Method for detecting vitamin A content in serum

Abstract

The present application relates to a method for detecting the content of vitamin A in serum, a vitamin A structural derivative and its application. The method for detecting the content of vitamin A in serum comprises the following steps: mixing the vitamin A structural derivative with a 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 using liquid phase-mass spectrometry technology for detection to obtain the content of vitamin A in the serum sample to be tested. In this detection method, a substitute for the vitamin A isotope internal standard is used for detection, which reduces the detection cost while achieving accurate quantitative detection of vitamin A in serum.

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), a fat-soluble vitamin, is an essential nutrient for the human body, playing multiple physiological roles, including promoting growth and reproduction, maintaining normal bone, epithelial tissue, vision, and mucosal secretion. Vitamin A deficiency is considered a public health concern and can lead to a variety of conditions, including night blindness, decreased immunity, reproductive decline, hair loss, muscle and visceral atrophy in children, poor bone growth and development, and female infertility. Therefore, accurate measurement of vitamin A levels in human serum is essential.

[0003] In clinical mass spectrometry applications, LC-MS / MS offers advantages such as high sensitivity, strong specificity, rapid analysis, and high throughput, making it the gold standard for quantitative analysis of biological samples. To accurately quantify the analyte, an internal standard is often required during LC-MS / MS testing. Its functions and principles include: first, calibrating for analyte loss and extraction efficiency during tedious sample pretreatment; second, calibrating for sample background differences caused by individual differences in biological sample constitution and diet; and third, calibrating for deviations arising from liquid phase injection, chromatographic separation, and mass spectrometry ionization. Therefore, internal standards are essential for accurate quantitative detection of analytes using the internal standard method.

[0004] Currently, for the LC-MS / MS quantitative detection of vitamin A, a stable isotope internal standard is usually required as the internal standard. The isotope internal standard and the analyte are the same substance, except that several hydrogen atoms are replaced by deuterium atoms, or 12C is replaced by 13C. The physical and chemical properties remain basically unchanged, thus achieving good calibration and accurate quantification of 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. In addition, 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, further increasing the cost and hindering its popularization and application. Summary of the Invention

[0005] Based on this, the present application provides a method for detecting the content of vitamin A 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 while reducing detection costs.

[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 a first solvent to prepare an internal standard solution;

[0008] Mixing the vitamin A standard with the 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 detecting them by liquid chromatography-mass spectrometry;

[0010] Constructing a standard curve based on 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 column;

[0015] (2) Mobile phase A was a 0.08% to 0.12% by volume formic acid aqueous solution, and mobile phase B was a 0.08% to 0.12% by volume formic acid methanol solution. Gradient elution was used, 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 changed from 20% to 1%, and the volume percentage of mobile phase B 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 99%; 3.5 min-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%; 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 80%.

[0016] In one embodiment, the HPLC detection conditions 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] Curtain air: 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 sample 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 sample to be tested.

[0030] In one embodiment, the concentration distribution range of vitamin A in the standard solution of different concentrations is 15 ng / mL~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 a 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 uses a specific structure of vitamin A structural derivative as an internal standard and combines it with liquid chromatography-mass spectrometry technology to detect the vitamin A content in serum. It can avoid the use of stable isotope internal standards of vitamin A, greatly reducing the detection cost of vitamin A content in serum. At the same time, the test results are 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 The figure shows 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 analogue 3 in 30 groups of serum samples.

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

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

[0047] The following detailed description of the present invention's methods for detecting serum vitamin A levels, vitamin A structural derivatives, and their applications is provided below in conjunction with specific examples. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[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 pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only 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, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, terms such as "first," "second," "third," and "fourth" serve only as non-exhaustive enumeration and description, and should not constitute a closed-ended limitation on quantity.

[0051] In this 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 referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0053] Unless otherwise specified, the percentage contents mentioned in this application refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[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 for temperature fluctuations within the precision range of instrument control.

[0056] The room temperature in this application generally refers to 4°C to 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 seek 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 real samples within the reference range, and their consistency analysis meets the statistical algorithm and there is no obvious linear deviation. Among them, consistency is the key to determining whether a compound can replace the isotope internal standard as an internal standard.

[0058] After research, this applicant found that two vitamin A structural derivatives 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 convenient for promotion and application.

