Method for detecting content of vitamin A in serum, vitamin A structure derivative and application of vitamin A structure derivative

Abstract

The invention relates to a method for detecting the content of vitamin A in serum, a vitamin A structure derivative and application of the vitamin A structure derivative. The method for detecting the content of vitamin A in serum comprises the following steps: mixing a vitamin A structure derivative with a first solvent to prepare an internal standard solution; mixing a vitamin A standard substance with a second solvent to prepare standard substance solutions with different concentrations; respectively adding the same amount of the internal standard solution into the standard substance solutions with different concentrations and the serum sample to be detected, and then detecting by adopting a liquid chromatography-mass spectrometry technology to obtain the content of the vitamin A in the serum sample to be detected. According to the detection method, a substitute of a vitamin A isotope internal standard substance is adopted for detection, so that the detection cost is reduced while accurate and quantitative detection of vitamin A in serum is realized.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a method for detecting the content of vitamin A in serum, a vitamin A structural derivative and an application thereof. Background Art

[0002] Vitamin A (VA) is a fat-soluble vitamin and one of the essential nutrients for the human body. It has many physiological functions, such as promoting growth and reproduction, maintaining bones, epithelial tissues, vision and normal secretion of mucosal epithelium. Vitamin A deficiency is considered a public health problem, which can lead to night blindness, decreased body resistance, reproductive function decline, hair loss, muscle and visceral atrophy in children, poor bone growth and growth retardation, female infertility and other diseases. Therefore, it is necessary to accurately detect the content of vitamin A in human serum.

[0003] In clinical mass spectrometry applications, LC-MS / MS has the advantages of high sensitivity, strong specificity, fast analysis speed and high throughput, and has become the gold standard for quantitative analysis of biological samples. In order to accurately quantify the analyte, internal standards are usually required in the LC-MS / MS detection process. Its functions and principles include: first, calibrating the loss and extraction efficiency of the analyte during the tedious sample pretreatment process; second, calibrating the sample background differences caused by differences in individual physique, diet, etc. of biological samples; third, calibrating the deviations caused by liquid phase injection, chromatographic separation and mass spectrometry ionization. Therefore, internal standards are indispensable in the accurate quantitative detection of analytes using the internal standard method.

[0004] At present, for the LC-MS / MS quantitative detection of vitamin A, it is usually necessary to use a stable isotope internal standard as the internal standard. The isotope internal standard and the analyte are the same substance, but several hydrogen atoms are replaced by deuterium atoms, or 12C is replaced by 13C. The physical and chemical properties remain basically unchanged, so it can achieve a good calibration effect and accurately quantify the vitamin A in serum. However, the stable isotope internal standard of vitamin A currently mainly comes from three import companies: CIL in the United States, Sigma-Aldrich of Merck in Germany, and TRC in Canada. The cost is relatively high, and the reference range of vitamin A in the normal population is between 160ng / mL and 1200ng / mL. It is a fat-soluble vitamin with a high content in common clinical tests. The amount of internal standard used in combination is also large, which further increases the cost and is not conducive to promotion and application. Summary of the invention

[0005] Based on this, the present application provides a method for detecting the vitamin A content in serum, in which a substitute for the vitamin A isotope internal standard is used for detection, thereby achieving accurate quantitative detection of vitamin A in serum and reducing the detection cost.

[0006] In a first aspect of the present application, a method for detecting the content of vitamin A in serum is provided, comprising the following steps:

[0007] Mixing the vitamin A structural derivative with the first solvent to prepare an internal standard solution;

[0008] Mixing a vitamin A standard with a second solvent to prepare standard solutions of different concentrations;

[0009] Adding the same amount of the internal standard solution to the standard solutions of different concentrations and the serum sample to be tested respectively, and then performing detection by liquid chromatography-mass spectrometry;

[0010] Constructing a standard curve according to the test results of standard solutions of different concentrations after adding the internal standard solution, substituting the test results of the serum sample to be tested after adding the internal standard solution into the standard curve to obtain the content of vitamin A in the serum sample to be tested;

[0011] Wherein, the vitamin A structural derivative has the following structure:

[0012] or .

