Vitamin detection method

Abstract

The invention relates to a method for detecting vitamins. The method comprises the following steps: pre-treating a sample, and carrying out liquid chromatography-tandem mass spectrometry detection on the pre-treated sample; the pretreatment method comprises the following steps: mixing a sample with water and an internal standard working solution, adding an extracting solution, centrifugally collecting a supernatant, drying, adding a reconstitution fluid, centrifugally collecting the supernatant; the internal standard working solution contains a precipitator, a vitamin standard substance, estriol and 2, 6-di-tert-butyl-4-methylphenol; the reconstitution fluid is prepared from methanol, estriol, 2, 6-di-tert-butyl-4-methylphenol and ammonium formate. According to the present invention, the vitamin A, the vitamin D (25-OH-D2, 25-OH-D3) and the vitamin E can be simultaneously detected, the specificity, the sensitivity and the accuracy are high, the sample demand is low, the derivatization treatment is not required, the operation is simple and convenient, and the popularization and the application are easily achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection and relates to a method for detecting vitamins. Background Art

[0002] Fat-soluble vitamins are micronutrients necessary for the human body to maintain an optimal metabolic state. With the deepening of research, the role of fat-soluble vitamins is no longer limited to people's initial understanding, such as night blindness (vitamin A deficiency), osteomalacia (vitamin D deficiency), abnormal oxidative stress (vitamin E deficiency), etc., but it has been found that abnormal changes in fat-soluble vitamins are related to more and more diseases and other physiological conditions. Studies have shown that vitamin A can promote the development of the immune system and enhance immunity. Its low content is even related to early adult obesity; vitamin D has a certain effect on the prevention of certain mental illnesses and diabetes; and appropriate concentrations of vitamin E are helpful for cancer prevention and the treatment of diabetic nephropathy and non-alcoholic fatty liver disease. With the increasingly clear relationship between vitamins and diseases and people's close attention to health issues, accurately, quickly and conveniently measuring the content of vitamins in the human body has become an urgent problem to be solved in the field of vitamin testing.

[0003] Traditional detection methods such as radioimmunoassay and enzyme-linked immunosorbent assay for vitamin detection are prone to cross-reaction with other non-target compounds, resulting in poor specificity. For example, it is difficult to distinguish between 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3, and there is a potential risk of radioactive contamination. Compared with traditional methods, liquid chromatography-tandem mass spectrometry has the advantages of high specificity, high sensitivity, and high accuracy, which has led to its rapid promotion in the field of vitamin detection. For example, LC-MS can simultaneously detect the precise concentrations of 25-hydroxyvitamin D2 (25-OH-D2) and 25-hydroxyvitamin D3 (25-OH-D3), making it a recognized gold standard for vitamin D detection. However, this method also has the problems of large sample volume (usually 2 mL of venous blood needs to be collected, and children and other patients have poor compliance), complex and time-consuming pretreatment, and complex instrument operation. It is currently mostly used for venous blood vitamin detection.

[0004] In summary, the current methods for detecting vitamins in the blood have problems such as low sensitivity, poor specificity, large sample volume required, and cumbersome operation. That is, how to develop a vitamin detection method that meets multiple requirements at the same time is still one of the urgent problems to be solved in the field of vitamin detection. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for detecting vitamins, so as to achieve rapid detection of multiple vitamins at the same time.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for detecting vitamins, the method comprising: pre-treating a sample, and performing liquid chromatography-tandem mass spectrometry detection on the pre-treated sample;

[0008] The pretreatment method comprises: taking a sample, mixing it with water and an internal standard working solution, adding an extracting solution, centrifugally collecting the upper clear liquid and drying it, adding a reconstitution solution, and centrifugally collecting the supernatant; the internal standard working solution contains a precipitant, a vitamin standard, estriol and 2,6-di-tert-butyl-4-methylphenol; and the reconstitution solution contains methanol, estriol, 2,6-di-tert-butyl-4-methylphenol and ammonium formate.

[0009] In the present invention, a new vitamin liquid chromatography-tandem mass spectrometry detection method is designed, a specific sample pretreatment process is designed, the sample is mixed with water to facilitate subsequent precipitation, effectively reduce the matrix effect, the precipitant is combined with the internal standard, and a specific internal standard working solution formula combination is designed. While simplifying the sample processing steps, the structure of the analyte is protected and the accuracy of detection is improved. A specific complex solution formula is designed to promote dissolution and effectively protect multiple vitamins in the sample, improve detection sensitivity and accuracy, and overall synergy is achieved to achieve rapid and accurate detection.

[0010] Preferably, the sample includes serum and / or plasma, etc.

[0011] Preferably, the sample volume is ≥10 μL.

[0012] In the present invention, a specific sample pretreatment process is designed based on the present invention, which can effectively reduce the sample requirement, and 10 μL of sample can meet the detection requirements.

[0013] Preferably, the vitamins include at least one of vitamin A, vitamin E, vitamin D2 or vitamin D3.

[0014] The present invention designs a vitamin LC-MS method with high specificity and sensitivity, which can realize the simultaneous detection of vitamin A, vitamin E, vitamin D2 and vitamin D3.

