Detection of fat-soluble vitamins ADE by magnetic solid phase extraction combined with single-rod mass spectrometry

Abstract

The present invention relates to a method for detecting fat-soluble vitamins ADE by combining magnetic solid phase extraction with single-rod mass spectrometry. The present invention uses a magnetic solid phase extractant to extract and enrich fat-soluble vitamins on a fully automated pre-treatment instrument, and the entire pre-treatment process can be automated; the entire automation process does not require lengthy and cumbersome experimental operations such as centrifugation, filtration, and nitrogen blowing concentration, which greatly improves experimental efficiency; reduces experimental errors due to manual operation, saves manpower, and improves the accuracy of analysis and detection; the extracted sample is then detected by a single quadrupole mass spectrometry, and the single quadrupole mass spectrometry has a simple structure, stable performance, simple operation, convenient maintenance, low procurement cost, and both accuracy and sensitivity can achieve the effect of a triple quadrupole, which is relatively more suitable for clinical detection. The present invention can simultaneously and efficiently and quickly detect vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E.

Description

Technical Field

[0001] The invention belongs to the technical field of vitamin detection, and specifically relates to a method for detecting fat-soluble vitamins ADE by combining magnetic solid phase extraction with single-rod mass spectrometry. Background Art

[0002] At present, the main methods for detecting fat-soluble vitamins include enzyme-linked immunosorbent assay, fluorescence analysis, liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), among which liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been widely used in the field of clinical testing due to its high sensitivity and good specificity. The methods for extracting and enriching fat-soluble vitamins in the existing technology mainly include protein precipitation method (PPT), liquid-liquid extraction (LLE), solid phase extraction (SPE) and solid-liquid extraction (SLE), etc. The above methods are not easy to realize automated operation, and a large amount of volatile organic reagents are required during the experiment, which is not conducive to the health of experimental operators.

[0003] Liquid chromatography tandem mass spectrometry (LC-MS / MS) is also known as triple quadrupole mass spectrometry. The price of this instrument is relatively high, which will increase the cost of clinical testing. In addition, its structure is more complex, and its volume and weight are larger than those of a single-stage quadrupole mass spectrometer, and it takes up a large space when installed. In addition, this instrument has strict requirements on gas pipelines, and argon gas is used when it is used, which has a high cost.

[0004] Patent CN115524425A discloses a kit and a detection method for detecting fat-soluble vitamins in serum and plasma. The method uses methyl tert-butyl ether as an extractant to extract fat-soluble vitamins, and nitrogen blows and re-dissolves them for machine detection after extraction. Patent CN115184482A discloses a method for a kit for LC-MS / MS detection of fat-soluble vitamins. The patent first uses acetonitrile and zinc sulfate as protein precipitants to precipitate proteins, then adds n-hexane for liquid-liquid extraction, and nitrogen blows and re-dissolves them for machine detection. Although the pre-treatment process of these two patents is simple and easy, the whole process involves multiple vortex oscillations and high-speed centrifugation steps, which require a lot of manual operation and waiting time for centrifugation, resulting in low experimental efficiency. Moreover, the published method must be nitrogen blown and re-dissolved before it can be tested on the machine, which is a time-consuming and laborious experimental operation, and is not conducive to automation. In addition, the volatile organic extractant used is also not conducive to the health of the experimenter. The currently commonly used solid phase extraction (SPE) method also has certain problems. This method usually requires the use of a negative pressure device, which increases the pre-treatment cost. In addition, serum samples are relatively viscous, and the flow rate is slow when passing through the column, which is prone to clogging and poor reproducibility.

