Method for detecting fat-soluble vitamins and carotenoids

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for detecting fat-soluble vitamins and carotenoids. The detection method comprises the following steps: mixing a to-be-detected sample with pancreatic lipase for enzyme treatment, adding a protein precipitant for protein precipitation, adding an extract liquor for extraction, taking supernate, carrying out nitrogen blowing drying, redissolving with an alcohol solvent, and detecting by adopting liquid chromatography-tandem mass spectrometry, the protein precipitant is a mixture of methanol, acetonitrile and isopropanol; the extract liquor is a mixture of BHT and normal hexane. The detection method provided by the invention can realize quantitative detection of various fat-soluble vitamins and carotenoids at the same time, the pretreatment method is simple and easy to implement, the pretreatment effect is good, and the subsequent liquid chromatography-tandem mass spectrometry detection time can be shortened; the method has the advantages of multiple detection items, accurate detection result, high detection speed, high detection flux, simplicity and convenience in detection operation, high detection repeatability and low detection cost, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical detection technology, and in particular to a detection method of fat-soluble vitamins and carotenoids. BACKGROUND

[0002] Fat-soluble nutrients in serum mainly include fat-soluble vitamins and carotenoids. Fat-soluble vitamins mainly include vitamins A, D, E and K, which all contain ring structures and long aliphatic hydrocarbon chains, are insoluble in water or glycerol, and are easily soluble in anhydrous ethanol, methanol, chloroform, diethyl ether and oil. Vitamins A and E are common antioxidants that can prevent the oxidative damage of unsaturated fatty acids and other easily oxidized substances in cells, and play a role in protecting cell membranes from being damaged. Vitamin D is a sterol derivative with an anti-rickets effect, also known as anti-rickets vitamin. Vitamin K is a general term for a class of menaquinone derivatives, which has physiological effects such as promoting normal blood clotting. Carotenoids are lipophilic isoprene plant pigments that exist in red, yellow, orange and dark green fruits and vegetables. Among the more than 750 carotenoids in nature, about 15 exist in human serum, and the content of lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, lycopene and the like is relatively high. Due to the presence of unsaturated double bonds in the chemical structure, carotenoids are effective antioxidants. Many fat-soluble nutrients are involved in the homeostatic regulation of whole-body processes closely related to systemic metabolism, including redox and inflammatory pathways, and are essential micronutrients for maintaining human health. The determination of the content of fat-soluble nutrients in serum is crucial in the study of nutrition, the treatment of cancer or non-cancer related diseases. Therefore, the development of a rapid and sensitive method to measure the serum concentration of these nutrients is of great significance for the evaluation of nutritional status, the diagnosis of malnutrition, the development of intervention programs and the study of oxidative or inflammatory reactions that may be involved or changed by micronutrients.

[0003] CN104155385A discloses a method for detecting 9 kinds of fat-soluble vitamins in serum by using a liquid chromatography method, and the detection time is 12.5 min. The detection method simultaneously detects vitamin A, alpha-tocopherol, gamma-tocopherol, delta-tocopherol, alpha-carotene, beta-carotene, coenzyme Q10, lycopene and lutein. This detection method is relatively simple, but does not simultaneously detect vitamin D and vitamin K, and uses an external standard method for quantification, which cannot effectively correct the influence of the matrix. CN111141840A discloses a method for simultaneously detecting fat-soluble vitamins and carotenoids in serum, wherein the fat-soluble vitamins mainly include vitamin A, vitamin D (vitamin D2, vitamin D3 and metabolites 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2), vitamin E (alpha-tocopherol and gamma-tocopherol) and vitamin K (vitamin K1 and vitamin K2), and the carotenoids mainly include lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, beta-carotene and lycopene. However, the simultaneous detection of fat-soluble vitamins and carotenoids needs to be completed within 40 minutes, which is too long. SUMMARY

[0004] In order to solve the defects of the prior art, the present application provides a detection method for fat-soluble vitamins and carotenoids, which comprises: mixing the sample to be tested with pancrelipase for enzyme treatment, adding a protein precipitant for protein precipitation, then adding an extraction liquid for extraction, taking the supernatant for nitrogen blowing drying, redissolving with an alcohol solvent, and then detecting by liquid chromatography tandem mass spectrometry; the protein precipitant is a mixture of methanol, acetonitrile and isopropanol; and the extraction liquid is a mixture of BHT and n-hexane.