[0015] Preferably, the vitamin standard includes at least one of isotope-labeled vitamin A (VA), vitamin E9 (VE), vitamin D2 (25-OH-D2) or vitamin D3 (25-OH-D3).

[0016] Preferably, the vitamin standard includes at least one of vitamin A-d6, vitamin E-d6, vitamin D2-d3 or vitamin D3-d6.

[0017] Preferably, the concentration of vitamin A-d6 in the internal standard working solution is 0.1-0.3 ng / mL, for example, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26 or 0.28 ng / mL, and the concentration of vitamin E-d6 is 0.8-1.6 ng / mL, for example, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35 , 1.4, 1.45, 1.5 or 1.55 ng / mL, etc., the concentration of vitamin D2-d3 is 0.01~0.1 ng / mL, for example, it can be 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 or 0.09 mg / mL, etc., and the concentration of vitamin D3-d6 is 0.01~0.1 ng / mL, for example, it can be 0.03, 0.04, 0.05, 0.06, 0.07, 0.08 or 0.09 ng / mL, etc.

[0018] Preferably, the precipitant comprises at least one of acetonitrile, methanol or ethanol.

[0019] Preferably, the internal standard working solution uses the precipitant as solvent.

[0020] Preferably, the concentration of estriol in the internal standard working solution is 0.9-1.5 μL / mL, for example, 1.0, 1.1, 1.2, 1.3 or 1.4 μL / mL.

[0021] Preferably, the concentration of 2,6-di-tert-butyl-4-methylphenol in the internal standard working solution is 0.4-1.0 mg / mL, for example, 0.5, 0.6, 0.7, 0.8 or 0.9 mg / mL.

[0022] Preferably, the extract comprises n-hexane and / or methyl tert-butyl ether.

[0023] Preferably, the reconstituted solution uses an aqueous solution with a methanol content of 70% to 90% (for example, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86% or 88%) as a solvent, the concentration of estriol is 0.9 to 1.5 μL / mL, for example, 1.0, 1.1, 1.2, 1.3 or 1.4 μL / mL, the concentration of 2,6-di-tert-butyl-4-methylphenol is 0.4 to 1.0 mg / mL, for example, 0.5, 0.6, 0.7, 0.8 or 0.9 mg / mL, and the concentration of ammonium formate is 2.0 to 3.0 mmol / L, for example, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 mmol / L, etc.

[0024] Preferably, the volume ratio of the sample, water, internal standard working solution, extract and reconstitution solution is 1:(1-5):(5-10):(30-50):(5-10), for example, it can be 1:3:7:40:7, 1:2:9:35:9, 1:4:6:45:6 or 1:3:8:48:8, etc.

[0025] Preferably, the liquid chromatography-tandem mass spectrometry detection utilizes isotope internal standard method for quantification.

[0026] Preferably, the isotope internal standard method quantification includes establishing a calibration curve with the concentration ratio of the standard to the internal standard as the X-axis and the peak area ratio of the standard to the internal standard as the Y-axis to calculate the concentration of the vitamin.

[0027] Preferably, the mobile phase of the liquid chromatography comprises an organic phase and an aqueous phase, the organic phase comprises a methanol solution containing ammonium formate and formic acid, and the aqueous phase comprises an aqueous solution containing ammonium formate and formic acid.

[0028] Preferably, the concentration of ammonium formate in the organic phase is 2.0-3.0 mmol / L, for example, it can be 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 mmol / L, and the concentration of formic acid is 0.1%-2%, for example, it can be 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 1.2%, 1.4%, 1.6% or 1.8%, etc.

[0029] Preferably, the concentration of ammonium formate in the aqueous phase is 2.0-3.0 mmol / L, for example, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8 or 2.9 mmol / L, and the concentration of formic acid is 0.1%-2%, for example, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1%, 1.2%, 1.4%, 1.6% or 1.8%, etc.

[0030] More preferably, the liquid chromatography adopts a gradient elution mode, and the gradient elution conditions are as follows:

[0031]

[0032]

[0033] Preferably, the mass spectrometry conditions include: using an electrospray ion source, in positive ion mode, and detecting in multiple reaction monitoring mode.

[0034] Preferably, the conditions of the mass spectrometry include: an ion spray voltage of 4500-5500 V, for example, 4600, 4700, 4800, 5000, 5100, 5300 or 5400 V, etc., a temperature of 400-550° C., for example, 420, 450, 480, 500, 520, 530 or 540° C., etc., a declustering voltage of 55.0-65.0 V, for example, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 V, and an inlet voltage of 8.0-12.0 V, for example, 9, 10 or 11 V, etc.