[0005] In addition, the monoclonal antibodies used in the currently commonly used immune antibody method are highly uniform antibodies produced by a single B cell clone and only target a specific antigen epitope. They can only bind to a certain substance. The immune antibody method can only detect VD, because antibodies are specific. VD antibodies can only detect VD, and cannot detect other substances, even if they are all fat-soluble vitamins. After all, the structures of different substances are still very different. For example, the structural formulas of vitamin A (VitaminA) and vitamin E (VitaminE) and 25-hydroxyvitamin D (25 (OH) VD) are quite different, among which 25-hydroxyvitamin D2 (25 (OH) VD2) and 25-hydroxyvitamin D3 (25 (OH) VD3) are relatively close in structural difference. Therefore, the antibody of VD cannot capture vitamin A and vitamin E. The concentration levels of vitamin A and vitamin E in the human body are relatively high, and the amount of antibodies corresponding to them is relatively large, and the cost is relatively expensive. How to realize the automated detection of fat-soluble vitamins ADE efficiently and conveniently is a technical problem that industry insiders urgently need to solve. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a method for detecting fat-soluble vitamins ADE by combining magnetic solid phase extraction with single-rod mass spectrometry. The present invention extracts and enriches fat-soluble vitamins in samples based on magnetic solid phase extraction, and uses single quadrupole mass spectrometry to simultaneously and efficiently and quickly detect vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E.

[0007] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0008] A method for detecting fat-soluble vitamins ADE by magnetic solid phase extraction combined with single-rod mass spectrometry comprises the following steps:

[0009] (1) Prepare the calibration curve: the calibration materials include vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E;

[0010] (2) Prepare fat-soluble vitamin internal standard solution;

[0011] (3) Pretreatment step: Using a magnetic solid phase extractant on a fully automatic mass spectrometer pretreatment instrument to extract and enrich fat-soluble vitamins;

[0012] (4) Detection: The extracted samples are then subjected to single quadrupole mass spectrometry to detect vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E;

[0013] (5) Detection data processing: By substituting the ratio of the peak area of ​​the sample to be tested and the peak area of ​​the internal standard into the calibration curve equation, the concentration of fat-soluble vitamins ADE can be obtained.

[0014] The present invention can simultaneously detect several compounds such as lipid, vitamin A, and E in serum by using a single-rod mass spectrometer through coordinated control of the conditions of each step, and is implemented on an automated instrument, which can save 50% of the time compared with traditional methods. The method of the present invention is highly efficient and convenient.

[0015] Preferably, step (1) is specifically:

[0016] (i) Prepare standard solutions: prepare different concentrations of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E calibrant intermediate solutions for later use;

[0017] (ii) Prepare the calibrant diluent: weigh bovine serum albumin (BSA), add it to water and dissolve it by sonication to obtain the calibrant diluent;

[0018] (iii) Prepare the calibration curve for fat-soluble vitamins: pipette the calibrant intermediate solution and add the calibrant diluent to prepare multiple calibration lines with different concentrations; among which, the concentration of vitamin A is 40-2000 ng / mL, the concentration of 25-hydroxyvitamin D2 is 2.5-125 ng / mL, the concentration of 25-hydroxyvitamin D3 is 2.5-125 ng / mL, and the concentration of vitamin E is 0.4-20 μg / mL.

[0019] Preferably, step (2) is specifically as follows: using 0.1% by mass 2,6-di-tert-butyl-4-methylphenol (BHT) methanol as a solvent, preparing isotopic internal standards corresponding to vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E, namely vitamin A-d6, 25-hydroxyvitamin D2-d3, 25-hydroxyvitamin D3-d3, and vitamin E-d6; the concentrations of the internal standard mixed working solution are: 800 to 1200 ng / mL for vitamin A-d6, 30 to 90 ng / mL for 25-hydroxyvitamin D2-d3, 30 to 90 ng / mL for 25-hydroxyvitamin D3-d3, and 1 to 5 μg / mL for vitamin E-d6.

[0020] Preferably, step (3) is specifically as follows:

[0021] (a) Preparation of sample kit

[0022] Prepare a 96-well plate, add diluent to wells 1 and 7, add magnetic bead solution to wells 2 and 8, add cleaning solution to wells 3, 9, 4, 10, 5 and 11, add eluent to wells 6 and 12, add internal standard mixed working solution and online diluent to the reagent compartment, respectively; wherein the diluent composition includes 50 vol% methanol water, the magnetic bead solution composition includes C18 silica gel magnetic adsorption microspheres (C18 magnetic beads), the cleaning solution composition includes 20 vol% methanol, 10 vol% ethanol, 0.2 vol% glacial acetic acid, and the balance is water, the eluent is acetonitrile, and the online diluent is water;

[0023] (b) Sample preparation

[0024] Transfer the sample to be tested and the mark line into a 1.5 mL microcentrifuge tube (EP tube), place the microcentrifuge tube on the sample rack, and place the sample rack in the sample rack placement area of ​​the fully automatic mass spectrometer pre-processor;

[0025] (c) Operation of the fully automatic mass spectrometer pre-treatment instrument

[0026] Transfer the liquid treated by the sample kit into a 96-well microplate through pre-treatment;

[0027] After the pretreatment process is completed, the 96-well microplate is taken out and tested on a mass spectrometer.