[0005] Through a large number of combinations and optimizations of the pretreatment conditions, it is found that by using the above-mentioned specific type of lipase incubation combined with a specific protein precipitant for protein precipitation, and further combined with a specific type of extraction liquid extraction, the extraction efficiency of fat-soluble vitamins and carotenoids can be maximized.

[0006] In some embodiments, the volume ratio of the sample to be tested mixed with pancrelipase is (1-4):1, preferably (1.8-2.2):1.

[0007] By using the above-mentioned ratio, higher extraction efficiency can be obtained with the least amount of pancrelipase, and the baseline can be reduced and the peaks near the retention time of the test substance have less interference.

[0008] In some embodiments, the enzyme treatment is incubation at 35-40°C (preferably 36-38°C) (preferably for more than 1 hour).

[0009] In some embodiments, the volume ratio of methanol, acetonitrile and isopropanol in the protein precipitation agent is (1-10):(0.5-5):(0.1-1), preferably (3-8):(1-4):(0.2-0.7).

[0010] In some embodiments, the volume ratio of BHT and n-hexane is (0.2-0.8):1, preferably (0.3-0.5):1.

[0011] In some embodiments, the amount of protein precipitation agent added is 180-220 μL per 100 μL of sample to be tested. Preferably, the amount of extraction solution added is 700-900 μL per 100 μL of sample to be tested. Preferably, the extraction is performed twice, and the supernatant after extraction is combined and dried by nitrogen blowing.

[0012] Under the above dosage relationship, the best extraction efficiency can be obtained with less amount of reagents.

[0013] In some embodiments, the alcohol solvent is ethanol.

[0014] As a preferred embodiment, the detection method comprises: vortexing the sample to be tested with pancreatic lipase and performing enzyme treatment at 35-40°C (preferably 36-38°C), adding the protein precipitation agent (preferably with an internal standard added) and vortexing and centrifuging, adding the extraction solution and vortexing and centrifuging, taking the supernatant and drying by nitrogen blowing, redissolving with ethanol, and then detecting by liquid chromatography tandem mass spectrometry; the protein precipitation agent is a mixture of methanol, acetonitrile and isopropanol; and the extraction solution is a mixture of BHT and n-hexane.

[0015] In some embodiments, the detection conditions of the liquid chromatography tandem mass spectrometry comprise: The specification of the chromatographic column is Phenomenex Kinetex® 2.6µm 3.0 100mm F5 100 A; the column temperature is 40-42°C (preferably 40°C); the mobile phase A is water, and the mobile phase B is methanol; both the mobile phase A and the mobile phase B contain 0.2% formic acid; The elution conditions are as follows:

[0016] Preferably, the injection volume is 10 μL.

[0017] The present application also optimizes screening of different brand specifications of chromatographic columns, and respectively selects ThermoAccucore™ PFP HPLC 2.6µm 2.1x100mm, Shim-pack Velox PFPP 2.7μm, 2.1x100mm and the above-mentioned PFPP chromatographic column of Finnigent to perform comparison under the same test conditions. The results show that, using the ThermoAccucore™ PFPP chromatographic column, even if the elution gradient is optimized, the separation degree of 25-hydroxyvitamin D3 and the isomer 3-EPI-25-hydroxyvitamin D3 is still poor, and the separation degree of β-carotene and the isomers α-carotene and lycopene is also poor. When the Shim-pack Velox PFPP chromatographic column is used, the sensitivity is not up to standard on a low-end machine due to the low carbon loading of the chromatographic column. When the above-mentioned PFPP chromatographic column of Finnigent is used for chromatographic condition optimization, the detection requirements can be better met.

[0018] In some embodiments, the detection conditions of the liquid chromatography tandem mass spectrometry method further comprise mass spectrometry conditions as follows:

[0019] Preferably, the scanning mode in the mass spectrometry is a positive ion scanning mode.