[0035] Preferably, the mass spectrometry parameters are as follows:

[0036] Object to be tested Precursor ion (m / z) Product ion (m / z) Collision Energy Collision cell exit voltage 25-OH-D2 413.2 395.4 15.0～16.0 6～10 25-OH-D2 internal standard 416.2 398.4 15.0～16.0 6～10 25-OH-D3 401.3 383.4 12.0～15.0 6～10 25-OH-D3 internal standard 407.3 389.4 12.0～15.0 6～10 VA 269.2 93.1 20.0～25.0 6～10 VA internal standard 275.2 96.1 20.0～25.0 6～10 VE 431.4 165.1 68.0～72.0 6～10 VE internal standard 437.4 171.1 68.0～72.0 6～10

[0037] In conventional peripheral blood vitamin D detection methods, due to the low content of the analyte, a derivatization method is often used to improve the mass spectrometry response value. In the present invention, specific mass spectrometry parameters are designed to achieve the same sensitivity and accuracy without derivatization, and the chromatographic peak shape and separation are better, which simplifies the operation steps and improves the detection efficiency.

[0038] Preferably, the liquid chromatography-tandem mass spectrometry analysis system includes a SCIEX MPX system. Based on the SCIEX MPX system, two liquid phase systems can detect samples alternately, and the average detection time for each sample is 3 minutes.

[0039] As a preferred technical solution, the method comprises the following steps:

[0040] (1) taking a sample, adding deionized water to mix, and adding an internal standard working solution to mix, wherein the internal standard working solution contains a precipitant, a vitamin standard, estriol and 2,6-di-tert-butyl-4-methylphenol;

[0041] (2) adding an extracting solution and mixing, wherein the extracting solution includes n-hexane;

[0042] (3) centrifuging and drying the supernatant;

[0043] (4) adding a reconstitution solution and mixing, wherein the reconstitution solution contains methanol, estriol, 2,6-di-tert-butyl-4-methylphenol and ammonium formate;

[0044] (5) collecting the supernatant by centrifugation;

[0045] (6) taking the supernatant of step (5) for liquid chromatography-tandem mass spectrometry analysis; the mobile phase of the liquid chromatography comprises an organic phase and an aqueous phase, the organic phase comprises a methanol solution containing ammonium formate and formic acid, the aqueous phase comprises an aqueous solution containing ammonium formate and formic acid, and the mass spectrometry conditions include: using an electrospray ion source, in positive ion mode, using multiple reaction monitoring mode for detection, and using isotope internal standard method for quantification.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] The present invention designs a new vitamin liquid chromatography-tandem mass spectrometry detection method, designs a specific sample pretreatment process, and overall synergy, which can realize the simultaneous detection of vitamin A, vitamin D (25-OH-D2, 25-OH-D3) and vitamin E, and has high specificity, sensitivity and accuracy. At the same time, the sample requirement is low, and accurate measurement can be performed using only 10 μL of sample. In addition, the mass spectrometry parameters are further designed to achieve the same sensitivity and accuracy after derivatization without derivatization, and the chromatographic peak shape and separation are better, which simplifies the operation steps, improves the detection efficiency, and is conducive to popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Standard curve for vitamin A detection;

[0049] Figure 2 This is the standard curve for vitamin E detection;

[0050] Figure 3 Standard curve for vitamin D2 detection;

[0051] Figure 4 Standard curve for vitamin D3 detection;

[0052] Figure 5A-5C For the chromatogram using the detection method of the present invention, Figure 5A Total ion current diagram of vitamins A, D, and E. Figure 5B Vitamin D3 chromatogram, Figure 5C is the chromatogram of vitamin D2;

[0053] Figure 6A-6C This is the chromatogram when vitamin D is detected using the derivatization method. Fig. 6A Total ion current of vitamin D Figure 6B Vitamin D3 chromatogram, Figure 6C This is a chromatogram of vitamin D2. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0055] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0056] Example 1

[0057] This embodiment provides a method for simultaneously detecting the levels of vitamins A, D, and E in blood.

[0058] (I) Reagent preparation

[0059] 1) Configuration of standard working curve

[0060] Weigh 8.95 g of disodium hydrogen phosphate using a balance and dissolve it in 1 L of deionized water, and adjust the pH to 7.5 to obtain a phosphate buffer solution;

[0061] 40 g of bovine serum albumin (BSA) was weighed on a balance and added to the above phosphate buffer, and then 1 mL of estriol (1.0 mg / mL) was added.

[0062] VA, VD (25-OH-D2, 25-OH-D3), VE self-prepared standard working solution: (taking the highest concentration of the standard curve point as 25-OH-D2: 120ng / mL, 25-OH-D3: 120ng / mL, VA: 1.1mg / L, VE: 22mg / L as an example), the curve blank matrix is ​​4% BSA (containing 1.0μg / mL estriol) solution. S1~S6 added 25μL, 50μL, 100μL, 200μL, 480μL, 960μL of 12.5μg / mL 25-OH-D2 intermediate stock solution respectively; S1~S6 added 25μL, 50μL, 100μL, 200μL, 480μL, 960μL of 12.5μg / mL 25-OH-D3 intermediate stock solution respectively; S1~S6 added 200mg / L 15 μL, 30 μL, 60 μL, 120 μL, 275 μL, 550 μL of VA intermediate stock solution; 15 μL, 30 μL, 60 μL, 120 μL, 275 μL, 550 μL of 4000 mg / L VE intermediate stock solution were added to S1-S6, respectively; S1-S6 were respectively diluted to 100 mL with 4% BSA (containing 1.0 μg / mL estriol) solution, and the concentrations were VD The standard curve points are 3.125ng / mL, 6.25ng / mL, 12.5ng / mL, 25ng / mL, 60ng / mL, 120ng / mL, VA 0.03mg / L, 0.06mg / L, 0.12mg / L, 0.24mg / L, 0.55mg / L, 1.10mg / L, VE 0.6mg / L, 1.2mg / L, 2.4mg / L, 4.8mg / L, 11.0mg / L, 22.0mg / L, the zero point of the standard curve is 4% BSA, and the concentration of the standard curve points shall be subject to the actual preparation operation.