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

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

[0061] Mixing the vitamin A standard with the 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 detecting them by liquid chromatography-mass spectrometry;

[0063] Constructing a standard curve based on 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 is understood that "constructing a standard curve based on the test results of standard solutions of varying concentrations after adding the internal standard solution" means constructing a standard curve based on the peak area ratio of the vitamin A standard to the vitamin A structural derivative (internal standard) obtained after detection using liquid chromatography-mass spectrometry, where the concentration of the vitamin A standard is on the X-axis and the peak area ratio of the vitamin A standard to the vitamin A structural derivative is on the Y-axis. Accordingly, "substituting the test results of the serum sample to be tested after adding the internal standard solution into the standard curve" means substituting the peak area ratio of the analyte to the vitamin A structural derivative (internal standard) obtained after detection using liquid chromatography-mass spectrometry into the standard curve to obtain the concentration of the analyte in the serum sample to be tested.

[0067] In some examples, 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:

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

[0069] (2) Mobile phase A was a 0.08% to 0.12% by volume formic acid aqueous solution, and mobile phase B was a 0.08% to 0.12% by volume formic acid methanol solution. Gradient elution was used, 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 changed from 20% to 1%, and the volume percentage of mobile phase B 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 99%; 3.5 min-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%; 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 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 HPLC detection conditions 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 HPLC detection conditions further 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 HPLC detection conditions further include: an injection volume of 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 HPLC detection conditions also include: the dimensions of the chromatographic column include: a length of 45 mm to 55 mm, a diameter of 2 mm to 2.3 mm, and a filler particle size of 2.5 μm to 3 μm. Furthermore, the dimensions of the chromatographic column include: a length of 50 mm, a diameter of 2.1 mm, and a filler particle size of 2.7 μm.

[0076] In some 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. The detection conditions of the mass spectrometer include:

[0077] Ion source: atmospheric pressure chemical ionization;

[0078] Detection method: Multiple reaction monitoring;

[0079] Curtain air: 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 needed, the preparation of each solution can be facilitated by methods such as vortex oscillation.

[0084] In some 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 sample 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 sample 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 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 should be made to the guidelines provided 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 the experimental conditions recommended by the manufacturer. Unless otherwise specified, the raw materials and reagents involved in the following specific examples are commercially available or can be prepared by those skilled in the art according to known methods.

[0092] Preparation Example 1

[0093] Synthesis of Vitamin A structural derivative 1:

[0094]

[0095] Preparation of intermediate 2:

[0096] Raw material 1 (28.5 mg, 0.0912 mmol) was dissolved in 1.7 mL of CH2Cl2, cooled to 0°C, and MnO2 (0.143, 1.64 mmol) and Na2CO3 (0.174 g, 1.64 mmol) were added. The mixture was then heated to 25°C and stirred for 5 h. The reaction solution was filtered, 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 in the absence of light and under the protection of an inert gas. 90 mg of the intermediate product 2 was dissolved in 8 mL of tetrahydrofuran, cooled to -78°C, and a methyllithium solution (1.6 M in ether, 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. Approximately 8 mL of a saturated aqueous solution of NH4Cl was added to quench the reaction. The solution was extracted with ether, washed with brine, dried over 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 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 intermediate 4 and the reaction was continued at room temperature for 45 min to obtain vitamin A structure derivative 2. Its mass spectrum and nuclear magnetic resonance spectrum are shown in FIG. Figure 3 and Figure 4 shown.

[0109] Example 1

[0110] This example investigates 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] From 0 to 0.2 min, 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 0.2 min to 2.5 min, 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] 2.5 min to 3.5 min, maintain the volume percentage of mobile phase A at 1% and the volume percentage of mobile phase B at 99%;

[0123] From 3.5 min 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 3.51 min to 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%.

[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, including 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 10 mL to prepare a 1 mg / mL VA stock solution.

[0133] When used, 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 the VA isotope internal standard (VA-d5 isotope) and add acetonitrile to 10 mL to prepare a 1 mg / mL VA-d5 stock solution. Dilute the stock solution with acetonitrile at a volume ratio of 5:200 to prepare a VA isotope internal standard working solution (concentration: 250 ng / mL).

[0138] Weigh 10 mg of VA structure derivative 1 dry powder and add acetonitrile to 10 mL to prepare a 1 mg / mL VA structure derivative 1 stock solution. Dilute this stock solution with acetonitrile at a volume ratio of 5:200 to prepare a VA structure derivative 1 internal standard working solution (concentration: 250 ng / mL).

[0139] Vitamin A structural derivatives 1.

[0140] Weigh 10 mg of VA structure derivative 2 dry powder and add acetonitrile to 10 mL to prepare a 1 mg / mL VA structure derivative 2 stock solution. Dilute this stock solution with acetonitrile at a volume ratio of 5:200 to prepare a VA structure derivative 2 internal standard working solution (concentration: 250 ng / mL).

[0141] Vitamin A structural derivatives 2.

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

[0143] Vitamin A structural analogue 3.