[0028] Preferably, the detection conditions in step (4) are controlled as follows:

[0029] Liquid phase detection conditions:

[0030] Chromatographic column: Waters C18 column; column temperature: 45°C; mobile phase: aqueous phase is 0.1% formic acid aqueous solution, organic phase is 2Mm ammonium formate and 0.1% formic acid methanol solution; injection volume: 10μL; time: 0-5.5min; flow rate: 0.4mL / min; aqueous phase volume ratio: 0-25%; organic phase volume ratio: 75-100%;

[0031] Mass spectrometry detection conditions:

[0032] Ionization mode: electrospray ionization positive mode; Scanning mode: single ion scanning monitoring; Capillary voltage: 0.8Kv; Desolvation gas temperature: 400℃; Desolvation gas flow rate: 800L / Hr; Ion source temperature: 120℃;

[0033] Preferably, in step (5), the calibration curve is obtained by drawing the calibration curve by using the labeled concentration of the calibration curve as the abscissa and the ratio of the actual detected peak area of ​​the calibration curve samples to the peak area of ​​the respective internal standards as the ordinate.

[0034] Preferably, the fully automatic mass spectrometry pre-processing instrument is a mass spectrometry pre-processing instrument IMS5000 (IMS5000 instrument), which is automatically completed by the mass spectrometry pre-processing instrument IMS5000 of Hangzhou Baichen Medical Laboratory Co., Ltd.

[0035] Preferably, in step (i), 0.1wt% 2,6-di-tert-butyl-4-methylphenol (BHT) in methanol is used as a solvent to prepare calibration intermediate solutions of different concentrations, wherein the concentration of vitamin A is 800-40000 ng / mL, the concentration of 25-hydroxyvitamin D2 is 50-2500 ng / mL, the concentration of 25-hydroxyvitamin D3 is 50-2500 ng / mL, and the concentration of vitamin E is 8-400 μg / mL.

[0036] The present invention uses a magnetic solid phase extraction method to extract and enrich fat-soluble vitamins in samples on a fully automatic mass spectrometry preprocessor, and uses a single quadrupole mass spectrometer Waters QDa to detect vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E. The reliability and robustness of this automated method are evaluated by testing the precision, accuracy, and sensitivity of the method. The instrument sensitivity and quantitative accuracy of the single quadrupole mass spectrometer and the triple quadrupole mass spectrometer are compared experimentally, thereby proving that the low-cost single quadrupole mass spectrometer can also meet the needs of clinical testing.

[0037] The beneficial effects of the present invention are:

[0038] On a fully automated pre-treatment instrument, through the control of the conditions in the steps, a magnetic solid phase extractant is used to extract and enrich fat-soluble vitamins, and the entire pre-treatment process can be automated; the entire automated process does not require lengthy and cumbersome experimental operations such as centrifugation, filtration, and nitrogen blowing concentration, which greatly improves the experimental efficiency; reduces the experimental error caused by manual operation, saves manpower, and improves the accuracy of analytical detection; the extracted sample is then detected by a single quadrupole mass spectrometer, which has a simple structure, stable performance, simple operation, convenient maintenance, low procurement cost, and both accuracy and sensitivity can achieve the effect of a triple quadrupole, which is relatively more suitable for clinical detection. The present invention is based on a magnetic solid phase extraction method to extract and enrich fat-soluble vitamins in samples, and uses a single quadrupole mass spectrometer to simultaneously and efficiently and quickly detect vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E, with efficient and convenient performance, greatly improving the practical application value of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the appearance of the IMS5000.

[0040] Figure 2 This is a schematic diagram of the IMS5000 movement structure.

[0041] Figure 3 This is a schematic diagram of the second structure of the IMS5000 movement;

[0042] Figure 4 This is the calibration curve of vitamin A;

[0043] Figure 5 This is the calibration curve of 25-hydroxyvitamin D2;

[0044] Figure 6 This is the calibration curve of 25-hydroxyvitamin D3;

[0045] Figure 7 This is the calibration curve of vitamin E.