[0063] 2) Internal standard working solution

[0064] Isotope VA, VD, and VE internal standard working solutions: respectively pipette 1 μL of d3-25-OH-D2 stock solution (1.0 mg / mL), 1 μL of d6-25-OH-D3 stock solution (1.0 mg / mL), 150 μL of VA-d6 (0.145 mg / mL) standard stock solution, 8 μL of VE-d6 (10.0 mg / mL) standard stock solution, 0.04 g of 2,6-di-tert-butyl-4-methylphenol (BHT), and 0.1 mL of estriol stock solution (1.0 mg / mL) into a 100 mL volumetric flask and make up to volume with precipitant acetonitrile.

[0065] 3) Reconstitution solution

[0066] Reconstitution solution (80% methanol aqueous solution: containing 2.5mmol / L ammonium formate, 0.4mg / mL BHT): Measure 160mL of methanol into a reagent bottle, add 0.08g of BHT, then measure 40mL of distilled water into the same reagent bottle, add 200μL of 2.5mol / L ammonium formate buffer solution and 200μL of 1.0mg / mL estriol solution.

[0067] 4) Mobile phase

[0068] Organic phase: 1L of chromatography-grade methanol, add 1mL of 2.5mol / L ammonium formate buffer solution and 1mL of formic acid;

[0069] Aqueous phase (containing 2.5 mmol / L ammonium formate, 0.1% formic acid): Use a measuring cylinder to measure 1 L of ultrapure water into a reagent bottle, and add 1 mL of 2.5 mol / L ammonium formate buffer solution and 1 mL of formic acid.

[0070] (II) Sample processing:

[0071] 1. After the sample returns to room temperature, centrifuge and reversely pipette 10 μL of serum into a 1.5 mL centrifuge tube;

[0072] 2. Add 40 μL of deionized water;

[0073] 3. Add 100 μL of internal standard working solution and vortex for 5 minutes;

[0074] 4. Add 500 μL of n-hexane extract and vortex for 10 min;

[0075] 5. Centrifuge at 10,000 rpm and 25°C for 10 min.

[0076] 6. Transfer 450 μL of the upper layer of n-hexane into a 1.5 mL centrifuge tube and blow dry with nitrogen;

[0077] 7. Add 75 μL of reconstitution solution and vortex for 5 minutes;

[0078] 8. Centrifuge at 10,000 rpm, 25°C for 5 min;

[0079] 9. Transfer the sample to the liquid injection bottle and test it on the machine;

[0080] 10. Calculate the results and compare them with the standard working curve.

[0081] (III) Chromatographic column: Sigma C18 5.0cm×2.1mm 2.7μm

[0082] (IV) Detection instrument: SCIEX 4500 LC-MS / MS

[0083] (V) Mass spectrometry conditions

[0084] Ion source: electrospray ion source (ESI), in positive ion mode, MRM multiple reaction monitoring mode was used for detection, ion pairs as shown in Table 1, ion spray voltage (IS) was 5500 V, temperature (TEM) was 400 °C, declustering potential (DP) was 60.0 V, and entrance potential (EP) was 10.0 V.

[0085] Table 1

[0086]

[0087] (VI) Liquid phase conditions

[0088] Organic phase: methanol, containing 2.5mmol / L ammonium formate, 0.1% formic acid.

[0089] Aqueous phase: 100% deionized water, containing 2.5 mmol / L ammonium formate, 0.1% formic acid (filtered with a 0.22 μm filter membrane after preparation).

[0090] Needle washing solution: an aqueous solution containing 25% methanol, 25% acetonitrile and 25% isopropanol.

[0091] Column temperature: 40°C, injector temperature: 10°C, injection volume: 10 mL, flow rate: 0.4 mL / min.

[0092] Elution gradient: Gradient elution. The gradient elution conditions are shown in Table 2.

[0093] Table 2

[0094] Time (min) Organic phase ratio (%) Flow rate (mL / min) 0.00 40 0.40 0.50 40 0.40 2.00 90 0.40 3.49 90 0.40 3.50 100 0.60 5.00 100 0.60 5.01 40 0.40 6.00 40 0.40

[0095] Example 2

[0096] This embodiment provides a method for simultaneously detecting the levels of vitamins A, D, and E in blood.

[0097] (I) Reagent preparation

[0098] 1) Configuration of standard working curve

[0099] Refer to Example 1.

