A method for detecting water-soluble vitamin metabolites in plants

Abstract

The present invention belongs to the technical field of material analysis and detection, and specifically discloses a method for detecting water-soluble vitamin metabolites in plants, which comprises the following steps: preparing a plurality of standard stock solutions containing different water-soluble vitamin metabolites; diluting the standard stock solution to prepare a standard solution, subjecting the standard solution to liquid chromatography tandem mass spectrometry detection, using the peak area of ​​the quantitative ion peak of the corresponding target compound and the corresponding target compound concentration as the coordinate system, and drawing a standard working curve; preparing a sample solution to be tested, and performing liquid chromatography tandem mass spectrometry detection; according to the peak area of ​​the quantitative ion peak of the target compound in the sample solution to be tested and the standard working curve, the content of a plurality of water-soluble vitamin metabolites in the test object is obtained. The present invention can realize simultaneous detection and quantification of 30 water-soluble vitamin metabolites in plants, with high sensitivity, accurate and reliable results, and can provide help for studying the metabolic process of water-soluble vitamins in plants.

Description

Technical Field

[0001] The present invention relates to the technical field of material analysis and detection, and in particular to a method for detecting water-soluble vitamin metabolites in plants. Background Art

[0002] Vitamins can be divided into two categories according to their solubility: fat-soluble and water-soluble. Water-soluble vitamins include B vitamins and vitamin C (L-ascorbic acid). B vitamins include vitamin B1 (thiamine), B2 (riboflavin), B3 (niacin and niacinamide), B5 (pantothenic acid), B6 ​​(pyridoxine), B7 (biotin), B9 (folic acid) and B12 (cyanocobalamin). Unlike fat-soluble vitamins, water-soluble vitamins are stored less in the human body and are easily excreted due to their water-soluble properties. Water-soluble vitamins mostly participate in a variety of important metabolic processes in the human body as coenzymes, so insufficient intake of water-soluble vitamins can lead to various deficiency diseases in the human body.

[0003] Plants are an important source of water-soluble vitamins for the human body, and there are many water-soluble vitamin synthesis and metabolic processes in their bodies. For example, vitamin B1 thiamine in plants mainly exists in three forms: free thiamine, thiamine monophosphate and thiamine pyrophosphate. The synthesis process of thiamine is mainly formed by the coupling of pyrimidine and thiazole to form thiamine pyrophosphate. With the rapid development of modern agriculture and biotechnology, humans can affect the synthesis and metabolism of water-soluble vitamins in crops by regulating environmental factors, hormones or gene expression, thereby changing the composition and content of water-soluble vitamins in crops. Therefore, the development of an efficient and accurate method for detecting water-soluble vitamin metabolites in plants can provide ideas and methods for studying the metabolic process of water-soluble vitamins in plants, which is of great significance for promoting research in plant food science and related fields.

[0004] Currently, most existing methods for detecting water-soluble vitamins focus on using high performance liquid chromatography (HPLC) or liquid chromatography tandem mass spectrometry (LC-MS) to detect several common water-soluble vitamins, while ignoring the detection of metabolites produced during the metabolism of water-soluble vitamins. In order to better understand the metabolic process of water-soluble vitamins in plants, it is urgent to establish a method that can quickly and high-throughput detect multiple water-soluble vitamin metabolites. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting water-soluble vitamin metabolites in plants in view of the above problems, to achieve rapid and high-throughput detection of multiple water-soluble vitamin metabolites, and to provide assistance for studying the metabolic process of water-soluble vitamins in plants.

[0006] To achieve the above-mentioned purpose, the present invention discloses a method for detecting water-soluble vitamin metabolites in plants, which method comprises the following steps: step S100, preparing a plurality of standard stock solutions containing different water-soluble vitamin metabolites; step S200, diluting the standard stock solutions to prepare standard solutions, subjecting the standard solutions to liquid chromatography-tandem mass spectrometry detection, and drawing a standard working curve with the peak area of ​​the quantitative ion peak of the corresponding target compound of the water-soluble vitamin metabolite as the first coordinate and the concentration of the corresponding target compound as the second coordinate; step S300, taking the object to be tested to prepare a sample solution to be tested, subjecting the sample solution to liquid chromatography-tandem mass spectrometry detection, and obtaining the peak area of ​​the quantitative ion peak of the target compound in the sample solution to be tested; and step S400, obtaining the content of a plurality of water-soluble vitamin metabolites in the object to be tested according to the peak area of ​​the quantitative ion peak of the target compound in the sample solution to be tested and the standard working curve.

[0007] Preferably, in the liquid chromatography tandem mass spectrometry detection: the liquid chromatography conditions include mobile phase A being ammonium acetate solution, mobile phase B being a mixed solution of methanol and formic acid, and gradient elution; and the mass spectrometry conditions include using a HESI ion source and an MRM scanning mode.