[0013] In one embodiment, the liquid phase in the liquid chromatography-mass spectrometry technique is high performance liquid chromatography, and the detection conditions of the high performance liquid chromatography include:

[0014] (1) The chromatographic column is a C18 chromatographic column;

[0015] (2) Mobile phase A was an aqueous solution of formic acid with a volume percentage of 0.08%-0.12%, and mobile phase B was a methanol solution of formic acid with a volume percentage of 0.08%-0.12%. Gradient elution was adopted, including the following procedures: 0-0.2 min, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was 80%; 0.2 min-2.5 min, the volume percentage of mobile phase A was changed from 20% to 1%, and the volume percentage of mobile phase B was changed from 80% to 99%; 2.5 min-3.5 min, the volume percentage of mobile phase A was maintained at 1%, and the volume percentage of mobile phase B was maintained at 99%; 3.5 min-3.51 min, the volume percentage of mobile phase A was changed from 1% to 20%, and the volume percentage of mobile phase B was changed from 99% to 80%; 3.51 min-4.5 min, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was maintained at 80%.

[0016] In one embodiment, the detection conditions of the HPLC further include one or more of the following:

[0017] (1) Column temperature is 39°C~45°C;

[0018] (2) Flow rate: 0.3 mL / min~0.8 mL / min;

[0019] (3) The injection volume is 8 μL~15 μL;

[0020] (4) The dimensions of the chromatographic column include: length of 45 mm to 55 mm, diameter of 2 mm to 2.3 mm, and filler particle size of 2.5 μm to 3 μm.

[0021] In one embodiment, the mass spectrometer in the liquid chromatography-mass spectrometry technique is a triple quadrupole mass spectrometer, and the detection conditions of the mass spectrometer include:

[0022] Ion source: atmospheric pressure chemical ionization;

[0023] Detection method: Multiple reaction monitoring;

[0024] Air curtain gas: 33psi~37psi;

[0025] Atomizing gas: 38psi~42psi;

[0026] Temperature: 370℃~430℃;

[0027] Collision gas: 7psi~9psi.

[0028] In one embodiment, the first solvent and the second solvent each independently include one or both of acetonitrile and bovine serum albumin aqueous solution.

[0029] In one embodiment, the concentration of the vitamin A structural derivative in the internal standard solution is 200 ng / mL~500 ng / mL, and the amount of the internal standard solution added to the standard solutions of different concentrations and the serum samples to be tested is: 150 μL~250 μL of the internal standard solution is added for every 50 μL of the standard solutions of different concentrations and the serum samples to be tested.

[0030] In one embodiment, the concentration distribution range of vitamin A in standard solutions of different concentrations is 15 ng / mL to 2000 ng / mL.

[0031] In a second aspect of the present application, a vitamin A structural derivative is provided, having the following structural characteristics:

[0032] .

[0033] In the third aspect of the present application, a vitamin A structural derivative is provided, having the following structural characteristics:

[0034] .

[0035] The fourth aspect of the present application provides the use of the vitamin A structural derivatives described in the second and third aspects as internal standards in the detection of vitamin A content in serum.

[0036] The above detection method adopts a specific structure of vitamin A structural derivative as an internal standard and combines it with liquid chromatography-mass spectrometry to detect the vitamin A content in serum. It can avoid the use of stable isotope internal standard of vitamin A, greatly reduce the detection cost of vitamin A content in serum, and at the same time, the detection result is accurate, which is conducive to promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the mass spectrum of vitamin A structural derivative 1.

[0038] Figure 2 This is the nuclear magnetic resonance spectrum of vitamin A structural derivative 1.

[0039] Figure 3 This is the mass spectrum of vitamin A structural derivative 2.

[0040] Figure 4 This is the nuclear magnetic resonance spectrum of vitamin A structural derivative 2.

[0041] Figure 5 This is the fitting curve of the VA isotope internal standard test results of 30 groups of serum samples.