[0100] 2) Internal standard working solution

[0101] Isotope VA, VD, and VE internal standard working solutions: respectively pipette 10 μL of d3-25-OH-D2 stock solution (1.0 mg / mL), 10 μL of d6-25-OH-D3 stock solution (1.0 mg / mL), 200 μL of VA-d6 (0.145 mg / mL) standard stock solution, 16 μL of VE-d6 (10.0 mg / mL) standard stock solution, 0.1 g of 2,6-di-tert-butyl-4-methylphenol (BHT), and 0.09 mL of estriol stock solution (1.0 mg / mL) into a 100 mL volumetric flask and make up to volume with precipitant methanol.

[0102] 3) Reconstitution solution

[0103] The reconstitution solution was prepared using 70% methanol aqueous solution as solvent, containing 0.4 mg / mL BHT, 3.0 mmol / L ammonium formate and 0.9 μL / mL estriol.

[0104] 4) Mobile phase

[0105] Organic phase: Use chromatography grade methanol to prepare a solution containing 2 mmol / L ammonium formate and 2% formic acid;

[0106] Aqueous phase: prepare an aqueous solution containing 3 mmol / L ammonium formate and 1% formic acid.

[0107] (III) Sample processing:

[0108] 1. After the sample returns to room temperature, centrifuge and reversely pipette 10 μL of serum into a 1.5 mL centrifuge tube;

[0109] 2. Add 10 μL of deionized water;

[0110] 3. Add 50 μL of internal standard working solution and vortex for 5 minutes;

[0111] 4. Add 300 μL of n-hexane extract and vortex for 10 min;

[0112] 5. Centrifuge at 10,000 rpm and 25°C for 10 min.

[0113] 6. Transfer 400 μL of the upper layer of n-hexane into a 1.5 mL centrifuge tube and blow dry with nitrogen;

[0114] 7. Add 50 μL of reconstitution solution and vortex for 5 minutes;

[0115] 8. Centrifuge at 10,000 rpm, 25°C for 5 min;

[0116] 11. Transfer the sample to a liquid injection bottle and test it on the machine;

[0117] 12. Calculate the results and compare them with the standard working curve.

[0118] (III) Chromatographic column: Sigma C18 5.0cm×2.1mm 2.7μm

[0119] (IV) Detection instrument: SCIEX 4500 LC-MS / MS

[0120] (V) Mass spectrometry conditions

[0121] Ion source: electrospray ion source (ESI), in positive ion mode, MRM multiple reaction monitoring mode was used for detection, ion pairs as shown in Table 3, ion spray voltage (IS) was 4500 V, temperature (TEM) was 500 °C, declustering potential (DP) was 55.0 V, and entrance potential (EP) was 8.0 V.

[0122] Table 3

[0123]

[0124] (VI) Liquid phase conditions

[0125] Organic phase: methanol, containing 2mmol / L ammonium formate, 2% formic acid.

[0126] Aqueous phase: 100% deionized water, containing 3 mmol / L ammonium formate, 1% formic acid (filtered with a 0.22 μm filter membrane after preparation).

[0127] Needle washing solution: an aqueous solution containing 25% methanol, 25% acetonitrile and 25% isopropanol.

[0128] Column temperature: 40°C, injector temperature: 10°C, injection volume: 10 mL, flow rate: 0.4 mL / min.

[0129] Elution gradient: Gradient elution. The gradient elution conditions are shown in Table 4.

[0130] Table 4

[0131]

[0132]

[0133] Example 3

[0134] This embodiment provides a method for simultaneously detecting the levels of vitamins A, D, and E in blood.

[0135] (I) Reagent preparation

[0136] 1) Configuration of standard working curve

[0137] Refer to Example 1.

[0138] 2) Internal standard working solution

[0139] Isotope VA, VD, and VE internal standard working solutions: respectively pipette 1 μL of d3-25-OH-D2 stock solution (1.0 mg / mL), 1 μL of d6-25-OH-D3 stock solution (1.0 mg / mL), 100 μL of VA-d6 (0.145 mg / mL) standard stock solution, 8 μL of VE-d6 (10.0 mg / mL) standard stock solution, 0.08 g of 2,6-di-tert-butyl-4-methylphenol (BHT), and 0.15 mL of estriol stock solution (1.0 mg / mL) into a 100 mL volumetric flask and make up to volume with precipitant methanol.

[0140] 3) Reconstitution solution

[0141] The reconstitution solution was prepared using 90% methanol aqueous solution as solvent, containing 1.0 mg / mL BHT, 2.0 mmol / L ammonium formate and 1.5 μL / mL estriol.

[0142] 4) Mobile phase

[0143] Organic phase: Use chromatography grade methanol to prepare a solution containing 3 mmol / L ammonium formate and 1% formic acid;

[0144] Aqueous phase: prepare an aqueous solution containing 2 mmol / L ammonium formate and 2% formic acid.