[0008] Preferably, the multiple water-soluble vitamin metabolites in step S100 include any two or more of thiamine or thiamine monophosphate or thiamine pyrophosphate or 4-methyl-5-(2-hydroxyethyl)thiazole or 4-amino-5-hydroxymethyl-2-methylpyrimidine or riboflavin or flavin mononucleotide or flavin adenine dinucleotide or nicotinic acid or nicotinamide or nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate or 1-methylnicotinamide or nicotinylglycine or 1-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide or trigonelline or pantothenic acid or D-panthenol or pyridoxamine or pyridoxine or pyridoxal or 5'-pyridoxal phosphate or biotin or desthiobiotin or folic acid or 5-methyltetrahydrofolate or 5-formyltetrahydrofolate or cyanocobalamin or orotic acid or ascorbic acid.

[0009] Preferably, in the liquid chromatography tandem mass spectrometry detection: the concentration of the ammonium acetate solution used in the mobile phase A is 5-15 mM; the formic acid content used in the mobile phase B is 0.05%-0.15%.

[0010] Preferably, the chromatographic column model in the liquid chromatography conditions is: Waters ACQUITY UPLC HSS T3, 2.1×150 mm, 1.8 μm, column temperature 30~40°C, mobile phase flow rate 0.1~0.3 mL / min, injection volume 1~3 μL; the elution gradient includes 0.0-3.0 min, the volume percentage of mobile phase A is 100%; 3.0-7.0 min, the volume percentage of mobile phase A changes from 100% to 5% at a uniform rate; 7.0-11.0 min, the volume percentage of mobile phase A is 5%; 11.0-11.1 min, the volume percentage of mobile phase A changes from 5% to 100% at a uniform rate; 11.1-15.0 min, the volume percentage of mobile phase A is 100%.

[0011] Preferably, the ion parameters in the mass spectrometry conditions are: electrospray voltage is -3000 / 4000 V, ion transfer tube temperature is 350° C., vaporization chamber temperature is 350° C., sheath gas is 35 Arb, and auxiliary gas is 10 Arb.

[0012] Preferably, the MRM scanning parameters in the mass spectrometry conditions are:

[0013] Compound Start Time(min) End Time(min) Polarity Precursor(m / z) Product (m / z) Collision Energy(V) RF Lens(V) Thiamine 0 15 Positive 264.967 121.967* 14.91 33 Thiamine 0 15 Positive 264.967 143.967 13.8 33 Thiamine monophosphate 0 15 Positive 344.883 121.883* 17.43 50 Thiamine monophosphate 0 15 Positive 344.883 125.8 28.86 50 Thiamine pyrophosphate 0 15 Positive 424.883 121.883* 22.03 68 Thiamine pyrophosphate 0 15 Positive 424.883 303.8 17.08 68 4-Methyl-5-(2-hydroxyethyl)thiazole 0 15 Positive 143.967 112.883* 23.9 52 4-Methyl-5-(2-hydroxyethyl)thiazole 0 15 Positive 143.967 125.883 18.5 52 4-Amino-5-hydroxymethyl-2-methylpyrimidine 0 15 Positive 139.967 80.883* 19.1 45 4-Amino-5-hydroxymethyl-2-methylpyrimidine 0 15 Positive 139.967 121.967 13.29 45 Riboflavin 0 15 Positive 377.05 171.967 39.16 72 Riboflavin 0 15 Positive 377.05 242.967* 23.69 72 Flavin mononucleotide 0 15 Positive 456.967 358.883 22.34 89 Flavin mononucleotide 0 15 Positive 456.967 438.883* 16.22 89 Flavin adenine dinucleotide 0 15 Positive 785.883 347.883* 19.81 96 Flavin adenine dinucleotide 0 15 Positive 785.883 438.967 24.87 96 niacin 0 15 Positive 123.883 77.883 23.5 61 niacin 0 15 Positive 123.883 79.883* 21.33 61 Niacinamide 0 15 Positive 122.883 77.883 24.11 56 Niacinamide 0 15 Positive 122.883 79.883* 20.82 56 Nicotinamide adenine dinucleotide 0 15 Positive 663.967 427.8* 25.88 75 Nicotinamide adenine dinucleotide 0 15 Positive 663.967 523.883 17.54 75 Nicotinamide adenine dinucleotide phosphate 0 15 Positive 743.883 135.883* 50.9 83 Nicotinamide adenine dinucleotide phosphate 0 15 Positive 743.883 603.8 19 83 1-Methylnicotinamide 0 15 Positive 136.883 91.967 21.88 55 1-Methylnicotinamide 0 15 Positive 136.883 93.967* 21.02 55 Nicotinyl Glycine 0 10 Positive 180.883 78.883 40.18 51 Nicotinyl Glycine 0 10 Positive 180.883 134.967* 17.49 51 1-Methyl-6-oxo-1,6-dihydropyridine-3-carboxamide 0 15 Positive 152.967 107.883* 22.59 68 1-Methyl-6-oxo-1,6-dihydropyridine-3-carboxamide 0 15 Positive 152.967 109.883 22.4 68 Trigonelline 0 15 Positive 137.967 91.883* 21.98 60 Trigonelline 0 15 Positive 137.967 93.883 21.48 60 Pantothenic acid 0 15 Positive 219.967 89.883* 14.55 39 Pantothenic acid 0 15 Positive 219.967 201.967 12.28 39 D-Panthenol 0 15 Positive 205.967 75.967* 15.87 38 D-Panthenol 0 15 Positive 205.967 187.967 13.74 38 Pyridoxine 0 15 Positive 168.967 133.883 22.34 31 Pyridoxine 0 15 Positive 168.967 151.967* 12.83 31 Pyridoxine 0 15 Positive 169.967 133.883 22.09 38 Pyridoxine 0 15 Positive 169.967 151.967* 13.59 38 Pyridoxal 0 15 Positive 167.883 93.883 24.41 32 Pyridoxal 0 15 Positive 167.883 149.883* 12.28 32 Pyridoxal 5'-phosphate 0 15 Positive 247.883 93.883 29.99 52 Pyridoxal 5'-phosphate 0 15 Positive 247.883 149.967* 17.25 52 Biotin 0 15 Positive 245.05 96.883 33.21 39 Biotin 0 15 Positive 245.05 226.967* 14.45 39 Desthiobiotin 0 15 Positive 215.05 178.967 18.29 30 Desthiobiotin 0 15 Positive 215.05 196.967* 13.39 30 Folic acid 0 15 Positive 441.967 175.883 39.42 58 Folic acid 0 15 Positive 441.967 294.967* 15.82 58 5-Methyltetrahydrofolate 0 15 Positive 460.133 179.967 41.45 54 5-Methyltetrahydrofolate 0 15 Positive 460.133 313.05* 20.04 54 5-Formyltetrahydrofolate 0 15 Positive 473.967 298.967 32.45 63 5-Formyltetrahydrofolate 0 15 Positive 473.967 326.967* 19.61 63 Cyanocobalamin 0 15 Positive 678.3 146.967* 39.52 85 Cyanocobalamin 0 15 Positive 678.3 358.883 23.4 85 Orotic acid 0 15 Negative 154.883 41.883 25.98 30 Orotic acid 0 15 Negative 154.883 110.883* 10.51 30 ascorbic acid 0 15 Negative 174.883 86.883* 19 38 ascorbic acid 0 15 Negative 174.883 114.883 19 38

[0014] * is the quantifier ion.

[0015] Preferably, the concentration of the standard stock solution in step S100 is 1-10 mg / mL; the concentration of the standard solution in step S200 is 2000 ng / mL, 1500 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, 0.1 ng / mL.

[0016] Preferably, the linear correlation coefficient R of the standard working curve is 2 ≥0.99.

[0017] Preferably, the preparation of the sample solution to be tested in step S300 comprises the following steps: weighing 10-30 mg of the sample lyophilized powder, adding a methanol aqueous solution containing 0.5 mg / mL of BHT at a mass volume ratio of 20 mg / mL, with the volume ratio of methanol to water being 1:1, mixing, ultrasonic extraction in an ice-water bath for 15-30 min, centrifuging to obtain the supernatant, filtering through a 0.22 μm organic phase filter membrane, and then diluting.

[0018] In summary, the beneficial effects of the present invention are as follows: the present invention provides a method for detecting a variety of metabolites produced during the metabolism of water-soluble vitamins in plants based on liquid chromatography-tandem mass spectrometry, which can realize the simultaneous detection and quantification of 30 water-soluble vitamin metabolites in plants, with high sensitivity and accurate and reliable results. Compared with the prior art, in addition to being able to simultaneously detect more water-soluble vitamins, the present invention can also simultaneously detect a variety of water-soluble vitamin metabolites. This method can subsequently be applied to the detection of water-soluble vitamin compounds in plants. On the one hand, it can provide ideas and methods for studying the metabolic process of water-soluble vitamins in plants. On the other hand, it can also provide help for studying the metabolic process of water-soluble vitamins in plants, which is of great significance for promoting research in plant food science and related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 are the extracted ion chromatograms of riboflavin, flavin mononucleotide, flavin adenine dinucleotide, nicotinic acid, and nicotinamide;

[0020] Figure 2 are extracted ion chromatograms of thiamine, thiamine monophosphate, thiamine pyrophosphate, 4-methyl-5-(2-hydroxyethyl)thiazole, and 4-amino-5-hydroxymethyl-2-methylpyrimidine;