[0042] Figure 6 This is the fitting curve of the test results of vitamin A structural derivative 1 in 30 groups of serum samples.

[0043] Figure 7 This is the fitting curve of the test results of vitamin A structural derivative 2 in 30 groups of serum samples.

[0044] Figure 8 This is the fitting curve of the test results of vitamin A structural analog 3 in 30 groups of serum samples.

[0045] Fig. 9 This is the fitting curve of the test results of vitamin A structural analog 4 in 30 groups of serum samples.

[0046] Fig.10 This is the fitting curve of the test results of vitamin A structural analog 5 in 30 groups of serum samples. DETAILED DESCRIPTION

[0047] The following is a further detailed description of the detection method of the vitamin A content in serum of the present application, the vitamin A structural derivatives and their applications in conjunction with specific examples. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0049] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0050] In this application, "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0051] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0052] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0053] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0054] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0055] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.

[0056] The room temperature in this application generally refers to 4°C~30°C, preferably 20±5°C.

[0057] At present, the detection of vitamin A content in serum usually uses isotope internal standards, which have the problem of high cost. Therefore, it is necessary to actively find low-cost compounds that can effectively replace isotope internal standards to achieve accurate detection of vitamin A content in serum. Whether a compound can replace isotope internal standards mainly needs to meet the following three points: (1) matrix effect, the matrix effect of the compound and the isotope internal standard should be as consistent as possible; (2) extraction efficiency, the extraction efficiency of the compound and the isotope internal standard should be as consistent as possible; (3) real sample measurement value fitting, the compound and the isotope internal standard are used to test the real samples within the reference range respectively, and the consistency analysis meets the statistical algorithm, and there is no obvious linear deviation. Among them, consistency is the key to determine whether a compound can replace the isotope internal standard as an internal standard.

[0058] After research, the present applicant discovered two vitamin A structural derivatives, which have good consistency with the detection results of vitamin A isotope internal standard in the detection of vitamin A content in serum, and the matrix effect is also consistent. They can effectively replace the isotope internal standard and realize accurate detection of vitamin A content in serum. At the same time, the preparation cost is low, which is easy to promote and apply.

[0059] Based on this, the present application provides a method for detecting the content of vitamin A in serum, comprising the following steps:

[0060] Mixing the vitamin A structural derivative with the first solvent to prepare an internal standard solution;

[0061] Mixing a vitamin A standard with a second solvent to prepare standard solutions of different concentrations;

[0062] Adding the same amount of the internal standard solution to the standard solutions of different concentrations and the serum sample to be tested respectively, and then performing detection by liquid chromatography-mass spectrometry;

[0063] Constructing a standard curve according to the test results of standard solutions of different concentrations after adding the internal standard solution, substituting the test results of the serum sample to be tested after adding the internal standard solution into the standard curve to obtain the content of vitamin A in the serum sample to be tested;

[0064] Wherein, the vitamin A structural derivative has the following structure:

[0065] or .

[0066] It can be understood that "constructing a standard curve based on the test results of the standard solution of different concentrations after adding the internal standard solution" means that after the liquid phase-mass spectrometry technology is used for detection, the peak area ratio of the vitamin A standard to the vitamin A structural derivative (internal standard) is obtained, and the standard curve is constructed according to the different concentrations of the vitamin A standard and the peak area ratio of the vitamin A standard to the vitamin A structural derivative, wherein the concentration of the vitamin A standard is the X-axis, and the peak area ratio of the vitamin A standard to the vitamin A structural derivative is the Y-axis. Correspondingly, "substituting the test results of the serum sample to be tested after adding the internal standard solution into the standard curve" means that after the liquid phase-mass spectrometry technology is used for detection, the peak area ratio of the analyte to the vitamin A structural derivative (internal standard) is obtained, and after substituting it into the standard curve, the concentration of the analyte in the serum sample to be tested can be obtained.

