Kit for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry) and detection method

Abstract

The invention relates to the technical field of analysis and detection, in particular to a kit for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry) and a detection method. The kit comprises a to-be-detected sample, an internal standard, an extraction agent and a diluent, the extracting agent comprises a mixture of trichloroacetic acid, water and hydrochloric acid; in the extraction agent, the concentration of trichloroacetic acid is 11-13 wt%, and the concentration of hydrochloric acid is 0.1-0.5 M; the diluent is water; according to the method, a high performance liquid chromatography-mass spectrometry method is adopted to detect various water-soluble vitamins and organic acids in human blood, and peaks of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxine, vitamin B7, vitamin B9, 5-methyltetrahydrofolic acid and methylmalonic acid can be obtained at the same time through one-time detection; the kit has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a kit and a detection method for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS. Background Art

[0002] Vitamins are small organic molecules essential for maintaining normal physiological functions and specific metabolic reactions within cells. They are essential nutrients for human health. They are required in relatively small quantities, primarily from food, and play a vital role in regulating metabolism.

[0003] The human body is known to contain a variety of vitamins, which can be divided into two major categories based on their polarity: fat-soluble and water-soluble. Water-soluble vitamins include the B vitamins and vitamin C. B vitamins are a collective term for vitamins that share similar functions, structures, and physicochemical properties. All B vitamins contain nitrogen and are readily soluble in water. They primarily function as coenzymes in the synthesis and metabolism of various substances in the body and are closely associated with the formation of blood cells and energy release. B vitamin deficiency is closely associated with the development and progression of numerous diseases, including skin disorders, memory loss, fatigue, insomnia, and mental weakness. For example, vitamin B1 deficiency is commonly known as beriberi, vitamin B2 deficiency is also known as "oral genital syndrome," vitamin B6 deficiency can lead to seborrheic dermatitis, vitamin B9 deficiency can cause hyperhomocysteinemia and neural tube defects in the fetus, and vitamin C deficiency can cause scurvy and a weakened immune system. Therefore, monitoring the levels of water-soluble vitamins in the human body helps assess nutritional status and monitor the progress of disease diagnosis and treatment, and is of great clinical significance.

[0004] The main methods for clinical detection of water-soluble vitamins in the human body include immunoassay, liquid chromatography and liquid chromatography tandem mass spectrometry. Immunoassay is a method that uses immunological principles to determine the content of the substance to be tested in the sample by using the substance to be tested as an antigen or antibody. It is usually simple to operate and does not require complex sample pretreatment steps, but it does not have the specific analysis of the substance to be tested and high-throughput detection; liquid chromatography is to achieve separation and detection through the interaction between the analyte and the mobile phase and the stationary phase. Compared with immunoassay, it has better separation ability and analytical specificity; liquid chromatography-tandem mass spectrometry (LC-MS / MS) combines the chromatographic separation characteristics and the qualitative function of mass spectrometry, and is more accurate in the quantitative analysis of complex mixtures, and can achieve high-throughput one-time testing of multiple vitamins. However, the existing methods have a low response to methylmalonic acid in blood samples and require derivatization, which has complicated steps and is not conducive to actual production operations. Summary of the Invention

[0005] The present invention provides a kit and a detection method for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS, which can meet the requirements of relevant regulations on linearity, accuracy, precision, residue and recovery rate for the detection of multiple water-soluble vitamins and organic acids.

[0006] In the present invention, vitamin B6-pyridoxal phosphate usually needs to be extracted with a strong acid such as trichloroacetic acid, but the acidity is too strong and the response is extremely low in the methylmalonic acid negative ion mode, so it is necessary to adjust the pH value before loading to balance the response of each substance. However, the present invention finds that the ideal effect cannot be achieved by adjusting the pH value by adding sodium hydroxide or ammonia. After a lot of attempts, the present invention unexpectedly finds that the use of water temperature and adjusting the pH value can greatly improve the response of methylmalonic acid without affecting the response of other substances. At the same time, the present invention further finds that when trichloroacetic acid solution is used as the extraction agent, the methylmalonic acid baseline interference in the organic acid is very serious, and even belongs to the unusable situation. The present invention finds that the addition of hydrochloric acid can greatly optimize the peak shape of methylmalonic acid, and the response of succinic acid can be appropriately depressed when the hydrochloric acid concentration increases while the methylmalonic acid response remains unchanged.

[0007] Based on this, the present invention has the following technical solutions: In a first aspect, the present invention provides a kit for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS, comprising: a sample to be tested, an internal standard, an extractant, and a diluent; The extractant includes a mixture of trichloroacetic acid, water and hydrochloric acid; in the extractant, the concentration of trichloroacetic acid is 11wt%~13wt%, and the concentration of hydrochloric acid is 0.1M~0.5M; The diluent is water; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxic acid, vitamin B7 and vitamin B9; the organic acids include 5-methyltetrahydrofolate, methylmalonic acid and succinic acid.

[0008] After extensive research, the present invention found that adding the above-mentioned concentration of hydrochloric acid to the extractant can significantly optimize the peak shape of methylmalonic acid, greatly improve the stability of detection, and reduce baseline interference.

[0009] In the present invention, the extraction agent in the kit can be trichloroacetic acid, water and hydrochloric acid packaged separately and prepared before use, or it can be a prepared extraction agent containing 11wt%~13wt% trichloroacetic acid and 0.1M~0.5M hydrochloric acid, which is not limited here.

[0010] According to the LC-MS / MS kit for simultaneous detection of water-soluble vitamins and organic acids provided by the present invention, the kit also contains a mobile phase, the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a mixture of ammonium acetate, formic acid and water, the concentration of ammonium acetate in mobile phase A is 5mM~10mM, and the concentration of formic acid is 0.002wt%~0.005wt%; the mobile phase B is methanol.