[0020] In some embodiments, the fat-soluble vitamins and carotenoids include vitamin A (retinol, VA), 25-hydroxyvitamin D2 (25-OH-VD2), 25-hydroxyvitamin D3 (25-OH-VD3), 3-epi-25-hydroxyvitamin D3 (3-epi-25-OH-VD3), vitamin E (α-tocopherol, VE), vitamin K1 (VK1), vitamin K2 (MK4, VK2), α-carotene (α-carotene), β-carotene (β-carotene) and lycopene (Lycopene).

[0021] Preferably, the extraction solution is subjected to freezing treatment before use.

[0022] Preferably, the pure solvent is subjected to freezing treatment before use.

[0023] In specific embodiments, the sample to be tested includes at least one of serum, plasma, cerebrospinal fluid, amniotic fluid, artificial simulated serum, artificial simulated plasma, artificial simulated cerebrospinal fluid and artificial simulated amniotic fluid.

[0024] In specific embodiments, a standard curve can be established by an internal standard method or an external standard method to obtain the content of the target substance in the sample to be tested.

[0025] Preferably, the standard curve is established with the standard target value concentration as the X axis and the peak area ratio of the standard to the internal standard as the Y axis; the peak area ratio of the sample to be tested and the corresponding internal standard is substituted into the standard curve to calculate the concentration of the fat-soluble vitamins and carotenoids in the sample to be tested.

[0026] Compared with the prior art, the present application has the advantages that: The detection method of the present application can simultaneously realize quantitative detection of multiple fat-soluble vitamins and carotenoids, and the pretreatment method is simple and easy to operate, has good pretreatment effect, strong impurity removal capacity, and can speed up the subsequent liquid chromatography tandem mass spectrometry detection time. The detection method of the present application has the advantages of multiple detection items, accurate detection results, fast detection speed, high detection flux, simple detection operation, high detection repeatability and low detection cost, and has a wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the chromatogram of Example 1 of the present application. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below. Obviously, the described embodiments are some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. In the embodiments provided in the present specification, the specific techniques or conditions are not specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased through regular channels. The pancrelipase in the following examples is purchased from Sigma-Aldrich, item number: L0382; Amano lipase A is derived from Aspergillus niger and is purchased from Sigma-Aldrich, item number: 534781.

[0029] Example 1 The present embodiment provides a detection kit for fat-soluble vitamins and carotenoids, and the components are shown in Table 1.

[0030] Table 1 Kit composition and main components

[0031] Specifically, the calibration sample includes 6 concentrations of calibration samples in gradient; the gradient concentrations of vitamin A (retinol) in the calibration sample are respectively: 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 1000 ng / mL and 2000 ng / mL; the gradient concentrations of 25-hydroxyvitamin D2 in the calibration sample are respectively: 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 100 ng / mL, 200 ng / mL; the gradient concentrations of 25-hydroxyvitamin D3 in the calibration sample are respectively: 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 100 ng / mL, 200 ng / mL; the gradient concentrations of 3-epi-25-hydroxyvitamin D3 in the calibration sample are respectively: 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 100 ng / mL, 200 ng / mL; the gradient concentrations of vitamin E (alpha-tocopherol) in the calibration sample are respectively: 0.4 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, 20 μg / mL, 40 μg / mL; the gradient concentrations of vitamin K1 in the calibration sample are respectively: 0.05 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL; the gradient concentrations of vitamin K2 (MK4) in the calibration sample are respectively: 0.05 ng / mL, 0.125 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL; the gradient concentrations of alpha-carotene in the calibration sample are respectively: 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL; the gradient concentrations of beta-carotene in the calibration sample are respectively: 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 1000 ng / mL and 2000 ng / mL; the gradient concentrations of lycopene in the calibration sample are respectively: 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 1000 ng / mL and 2000 ng / mL.