[0067] In some examples, the liquid phase in the liquid phase-mass spectrometry technique is high performance liquid chromatography, and the detection conditions of the high performance liquid chromatography include:

[0068] (1) The chromatographic column is a C18 chromatographic column;

[0069] (2) Mobile phase A was an aqueous solution of formic acid with a volume percentage of 0.08%-0.12%, and mobile phase B was a methanol solution of formic acid with a volume percentage of 0.08%-0.12%. Gradient elution was adopted, including the following procedures: 0-0.2 min, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was 80%; 0.2 min-2.5 min, the volume percentage of mobile phase A was changed from 20% to 1%, and the volume percentage of mobile phase B was changed from 80% to 99%; 2.5 min-3.5 min, the volume percentage of mobile phase A was maintained at 1%, and the volume percentage of mobile phase B was maintained at 99%; 3.5 min-3.51 min, the volume percentage of mobile phase A was changed from 1% to 20%, and the volume percentage of mobile phase B was changed from 99% to 80%; 3.51 min-4.5 min, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was maintained at 80%.

[0070] Specifically, the volume percentage of the formic acid aqueous solution in the mobile phase A includes, but is not limited to, 0.08%, 0.09%, 0.1%, 0.11%, 0.12% or a range between any two of the foregoing.

[0071] Specifically, the volume percentage of the formic acid methanol solution in the mobile phase B includes, but is not limited to, 0.08%, 0.09%, 0.1%, 0.11%, 0.12% or a range between any two of the foregoing.

[0072] In some examples, the detection conditions of the HPLC further include: a column temperature of 39° C. to 45° C. Specifically, the column temperature includes but is not limited to: 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., or a range between any two of the foregoing.

[0073] In some examples, the detection conditions of the HPLC also include: a flow rate of 0.3 mL / min to 0.8 mL / min. Specifically, the flow rate includes but is not limited to: 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min or a range between any two of the foregoing.

[0074] In some examples, the detection conditions of the HPLC also include: the injection volume is 8 μL to 15 μL. Specifically, the injection volume includes but is not limited to: 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL or a range between any two of the foregoing.

[0075] In some examples, the detection conditions of the HPLC also include: the dimensions of the chromatographic column include: length of 45 mm to 55 mm, diameter of 2 mm to 2.3 mm, and filler particle size of 2.5 μm to 3 μm. Further, the dimensions of the chromatographic column include: length of 50 mm, diameter of 2.1 mm, and filler particle size of 2.7 μm.

[0076] In some of the examples, the mass spectrometer in the liquid chromatography-mass spectrometry technique is a triple quadrupole mass spectrometer, and the mass spectrometer in the liquid chromatography-mass spectrometry technique is a triple quadrupole mass spectrometer, and the detection conditions of the mass spectrometer include:

[0077] Ion source: atmospheric pressure chemical ionization;

[0078] Detection method: Multiple reaction monitoring;

[0079] Air curtain gas: 33psi~37psi;

[0080] Atomizing gas: 38psi~42psi;

[0081] Temperature: 370℃~430℃;

[0082] Collision gas: 7psi~9psi.

[0083] In some examples, the first solvent and the second solvent each independently include one or both of acetonitrile and bovine serum albumin aqueous solution. Without limitation, as required, the dissolution can be promoted by vortex oscillation during the preparation of each solution.

[0084] In some of the examples, the concentration of the vitamin A structural derivative in the internal standard solution is 200 ng / mL~500 ng / mL, and the amount of the internal standard solution added to the standard solutions of different concentrations and the serum samples to be tested is: 150 μL~250 μL of the internal standard solution is added for every 50 μL of the standard solutions of different concentrations and the serum samples to be tested.

[0085] In some of these examples, the concentration distribution of vitamin A standards in standard solutions of different concentrations ranges from 15 ng / mL to 2000 ng / mL.

[0086] In some other examples of the present application, a vitamin A structural derivative is provided, which has the following structural features:

[0087] .

[0088] In some other examples of the present application, a vitamin A structural derivative is provided, which has the following structural features:

[0089] .

[0090] In some other examples of the present application, the use of the vitamin A structural derivative as described above as an internal standard in the detection of vitamin A content in serum is provided.

[0091] For experimental parameters not specified in the following specific examples, reference is made to the instructions given in this application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by manufacturers. Unless otherwise specified, the raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known means.