[0144] Weigh 10 mg of VA structural analogue 4 dry powder and add acetonitrile to 10 mL to prepare a 1 mg / mL VA structural analogue 4 stock solution. Dilute this stock solution with acetonitrile at a volume ratio of 5:200 to prepare a VA structural analogue 4 internal standard working solution (concentration: 250 ng / mL).

[0145] Vitamin A structural analogue 4.

[0146] Weigh 10 mg of VA structural analogue 5 dry powder and add acetonitrile to 10 mL to prepare a 1 mg / mL VA structural analogue 5 stock solution. Dilute this stock solution with acetonitrile at a volume ratio of 5:200 to prepare a VA structural analogue 5 internal standard working solution (concentration: 250 ng / mL).

[0147] Vitamin A structural analogue 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 internal standard working solution (isotope, derivative or structural analog), vortex for 10 minutes, centrifuge at high speed (13000 rpm) for 10 minutes, take 100 μL of the supernatant for detection on the machine, use different concentrations of standard to draw a standard curve, and determine the concentration of the serum sample.

[0150] 3. Consistency Examination

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

[0152] Can be used as a standard for VA isotope internal standard substitutes: the standard curve working solution with VA isotope internal standard added and different ion pairs in the serum sample to be tested are used as the control group, and the standard curve working solution with vitamin A structural derivatives 1, 2 or analogs 3, 4, 5 added 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 sample to be tested.

[0153] Testing steps:

[0154] 200 μL of internal standard working solution (isotope, derivative, or structural analog) was added to 50 μL of standard curve working solution and serum sample, respectively, and the samples were injected into the AB Sciex 4500 tandem triple quadrupole mass spectrometer-high performance liquid chromatography for detection. A standard curve was constructed based on the test results and concentration of the standard curve working solution after adding the internal standard working solution. The test results of the serum sample 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 analog 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 those of VA isotope internal standard, with slopes of 1.01, 0.97, and 0.97, respectively, and R 2 The relative molecular weights of the VA isotope internal standard are 0.9913, 0.9939, and 0.9893, respectively, which can be used as a substitute for the VA isotope internal standard 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 and shake for 10 minutes, centrifuge at high speed (13000 rpm) for 10 minutes, take 100 μL of supernatant for detection on the instrument, 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 and shake for 10 minutes, centrifuge at high speed (13000 rpm) for 10 minutes, take 100 μL of supernatant for detection on the machine, 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 and shake for 10 minutes, centrifuge at high speed (13000 rpm) for 10 minutes, take 100 μL of supernatant for detection on the machine, 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 and shake for 10 minutes, centrifuge at high speed (13000 rpm) for 10 minutes, 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 vitamin A structural derivative 1 as an internal standard to test the VA content in serum samples.

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

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

[0172] Table 4

[0173]

[0174] Example 3

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

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

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

[0178] Table 5

[0179]

[0180] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned 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 embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they 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 variations and improvements can be made, which all fall within 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 application 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 following steps are involved: 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 structure shown below: 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 used, including the following procedures: 0-0.2 min, maintaining the volume percentage of mobile phase A at 20%, and the volume percentage of mobile phase B at 80%; 0.2 min-2.5 min, 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%; 2.5 min-3.5 min, maintaining the volume percentage of mobile phase A at 1%, and the volume percentage of mobile phase B at 99%; 3.5 min-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%; 3.51 min-4.5 min, maintaining the volume percentage of mobile phase A at 20%, and the volume percentage of mobile phase B at 80%.

3. The method for detecting the content of vitamin A in serum according to claim 2, characterized in that: The column temperature is 39℃~45℃.

4. The method for detecting the content of vitamin A in serum according to claim 2, characterized in that: The flow rate is 0.3mL / min~0.8mL / min.

5. The method for detecting the content of vitamin A in serum according to claim 2, characterized in that: The injection volume is 8μL~15μL.

6. The method for detecting the content of vitamin A in serum according to claim 2, characterized in that: The dimensions of the chromatographic column include: a length of 45 mm to 55 mm, a diameter of 2 mm to 2.3 mm, and a filler particle size of 2.5 μm to 3 μm.

7. The method for detecting the content of vitamin A in serum according to any one of claims 1 to 6, 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.

8. The method for detecting the content of vitamin A in serum according to any one of claims 1 to 6, characterized in that: The first solvent and the second solvent each independently include one or both of acetonitrile and bovine serum albumin aqueous solution.

9. The method for detecting the content of vitamin A in serum according to any one of claims 1 to 6, 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.

10. The method for detecting the content of vitamin A in serum according to any one of claims 1 to 6, characterized in that: The concentration distribution range of vitamin A in standard solutions of different concentrations is 15ng / mL~2000ng / mL.

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