[0011] In the present invention, a conventional mobile phase in the prior art (such as the mobile phase for detecting methylmalonic acid disclosed in CN 115508483 A) is used, but the separation between methylmalonic acid and succinic acid is poor, affecting the detection accuracy. The present invention subsequently unexpectedly discovered that reducing the concentration of formic acid in the mobile phase to the above range can effectively improve the separation between methylmalonic acid and succinic acid, while further improving the response value of methylmalonic acid.

[0012] According to the LC-MS / MS kit for simultaneously detecting water-soluble vitamins and organic acids provided by the present invention, the kit further contains a protective agent, which includes one or more of ascorbic acid, citric acid, dithiothreitol, tea polyphenols and chlorogenic acid.

[0013] Preferably, the protective agent comprises ascorbic acid, dithiothreitol and citric acid in a mass ratio of (1-3):(1-3):1.

[0014] According to the LC-MS / MS kit for simultaneously detecting water-soluble vitamins and organic acids provided by the present invention, the samples to be tested include calibrators, quality control products and blood.

[0015] Preferably, in the calibrator, the concentration of vitamin B1 is 0.5~25 ng / mL; the concentration of vitamin B2 is 2~100 ng / mL; the concentration of vitamin B3 is 2~100 ng / mL; the concentration of vitamin B5 is 10~500 ng / mL; the concentration of vitamin B6-pyridoxal phosphate is 4~200 ng / mL; the concentration of vitamin B6-pyridoxal acid is 1~50 ng / mL; the concentration of vitamin B7 is 1~50 ng / mL; the concentration of vitamin B9 is 2~100 ng / mL; the concentration of 5-methyltetrahydrofolate is 2~100 ng / mL; the concentration of methylmalonic acid is 20~1000 ng / mL; and the concentration of succinic acid is 20~1000 ng / mL.

[0016] Preferably, in the quality control product, the concentration of vitamin B1 is 1.5~18.75ng / mL; the concentration of vitamin B2 is 6~75ng / mL; the concentration of vitamin B3 is 6~75ng / mL; the concentration of vitamin B5 is 30~375ng / mL; the concentration of vitamin B6-pyridoxal phosphate is 12~150ng / mL; the concentration of vitamin B6-pyridoxal acid is 3~37.5ng / mL; the concentration of vitamin B7 is 3~37.5ng / mL; the concentration of vitamin B9 is 6~75ng / mL; the concentration of 5-methyltetrahydrofolate is 6~75ng / mL; the concentration of methylmalonic acid is 60~750ng / mL; and the concentration of succinic acid is 60~750ng / mL.

[0017] Preferably, the internal standard for vitamin B1 is thiamine-4-methyl- 13 C-thiazol-5-yl- 13 C3 hydrochloride; the internal standard for vitamin B2 is vitamin B2- 13 C4, 15 N2; the internal standard for vitamin B3 is vitamin B3-nicotinamide-D4; the internal standard for vitamin B5 is vitamin B5, calcium salt: H2O-β-alanyl- 13 C3; the internal standard for vitamin B6-pyridoxal phosphate is vitamin B6-pyridoxal-d3 phosphate; the internal standard for vitamin B6-pyridoxal acid is vitamin B6-4-pyridoxal-d3; the internal standard for vitamin B7 is vitamin B7-RAC biotin-d4; the internal standard for vitamin B9 is folic acid-glutamic acid- 13 C5; The internal standard of 5-methyltetrahydrofolate is vitamin-5-methyltetrahydrofolate- 13 C5; the internal standard for methylmalonic acid is methylmalonic acid-d3.

[0018] In a second aspect, the present invention provides a method for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS, comprising: S1: mixing a sample to be tested, a mixed internal standard, and an extractant, and centrifuging the resulting mixed solution at a temperature below 10° C.; the extractant comprises a mixture of trichloroacetic acid, water, and hydrochloric acid, wherein the concentration of trichloroacetic acid in the extractant is 11 wt % to 13 wt %, and the concentration of hydrochloric acid is 0.1 M to 0.5 M; S2: The pH value of the supernatant is adjusted to 1-2 with water, and then tested on the machine; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxic acid, vitamin B7 and vitamin B9; and the organic acids include 5-methyltetrahydrofolate, methylmalonic acid and succinic acid.

[0019] According to the LC-MS / MS method for simultaneous detection of water-soluble vitamins and organic acids provided by the present invention, the volume ratio of the sample to be tested to the extractant is (4-6): (1-3).

[0020] According to the LC-MS / MS method for simultaneous detection of water-soluble vitamins and organic acids provided by the present invention, the volume ratio of the sample to be tested to the mixed internal standard is (9-10): (1-2).

[0021] According to the LC-MS / MS method for simultaneous detection of water-soluble vitamins and organic acids provided by the present invention, the mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein the mobile phase A is a mixture of ammonium acetate, formic acid and water, wherein the concentration of ammonium acetate in the mobile phase A is 5 mM to 10 mM, and the concentration of formic acid is 0.002 wt% to 0.005 wt%; the mobile phase B is methanol.

[0022] According to the LC-MS / MS method for simultaneous detection of water-soluble vitamins and organic acids provided by the present invention, the gradient elution conditions for liquid chromatography detection include:

[0023] Wherein, % represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

[0024] Preferably, the gradient elution conditions for liquid chromatography detection include:

[0025] Here, % represents volume percentage.

[0026] Preferably, the chromatographic column is a C18 column.

[0027] Preferably, the flow rate of the mobile phase is 0.3 mL / min; the column temperature is 35° C.; the temperature of the automatic sampler is 8° C.; the needle washing solution is methanol-water solution; and the injection volume is 10 μL.