[0021] Figure 3 It is the extracted ion chromatogram of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, 1-methylnicotinamide, nicotinylglycine, and 1-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide;

[0022] Figure 4 The extracted ion chromatograms are trigonelline, pantothenic acid, D-panthenol, pyridoxamine, and pyridoxine;

[0023] Figure 5 are the extracted ion chromatograms of pyridoxal, pyridoxal 5'-phosphate, biotin, desthiobiotin, and folic acid;

[0024] Figure 6 It is the extracted ion chromatogram of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, cyanocobalamin, orotic acid and ascorbic acid. DETAILED DESCRIPTION

[0025] In the study of the metabolic process of water-soluble vitamins in plants, in order to have a more comprehensive understanding of the metabolism of water-soluble vitamins in plants and to make up for the deficiency of existing detection technologies that only focus on the detection of several common water-soluble vitamins and ignore the detection of their metabolites, the present invention provides a method for detecting water-soluble vitamin metabolites in plants, aiming to achieve rapid and high-throughput detection of multiple water-soluble vitamin metabolites, and to provide assistance for studying the metabolic process of water-soluble vitamins in plants.

[0026] The present invention provides a method for detecting water-soluble vitamin metabolites in plants, the method comprising the following steps:

[0027] Step S100: preparing a standard stock solution containing water-soluble vitamin metabolites;

[0028] Step S200: dilute the standard stock solution to prepare a standard solution, perform liquid chromatography tandem mass spectrometry on the standard solution, and draw a standard working curve using the peak area of ​​the quantitative ion peak of the corresponding target compound of various water-soluble vitamin metabolites as the first coordinate and the corresponding target compound concentration as the second coordinate.

[0029] Step S300: preparing a sample solution by taking the test object, and subjecting the sample solution to liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​the quantitative ion peak of the target compound in the sample solution;

[0030] Step S400: deriving the contents of multiple water-soluble vitamin metabolites in the sample to be tested based on the peak area of ​​the quantitative ion peak of the target compound in the sample solution to be tested and the standard working curve.

[0031] In the present method, step S100 and step S200 involve the construction process of a liquid chromatography tandem mass spectrometry detection method for water-soluble vitamin metabolites. Next, a detailed explanation will be given in conjunction with the accompanying drawings and specific embodiments.

[0032] This method for detecting water-soluble vitamin metabolites in plants can be used to detect 30 water-soluble vitamin metabolites, including 5 vitamin B1 metabolites: thiamine, thiamine monophosphate, thiamine pyrophosphate, 4-methyl-5-(2-hydroxyethyl)thiazole and 4-amino-5-hydroxymethyl-2-methylpyrimidine; 3 vitamin B2 metabolites: riboflavin, flavin mononucleotide, flavin adenine dinucleotide; 8 vitamin B3 metabolites: niacin, nicotinamide, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate Acid, 1-methylnicotinamide, nicotinylglycine, 1-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide and trigonelline; 2 vitamin B5 metabolites: pantothenic acid, D-panthenol; 4 vitamin B6 metabolites: pyridoxamine, pyridoxine, pyridoxal, 5'-pyridoxal phosphate; 2 vitamin B7 metabolites: biotin, desthiobiotin; 3 vitamin B9 metabolites: folic acid, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate; vitamin B12 cyanocobalamin, vitamin B13 orotic acid, vitamin C ascorbic acid.

[0033] Example 1: Establishment of a liquid chromatography tandem mass spectrometry detection method for water-soluble vitamin metabolites. The specific steps are as follows.

[0034] Prepare standard solutions. The specific preparation method is as follows: take an appropriate amount of each standard, accurately weigh, place in a 1.5 mL centrifuge tube, add solvent, and dissolve by ultrasonic to prepare a 1-10 mg / mL standard stock solution. Dilute the standard stock solution to prepare the standard solution. The concentrations of the standard solutions are 2000 ng / mL, 1500 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.1 ng / mL.

[0035] Determine the parameters of the liquid chromatography tandem mass spectrometry detection method:

[0036] Liquid chromatography conditions: mobile phase A is 10 mM ammonium acetate solution (in other embodiments, the concentration of ammonium acetate solution can be 5 mM or 15 mM), mobile phase B is methanol containing 0.1% formic acid (in other embodiments, the formic acid content can be 0.05% or 0.15%); the chromatographic column is a Waters ACQUITY UPLC HSS T3 chromatographic column with a specification of i.d. 2.1×150 mm, 1.8 μm; the elution gradient is: 0.0~3.0 min, the volume percentage of mobile phase A is 100%; 3.0~7.0 min, the volume percentage of mobile phase A changes from 100% to 5% at a uniform rate; 7.0~11.0 min, the volume percentage of mobile phase A is 5%; 11.0~11.1 min, the volume percentage of mobile phase A changes from 5% to 100% at a uniform rate; 11.1~15.0 min, the volume percentage of mobile phase A is 100%; column temperature is 35°C (in other embodiments, the column temperature can be 30°C or 40°C), mobile phase flow rate is 0.2 ml / min (in other embodiments, the mobile phase flow rate can be 0.1 mL / min or 0.3 mL / min), and injection volume is 2 μl (in other embodiments, the injection volume can be 1 μL or 3 μL). Mass spectrometry conditions: Thermo Scientific TSQ Altis Plus mass spectrometer equipped with H-ESI ionization source is used; electrospray voltage is -3000 / 4000 V, ion transfer tube temperature is 350°C, vaporization chamber temperature is 350°C, sheath gas is 35 Arb, and auxiliary gas is 10 Arb. The MRM analysis parameters of each compound are determined using the diluted standard solution. The specific parameters are shown in Table 1:

[0037] Table 1. Analysis parameters of each compound in the standard solution

[0038] Compound Start Time(min) End Time(min) Polarity Precursor(m / z) Product (m / z) Collision Energy(V) RF Lens(V) Thiamine 0 15 Positive 264.967 121.967* 14.91 33 Thiamine 0 15 Positive 264.967 143.967 13.8 33 Thiamine monophosphate 0 15 Positive 344.883 121.883* 17.43 50 Thiamine monophosphate 0 15 Positive 344.883 125.8 28.86 50 Thiamine pyrophosphate 0 15 Positive 424.883 121.883* 22.03 68 Thiamine pyrophosphate 0 15 Positive 424.883 303.8 17.08 68 4-Methyl-5-(2-hydroxyethyl)thiazole 0 15 Positive 143.967 112.883* 23.9 52 4-Methyl-5-(2-hydroxyethyl)thiazole 0 15 Positive 143.967 125.883 18.5 52 4-Amino-5-hydroxymethyl-2-methylpyrimidine 0 15 Positive 139.967 80.883* 19.1 45 4-Amino-5-hydroxymethyl-2-methylpyrimidine 0 15 Positive 139.967 121.967 13.29 45 Riboflavin 0 15 Positive 377.05 171.967 39.16 72 Riboflavin 0 15 Positive 377.05 242.967* 23.69 72 Flavin mononucleotide 0 15 Positive 456.967 358.883 22.34 89 Flavin mononucleotide 0 15 Positive 456.967 438.883* 16.22 89 Flavin adenine dinucleotide 0 15 Positive 785.883 347.883* 19.81 96 Flavin adenine dinucleotide 0 15 Positive 785.883 438.967 24.87 96 niacin 0 15 Positive 123.883 77.883 23.5 61 niacin 0 15 Positive 123.883 79.883* 21.33 61 Niacinamide 0 15 Positive 122.883 77.883 24.11 56 Niacinamide 0 15 Positive 122.883 79.883* 20.82 56 Nicotinamide adenine dinucleotide 0 15 Positive 663.967 427.8* 25.88 75 Nicotinamide adenine dinucleotide 0 15 Positive 663.967 523.883 17.54 75 Nicotinamide adenine dinucleotide phosphate 0 15 Positive 743.883 135.883* 50.9 83 Nicotinamide adenine dinucleotide phosphate 0 15 Positive 743.883 603.8 19 83 1-Methylnicotinamide 0 15 Positive 136.883 91.967 21.88 55 1-Methylnicotinamide 0 15 Positive 136.883 93.967* 21.02 55 Nicotinyl Glycine 0 10 Positive 180.883 78.883 40.18 51 Nicotinyl Glycine 0 10 Positive 180.883 134.967* 17.49 51 1-Methyl-6-oxo-1,6-dihydropyridine-3-carboxamide 0 15 Positive 152.967 107.883* 22.59 68 1-Methyl-6-oxo-1,6-dihydropyridine-3-carboxamide 0 15 Positive 152.967 109.883 22.4 68 Trigonelline 0 15 Positive 137.967 91.883* 21.98 60 Trigonelline 0 15 Positive 137.967 93.883 21.48 60 Pantothenic acid 0 15 Positive 219.967 89.883* 14.55 39 Pantothenic acid 0 15 Positive 219.967 201.967 12.28 39 D-Panthenol 0 15 Positive 205.967 75.967* 15.87 38 D-Panthenol 0 15 Positive 205.967 187.967 13.74 38 Pyridoxine 0 15 Positive 168.967 133.883 22.34 31 Pyridoxine 0 15 Positive 168.967 151.967* 12.83 31 Pyridoxine 0 15 Positive 169.967 133.883 22.09 38 Pyridoxine 0 15 Positive 169.967 151.967* 13.59 38 Pyridoxal 0 15 Positive 167.883 93.883 24.41 32 Pyridoxal 0 15 Positive 167.883 149.883* 12.28 32 Pyridoxal 5'-phosphate 0 15 Positive 247.883 93.883 29.99 52 Pyridoxal 5'-phosphate 0 15 Positive 247.883 149.967* 17.25 52 Biotin 0 15 Positive 245.05 96.883 33.21 39 Biotin 0 15 Positive 245.05 226.967* 14.45 39 Desthiobiotin 0 15 Positive 215.05 178.967 18.29 30 Desthiobiotin 0 15 Positive 215.05 196.967* 13.39 30 Folic acid 0 15 Positive 441.967 175.883 39.42 58 Folic acid 0 15 Positive 441.967 294.967* 15.82 58 5-Methyltetrahydrofolate 0 15 Positive 460.133 179.967 41.45 54 5-Methyltetrahydrofolate 0 15 Positive 460.133 313.05* 20.04 54 5-Formyltetrahydrofolate 0 15 Positive 473.967 298.967 32.45 63 5-Formyltetrahydrofolate 0 15 Positive 473.967 326.967* 19.61 63 Cyanocobalamin 0 15 Positive 678.3 146.967* 39.52 85 Cyanocobalamin 0 15 Positive 678.3 358.883 23.4 85 Orotic acid 0 15 Negative 154.883 41.883 25.98 30 Orotic acid 0 15 Negative 154.883 110.883* 10.51 30 ascorbic acid 0 15 Negative 174.883 86.883* 19 38 ascorbic acid 0 15 Negative 174.883 114.883 19 38