[0046] Illustrations: touch screen 1, reagent compartment door 2, power switch 3, transparent compartment door 4, sample rack placement area 5, TIP head recovery box 6, reagent kit injection placement area 7, reagent kit 8, consumables platform 9, 96 microplate 10, TIP box 11, sampling needle cleaner 12, reagent compartment 13, sample rack 14, sampling box grabbing robot 15, sampling needle 16, pipetting robot 17, pipette 18. DETAILED DESCRIPTION

[0047] The technical scheme of the present invention is further specifically described below through examples, which are provided for the purpose of explaining the present invention, not for limiting the present invention. Based on the examples in this application, all other examples obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0048] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0049] Standards: Vitamin A (Sigma-Aldrich); 25-hydroxyvitamin D2 (Bepure); 25-hydroxyvitamin D3 (Bepure); Vitamin E (Sigma-Aldrich); Vitamin A-d6 (Bepure); 25-hydroxyvitamin D2-d3 (Bepure); 25-hydroxyvitamin D3-d3 (Bepure); Vitamin E-d6 (Bepure).

[0050] Main reagents: methanol (B&K); acetonitrile (Sigma-Aldrich); isopropanol (B&K); bovine serum albumin (Sinopharm); formic acid (Aladdin); acetic acid (Aladdin); ammonium formate (Honeywell).

[0051] Example 1

[0052] 1. Configuration of calibration curve

[0053] Prepare standard solutions of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E of different concentrations for later use. Use 0.1% BHT methanol as solvent to prepare 6 different concentrations of calibration intermediate solutions (named S1-S6). The specific concentrations are shown in Table 1 below:

[0054] Table 1 Concentration of calibrant intermediate solution

[0055]

[0056] Prepare the calibration dilution solution: accurately weigh 5 g BSA, add it to 100 ml water and dissolve it by ultrasonic for 5 minutes to obtain the calibration dilution solution.

[0057] Preparation of the calibration curve for fat-soluble vitamins: Accurately pipette 50 μL of the above calibration intermediate solution (S1-S6), add 950 μL of calibration diluent, and prepare 6 different concentration lines (named J1-J6). The specific concentrations are shown in Table 2:

[0058] Table 2 Lipid calibration curve

[0059]

[0060] 2. Preparation of fat-soluble vitamin internal standard solution

[0061] Using 0.1wt% BHT methanol as solvent, the isotope internal standard mixed solution corresponding to each compound was prepared. The final concentration of the internal standard mixed working solution was: VA-d6 was 1000ng / mL, 25-hydroxyvitamin D2-d3 was 60ng / mL, 25-hydroxyvitamin D3-d3 was 60ng / mL, and vitamin E-d6 was 3μg / mL.

[0062] 3. Pre-processing steps

[0063] Sample kit preparation

[0064] Prepare a 96-well plate, add the corresponding reagents as shown in Table 3 below, and seal it with an aluminum mold:

[0065] Table 3 Reagent composition of 96-well plate and reagent chamber

[0066]

[0067] Sample preparation

[0068] Pipette 300 μL of the sample to be tested and the mark line into a 1.5 mL EP tube, place the EP tube on the sample rack, and place the sample rack in the sample rack placement area of ​​the IMS5000 instrument.

[0069] IMS5000 instrument operation

[0070] Reference Figure 1-Figure 3 The mass spectrometer preprocessor IMS5000 comes with a 12-inch touch screen 1 (upper right corner), which can be used to control the instrument; the reagent compartment door 2 is on the left side of the instrument, and the corresponding reagents can be replaced by opening the reagent compartment door 2; the power switch 3 is on the right side of the instrument. The front of the instrument is equipped with a transparent compartment door 4, which can be rotated open to replace the corresponding consumables; the opening at the lower left corner of the instrument is equipped with a sample rack placement area 5 (insert the sample rack to be processed for sample loading); the lower right corner of the instrument is equipped with a TIP head recovery box 6, which is used to recycle waste TIP boxes.