[0032] Specifically, the quality control samples include quality control sample C1, quality control sample C2 and quality control sample C3; the quality control sample C1 includes 100 ng / mL vitamin A, 10 ng / mL 25-hydroxyvitamin D2, 10 ng / mL 25-hydroxyvitamin D3, 10 ng / mL 3-epi-25-hydroxyvitamin D3, 2 μg / mL vitamin E, 0.25 ng / mL vitamin K1, 0.25 ng / mL vitamin K2 (MK4), 50 ng / mL α-carotene, 100 ng / mL β-carotene and 100 ng / mL lycopene; the quality control sample C2 includes 500 ng / mL vitamin A, 50 ng / mL 25-hydroxyvitamin D2, 50 ng / mL 25-hydroxyvitamin D3, 50 ng / mL 3-epi-25-hydroxyvitamin D3, 10 μg / mL vitamin E, 1.25 ng / mL vitamin K1, 1.25 ng / mL vitamin K2 (MK4), 250 ng / mL α-carotene, 500 ng / mL β-carotene and 500 ng / mL lycopene; and the quality control sample C3 includes 1500 ng / mL vitamin A, 150 ng / mL 25-hydroxyvitamin D2, 150 ng / mL 25-hydroxyvitamin D3, 150 ng / mL 3-epi-25-hydroxyvitamin D3, 30 μg / mL vitamin E, 3.75 ng / mL vitamin K1, 3.75 ng / mL vitamin K2 (MK4), 750 ng / mL α-carotene, 1500 ng / mL β-carotene and 1500 ng / mL lycopene.

[0033] Further, the embodiment provides a method for detecting fat-soluble vitamins and carotenoids by using the above-mentioned detection kit, and the steps are as follows: 1. Pretreatment 100 μL of the sample (serum) to be detected, the calibration sample and the quality control sample are respectively added to 50 μL of pancrelipase in a 2 mL EP tube, vortexed at 2000 rpm for 5 min, incubated in a 37°C water bath for 1 h, then 200 μL of protein precipitant containing an internal standard is added, vortexed at 2000 rpm for 5 min, 800 μL of extraction solution is added and vortexed, vortexed at 2000 rpm for 10 min, and the supernatant is taken after centrifugation; the precipitate is added with 800 μL of extraction solution, vortexed, vortexed at 2000 rpm for 10 min, and the supernatant is taken after centrifugation; the two supernatants are combined, dried by nitrogen blowing, and then dissolved with 100 μL of chilled ethanol before being detected by a machine.

[0034] 2. Detection by liquid chromatography tandem mass spectrometry The specifications of the chromatographic column were Phenomenex Kinetex® 2.6µm 3.0*100mm F5 100 A; the column temperature was 40℃; the mobile phase A was water, and the mobile phase B was methanol; both the mobile phase A and the mobile phase B contained 0.2% formic acid; the injection volume was 10µL; and the injection temperature was 10℃. The elution conditions are shown in Table 2. The mass spectrometry conditions are shown in Table 3.

[0035] Table 2 Elution conditions

[0036] Table 3 Mass spectrometry conditions

[0037] The ion pair parameters from 0 to 5.5min are shown in Table 4. The ion pair parameters from 5.5 to 15min are shown in Table 5.

[0038] Table 4 Ion pair parameters from 0 to 5.5min

[0039] Table 5 Ion pair parameters from 5.5 to 15min

[0040] 3. Result analysis The standard curve was drawn and the regression equation of the curve was calculated with the indicated concentrations of the 6 calibration samples as the abscissa (X) and the ratio of the actual detection peak area of the 6 calibration samples to the peak area of the internal standard as the ordinate (Y), so that the concentration values of vitamin A (retinol), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, vitamin E (α-tocopherol), vitamin K1, vitamin K2 (MK4), α-carotene, β-carotene, and lycopene in the sample to be measured can be calculated.

[0041] The chromatogram of this example is shown in Figure 1 .

[0042] Example 2 This example provides a method for detecting fat-soluble vitamins and carotenoids, and the only difference between the steps and those of Example 1 is that the amount of pancrelipase is replaced by 25µL, 75µL, and 100µL, respectively. In addition, Amano lipase A is used to detect at different amounts. The experimental groups are shown in Table 6.

[0043] Table 6 Experimental groups

[0044] The same sample was detected by the detection method of Example 1 and Example 2.

[0045] The test results are shown in Table 7. It is found that the extraction efficiency is higher when pancrelipase is used than when Amano lipase A is used, and the extraction efficiency is the highest when 50 μL of pancrelipase is used, and the baseline can be reduced to a certain extent and the peaks near the retention time of the analyte have less interference.