[0145] (IV) Sample processing:

[0146] 1. After the sample returns to room temperature, centrifuge and reversely pipette 10 μL of serum into a 1.5 mL centrifuge tube;

[0147] 2. Add 10 μL of deionized water;

[0148] 3. Add 50 μL of internal standard working solution and vortex for 5 minutes;

[0149] 4. Add 300 μL of n-hexane extract and vortex for 10 min;

[0150] 5. Centrifuge at 10,000 rpm and 25°C for 10 min.

[0151] 6. Transfer 400 μL of the upper layer of n-hexane into a 1.5 mL centrifuge tube and blow dry with nitrogen;

[0152] 7. Add 50 μL of reconstitution solution and vortex for 5 minutes;

[0153] 8. Centrifuge at 10,000 rpm, 25°C for 5 min;

[0154] 13. Transfer the sample to a liquid injection bottle and test it on the machine;

[0155] 14. Calculate the results and compare them with the standard working curve.

[0156] (III) Chromatographic column: Sigma C18 5.0cm×2.1mm 2.7μm

[0157] (IV) Detection instrument: SCIEX 4500 LC-MS / MS

[0158] (V) Mass spectrometry conditions

[0159] Ion source: electrospray ion source (ESI), in positive ion mode, MRM multiple reaction monitoring mode was used for detection, ion pairs as shown in Table 5, ion spray voltage (IS) was 4500 V, temperature (TEM) was 500 °C, declustering potential (DP) was 55.0 V, and entrance potential (EP) was 8.0 V.

[0160] Table 5

[0161]

[0162]

[0163] (VI) Liquid phase conditions

[0164] Organic phase: methanol, containing 3mmol / L ammonium formate, 1% formic acid.

[0165] Aqueous phase: 100% deionized water, containing 2 mmol / L ammonium formate, 2% formic acid (filtered with a 0.22 μm filter membrane after preparation).

[0166] Needle washing solution: an aqueous solution containing 25% methanol, 25% acetonitrile and 25% isopropanol.

[0167] Column temperature: 40°C, injector temperature: 10°C, injection volume: 10 mL, flow rate: 0.4 mL / min.

[0168] Elution gradient: Gradient elution. The gradient elution conditions are shown in Table 6.

[0169] Table 6

[0170] Time (min) Organic phase ratio (%) Flow rate (mL / min) 0.00 40 0.40 0.50 40 0.40 2.00 90 0.40 3.49 90 0.40 3.50 100 0.60 5.00 100 0.60 5.01 40 0.40 6.00 40 0.40

[0171] Comparative Example 1

[0172] The method of Example 1 was used, except that the reconstitution solution was 50% methanol aqueous solution and 80% methanol aqueous solution. The results are shown in Table 7 (wherein, VD-Q1 and VD-Q2 refer to the concentrations of vitamin D quality control 1 and vitamin D quality control 2, and VAE-Q1 and VAE-Q2 refer to the concentrations of vitamin AE quality control 1 and vitamin AE quality control 2). Compared with the results of Example 1, the response value of the vitamin E internal standard is lower.

[0173] Table 7

[0174]

[0175]

[0176] Comparative Example 2

[0177] Referring to the method of Example 1, the only difference is that 40 μL of deionized water is not added in step 2 of sample processing. The test results show that the vitamin E matrix effect is strong, the quality control is not under control, and the accuracy cannot meet the standard (Table 8).

[0178] Table 8

[0179] Sample name Measured Theoretical value Offset VAE-Q1 5.37 10.0 -46％ VAE-Q2 7.73 20.0 -61％ VAE-Q1 5.37 10.0 -47％ VAE-Q2 7.73 20.0 -57％

[0180] Test Case

[0181] This test example uses the scheme in Example 1 as an example to verify the method and analyze the detection results in Example 1.

[0182] (1) Analytical sensitivity (quantitation limit) and analytical measurement range. The results are shown in Tables 9, 10 and 11. The results show that the detection method of the present invention has good linear performance when the vitamin D2 concentration range is 2.60-99.60 ng / mL, the vitamin D3 concentration range is 3.74-144.62 ng / mL, the vitamin A concentration range is 0.026-0.968 mg / L, and the vitamin E concentration range is 0.53-19.36 ng / mL.

[0183] Table 9

[0184]

[0185]

[0186]

[0187] Table 10

[0188]

[0189]

[0190] Table 11

[0191]

[0192]

[0193] (2) With concentration as the horizontal axis and the ratio of the chromatographic peak area of ​​the analyte to the internal standard as the vertical axis, the curves were fitted and constructed. The linear results of the vitamin A curve, vitamin E curve, vitamin D2 curve, and vitamin D3 curve are shown in the following figure. Figure 1-Figure 4 As shown, the results show that within this concentration range, the detection of the four vitamins has good linear performance.

[0194] (3) Accuracy

[0195] At least 20 clinical samples with different concentration distributions were collected and processed to compare the detection method of the present invention with the existing detection method.