[0028] According to the LC-MS / MS method for simultaneous detection of water-soluble vitamins and organic acids provided by the present invention, the mass spectrometry detection conditions include:

[0029] Preferably, the ion pair parameters include:

[0030] The method for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS provided by the present invention comprises the following steps: S0: Different water-soluble vitamins and organic acids are diluted with 40-60% methanol aqueous solution, and a protective agent is added to prepare the test sample; different water-soluble vitamins and organic acid internal standards are diluted with 40-60% methanol aqueous solution, and a protective agent is added, and then the different internal standard solutions are mixed to prepare a mixed internal standard; S1: Mixing a sample to be tested, a mixed internal standard, and an extractant in a volume ratio of (9-10):(1-2):(3-5), and centrifuging the resulting mixed solution at a temperature below 10°C; the extractant comprises a mixture of trichloroacetic acid, water, and hydrochloric acid, wherein the concentration of trichloroacetic acid in the extractant is 11 wt%-13 wt%, and the concentration of hydrochloric acid is 0.1 M-0.5 M; S2: The pH value of the supernatant is adjusted to 1-2 with water, and then tested on the machine; The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein the mobile phase A is a mixture of ammonium acetate, formic acid, and water, wherein the concentration of ammonium acetate in the mobile phase A is 5 mM to 10 mM, and the concentration of formic acid is 0.002 wt% to 0.005 wt%; the mobile phase B is methanol; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxic acid, vitamin B7 and vitamin B9; and the organic acids include 5-methyltetrahydrofolate, methylmalonic acid and succinic acid.

[0031] The present invention adopts high performance liquid chromatography-mass spectrometry to detect multiple water-soluble vitamins and organic acids in human blood. Peaks of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxalic acid, vitamin B7, vitamin B9, 5-methyltetrahydrofolate, and methylmalonic acid can be obtained simultaneously in one detection. The method has the characteristics of high sensitivity, good repeatability, high accuracy, good specificity, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The left picture is the peak diagram of VB1 in the serum of Example 1, and the right picture is the peak diagram of VB1 internal standard.

[0034] Figure 2The left figure is the peak diagram of VB2 in serum of Example 1, and the right figure is the peak diagram of VB2 internal standard.

[0035] Figure 3 The left figure is the peak diagram of VB3 in serum of Example 1, and the right figure is the peak diagram of VB3 internal standard.

[0036] Figure 4 The left figure is the peak diagram of VB5 in serum of Example 1, and the right figure is the peak diagram of VB5 internal standard.

[0037] Figure 5 The left figure is the peak diagram of VB6-CHO in the serum of Example 1, and the right figure is the peak diagram of VB6-CHO internal standard.

[0038] Figure 6 The left figure is the peak diagram of VB6-COOH in the serum of Example 1, and the right figure is the peak diagram of VB6-COOH internal standard.

[0039] Figure 7 The left figure is the peak diagram of VB7 in serum of Example 1, and the right figure is the peak diagram of VB7 internal standard.

[0040] Figure 8 The left figure is the peak diagram of VB9 in serum of Example 1, and the right figure is the peak diagram of VB9 internal standard.

[0041] Figure 9 The left figure is the peak diagram of 5-MTHF in serum of Example 1, and the right figure is the peak diagram of 5-MTHF internal standard.

[0042] Figure 10 The left figure is the peak diagram of MMA in serum of Example 1, and the right figure is the peak diagram of MMA internal standard.

[0043] Figure 11 The left figure is the peak diagram of MMA in the standard product of Example 2 under mobile phase A1, and the right figure is the peak diagram of MMA internal standard under mobile phase A1.

[0044] Figure 12 The left figure is the peak diagram of MMA in the standard product of Example 2 under mobile phase A2, and the right figure is the peak diagram of MMA internal standard product under mobile phase A2.

[0045] Figure 13 The left figure is the peak diagram of MMA in the standard product of Example 2 under mobile phase A3, and the right figure is the peak diagram of MMA internal standard under mobile phase A3.

[0046] Figure 14 The left figure is the peak diagram of methylmalonic acid in the serum of Example 3 when the extract is 12% trichloroacetic acid (0.1M hydrochloric acid), and the right figure is the peak diagram of methylmalonic acid internal standard when the extract is 12% trichloroacetic acid (0.1M hydrochloric acid).

[0047] Figure 15 The left figure is the peak diagram of methylmalonic acid in the serum of Example 3 when the extract is 12% trichloroacetic acid (0.2M hydrochloric acid), and the right figure is the peak diagram of methylmalonic acid internal standard when the extract is 12% trichloroacetic acid (0.2M hydrochloric acid).

[0048] Figure 16 The left figure is the peak diagram of methylmalonic acid in the serum of Example 3 when the extract is 12% trichloroacetic acid (0.3M hydrochloric acid), and the right figure is the peak diagram of methylmalonic acid internal standard when the extract is 12% trichloroacetic acid (0.3M hydrochloric acid).

[0049] Figure 17 The left figure is the peak diagram of methylmalonic acid in the serum of Example 3 when the extract is 12% trichloroacetic acid (0.4M hydrochloric acid), and the right figure is the peak diagram of methylmalonic acid internal standard when the extract is 12% trichloroacetic acid (0.4M hydrochloric acid).

[0050] Figure 18 The left figure is the peak diagram of methylmalonic acid in the serum of Example 3 when the extract is 12% trichloroacetic acid (0.5M hydrochloric acid), and the right figure is the peak diagram of methylmalonic acid internal standard when the extract is 12% trichloroacetic acid (0.5M hydrochloric acid).