[0039] * is the quantifier ion.

[0040] Figures 1 to 6 The following are the extracted ion chromatograms of 30 water-soluble vitamin metabolites under these conditions.

[0041] Methodological investigation: The analytical method of the target compound was validated by using matrix blank and gradient diluted standard solution. The validation parameters included specificity, linear range, detection limit and quantification limit. Specificity: Under the above instrumental analysis conditions, the matrix blank and standard were tested. The matrix blank had no interference on the analysis of the target, and the target compounds were well detected. Linear range, detection limit and quantification limit: With the standard concentration c (ng / mL) as the horizontal coordinate and the peak area of ​​each standard as the vertical coordinate, the standard curve of each standard was drawn, and the linear regression equation and correlation coefficient of the concentration-peak area of ​​each target compound were calculated. The detection limit was 3 times the signal-to-noise ratio, and the quantification limit was 10 times the signal-to-noise ratio. The standard curve, correlation coefficient, linear range, detection limit and quantification limit of this method are shown in Table 2. The linear correlation coefficients all meet the requirements of greater than 0.99. In this embodiment, when drawing the standard curve of each standard, the standard concentration c (ng / mL) is used as the horizontal coordinate, and the peak area of ​​each standard is used as the vertical coordinate; in other embodiments of the present invention, the peak area of ​​each standard is used as the horizontal coordinate, and the standard concentration c (ng / mL) is used as the vertical coordinate.

[0042] Table 2. Standard curves, correlation coefficients, linear ranges, detection limits and quantification limits of each standard

[0043] Compound Standard curve Correlation coefficient Detection limit (ng / mL) Limit of quantification (ng / mL) Linear range (ng / mL) Thiamine y=71263.6x-68283.5 0.9959 0.8 2 2-120 Thiamine monophosphate y=4482.42x-36337.1 0.9981 8 20 20-1200 Thiamine pyrophosphate y=1639.12x-13684.9 0.9985 15 40 40-1200 4-Methyl-5-(2-hydroxyethyl)thiazole y=66745.3x+2099.84 0.9983 0.01 0.03 0.03-240 4-Amino-5-hydroxymethyl-2-methylpyrimidine y=373749x-1495.38 0.9985 0.2 0.5 0.5-40 Riboflavin y=5989.99x+2478.93 0.9969 0.2 0.8 0.8-1200 Flavin mononucleotide y=2264.9x-8639.5 0.9946 8 20 20-2400 Flavin adenine dinucleotide y=1969.64x-12082.9 0.9970 8 20 20-2400 niacin y=16311x+839.701 0.9962 0.8 2 2-240 Niacinamide y=41546.3x-287.31 0.9961 0.5 1 1-240 Nicotinamide adenine dinucleotide y=626.765x-665.825 0.9991 3 10 10-2400 Nicotinamide adenine dinucleotide phosphate y=344.542x-69.4583 0.9978 2 8 8-2400 1-Methylnicotinamide y=107475x-2855.59 0.9981 0.1 0.3 0.3-80 Nicotinyl Glycine y=12320.8x-371.127 0.9979 1 3 3-240 1-Methyl-6-oxo-1,6-dihydropyridine-3-carboxamide y=9623.72x+21483.1 0.9991 1 3 3-240 Trigonelline y=87482.6x+1511.04 0.9973 0.05 0.1 0.1-40 Pantothenic acid y=4075.19x+831.87 0.9984 1 2 2-120 Panthenol y=27552x+76375.2 0.9994 0.5 1 1-240 Pyridoxine y=69206.5x+8035.89 0.9980 0.3 1 1-80 Pyridoxine y=197695x+21478.6 0.9980 0.1 0.3 0.3-80 Pyridoxal y=23291.2x+9747.05 0.9984 1 2 2-80 Pyridoxal 5'-phosphate y=2752.31x+1601.87 0.9981 2 4 4-1200 Biotin y=27685.8x-1385.88 0.9984 1 2 2-240 Desthiobiotin y=81708.4x-12686.4 0.9991 0.2 0.8 0.8-1200 Folic acid y=1774.94x-6473.5 0.9997 10 20 20-2400 5-Methyltetrahydrofolate y=726.441x-361.295 0.9953 3 8 8-800 5-Formyltetrahydrofolate y=2093.62x-107.876 0.9997 10 20 20-2400 Cyanocobalamin y=2352.28x-8054.32 0.9907 2 8 8-2400 Orotic acid y=7764.73x+1012.83 0.9973 0.5 1 1-80 ascorbic acid y=200.639x-397.235 0.9948 10 20 20-2000