[0092] Preparation Example 1

[0093] Synthesis of Vitamin A structural derivative 1:

[0094]

[0095] Preparation of intermediate 2:

[0096] The raw material 1 (28.5 mg, 0.0912 mmol) was dissolved in 1.7 mL CH2Cl2, cooled to 0°C, MnO2 (0.143, 1.64 mmol) and Na2CO3 (0.174 g, 1.64 mmol) were added, and then the temperature was raised to 25°C and stirred for reaction for 5 h; the reaction solution was filtered and dried, and purified by column chromatography (silica gel, eluent: 95:2:3 hexane:EtOAc:Et3N) to prepare intermediate 2.

[0097] Preparation of vitamin A structural derivative 1:

[0098] The reaction was carried out under light-proof and inert gas protection. 90 mg of the intermediate product 2 was dissolved in 8 mL of tetrahydrofuran, cooled to -78 °C, and a methyl lithium solution (ethyl ether, concentration of 1.6 M, 0.24 mL, 0.38 mmol) was added dropwise. The reaction was continued at -78 °C with stirring for 15 min, then the temperature was raised to room temperature and the reaction was continued for 1 h. About 8 mL of a saturated aqueous solution of NH4Cl was added to quench the reaction. The solution was extracted with ethyl ether, washed with brine, dried with magnesium sulfate, and concentrated to obtain a vitamin A structural derivative 1. Its mass spectrum and nuclear magnetic resonance spectrum are shown in FIG. Figure 1 and Figure 2 shown.

[0099] Preparation Example 2

[0100] Synthesis of Vitamin A structural derivative 2:

[0101]

[0102] The preparation steps are as follows:

[0103] Preparation of intermediate 2:

[0104] Same structure as vitamin A derivative 1.

[0105] Preparation of intermediate 4:

[0106] 90 mg of intermediate product 2 and 72 mg of raw material 3 were dissolved in 8 mL of tetrahydrofuran, cooled to 0°C, 200 mg of NaH was added, and then the temperature was raised to room temperature and stirred for reaction for 48 h. No post-treatment was required, and the reaction solution containing intermediate product 4 was directly used for the next step.

[0107] Preparation of Vitamin A structural derivative 2:

[0108] 500 mg of LiAlH4 was added to the reaction solution containing the intermediate product 4, and the reaction was continued at room temperature for 45 min to obtain the vitamin A structure derivative 2, whose mass spectrum and nuclear magnetic resonance spectrum are shown in Figure 3 and Figure 4 shown.

[0109] Example 1

[0110] This example is an investigation of alternatives to isotopic internal standards.

[0111] 1. Instruments and testing conditions:

[0112] Instrument: AB Sciex 4500 triple quadrupole mass spectrometer-high performance liquid chromatography (HPLC).

[0113] HPLC conditions:

[0114] (1) Chromatographic column: Yuexu Bolitimate C18 2.7μm 2.1*50mm;

[0115] (2) Mobile phase: A: 0.1% formic acid in water, B: 0.1% formic acid in methanol;

[0116] (3) Column temperature: 40°C;

[0117] (4) Injection volume: 10 μL;

[0118] (5) Flow rate: 0.5 mL / min;

[0119] (6) Elution gradient:

[0120] 0-0.2min, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was maintained at 80%;

[0121] From 0.2min to 2.5min, the volume percentage of mobile phase A changed from 20% to 1%, and the volume percentage of mobile phase B changed from 80% to 99%;

[0122] 2.5min~3.5min, keep the volume percentage of mobile phase A at 1% and the volume percentage of mobile phase B at 99%;

[0123] From 3.5 to 3.51 min, the volume percentage of mobile phase A changed from 1% to 20%, and the volume percentage of mobile phase B changed from 99% to 80%;

[0124] From 3.51min to 4.5min, the volume percentage of mobile phase A was maintained at 20% and the volume percentage of mobile phase B was maintained at 80%.