[0051] Figure 19 The left figure is the peak diagram of methylmalonic acid in the serum of Example 3 when the extract is 12% trichloroacetic acid, and the right figure is the peak diagram of methylmalonic acid internal standard when the extract is 12% trichloroacetic acid.

[0052] Figure 20 The left figure is a peak diagram of methylmalonic acid in the serum of Comparative Example 2 provided by the present invention, in which the extract is 12% trichloroacetic acid (0.4M sulfuric acid), and the right figure is a peak diagram of methylmalonic acid internal standard in which the extract is 12% trichloroacetic acid (0.4M sulfuric acid).

[0053] Figure 21 The left figure is a peak diagram of methylmalonic acid in the serum of Comparative Example 3 provided by the present invention, in which the extract is 12% trichloroacetic acid (0.4M formic acid), and the right figure is a peak diagram of methylmalonic acid internal standard in which the extract is 12% trichloroacetic acid (0.4M formic acid).

[0054] Figure 22 The left figure is a peak diagram of methylmalonic acid in the standard sample of Comparative Example 4 provided by the present invention, in which the extract is 12% trichloroacetic acid (0.4M hydrochloric acid) and the sample is directly loaded without dilution. The right figure is a peak diagram of methylmalonic acid internal standard in which the extract is 12% trichloroacetic acid (0.4M hydrochloric acid) and the sample is directly loaded without dilution.

[0055] Figure 23The left figure is a peak diagram of methylmalonic acid in the standard sample of Comparative Example 5 provided by the present invention, in which the extract is 12% trichloroacetic acid (0.4M hydrochloric acid) and the pH is adjusted with 0.1% sodium hydroxide and then loaded. The right figure is a peak diagram of methylmalonic acid internal standard in which the extract is 12% trichloroacetic acid (0.4M hydrochloric acid) and the pH is adjusted with 0.1% sodium hydroxide and then loaded.

[0056] Figure 24 The left figure is a peak diagram of methylmalonic acid in the standard sample of Comparative Example 6 provided by the present invention, in which the extract is 12% trichloroacetic acid (0.4M hydrochloric acid) and the pH is adjusted with 0.1% ammonia water and then loaded. The right figure is a peak diagram of methylmalonic acid internal standard in which the extract is 12% trichloroacetic acid (0.4M hydrochloric acid) and the pH is adjusted with 0.1% ammonia water and then loaded. DETAILED DESCRIPTION

[0057] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0058] Unless otherwise specified, the various raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0059] Example 1 This embodiment provides a kit and a method for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS, comprising: 1. Main compounds and reagents Table 1 Standards and reagents

[0060] The internal standard of vitamin B1 is thiamine hydrochloride-(4-methyl- 13 C-thiazol-5-yl- 13 C3) hydrochloride; the internal standard for vitamin B2 is vitamin B2- 13 C4, 15 N2; the internal standard for vitamin B3 is vitamin B3-nicotinamide-D4; the internal standard for vitamin B5 is vitamin B5, calcium salt: H2O-β-alanyl- 13 C3; the internal standard for vitamin B6-pyridoxal phosphate is vitamin B6-pyridoxal-[d3]phosphate; the internal standard for vitamin B6-pyridoxal acid is vitamin B6-4-pyridoxal-d3; the internal standard for vitamin B7 is vitamin B7-RAC-biotin-d4; the internal standard for vitamin B9 is folic acid-glutamic acid-13 C5; The internal standard of 5-methyltetrahydrofolate is vitamin-5-methyltetrahydrofolate- 13 C5; the internal standard for methylmalonic acid is methylmalonic acid-d3.

[0061] 2. Instruments and equipment required for the experiment Table 2 Instruments and equipment

[0062] 3. Preparation of standard products and quality control products: 3.1. Preparation of various water-soluble vitamin protective agents, diluents and extracts Preparation of various water-soluble vitamin protective agents: Use a 1 / 10,000 electronic balance to weigh 5g of ascorbic acid, citric acid, and dithiothreitol respectively, add 50ml of methanol and then 50ml of water to prepare a 50mg / mL protective agent, label it and store it at 4℃. When using, dilute it 50 times to 1mg / ml.

[0063] Preparation of PBS dilutions of various water-soluble vitamins: Take a bag of PBS powder, add 2L of ultrapure water, add 40ml of protective agent after fully dissolving, label and store at 4℃.

[0064] Preparation of various water-soluble vitamin extracts: Use a 1 / 10,000 electronic balance to weigh 60g of trichloroacetic acid (TCA) solid powder, add 300ml of ultrapure water, and then add 200ml of 1M standard hydrochloric acid to prepare an extract containing 12% trichloroacetic acid and 0.4M hydrochloric acid. Label and store at 4°C.

[0065] 3.2 Preparation of multiple water-soluble vitamin stock solutions Preparation of various water-soluble vitamin stock solutions: Use a 1 / 100,000 electronic balance to weigh appropriate amounts of vitamin B1 (VB1), vitamin B2 (VB2), vitamin B3 (VB3-NH2), vitamin B5 (VB5), vitamin B6-pyridoxal phosphate (VB6-CHO), vitamin B6-pyridoxal acid (VB6-COOH), vitamin B7 (VB7), vitamin B9 (VB9), 5-methyltetrahydrofolate (5-MTHF), and methylmalonic acid (MMA), place them in 10 mL volumetric flasks, dissolve them in 50% methanol (containing 1 mg / mL protective agent), and dilute to the scale to prepare a stock solution with a concentration of 1 mg / mL. Transfer them to 1.5 mL EP tubes, label them, and store them at -80°C.