[0071] The working principle of the instrument is as follows:

[0072] Open the transparent door on the front, put two new sample reagent kits 8 in the reagent kit injection area 7 (one reagent kit can process 16 samples), place a 96-well microplate 10 (to collect the processed sample liquid, that is, transfer the liquid processed by the sample kit to the 96-well microplate), a TIP box 11 (96 pieces), and a sealing strip carrier (12 strips) on the consumable platform 9; open the reagent compartment door 2 on the left side of the instrument and put the internal standard and diluent reagent bottles into the reagent compartment 13; insert two sample racks 14 (32 samples), and the entire pre-processing preparation is completed.

[0073] The sampling grab box robot arm 15 is equipped with a puncture needle, which first punctures the hole film of the reagent box 8 in the reagent box injection placement area 7 where the sample and reagent need to be added. The sampling grab box robot arm 15 is equipped with a sampling needle 16 (which can be moved up and down), and the sampling needle 16 is correspondingly provided with a sampling needle cleaner 12; the internal standard, diluent in the reagent compartment 13 and the sample in the sample rack 14 can be quantitatively transferred to the corresponding hole of the sample reagent box 8 in the reagent box injection placement area 7 through the sampling needle, and mixed with the reagent in the hole. After the 32 samples and the corresponding distribution are completed, the clamp on the sampling grab box robot arm 15 first transfers a reagent box (filled with samples and reagents) to the processing area of ​​the consumable platform 9 (on which a heating incubation module is provided). The consumable platform 9 moves horizontally to the TIP head position, the liquid transfer robot 17 moves forward and backward to the TIP head position, and the two groups of pipettes 18 on the liquid transfer robot 17 poke downward to take the first and second columns of TIP heads (a total of 16) on the TIP head box, and the liquid transfer robot 17 moves forward and backward to the processing position, and the consumable platform 9 moves horizontally to the processing position. At this time, the two groups of TIP heads on the two groups of pipettes are respectively aligned with the first and seventh columns of the reagent box on the consumable platform 9. The two groups of pipettes 18 move vertically downward at the same time, insert the two groups of TIP heads into the first and seventh columns of the reagent box, and mix the injected samples and reagents by suction and spitting. After the mixing is completed, the pipette quantitatively sucks the liquid in the hole into the TIP cavity and transfers it to other columns of holes or performs magnetic separation in the TIP cavity.

[0074] Magnetic separation is performed in the TIP cavity: the rear end of the pipetting mechanical arm 17 is equipped with a magnetic suction device (which can move forward and backward), which is located between the two groups of pipettes 18; after the TIP head absorbs the magnetic bead mixture, the pipette 18 moves vertically upward to the magnetic suction position, and the magnetic suction device moves forward to the magnetic suction position. At this time, the left and right rows of bar magnets on the magnetic suction device are respectively aligned with the two columns of TIP heads, and the magnetic beads in the TIP cavity are adsorbed on one side of the TIP inner wall by magnetic force. At this time, the plunger of the pipette 18 moves vertically downward to slowly discharge the liquid in the TIP cavity into the corresponding column of holes in the reagent kit, and the consumable platform 9 is moved, and the magnetic beads on the TIP head of the pipette 18 can be transferred to other columns of holes in the reagent kit. According to the processing flow, 16 samples can be processed, and the purified solution can be moved to the 96 microplates on the consumable platform 9 by the pipette 18. The pipette 18 is provided with a TIP head beating device, which can knock the processed TIP head into the TIP head recovery box (waste TIP box). Then, the right pipette 18 sucks the sealing strips on the consumable platform 9 and sticks them on the 96-well plate one by one to prevent the purified solution from volatilizing. At this point, the processing of 16 samples is completed, the corresponding reagent kit is clamped back to its original position, and the remaining 16 samples are processed in turn.

[0075] This method can detect 16 samples simultaneously in 15 minutes.

[0076] After the pretreatment process is completed, the 96-well microplate is taken out and detected by Waters QDa mass spectrometer.

