood according to claim 1, characterized in that: The derivatization reaction time is 10 min-40 min.

8. The method for detecting the content of fat-soluble vitamins A, D and E in blood according to claim 1, characterized in that: The concentration temperature of the concentrated drying is 30° C. to 45° C., and the concentration time is 15 min to 60 min.

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