[0066] 3.3 Preparation of secondary stock solutions of various water-soluble vitamins Use a pipette to transfer each stock solution to 50% methanol (containing 1 mg / ml protective agent) to prepare secondary stock solutions of 10ug / mL vitamin B1, 50ug / mL vitamin B2, 50ug / mL vitamin B3, 100ug / mL vitamin B5, 50ug / mL vitamin B6-pyridoxal phosphate, 10ug / mL vitamin B6-pyridoxalic acid, 10ug / mL vitamin B7, 50ug / mL vitamin B9, 50ug / mL 5-methyltetrahydrofolate, and 50ug / mL methylmalonic acid.

[0067] 3.4 Preparation of various water-soluble vitamin internal standard stock solutions Preparation of various water-soluble vitamin internal standard stock solutions: Use a 1 / 100,000 electronic balance to weigh appropriate amounts of vitamin B1 internal standard, vitamin B2 internal standard, vitamin B3 internal standard, vitamin B5 internal standard, vitamin B6-pyridoxal phosphate internal standard, vitamin B6-pyridoxalic acid internal standard, vitamin B7 internal standard, vitamin B9 internal standard, 5-methyltetrahydrofolate internal standard, and methylmalonic acid internal standard, and place them in 10 mL volumetric flasks were dissolved with 50% methanol (containing 1 mg / mL protective agent) and diluted to the mark to prepare internal standard stock solutions of vitamin B1 with an internal standard concentration of 200 μg / mL, vitamin B2 with an internal standard concentration of 100 μg / mL, vitamin B3 with an internal standard concentration of 1000 μg / mL, vitamin B5 with an internal standard concentration of 1000 μg / mL, vitamin B6-pyridoxal phosphate with an internal standard concentration of 100 μg / mL, vitamin B6-pyridoxal acid with an internal standard concentration of 100 μg / mL, vitamin B7 with an internal standard concentration of 100 μg / mL, vitamin B9 with an internal standard concentration of 100 μg / mL, 5-methyltetrahydrofolate with an internal standard concentration of 100 μg / mL, and methylmalonic acid with an internal standard concentration of 1000 μg / mL. The internal standard solutions were transferred to 1.5 mL EP tubes, labeled, and stored at -80°C.

[0068] Use a pipette to pipette 50uL of vitamin B1 internal standard stock solution, 200uL of vitamin B2 internal standard stock solution, 20uL of vitamin B3 internal standard stock solution, 20uL of vitamin B5 internal standard stock solution, 500uL of vitamin B6-pyridoxal phosphate internal standard stock solution, 100uL of vitamin B6-pyridoxalic acid internal standard stock solution, 100uL of vitamin B7 internal standard stock solution, 200uL of vitamin B9 internal standard stock solution, 200uL of 5-methyltetrahydrofolate internal standard stock solution, and 200uL of methylmalonic acid internal standard stock solution, then add 48.410mL 50% methanol (containing 1 mg / mL protective agent) was mixed evenly to prepare a water-soluble multivitamin internal standard mixed stock solution with the following concentrations: vitamin B1 internal standard 200 ng / mL, vitamin B2 internal standard 400 ng / mL, vitamin B3 internal standard 400 ng / mL, vitamin B5 internal standard 400 ng / mL, vitamin B6-pyridoxal phosphate internal standard 1000 ng / mL, vitamin B6-pyridoxalic acid internal standard 200 ng / mL, vitamin B7 internal standard 200 ng / mL, vitamin B9 internal standard 400 ng / mL, 5-methyltetrahydrofolate internal standard 400 ng / mL, and methylmalonic acid internal standard 4000 ng / mL. Label the solution and store at -80°C.

[0069] 3.5 Preparation of Calibrators S1 to S6 The standard samples were prepared in a high-to-low preparation mode, that is, S1 was prepared and diluted in proportion to prepare S2 to S5 and LQC and HQC.

[0070] Preparation of calibrator S1: Use a pipette to transfer 100uL of vitamin B1 secondary stock solution, 80uL of vitamin B2 secondary stock solution, 80uL of vitamin B3 secondary stock solution, 100uL of vitamin B5 secondary stock solution, 160uL of vitamin B6-pyridoxal phosphate secondary stock solution, 200uL of vitamin B6-pyridoxalic acid secondary stock solution, 200uL of vitamin B7 secondary stock solution, 80uL of vitamin B9 secondary stock solution, 80uLL of 5-methyltetrahydrofolate secondary stock solution, and 200uL of methylmalonic acid secondary stock solution, then add 38.720mL of PBS diluent and mix well to prepare water-soluble multivitamin calibrator S1 with the following concentrations: vitamin B1 25ng / mL, vitamin B2 100ng / mL, vitamin B3 100ng / mL, vitamin B5 250 ng / mL, vitamin B6-pyridoxal phosphate 200 ng / mL, vitamin B6-pyridoxal acid 50 ng / mL, vitamin B7 50 ng / mL, vitamin B9 100 ng / mL, 5-methyltetrahydrofolate 100 ng / mL, methylmalonic acid 1000 ng / mL, label, and store at -80 ℃.

[0071] To prepare calibrator S2: Accurately pipette 150uL of standard S1, add 150uL of PBS diluent, mix thoroughly, label, and store at -80°C. To prepare calibrator S3: Accurately pipette 120uL of standard S2, add 180uL of PBS diluent, mix thoroughly, label, and store at -80°C. To prepare calibrator S4: Accurately pipette 150uL of standard S3, add 150uL of PBS diluent, mix thoroughly, label, and store at -80°C. To prepare calibrator S5: Accurately pipette 120uL of standard S4, add 180uL of PBS diluent, mix thoroughly, label, and store at -80°C. To prepare calibrator S6: Accurately pipette 150uL of standard S4, add 150uL of PBS diluent, mix thoroughly, label, and store at -80°C.