[0125] Mass spectrometry conditions:

[0126] Ion source: atmospheric pressure chemical ionization (APCI); detection method: multiple reaction monitoring (MRM); curtain gas (CUR): 35 psi, nebulizer gas (GS1): 40 psi, temperature (TEM): 400 °C, collision gas (CAD): 8 psi.

[0127] The mass spectrometry parameters of each compound, such as parent ion, daughter ion, dwell time, cone voltage, collision energy, etc., are shown in Table 1 below:

[0128] Table 1

[0129]

[0130] 2. Preparation of working solution:

[0131] (1) Preparation of standard curve working solution:

[0132] Weigh 10 mg of VA standard powder and add acetonitrile to make up to 10 mL to prepare a 1 mg / mL VA stock solution;

[0133] When in use, VA matrix solution (2 μg / mL) was prepared with 5% bovine serum albumin aqueous solution, and then a series of standards were prepared according to the formula in Table 2 below for drawing the standard curve.

[0134] Table 2

[0135]

[0136] (2) Preparation of internal standard working solution:

[0137] Weigh 10 mg of dry powder of VA isotope internal standard (VA-d5 isotope), add acetonitrile to make up to 10 mL, prepare 1 mg / mL VA-d5 stock solution, dilute the stock solution with acetonitrile at a volume ratio of 5:200 to prepare VA isotope internal standard working solution (concentration is 250 ng / mL).

[0138] Weigh 10 mg of VA structure derivative 1 dry powder, add acetonitrile to make up to 10 mL, prepare 1 mg / mL VA structure derivative 1 stock solution, dilute the stock solution with acetonitrile at a volume ratio of 5:200 to prepare VA structure derivative 1 internal standard working solution (concentration of 250 ng / mL);

[0139] Vitamin A structural derivatives 1.

[0140] Weigh 10 mg of dry powder of VA structure derivative 2, add acetonitrile to make up to 10 mL, prepare 1 mg / mL VA structure derivative 2 stock solution, dilute the stock solution with acetonitrile at a volume ratio of 5:200 to prepare VA structure derivative 2 internal standard working solution (concentration of 250 ng / mL);

[0141] Vitamin A structural derivatives2.

[0142] Weigh 10 mg of dry powder of VA structural analogue 3, add acetonitrile to make the volume to 10 mL, prepare 1 mg / mL VA structural analogue 3 stock solution, dilute the stock solution with acetonitrile at a volume ratio of 5:200 to prepare VA structural analogue 3 internal standard working solution (concentration of 250 ng / mL);

[0143] Vitamin A structural analogues 3.

[0144] Weigh 10 mg of dry powder of VA structural analog 4, add acetonitrile to make the volume to 10 mL, prepare 1 mg / mL VA structural analog 4 stock solution, dilute the stock solution with acetonitrile at a volume ratio of 5:200 to prepare VA structural analog 4 internal standard working solution (concentration of 250 ng / mL);

[0145] Vitamin A structural analogues4.

[0146] Weigh 10 mg of VA structural analogue 5 dry powder, add acetonitrile to make up to 10 mL, prepare 1 mg / mL VA structural analogue 5 stock solution, dilute the stock solution with acetonitrile at a volume ratio of 5:200 to prepare VA structural analogue 5 internal standard working solution (concentration of 250 ng / mL);

[0147] Vitamin A structural analogues 5.

[0148] (3) Test objects: 30 groups of real serum samples, sourced from the Medical Laboratory of BGI Biotechnology Co., Ltd.

[0149] (4) Detection: Take 50 μL of the prepared standard and serum sample, add 200 μL of the internal standard working solution (isotope, derivative or structural analog), vortex for 10 min, centrifuge at high speed (13000 rpm) for 10 min, take 100 μL of the supernatant for detection, draw a calibration curve using standards of different concentrations, and determine the concentration of the serum sample.