[0077] The specific timing steps of IMS5000 are shown in Table 4 below:

[0078] Table 4 IMS5000 timing steps

[0079]

[0080] 4. Test condition control

[0081] Liquid phase detection conditions

[0082] 1. Chromatographic column: Waters BEH C18 (50mm*2.1mm, 1.7um)

[0083] 2 Column temperature: 45°C

[0084] 3 Mobile phase: Aqueous phase: 0.1% formic acid in water; Organic phase: 2Mm ammonium formate + 0.1% formic acid in methanol

[0085] 4 Injection volume: 10 μL

[0086] The mobile phase elution program is shown in Table 5 below:

[0087] Table 5 Mobile phase elution program

[0088]

[0089] Mass spectrometry detection conditions

[0090] 1 Ionization mode: electrospray ionization positive mode (ESI+)

[0091] 2 Scanning mode: single ion scanning monitoring (SIR mode)

[0092] 3 Capillary voltage: 0.8Kv

[0093] 4 Desolvation temperature: 400℃

[0094] 5 Desolvation gas flow rate: 800L / Hr

[0095] 6. Ion source temperature: 120°C

[0096] The mass numbers and cone voltages of each compound and internal standard are shown in Table 6 below:

[0097] Table 6 Mass numbers and cone voltages of various compounds and internal standards

[0098]

[0099] 5. Test data processing

[0100] Method for drawing the calibration curve: Draw the calibration curve with the labeled concentration of the calibration curve as the horizontal axis (x) and the ratio of the actual detection peak area of ​​the calibration curve sample to the peak area of ​​each internal standard as the vertical axis (y). The calibration curve of vitamin A is as follows: Figure 4 As shown, the correlation coefficient is: r=0.999511, r 2 =0.999022, calibration curve: 0.000840031*x+0.0132438; 25-hydroxyvitamin D2 calibration curve is as follows Figure 5 As shown, the correlation coefficient is: r=0.999759, r 2 =0.999518, calibration curve: 0.026779*x+0.0692571; 25-hydroxyvitamin D3 calibration curve is as follows Figure 6 As shown, the correlation coefficient is: r=0.999934, r 2 =0.999868, calibration curve: 0.0223403*x+0.0270099; Vitamin E calibration curve is as follows Figure 7 As shown, the correlation coefficient is: r=0.999886, r 2 =0.999772, calibration curve: 0.637972*x+0.155952.

[0101] The serum samples to be tested are injected sequentially to obtain the ratio of the peak area of ​​the test substance to that of the internal standard, which is then substituted into the calibration curve equation to obtain the concentration of the fat-soluble vitamins.

[0102] Example 2

[0103] Single quadrupole mass spectrometry precision verification experiment

[0104] Prepare low and high mixed standard solutions of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E. Take 20 μL of low and high mixed standard solutions of fat-soluble vitamins respectively, add them to 380 μL of 5% BSA matrix solution, vortex mix for 5 minutes, and prepare precision quality control verification samples. The specific concentrations are shown in Table 7:

[0105] Table 7 Low and high quality control samples

[0106]

[0107] Verification method: Test low and high quality control samples separately, repeat 3 times each day, and measure continuously for 5 days. The precision verification results are shown in Tables 8-11.

[0108] Table 8 Vitamin A precision verification results

[0109]

[0110] Table 9 25-Hydroxyvitamin D2 precision verification results

[0111]

[0112] Table 10 25-Hydroxyvitamin D3 precision verification results

[0113]

[0114] Table 11 Vitamin E precision verification results

[0115]

[0116] From the above results, it can be seen that the intra-batch CV values ​​of low quality control and high quality control of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E are all less than 6.25%, and the inter-batch CV values ​​are all less than 8.33%, which meet the requirements of clinical testing.

[0117] Example 3

[0118] Comparison of the accuracy of two mass spectrometers

[0119] Using 0.1% BHT methanol as solvent, prepare low, medium and high fat-soluble vitamin spike solutions, take 50 μL of the prepared spike solution and add it to 950 μL of real human serum sample to prepare the accuracy verification sample. The preparation process is shown in Table 12 below:

[0120] Table 12 Correctness sample configuration process

[0121]

[0122] Spiked recovery rate = (test value - basic sample value) / spiked value × 100%. The test result must meet the following requirements: 85% ≤ recovery rate ≤ 115%.

[0123] Verification method: The processed samples were tested by single-rod mass spectrometry and triple quadrupole mass spectrometry. The accuracy verification results are shown in Table 13-Table 14.