[0072] 3.6 Preparation of quality control products Preparation of low-concentration quality control (LQC): Accurately pipette 1.68 ml of standard S1, add 26.32 ml of PBS diluent, mix thoroughly, label, and store at -80°C. Preparation of high-concentration quality control (HQC): Accurately pipette 21 ml of standard S1, add 7 ml of PBS diluent, mix thoroughly, label, and store at -80°C.

[0073] 4. Preparation of mobile phase additives 2M ammonium acetate: Accurately weigh 62.66 g of ammonium acetate, add 400 mL of pure water, and mix thoroughly by ultrasonication.

[0074] 5. Mobile phase preparation Mobile phase A: Add 2.5 mL of 2M ammonium acetate and 25 μL of formic acid to 500 mL of pure water and mix thoroughly by ultrasonication. Mobile phase B: Filter the methanol solution, degas by ultrasonication, and set aside.

[0075] 6. Pre-treatment process: (1) Add calibrator and control solution: Pipette 100 μL of standard solution / control solution into new 1.5 ml centrifuge tubes respectively; (2) Add internal standard: Pipette 10 μL of internal standard mixed stock solution into the above centrifuge tubes and mix thoroughly; (3) Adding the extract: Pipette 40 μL of the extract into the above centrifuge tubes and mix thoroughly; (4) Centrifugation: Place the sample from step (3) in a centrifuge and centrifuge at 12,000 rpm and 4°C for 10 min; (5) Dilution: Pipette 50 μL of the supernatant from step (4), add 50 μL of ultrapure water, and mix until the pH of the supernatant is approximately 1.5; (6) Detection: Place the sample receiving plate in the instrument and use liquid chromatography-mass spectrometry AB4500 for detection.

[0076] 7. Liquid phase method 1) Chromatographic column: C18 (2.5 μm, 2.1 × 100 mm); 2) Mobile phase: Phase A: water (containing 10 mM ammonium acetate, 0.0025% formic acid); Phase B: methanol; 3) Elution gradient: Table 3 Gradient conditions

[0077] 4) Flow rate: 0.3 mL / min 5) Column temperature: 35°C 6) Automatic sampler temperature: 8°C 7) Needle wash solution: methanol: water (1:1, v / v) 8) Injection volume: 10 μL 8. Mass spectrometry Table 4

[0078] Table 5

[0079] 9. Method Validation (1) Linear range Prepare calibrators and quality control products, process the samples according to the pretreatment method, and perform linear regression based on the amount of standard added (x) and the peak area calculated by the instrument (y). The correlation coefficient of linear regression is required to be r ≥ 0.990.

[0080] The following regression equations were calculated from the experimental data: VB1: y = 0.0383 x + 0.00131 (r=0.9983), VB2: y = 0.0184 x + 0.00356 (r=0.9974), VB3: y = 0.0216 x + 0.00448 (r=0.9989), VB5: y = 0.0102 x + 0.00482 (r=0.9996), VB6-CHO: y = 0.00893 x + 0.00498 (r=0.9993), VB6-COOH: y = 0.055 x + 0.000675 (r=0.9990), VB7: y = 0.0555 x + 0.00231 (r=0.9994), VB9: y = Good linear relationships were observed for vitamin B1 (0.5-25 ng / mL), vitamin B2, vitamin B3, vitamin B9, and 5-methyltetrahydrofolate (2-100 ng / mL), vitamin B5 (10-500 ng / mL), vitamin B6 (pyridoxal acid) and vitamin B7 (1-50 ng / mL), vitamin B6 (pyridoxal phosphate) (4-200 ng / mL), and methylmalonic acid (20-1000 ng / mL).

[0081] (2) Precision and accuracy Prepare samples at the lower limit of quantification and a quality control sample at two concentrations. Process the samples according to the pretreatment method, process five replicates, and calculate precision and accuracy. Calculate the coefficient of variation and relative bias of the samples, requiring the coefficient of variation (CV) to be ≤15% and the relative bias (B) to be ≤±15%.

[0082] Experimental results: Table 6

[0083] Conclusion: The coefficient of variation (CV) of the lower limit of quantification, low-value quality control, mid-value quality control and high-value quality control of each compound was ≤15%, and the relative deviation (B) of accuracy was ≤±15%.

[0084] (3) Residue Prepare the upper limit of quantitation (S1) sample and blank sample, perform pretreatment, and evaluate the presence of carryover by first injecting the upper limit of quantitation (S1) sample, followed by three consecutive blank samples. The peak area of ​​the blank sample must be <20% of the LLOQ.

[0085] Experimental results: Table 7

[0086] Conclusion: The peak area of ​​blank samples of each compound was less than 20% LLOQ, indicating no residue.

[0087] (4) Recovery rate Prepare two standard mixtures (see Table 8 for standard concentrations), designated as 10x the mid- and high-value quality control solutions. Take 10 μL of the 10x mid- and high-value quality control solutions and add 90 μL of human serum to serve as spike recovery samples. Prepare six replicates of each level of spike recovery and process the samples according to the pretreatment protocol. Require a spike recovery of 80% to 120%.

[0088] Table 8

[0089] Experimental results: Table 9

[0090] Conclusion: The recoveries of the spiked samples with medium and high values ​​were both between 80% and 120%.

[0091] (5) Matrix effect Prepare low-value and high-value quality controls with the standard concentrations shown in Table 10. Matrix effects were assessed using a 1:1 serum matrix and surrogate matrix method. Add 50 μL of each low-value and high-value quality control to 50 μL of human serum to prepare six matrix effect samples. Prepare the samples according to the pretreatment protocol. The matrix effect was expected to be 80% to 120%.