[0046] Table 7 Influence of lipase type and amount

[0047] Example 3 The method for detecting fat-soluble vitamins and carotenoids provided in this example is different from Example 1 only in that the amount of protein precipitant is replaced by 50 μL, 150 μL, 250 μL, 400 μL and 500 μL, respectively.

[0048] The same sample was detected by the detection method of Example 1 and Example 3.

[0049] The test results are shown in Table 8. The extraction efficiency of all analytes is higher when 200 μL of protein precipitant is added.

[0050] Table 8 Influence of protein precipitant amount

[0051] Example 4 The method for detecting fat-soluble vitamins and carotenoids provided in this example is different from Example 1 only in that the amount of extraction solution is replaced by 250 μL, 500 μL and 1000 μL, respectively.

[0052] The same sample was detected by the detection method of Example 1 and Example 4.

[0053] The test results are shown in Table 9. The extraction efficiency of all analytes is higher when 800 μL of extraction solution is added.

[0054] Table 9 Influence of extraction solution amount

[0055] Comparative Example 1 The method for detecting fat-soluble vitamins and carotenoids provided in this comparative example is different from Example 1 only in that the protein precipitant is replaced by an equal amount of acetonitrile.

[0056] The same sample was detected by the detection method of Example 1 and Comparative Example 1.

[0057] The test results are shown in Table 10.

[0058] Table 10 Comparison of extraction effect with Example 1

[0059] It can be seen that the extraction effect of Comparative Example 1 is poor.

[0060] Comparative Example 2 This comparative example provides a method for detecting fat-soluble vitamins and carotenoids, the only difference in the steps being that the n-hexane in the extraction solution is replaced with an equal amount of ethyl acetate.

[0061] The same sample is detected using the detection methods of Example 1 and Comparative Example 2.

[0062] The test results are shown in Table 11.

[0063] Table 11 Comparison of extraction effect with Example 1

[0064] It can be seen that the extraction effect of Comparative Example 2 is poor.

[0065] Test Example This test example verifies the performance of the detection method of Example 1.

[0066] 1. Linearity Test method: The calibration solution is detected according to the detection method of Example 1, and each concentration is repeated 3 times.

[0067] Test results: The linearity of vitamin A (retinol), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, vitamin E (alpha-tocopherol), vitamin K1, vitamin K2 (MK4), alpha-carotene, beta-carotene, lycopene is 20-2000 ng / mL, 2-200 ng / mL, 2-200 ng / mL, 2-200 ng / mL, 0.4-40 μg / mL, 0.05-5 ng / mL, 0.05-5 ng / mL, 10-1000 ng / mL, 20-2000 ng / mL, 20-2000 ng / mL, respectively, and the correlation coefficient is greater than 0.99, meeting the acceptance criteria.

[0068] 2. Precision Test method: Prepare a low, medium and high concentration mixed sample, detect 5 times a day for 5 consecutive days, and evaluate the within-batch precision % and between-batch precision % of fat-soluble vitamins and carotenoids in the sample.

[0069] The test results are shown in Table 12. The intra-batch precision of vitamin A (retinol), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, vitamin E (a-tocopherol), vitamin K1, vitamin K2 (MK4), a-carotene, b-carotene, and lycopene is 2.5%-5.0%, 2.2%-6.6%, 4.8%-5.0%, 3.8%-8.8%, 2.9%-4.4%, 4.6%-8.1%, 5.0%-9.6%, 5.0%-7.6%, 5.1%-6.9%, and 5.9%-7.2%, respectively. The inter-batch precision is 5.4%-6.1%, 4.6%-7.2%, 5.4%-6.3%, 5.1%-9.4%, 5.9%-7.0%, 5.6%-9.3%, 6.2%-10.4%, 6.2%-8.5%, 6.0%-7.5%, and 6.7%-8.9%, respectively. In summary, the coefficient of variation (CV) of the repeatability of the low-value quality control product is less than or equal to 10.4%, and the coefficient of variation (CV) of the repeatability of the medium / high-value quality control product is less than or equal to 8.4%.

[0070] Table 12 Precision of fat-soluble vitamins and carotenoids

[0071] 3. Recovery and matrix effect Recovery test method: Add standard solutions at low, medium, and high concentrations to the mixed serum sample, respectively, and detect according to the detection method in Example 1.