[0124] Table 13 Single-rod mass spectrometer accuracy verification results

[0125]

[0126] Table 14 Triple quadrupole mass spectrometry accuracy verification results

[0127]

[0128] From the results in Table 13 and Table 14, it can be seen that the low, medium and high recoveries of each compound tested by single-rod mass spectrometry and triple quadrupole mass spectrometry are all between 85% and 115%, which meets the requirements of clinical testing. This shows that single-rod mass spectrometry can achieve the detection accuracy of triple quadrupole.

[0129] Example 4

[0130] Comparison of the lower limit of quantitation between two mass spectrometers

[0131] The J1 point in the marking line is used as the sample of the lower limit of quantification. After pre-treatment, it is tested by single-rod mass spectrometry and triple quadrupole mass spectrometry. The test results must meet the following requirements: CV≤20%, bias<15%. The verification results of the lower limit of quantification are shown in Tables 15 and 16.

[0132] Table 15 Single-rod mass spectrometry quantitative lower limit detection results

[0133]

[0134] Table 16 Triple quadrupole mass spectrometry quantitative lower limit detection results

[0135]

[0136] J1 in the calibration curve is used as the lower limit of quantification for detection. The detection results of each compound tested by single-rod mass spectrometry and triple quadrupole mass spectrometry all meet RE < ± 15%, and the coefficient of variation CV < 15%. Therefore, in this method, the lower limit of quantification of vitamin A is 40ng / mL, the lower limit of quantification of 25-hydroxyvitamin D2 is 2.5ng / mL, the lower limit of quantification of 25-hydroxyvitamin D3 is 2.5ng / mL, and the lower limit of quantification of vitamin E is 0.4μg / mL. The above lower limit results all meet the needs of clinical testing. It shows that the detection sensitivity of the triple quadrupole can be achieved in the process of simultaneously detecting lipid vitamins ADE in serum using a single-rod mass spectrometer.

[0137] The present invention realizes fully automated processing of samples based on magnetic solid phase extraction, improves detection throughput, reduces manual operations, and saves manpower. The detection goals of high throughput, high precision and accuracy can also be achieved by using a single-rod mass spectrometer. The analytical sensitivity and quantitative accuracy can also achieve the detection effect of a triple quadrupole, but the price is half that of this instrument, which reduces the detection cost in clinical use.