[0092] Table 10

[0093] Table 11

[0094] In the present invention, the linear range of vitamin B1 is 0.5-25 ng / mL; the linear range of vitamin B2, vitamin B3, vitamin B9, and 5-methyltetrahydrofolate is 2-100 ng / mL; the linear range of vitamin B5 is 10-500 ng / mL; the linear range of vitamin B6-pyridoxal acid and vitamin B7 is 1-50 ng / mL; the linear range of vitamin B6-pyridoxal phosphate is 4-200 ng / mL; and the linear range of methylmalonic acid is 20-1000 ng / mL. The correlation linear coefficients R2 are all ≥0.99, and the correlation coefficients r are all ≥0.990. The coefficient of variation (CV) of the precision of the quantitative lower limit, low value, and high value quality control products is ≤15%, the relative deviation (B) of the accuracy is ≤±15%, and the spike recovery rate is 85%-115%.

[0095] The peaks of VB1, VB2, VB3, VB5, VB6-CHO, VB6-COOH, VB7, VB9, 5-MTHF and MMA are shown in Figures 1 to 10 .

[0096] Example 2 This example provides a kit and method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS. The difference between this kit and Example 1 is that the mobile phase A is replaced by A1, A2, and A3, respectively. The peak results of methylmalonic acid under A1, A2, and A3 are shown in Tables 10 and 11. Figures 11 to 13 As shown: Table 12

[0097] The results showed that when the formic acid concentration in the mobile phase was high, the response of methylmalonic acid was reduced, and the separation between methylmalonic acid and succinic acid was reduced; increasing the ammonium acetate concentration in phase A could improve the response of methylmalonic acid.

[0098] Example 3 This embodiment provides a kit and method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS. The only difference between this kit and Example 1 is that the extractants are replaced with extractants 2 to 6, respectively. The peak results of methylmalonic acid under extractants 2 to 6 are shown in Tables 11 and 12. Figures 14 to 18 As shown: Table 13

[0099] According to the spectrum, the addition of hydrochloric acid can greatly optimize the peak shape of methylmalonic acid in serum.

[0100] Comparative Example 1 This comparative example provides a kit and a detection method for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS. The difference between the kit and the detection method is that the extraction agent is replaced with extraction solution 1, which is a 12% trichloroacetic acid solution and does not contain hydrochloric acid.

[0101] according to Figure 19 It can be seen that when there is only 12% trichloroacetic acid, methylmalonic acid and succinic acid in the serum are not completely separated, the two peaks interfere with each other, and are in an unusable state.

[0102] Comparative Example 2 This comparative example provides a kit and a detection method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS, which differs from Example 1 only in that the extraction solvent is replaced with 12% trichloroacetic acid containing 0.4M sulfuric acid.

[0103] according to Figure 20 It can be seen that when 12% trichloroacetic acid contains 0.4M sulfuric acid, the methylmalonic acid in the blood sample is similar to hydrochloric acid, but sulfuric acid is difficult to volatilize, so it is not used.

[0104] Comparative Example 3 This comparative example provides a kit and a detection method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS, which differs from Example 1 only in that the extraction solvent is replaced with 12% trichloroacetic acid containing 0.4M formic acid.

[0105] according to Figure 21 It can be seen that 12% trichloroacetic acid containing 0.4M formic acid and succinic acid does not form a peak and still interferes with methylmalonic acid and cannot be used.

[0106] Comparative Example 4 This comparative example provides a kit and method for simultaneously detecting water-soluble vitamins and organic acids by LC-MS / MS. The difference between the kit and the method in Example 1 is that the supernatant after centrifugation is directly loaded onto the instrument without dilution or pH adjustment.

[0107] according to Figure 22 It can be seen that the pH of the supernatant after centrifugation is too low when it is directly applied to the instrument, and the methylmalonic acid response is severely inhibited.

[0108] Comparative Example 5 This comparative example provides a kit and a detection method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS, which differs from Example 1 only in that the same volume of 0.1% sodium hydroxide is added to make the pH value of the supernatant approximately 1.5.

[0109] according to Figure 23 It can be seen that adding the same volume of 0.1% sodium hydroxide to the supernatant after centrifugation makes the pH value of the supernatant about 1.5, and methylmalonic acid interferes seriously.

[0110] Comparative Example 6 This comparative example provides a kit and a detection method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS, which differs from Example 1 only in that the same volume of 0.1% ammonia water is added to make the pH value of the supernatant approximately 1.5.

[0111] according to Figure 24 It can be seen that adding the same volume of 0.1% ammonia water to the supernatant after centrifugation makes the pH value of the supernatant about 1.5, and methylmalonic acid and succinic acid are not separated and cannot be used.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A kit for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS, characterized in that: include: Samples to be tested, internal standards, extractants and diluents; The extractant includes a mixture of trichloroacetic acid, water and hydrochloric acid; in the extractant, the concentration of trichloroacetic acid is 11wt%~13wt%, and the concentration of hydrochloric acid is 0.1M~0.5M; The diluent is water; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxic acid, vitamin B7 and vitamin B9; the organic acids include 5-methyltetrahydrofolate, methylmalonic acid and succinic acid.

2. The kit for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to claim 1, characterized in that: The kit also contains a mobile phase, which includes mobile phase A and mobile phase B. The mobile phase A is a mixture of ammonium acetate, formic acid and water. In the mobile phase A, the concentration of ammonium acetate is 5mM~10mM, and the concentration of formic acid is 0.002wt%~0.005wt%; the mobile phase B is methanol.