[0196] The existing method uses Shimadzu 8060 as the detection instrument and Waters ACQUITY UPLC BEH C185cm×2.1mm×1.7μm as the chromatographic column. Vitamins D, A, and E need to be detected separately, and vitamin D needs to be derivatized. The specific method is as follows:

[0197] Vitamin D detection method: 1) Pipette 10 μL sample into a 1.5 mL centrifuge tube; 2) Add 20 μL of internal standard working solution and vortex for 5 minutes; 3) Add 20 μL of 2% ammonia solution and vortex for 1 minute; 4) Add 500 μL of extract n-hexane and vortex for 10 minutes; 5) Centrifuge at 10,000 rpm for 10 minutes; 6) Pipette 450 μL of the upper n-hexane into a 1.5 mL centrifuge tube and heat at 60°C and blow dry with nitrogen; 7) Let cool, add 200 μL of derivatization, shake at 10,000 rpm for 1 hour, blow dry with nitrogen without heating for about 6 minutes; 8) Add 75 μL of reconstitution solution and vortex for 5 minutes; 9) Centrifuge at 10,000 rpm for 5 minutes; 10) Detect on the machine;

[0198] Vitamin A and E detection method: 1) Pipette 10 μL of sample into a 1.5 mL centrifuge tube, then add 40 μL of deionized water;

[0199] 2) Add 100 μL of internal standard working solution and vortex for 5 minutes; 3) Add 500 μL of extraction solution n-hexane and vortex for 10 minutes; 4) Centrifuge at 10000 rpm for 10 minutes; 5) Pipette 450 μL of the upper n-hexane layer into a 1.5 mL centrifuge tube and blow dry with nitrogen for about 5 minutes; 6) Add 75 μL of reconstitution solution and vortex for 5 minutes; 7) Centrifuge at 10000 rpm for 5 minutes; 8) Detect on the machine.

[0200] The results are shown in Tables 12-15 (detection system A is the existing detection method, and detection system B is the detection method of the present invention). There is no significant difference in detecting clinical patient samples by the two methods, indicating that the new method designed by the present invention can ensure accuracy and meet actual detection requirements.

[0201] Table 12

[0202]

[0203]

[0204] Table 13

[0205]

[0206] Table 14

[0207]

[0208]

[0209] Table 15

[0210]

[0211] (4) Precision

[0212] 1) Repeatability precision

[0213] High and low concentration samples were collected and mixed for repeatability precision verification. 20 samples of each level were processed in parallel and tested continuously, with each sample tested once. The repeatability precision results are shown in Table 16. The detection method of the present invention has high repeatability precision and low concentration CV<20%, which meets the requirements.

[0214] Table 16

[0215]

[0216]

[0217] 2) Intermediate precision

[0218] High and low concentration samples were selected to verify the intermediate precision. Four batches of samples at each level were processed every day and measured for five days, with a total of 20 test data accumulated. The intermediate precision results are shown in Table 17. The intermediate precision high and low concentration CVs were both less than 1 / 3ALE (8.3%), meeting the requirements.

[0219] Table 17

[0220]

[0221]

[0222] (5) Matrix specificity

[0223] The retention times of VD2, VD3, VA and VE were 3.30min, 3.22min, 3.49min and 4.71min respectively, while no peaks were detected in the blank matrix at the peaks of VA, VE, 25-OH-D2, 25-OH-D3 analytes and internal standard or the peaks were less than 20% of the quantitative lower limit peak area, indicating that the detection method of the present invention can effectively reduce the sample matrix effect and the matrix specificity meets the requirements.

[0224] (6) Matrix effect

[0225] The matrix for preparing the standard curve is used to dissolve at least two standard solutions L and H with different concentration levels, high and low; at least 6 patient matrix samples from different sources are collected and numbered AF; the standard solution and the 6 patient matrix samples from different sources are mixed in a ratio of 1:1 to obtain mixed samples LA-LF and HA-HF; 3 samples of the patient samples and the mixed samples are processed in parallel, and then tested on a machine; the difference between the response value (analyte / internal standard) of the mixed sample and the mean response value of the patient sample and the standard solution is less than 20% (the experimental data of the matrix effect of VA, VE, VD2 and VD3 are shown in Tables 18 to 21, respectively), indicating that the detection method of the present invention can effectively reduce the sample matrix effect.

[0226] Table 18

[0227]

[0228]

[0229] Table 19

[0230]

[0231]

[0232]

[0233] Table 20

[0234]

[0235]

[0236] Table 21

[0237]

[0238]

[0239]

[0240] (7) Carryover effect

[0241] The test was performed three times in the following cycle: C1 = sample 1 (LLOQ concentration) → C2 = sample 2 (high concentration) → C3 = sample 1 (LLOQ concentration); (C3-C1) / C1≤20%. The results are shown in Table 22, indicating that the system has no carryover effect.

[0242] Table 22

[0243]

[0244] In addition, in conventional peripheral blood vitamin D detection methods, due to the low content of the analyte, a derivatization method is often used to improve the mass spectrometry response value. However, after the specific mass spectrometry parameters are designed in the present invention, the same sensitivity and accuracy can be achieved without derivatization, and the chromatographic peak shape and separation are better. The chromatograms of non-derivatized and derivatized vitamin D detected by the same instrument are as follows: Figure 5A-5C and Figure 6A-6C As shown, Figure 5A-5C The chromatograms of the detection method of the present invention are respectively the total ion current of vitamins A, D, E, the chromatogram of vitamin D3, and the chromatogram of vitamin D2. Figure 6A-6C These are the chromatograms when vitamin D is detected using the derivatization method, which are the total ion current of vitamin D, the chromatogram of vitamin D3, and the chromatogram of vitamin D2, respectively.