[0138] Finally, it should be noted that the above are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for detecting fat-soluble vitamins ADE by magnetic solid phase extraction combined with single-rod mass spectrometry, characterized in that The steps include: (1) Prepare the calibration curve: the calibration materials include vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E; (2) Prepare fat-soluble vitamin internal standard solution; (3) Pretreatment step: Using a magnetic solid phase extractant on a fully automatic mass spectrometer pretreatment instrument to extract and enrich fat-soluble vitamins; (4) Detection: The extracted samples are then subjected to single quadrupole mass spectrometry using Waters QDa to detect vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, and vitamin E. (5) Detection data processing: The concentration of fat-soluble vitamins ADE is obtained by substituting the ratio of the peak area of ​​the sample to be tested and the peak area of ​​the internal standard into the calibration curve equation; Step (1) is as follows: (i) Prepare standard solutions: prepare different concentrations of vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E calibrant intermediate solutions for later use; (ii) Prepare the calibrant diluent: weigh bovine serum albumin, add it to water and dissolve it by sonication to obtain the calibrant diluent; (iii) Prepare a calibration curve for fat-soluble vitamins: pipette the calibration intermediate solution and add the calibration diluent to obtain multiple calibration lines with different concentrations; the concentration of vitamin A is 40 to 2000 ng / mL, the concentration of 25-hydroxyvitamin D2 is 2.5 to 125 ng / mL, the concentration of 25-hydroxyvitamin D3 is 2.5 to 125 ng / mL, and the concentration of vitamin E is 0.4 to 20 μg / mL; Step (2) is specifically as follows: using 0.1wt% 2,6-di-tert-butyl-4-methylphenol in methanol as a solvent, preparing isotope internal standards corresponding to vitamin A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and vitamin E, namely vitamin A-d6, 25-hydroxyvitamin D2-d3, 25-hydroxyvitamin D3-d3 and vitamin E-d6; the concentration of the internal standard mixed working solution is as follows: vitamin A-d6 is 800-1200ng / mL, 25-hydroxyvitamin D2-d3 is 30-90ng / mL, 25-hydroxyvitamin D3-d3 is 30-90ng / mL, and vitamin E-d6 is 1-5μg / mL; Step (3) is as follows: (a) Preparation of sample kit Prepare a 96-well plate, add diluent to wells 1 and 7, add magnetic bead solution to wells 2 and 8, add cleaning solution to wells 3, 9, 4, 10, 5 and 11, add eluent to wells 6 and 12, add internal standard mixed working solution and online diluent to the reagent compartment, respectively; wherein the diluent composition includes 50 vol% methanol water, the magnetic bead solution composition includes C18 silica gel magnetic adsorption microspheres, the cleaning solution composition includes 20 vol% methanol, 10 vol% ethanol, 0.2 vol% glacial acetic acid, and the balance is water, the eluent is acetonitrile, and the online diluent is water; (b) Sample preparation Transfer the sample to be tested and the mark line into a 1.5 mL microcentrifuge tube, place the microcentrifuge tube on the sample rack, and place the sample rack in the sample rack placement area of ​​the fully automatic mass spectrometer preprocessor; (c) Operation of the fully automatic mass spectrometer pre-treatment instrument Transfer the liquid treated by the sample kit into a 96-well microplate through pre-treatment; After the pretreatment process is completed, the 96-well microplate is taken out and tested on a mass spectrometer; The detection conditions in step (4) are controlled as follows: Liquid phase detection conditions: Chromatographic column: Waters C18 column; column temperature: 45°C; mobile phase: aqueous phase is 0.1% formic acid aqueous solution, organic phase is 2Mm ammonium formate and 0.1% formic acid methanol solution; injection volume: 10μL; flow rate: 0.4mL / min; mobile phase elution program: 0-0.5min, aqueous phase ratio is 25%, organic phase ratio is 75%; 0.5-2.5min, aqueous phase ratio is 25-5%, organic phase ratio is 75-95%; 2.5-5min, aqueous phase ratio is 5-0%, organic phase ratio is 95-100%; 5-5.5min, aqueous phase ratio is 0%, organic phase ratio is 100%; 5.5-5.6min, aqueous phase ratio is 0-25%, organic phase ratio is 100-75%; 5.6-6.5min, aqueous phase ratio is 25%, organic phase ratio is 75%; Mass spectrometry detection conditions: Ionization mode: electrospray ionization positive mode; scanning mode: single ion scanning monitoring; capillary voltage: 0.8Kv; desolvation gas temperature: 400℃; desolvation gas flow rate: 800L / Hr; ion source temperature: 120℃; the mass number and cone voltage of each compound and internal standard are: vitamin A, parent ion 269.2m / z, cone voltage 35V; vitamin A-d6, parent ion 275.2m / z, cone voltage 35V; 25-hydroxyvitamin D2, parent ion 39 5.2m / z, cone voltage 25V; 25-hydroxyvitamin D2-d3, parent ion 398.2m / z, cone voltage 25V; 25-hydroxyvitamin D3, parent ion 383.3m / z, cone voltage 25V; 25-hydroxyvitamin D3-d3, parent ion 386.3m / z, cone voltage 25V; vitamin E, parent ion 431.4m / z, cone voltage 40V; vitamin E-d6, parent ion 437.4m / z, cone voltage 40V; In step (5), the calibration curve is obtained by drawing the calibration curve by using the labeled concentration of the calibration curve as the horizontal axis and the ratio of the actual detected peak area of ​​the calibration curve sample to the peak area of ​​each internal standard as the vertical axis.

2. The method for detecting fat-soluble vitamins ADE according to claim 1, characterized in that: The fully automatic mass spectrometry pre-processing instrument is the mass spectrometry pre-processing instrument IMS5000.

3. The method for detecting fat-soluble vitamins ADE by magnetic solid phase extraction combined with single-rod mass spectrometry according to claim 1, characterized in that In step (i), 0.1wt% 2,6-di-tert-butyl-4-methylphenol in methanol is used as a solvent to prepare calibration intermediate solutions of different concentrations, wherein the concentration of vitamin A is 800-40000 ng / mL, the concentration of 25-hydroxyvitamin D2 is 50-2500 ng / mL, the concentration of 25-hydroxyvitamin D3 is 50-2500 ng / mL, and the concentration of vitamin E is 8-400 μg / mL.

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