[0072] Matrix effect test method: Select 6 serum samples, mix with standard solutions at a ratio of 1:1, respectively, and detect according to the detection method in Example 1.

[0073] Calculate the recovery rate of the spiked sample results. If the recovery rate is within the range of 85%-115%, the method is considered to be acceptable in terms of accuracy. The difference between the response value (analyte / internal standard) of the mixed sample and the average response value of the clinical sample and the standard solution should be less than 20%, and the relative matrix effect is considered to be acceptable.

[0074] The test results are shown in Table 13 and Table 14, the standard addition recoveries of vitamin A (retinol), 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, vitamin E (alpha-tocopherol), vitamin K1, vitamin K2 (MK4), alpha-carotene, beta-carotene, lycopene are 96.7%~102.1%, 89.4%~91.7%, 91.7%~100.1%, 90.4%~100.8%, 92.7%~98.0%, 92.3%~104.9%, 92.7%~98.0%, 90.5%~94.7%, 95.1%~98.0%, 90.2%~101.7% respectively; the matrix effects are -7.0%~0.0%, -7.0%~0.0%, -6.0%~0.0%, -10.0%~-1.0%, -8.0%~1.0%, -4.0%~3.0%, -1.0%~0.0%, -4.0%~2.0%, -4.0%~2.0%, -8.0%~1.0% respectively. In summary, the relative deviation (B) of accuracy is ≤ ±10%, the standard addition recovery is 89.4%~104.9%.

[0075] Table 13 recovery results

[0076] Table 14 matrix effect results

[0077] From the performance verification results, it can be seen that the detection method of the present application has good precision, good correlation in linear range, and the recovery rate and matrix effect meet the clinical detection requirements.

[0078] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting fat-soluble vitamins and carotenoids, characterized in that, include: The sample to be tested was mixed with pancreatic lipase for enzymatic treatment, a protein precipitant was added for protein precipitation, an extraction solution was added for extraction, the supernatant was dried by nitrogen blowing, and the sample was redissolved with an alcohol solvent and detected by liquid chromatography-tandem mass spectrometry. The protein precipitant was a mixture of methanol, acetonitrile and isopropanol; the extraction solution was a mixture of BHT and n-hexane.

2. The detection method according to claim 1, characterized in that, The volume ratio of the sample to be tested to pancreatic lipase is (1~4):1, preferably (1.8~2.2):

1.

3. The detection method according to claim 1 or 2, characterized in that, The enzyme treatment was performed by incubation at 35°C to 40°C.

4. The detection method according to claim 1 or 2, characterized in that, In the protein precipitant, the volume ratio of methanol, acetonitrile, and isopropanol is (1~10):(0.5~5):(0.1~1), preferably (3~8):(1~4):(0.2~0.7).

5. The detection method according to claim 1 or 2, characterized in that, The volume ratio of BHT to n-hexane is (0.2~0.8):1, preferably (0.3~0.5):

1.

6. The detection method according to claim 2, characterized in that, For every 100 μL of test sample, the amount of protein precipitant added is 180~220 μL; Preferably, the amount of extraction solution added is 700~900μL for every 100μL of sample to be tested; Preferably, the extraction is performed twice, and the supernatant after extraction is combined and dried by nitrogen blowing.

7. The detection method according to claim 1, characterized in that, The alcohol solvent is ethanol.

8. The detection method according to claim 1, characterized in that, The detection conditions for the liquid chromatography-tandem mass spectrometry method include: The chromatographic column specifications are Phenomenex Kinetex® 2.6µm 3.

0. 100mm F5 100 A; column temperature 40℃~42℃; mobile phase A is water, mobile phase B is methanol; both mobile phase A and mobile phase B contain 0.2% formic acid; The elution conditions are as follows: 。 9. The detection method according to claim 8, characterized in that, The detection conditions for the liquid chromatography-tandem mass spectrometry method also include the following mass spectrometry conditions: 。 10. The detection method according to any one of claims 1 to 9, characterized in that, The fat-soluble vitamins and carotenoids include: vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3, vitamin E, vitamin K1, vitamin K2, α-carotene, β-carotene, and lycopene.

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