[0044] In the present method, step S100 and step S200 involve the process of detecting the content of multiple water-soluble vitamin metabolites in a specific analyte. Next, a detailed explanation will be given through a specific embodiment.

[0045] Example 2: Analysis of the composition and content of water-soluble vitamin metabolites in watermelon. The specific steps are as follows.

[0046] Preparation of analytical samples: weigh about 20 mg of watermelon freeze-dried powder sample, add extract at a mass volume ratio of 20 mg / mL, the extract uses a methanol-water solution containing 0.5 mg / mL BHT, the volume ratio of methanol to water in the methanol-water solution is 1:1, after oscillation for 1 min, ultrasonication for 15 min under ice bath conditions, centrifugation at 14000 rpm for 15 min at 4°C, pass through a 0.22μm organic phase filter, dilute 10 times with water, centrifuge again, and finally transfer to a sample injection bottle.

[0047] Sample testing and data analysis: Prepare the corresponding mobile phase and set the detection parameters according to the technical plan; prepare standard working solutions of different concentrations and analyze them together with the samples. Use Quan Browser software to analyze the data files of standards and samples: qualitatively identify the sample mass spectrum peaks based on the retention time and daughter ion types of the standards, and quantitatively identify the substances detected in the samples based on the standard curve of the quantitative ion peak area of ​​the standards, so as to analyze the composition and content of water-soluble vitamin metabolites in the samples. The results are shown in the following table.

[0048] Example 3: Composition and content analysis of water-soluble vitamin metabolites in kiwifruit. The specific steps are as follows.

[0049] Preparation of analytical samples: weigh about 20 mg of kiwi freeze-dried powder sample, add extract at a mass volume ratio of 20 mg / mL, the extract uses a methanol-water solution containing 0.5 mg / mL BHT, the volume ratio of methanol to water in the methanol-water solution is 1:1, after oscillation for 1 min, ultrasonication for 15 min under ice bath conditions, centrifugation at 14000 rpm for 15 min at 4°C, pass through a 0.22μm organic phase filter, dilute 10 times with water, centrifuge again, and finally transfer to a sample injection bottle.

[0050] Sample detection and data analysis: Prepare the corresponding mobile phase and set the detection parameters according to the technical plan; prepare standard working solutions of different concentrations and analyze them together with the samples. Use Quan Browser software to analyze the data files of standards and samples: qualitatively identify the sample mass spectrum peaks according to the retention time and daughter ion types of the standards, and quantitatively identify the substances detected in the samples according to the standard curve of the quantitative ion peak area of ​​the standards, so as to analyze the composition and content of water-soluble vitamin metabolites in the samples. The results are shown in Table 3.

[0051] Table 3. Contents of various water-soluble vitamin metabolites in kiwifruit and watermelon samples

[0052] Compound Content in kiwi fruit (ug / g) Content in watermelon (ug / g) Thiamine 1.16 4.64 Thiamine monophosphate ND ND Thiamine pyrophosphate ND ND 4-Methyl-5-(2-hydroxyethyl)thiazole 0.02 ND 4-Amino-5-hydroxymethyl-2-methylpyrimidine ND ND Riboflavin ND 1.04 Flavin mononucleotide ND ND Flavin adenine dinucleotide 5.67 7.38 niacin ND ND Niacinamide 0.44 0.58 Nicotinamide adenine dinucleotide 36.8 73.57 Nicotinamide adenine dinucleotide phosphate 22.75 56.86 1-Methylnicotinamide ND ND Nicotinyl Glycine 0.61 ND 1-Methyl-6-oxo-1,6-dihydropyridine-3-carboxamide ND ND Trigonelline ND 104.17 Pantothenic acid 14.83 43.68 Panthenol ND ND Pyridoxine 0.36 5.34 Pyridoxine 0.18 0.23 Pyridoxal ND ND Pyridoxal 5'-phosphate 1.67 1.78 Biotin ND ND Desthiobiotin ND ND Folic acid ND ND 5-Methyltetrahydrofolate ND ND 5-Formyltetrahydrofolate ND 18.4 Cyanocobalamin ND ND Orotic acid ND 3.46 ascorbic acid 5940.4 819.1

[0053] ND: Not detected.