[0245] The present invention designs a new vitamin liquid chromatography-tandem mass spectrometry detection method, designs a specific sample pretreatment process, and overall synergy, which can realize the simultaneous detection of vitamin A, vitamin D (25-OH-D2, 25-OH-D3) and vitamin E, and has high specificity, sensitivity and accuracy. At the same time, the sample requirement is low, and only 10 μL of sample is used for accurate measurement. In addition, the mass spectrometry parameters are further designed to achieve the same sensitivity and accuracy after derivatization without derivatization, and the chromatographic peak shape and separation are better, which simplifies the operation steps, improves the detection efficiency, and is conducive to popularization and application.

[0246] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting vitamins, characterized in that: The method comprises: Pre-treating the samples, and performing liquid chromatography-tandem mass spectrometry detection on the pre-treated samples; The pretreatment method comprises: Take the sample and mix it with water and internal standard working solution, add the extraction solution, centrifuge to collect the supernatant and dry it, add the reconstitution solution, centrifuge to collect the supernatant; The internal standard working solution contains a precipitant, a vitamin standard, estriol and 2,6-di-tert-butyl-4-methylphenol; The reconstituted solution contains methanol, estriol, 2,6-di-tert-butyl-4-methylphenol and ammonium formate.

2. The method for detecting vitamins according to claim 1, characterized in that: The sample includes serum and / or plasma; Preferably, the sample volume is ≥10 μL; Preferably, the vitamins include at least one of vitamin A, vitamin E, vitamin D2 or vitamin D3; Preferably, the vitamin standard comprises at least one of isotope-labeled vitamin A, vitamin E, vitamin D2 or vitamin D3; Preferably, the vitamin standard includes at least one of vitamin A-d6, vitamin E-d6, vitamin D2-d3 or vitamin D3-d6.

3. The method for detecting vitamins according to claim 1, characterized in that: The precipitant comprises at least one of acetonitrile, methanol or ethanol; Preferably, the internal standard working solution uses the precipitant as solvent.

4. The method for detecting vitamins according to claim 1, characterized in that: The concentration of estriol in the internal standard working solution is 0.9-1.5 μL / mL; Preferably, the concentration of 2,6-di-tert-butyl-4-methylphenol in the internal standard working solution is 0.4-1.0 mg / mL.

5. The method for detecting vitamins according to claim 1, characterized in that: The extracting solution comprises n-hexane and / or methyl tert-butyl ether.

6. The method for detecting fat-soluble vitamins according to claim 1, characterized in that: The complex solution uses an aqueous solution with a methanol content of 70% to 90% as a solvent, an estriol concentration of 0.9 to 1.5 μL / mL, a 2,6-di-tert-butyl-4-methylphenol concentration of 0.4 to 1.0 mg / mL, and an ammonium formate concentration of 2.0 to 3.0 mmol / L.

7. The method for detecting fat-soluble vitamins according to claim 1, characterized in that: The volume ratio of the sample, water, internal standard working solution, extracting solution and reconstitution solution is 1:(1-5):(5-10):(30-50):(5-10).

8. The method for detecting vitamins according to claim 1, characterized in that: The liquid chromatography-tandem mass spectrometry detection utilizes isotope internal standard method for quantification; Preferably, the mobile phase of the liquid chromatography comprises an organic phase and an aqueous phase, the organic phase comprises a methanol solution containing ammonium formate and formic acid, and the aqueous phase comprises an aqueous solution containing ammonium formate and formic acid.

9. The method for detecting vitamins according to claim 1, characterized in that: The mass spectrometry conditions include: using an electrospray ion source, in a positive ion mode, and using a multiple reaction monitoring mode for detection.

10. The method for detecting fat-soluble vitamins in serum according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: (1) taking a sample, adding deionized water to mix, and adding an internal standard working solution to mix, wherein the internal standard working solution contains a precipitant, a vitamin standard, estriol and 2,6-di-tert-butyl-4-methylphenol; (2) adding an extracting solution and mixing, wherein the extracting solution includes n-hexane; (3) centrifuging and drying the supernatant; (4) adding a reconstitution solution and mixing, wherein the reconstitution solution contains methanol, estriol, 2,6-di-tert-butyl-4-methylphenol and ammonium formate; (5) collecting the supernatant by centrifugation; (6) taking the supernatant of step (5) for liquid chromatography-tandem mass spectrometry analysis; the mobile phase of the liquid chromatography comprises an organic phase and an aqueous phase, the organic phase comprises a methanol solution containing ammonium formate and formic acid, the aqueous phase comprises an aqueous solution containing ammonium formate and formic acid, and the mass spectrometry conditions include: using an electrospray ion source, in positive ion mode, using multiple reaction monitoring mode for detection, and using isotope internal standard method for quantification.

Images