3. The kit for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to claim 1 or 2, characterized in that: The kit further contains a protective agent, which includes one or more of ascorbic acid, citric acid, dithiothreitol, tea polyphenols and chlorogenic acid; preferably, the protective agent includes ascorbic acid, dithiothreitol and citric acid in a mass ratio of (1-3): (1-3): 1; And / or, the samples to be tested include calibrators, quality control materials and blood; Preferably, in the calibrator, the concentration of vitamin B1 is 0.5-25 ng / mL; the concentration of vitamin B2 is 2-100 ng / mL; the concentration of vitamin B3 is 2-100 ng / mL; the concentration of vitamin B5 is 10-500 ng / mL; the concentration of vitamin B6-pyridoxal phosphate is 4-200 ng / mL; the concentration of vitamin B6-pyridoxalic acid is 1-50 ng / mL; the concentration of vitamin B7 is 1-50 ng / mL; the concentration of vitamin B9 is 2-100 ng / mL; the concentration of 5-methyltetrahydrofolate is 2-100 ng / mL; the concentration of methylmalonic acid is 20-1000 ng / mL; the concentration of succinic acid is 20-1000 ng / mL; Preferably, in the quality control product, the concentration of vitamin B1 is 1.5~18.75ng / mL; the concentration of vitamin B2 is 6~75ng / mL; the concentration of vitamin B3 is 6~75ng / mL; the concentration of vitamin B5 is 30~375ng / mL; the concentration of vitamin B6-pyridoxal phosphate is 12~150ng / mL; the concentration of vitamin B6-pyridoxal acid is 3~37.5ng / mL; the concentration of vitamin B7 is 3~37.5ng / mL; the concentration of vitamin B9 is 6~75ng / mL; the concentration of 5-methyltetrahydrofolate is 6~75ng / mL; the concentration of methylmalonic acid is 60~750ng / mL; the concentration of succinic acid is 60~750ng / mL; Preferably, the internal standard for vitamin B1 is 4-methyl-thiamine hydrochloride. 13 C-thiazol-5-yl- 13 C3 hydrochloride; the internal standard for vitamin B2 is vitamin B2- 13 C4, 15 N2; the internal standard for vitamin B3 is vitamin B3-nicotinamide-D4; the internal standard for vitamin B5 is vitamin B5, calcium salt: H2O-β-alanyl- 13 C3; the internal standard for vitamin B6-pyridoxal phosphate is vitamin B6-pyridoxal-d3 phosphate; the internal standard for vitamin B6-pyridoxal acid is vitamin B6-4-pyridoxal-d3; the internal standard for vitamin B7 is vitamin B7-RAC biotin-d4; the internal standard for vitamin B9 is folic acid-glutamic acid- 13 C5; The internal standard of 5-methyltetrahydrofolate is vitamin-5-methyltetrahydrofolate- 13 C5; the internal standard for methylmalonic acid is methylmalonic acid-d3.

4. A method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS, characterized in that: include: S1: mixing a sample to be tested, a mixed internal standard, and an extractant, and centrifuging the resulting mixed solution at a temperature below 10° C.; the extractant comprises a mixture of trichloroacetic acid, water, and hydrochloric acid, wherein the concentration of trichloroacetic acid in the extractant is 11 wt % to 13 wt %, and the concentration of hydrochloric acid is 0.1 M to 0.5 M; S2: The pH value of the supernatant is adjusted to 1-2 with water, and then tested on the machine; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxic acid, vitamin B7 and vitamin B9; and the organic acids include 5-methyltetrahydrofolate, methylmalonic acid and succinic acid.

5. The method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to claim 4, characterized in that: The volume ratio of the sample to be tested to the extractant is (4-6): (1-3).

6. The method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to claim 4 or 5, characterized in that The volume ratio of the sample to be tested to the mixed internal standard is (9-10): (1-2).

7. The method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to any one of claims 4 to 6, characterized in that The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein mobile phase A is a mixture of ammonium acetate, formic acid and water, wherein the concentration of ammonium acetate in mobile phase A is 5mM~10mM, and the concentration of formic acid is 0.002wt%~0.005wt%; mobile phase B is methanol.

8. The method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to claim 7, characterized in that: Gradient elution conditions for liquid chromatography detection include: Wherein, % represents volume percentage, and the sum of the volume percentage of mobile phase A and the volume percentage of mobile phase B is 1.

9. The method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to any one of claims 4 to 8, characterized in that Mass spectrometry detection conditions include: Preferably, the ion pair parameters include: 。 10. The method for simultaneous detection of water-soluble vitamins and organic acids by LC-MS / MS according to any one of claims 4 to 9, characterized in that: include: S0: Different water-soluble vitamins and organic acids are diluted with 40-60% methanol aqueous solution, and a protective agent is added to prepare the test sample; different water-soluble vitamins and organic acid internal standards are diluted with 40-60% methanol aqueous solution, and a protective agent is added, and then the different internal standard solutions are mixed to prepare a mixed internal standard; S1: Mixing a sample to be tested, a mixed internal standard, and an extractant in a volume ratio of (9-10):(1-2):(3-5), and centrifuging the resulting mixed solution at a temperature below 10°C; the extractant comprises a mixture of trichloroacetic acid, water, and hydrochloric acid, wherein the concentration of trichloroacetic acid in the extractant is 11 wt%-13 wt%, and the concentration of hydrochloric acid is 0.1 M-0.5 M; S2: The pH value of the supernatant is adjusted to 1-2 with water, and then tested on the machine; The mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, wherein the mobile phase A is a mixture of ammonium acetate, formic acid, and water, wherein the concentration of ammonium acetate in the mobile phase A is 5 mM to 10 mM, and the concentration of formic acid is 0.002 wt% to 0.005 wt%; the mobile phase B is methanol; The water-soluble vitamins include vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6-pyridoxal phosphate, vitamin B6-pyridoxic acid, vitamin B7 and vitamin B9; and the organic acids include 5-methyltetrahydrofolate, methylmalonic acid and succinic acid.

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