Claims

1. A method for detecting water-soluble vitamin metabolites in plants, characterized in that: The method comprises the following steps: Step S100: preparing a plurality of standard stock solutions containing different water-soluble vitamin metabolites, wherein the plurality of water-soluble vitamin metabolites include thiamine and thiamine monophosphate and thiamine pyrophosphate and 4-methyl-5-(2-hydroxyethyl)thiazole and 4-amino-5-hydroxymethyl-2-methylpyrimidine and riboflavin and flavin mononucleotide and flavin adenine dinucleotide and nicotinic acid and nicotinamide and nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate and 1-methylnicotinamide and nicotinylglycine and 1-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide and trigonelline and pantothenic acid and D-panthenol and pyridoxamine and pyridoxine and pyridoxal and 5'-pyridoxal phosphate and biotin and desthiobiotin and folic acid and 5-methyltetrahydrofolate and 5-formyltetrahydrofolate and cyanocobalamin and orotic acid and ascorbic acid; Step S200: diluting the standard stock solution to prepare a standard solution, subjecting the standard solution to liquid chromatography tandem mass spectrometry detection, and drawing a standard working curve using the peak area of ​​the quantitative ion peak of the corresponding target compound of the water-soluble vitamin metabolite as the first coordinate and the corresponding target compound concentration as the second coordinate; Step S300: preparing a sample solution by taking the test object, and subjecting the sample solution to liquid chromatography-tandem mass spectrometry to obtain the peak area of ​​the quantitative ion peak of the target compound in the sample solution; Step S400: obtaining the contents of multiple water-soluble vitamin metabolites in the test object according to the peak area of ​​the quantitative ion peak of the target compound in the test sample solution and the standard working curve; In the liquid chromatography tandem mass spectrometry detection: The liquid chromatography conditions include: mobile phase A is an ammonium acetate solution with a concentration of 5 to 15 mM, mobile phase B is a methanol-formic acid mixed solution with a formic acid content of 0.05% to 0.15%, and gradient elution is used; In the liquid chromatography conditions: Chromatographic column model: Waters ACQUITY UPLC HSS T3, 2.1×150 mm, 1.8 μm, column temperature 30~40℃, mobile phase flow rate 0.1~0.3 mL / min, injection volume 1~3 μL; Elution gradient: 0.0-3.0 min, the volume percentage of mobile phase A is 100%; 3.0-7.0 min, the volume percentage of mobile phase A changes from 100% to 5% at a constant rate; 7.0-11.0 min, the volume percentage of mobile phase A is 5%; 11.0-11.1 min, the volume percentage of mobile phase A changes from 5% to 100% at a constant rate; 11.1-15.0 min, the volume percentage of mobile phase A is 100%; Mass spectrometry conditions included: using a HESI ion source and MRM scanning mode; The ion parameters in the mass spectrometry conditions are: electrospray voltage is -3000 / 4000 V, ion transfer tube temperature is 350°C, vaporization chamber temperature is 350°C, sheath gas is 35 Arb, auxiliary gas is 10 Arb; The MRM scanning parameters in the mass spectrometry conditions are: * is the quantifier ion.

2. The method for detecting water-soluble vitamin metabolites in plants according to claim 1, characterized in that: The concentration of the standard stock solution in step S100 is 1-10 mg / mL; the concentration of the standard solution in step S200 is 2000 ng / mL, 1500 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.1 ng / mL.

3. The method for detecting water-soluble vitamin metabolites in plants according to claim 1, characterized in that: The linear correlation coefficient R of the standard working curve 2 ≥0.

99.

4. The method for detecting water-soluble vitamin metabolites in plants according to claim 1, characterized in that: The preparation of the sample solution to be tested in step S300 includes the following steps: weighing 10-30 mg of the sample lyophilized powder, adding a methanol aqueous solution containing 0.5 mg / mL of BHT at a mass volume ratio of 20 mg / mL, wherein the volume ratio of methanol to water in the methanol aqueous solution is 1:1, mixing, ultrasonic extraction in an ice-water bath for 15-30 min, centrifuging to obtain the supernatant, filtering through a 0.22 μm organic phase filter membrane, and then diluting.

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