[0150] 3. Consistency Inspection

[0151] (1) Fitting of real sample measurement values

[0152] The standard that can be used as a substitute for the VA isotope internal standard is as follows: the standard curve working solution with the addition of the VA isotope internal standard and different ion pairs in the serum sample to be tested are used as the control group, and the standard curve working solution with the addition of the vitamin A structural derivatives 1, 2 or analogs 3, 4, 5 and different ion pairs in the serum sample to be tested are used as the experimental group. The slope of the linear fit between the experimental group and the control group is required to be between 0.85 and 1.15, and R 2 ≥0.95, which means that it can be used as a substitute for VA isotope internal standard to accurately quantify the VA content in the serum samples to be tested.

[0153] Testing steps:

[0154] 200 μL of internal standard working solution (isotope, derivative or structural analogue) was added to 50 μL of standard curve working solution and serum sample, respectively, and then injected into AB Sciex 4500 tandem triple quadrupole mass spectrometer-high performance liquid chromatograph for detection. A standard curve was constructed based on the detection results and concentration of the standard curve working solution after adding the internal standard working solution. The detection results of the serum samples to be tested after adding the internal standard working solution were substituted into the standard curve to calculate the content. Then, the test results of VA isotope as internal standard of 30 serum samples were linearly fitted with the test results of vitamin A structural derivative or vitamin A structural analogue as internal standard. The results are shown in Figure 2. Figures 5 to 10 shown.

[0155] It can be seen that the test results of vitamin A structural derivatives 1 and 2 are highly consistent with the VA isotope internal standard, with slopes of 1.01, 0.97, and 0.97, respectively, and R 2 The values ​​of VA are 0.9913, 0.9939 and 0.9893 respectively, which can be used as substitutes for VA isotope internal standards to accurately quantify the VA content in serum samples. However, vitamin A structural analogs 3, 4 and 5 are difficult to replace.

[0156] (2) Matrix effect and extraction efficiency

[0157] Testing steps:

[0158] Take 50 μL of standard curve working solution L8, add 200 μL of internal standard working solution (isotope or derivative), vortex for 10 min, centrifuge at high speed (13000 rpm) for 10 min, take 100 μL of supernatant for detection, and record the internal standard peak area in the sample as S1.

[0159] Take 50 μL of serum sample, add 200 μL of internal standard working solution (isotope or derivative), vortex for 10 min, centrifuge at high speed (13000 rpm) for 10 min, take 100 μL of supernatant for detection, and record the internal standard peak area in the sample as S0.

[0160] S1 / S0 is the matrix effect.

[0161] Extraction efficiency: Take 50 μL of serum sample, add 200 μL of internal standard working solution (isotope or derivative), vortex for 10 min, centrifuge at high speed (13000 rpm) for 10 min, take 100 μL of supernatant for detection, and record the internal standard peak area in the sample as S2.

[0162] Take 50 μL of serum sample, add 200 μL of working solution without internal standard, vortex for 10 min, centrifuge at high speed (13000 rpm) for 10 min, take 90 μL of supernatant and add 10 uL of internal standard working solution for detection on the machine, and the internal standard peak area in the sample is recorded as S3.

[0163] S2 / S3 is the extraction efficiency.

[0164] The test results are shown in Table 3 below:

[0165] Table 3

[0166]

[0167] It can be seen that the matrix effect, extraction efficiency and isotope deviation meet the requirements.

[0168] Example 2

[0169] This example uses the vitamin A structural derivative 1 as an internal standard to test the VA content in the serum sample.

[0170] The instrument and detection conditions, preparation of the standard curve working solution, preparation and detection of the internal standard working solution of the VA structure derivative 1 refer to Example 1. The standard curve constructed is: y=0.9744x+9.1894, R 2 =0.9939.

[0171] The calculated VA contents in 30 groups of serum samples are shown in Table 4 below:

[0172] Table 4

[0173]

[0174] Example 3

[0175] This example uses the vitamin A structural derivative 2 as an internal standard to test the VA content in the serum sample.

[0176] The instrument and detection conditions, preparation of the standard curve working solution, preparation and detection of the VA structure derivative 2 internal standard working solution refer to Example 1. The standard curve constructed is: y=0.9741x+14.276, R 2 =0.9893.

[0177] The calculated VA contents in 30 groups of serum samples are shown in Table 5 below:

[0178] Table 5

[0179]

[0180] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0181] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for detecting the content of vitamin A in serum, characterized in that: The steps include: Mixing the vitamin A structural derivative with the first solvent to prepare an internal standard solution; Mixing a vitamin A standard with a second solvent to prepare standard solutions of different concentrations; Adding the same amount of the internal standard solution to the standard solutions of different concentrations and the serum sample to be tested respectively, and then performing detection by liquid chromatography-mass spectrometry; Constructing a standard curve according to the test results of standard solutions of different concentrations after adding the internal standard solution, substituting the test results of the serum sample to be tested after adding the internal standard solution into the standard curve to obtain the content of vitamin A in the serum sample to be tested; Wherein, the vitamin A structural derivative has the following structure: or .

2. The method for detecting the vitamin A content in serum according to claim 1, characterized in that: The liquid phase in the liquid phase-mass spectrometry technique is high performance liquid chromatography, and the detection conditions of the high performance liquid chromatography include: (1) The chromatographic column is a C18 chromatographic column; (2) Mobile phase A was an aqueous solution of formic acid with a volume percentage of 0.08%-0.12%, and mobile phase B was a methanol solution of formic acid with a volume percentage of 0.08%-0.12%. Gradient elution was adopted, including the following procedures: 0-0.2 min, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was 80%; 0.2 min-2.5 min, the volume percentage of mobile phase A was changed from 20% to 1%, and the volume percentage of mobile phase B was changed from 80% to 99%; 2.5 min-3.5 min, the volume percentage of mobile phase A was maintained at 1%, and the volume percentage of mobile phase B was maintained at 99%; 3.5 min-3.51 min, the volume percentage of mobile phase A was changed from 1% to 20%, and the volume percentage of mobile phase B was changed from 99% to 80%; 3.51 min-4.5 min, the volume percentage of mobile phase A was maintained at 20%, and the volume percentage of mobile phase B was maintained at 80%.

3. The method for detecting the content of vitamin A in serum according to claim 2, characterized in that: The detection conditions of the high performance liquid chromatography also include one or more of the following: (1) Column temperature is 39°C~45°C; (2) Flow rate: 0.3 mL / min~0.8 mL / min; (3) The injection volume is 8 μL~15 μL; (4) The dimensions of the chromatographic column include: length of 45 mm to 55 mm, diameter of 2 mm to 2.3 mm, and filler particle size of 2.5 μm to 3 μm.

4. The method for detecting the vitamin A content in serum according to any one of claims 1 to 3, characterized in that: The mass spectrometer in the liquid chromatography-mass spectrometry technology is a triple quadrupole mass spectrometer, and the detection conditions of the mass spectrometer include: Ion source: atmospheric pressure chemical ionization; Detection method: Multiple reaction monitoring; Air curtain gas: 33psi~37psi; Atomizing gas: 38psi~42psi; Temperature: 370℃~430℃; Collision gas: 7psi~9psi.

5. The method for detecting the content of vitamin A in serum according to any one of claims 1 to 3, characterized in that: The first solvent and the second solvent each independently include one or both of acetonitrile and bovine serum albumin aqueous solution.

6. The method for detecting the vitamin A content in serum according to any one of claims 1 to 3, characterized in that: The concentration of the vitamin A structural derivative in the internal standard solution is 200ng / mL~500ng / mL, and the amount of the internal standard solution added to the standard solutions of different concentrations and the serum samples to be tested is: 150μL~250μL of the internal standard solution is added for every 50μL of the standard solutions of different concentrations and the serum samples to be tested.

7. The method for detecting the content of vitamin A in serum according to any one of claims 1 to 3, characterized in that: The concentration distribution range of vitamin A in standard solutions of different concentrations is 15ng / mL~2000ng / mL.

8. A vitamin A structural derivative having the following structural characteristics: 。 9. A vitamin A structural derivative having the following structural characteristics: 。 10. Use of the vitamin A structural derivatives according to claims 8 and 9 as internal standards in the detection of vitamin A content in serum.

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