Method for determining amino acid content in crops based on LC-MS / MS

Abstract

The invention discloses a method for determining the content of amino acid in crops based on LC-MS / MS. The method comprises the following steps: 1) crushing and sieving crop grains or tissues, mixing with an acid solution, hydrolyzing, filtering the obtained hydrolysate, taking the supernatant, and filtering with a filter membrane to obtain a sample to be detected; and 2) determining the to-be-detected sample by using the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, detecting the amino acid mixed standard solution by using the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry under the same condition, and calculating the concentration of the amino acid in the to-be-detected sample through a peak area by using an external standard method, thereby obtaining the content of the amino acid in the crops. The method can be used for rapidly and accurately detecting the content of amino acids in crops with a good separation effect.

Description

Technical Field

[0001] The invention belongs to the technical field of amino acid quantitative analysis and relates to a method for measuring the amino acid content in crops based on LC-MS / MS. Background Art

[0002] Crops are rich in various amino acids. The content of amino acids affects the quality and nutritional content of crops. Therefore, it is very important to determine the amino acid content in crops. For example, soybeans are rich in amino acids. At present, there are mainly the following methods for the determination and analysis of amino acids in crops and foods such as soybeans: (1) using amino acid analyzers or liquid chromatography for pre-column or post-column derivatization; (2) directly using LC-MS / MS instruments and equipment to configure corresponding chromatographic columns for analysis and determination without derivatization. According to the chromatographic columns commonly used for amino acid analysis in the world, the separation effect and retention effect are poor. Therefore, it is necessary to develop a method for detecting the amino acid content in crops with good separation effect, short analysis time, high efficiency and accuracy. Summary of the invention

[0003] The purpose of the present invention is to provide a method for determining the amino acid content in crops based on LC-MS / MS. The present invention can detect the amino acid content in crops quickly, accurately and with good separation effect.

[0004] The present invention provides a method for determining the amino acid content in crops based on LC-MS / MS, comprising the following steps: 1) crushing and sieving the grains or tissues of the crops, mixing with an acidic solution for hydrolysis, filtering the obtained hydrolyzate, and filtering the supernatant through a filter membrane to obtain a sample to be tested; 2) The sample to be tested is subjected to ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, and the amino acid mixed standard solution is detected by the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry under the same conditions, and the concentration of amino acids in the sample to be tested is calculated by peak area using the external standard method to obtain the amino acid content in the crop.

[0005] In the above method, the crop includes at least one of soybean, corn, wheat, rice, oats and buckwheat.

[0006] In the above method, the crop seeds or tissues are crushed through a 60-mesh sieve; The acidic solution is 6 mol / L hydrochloric acid solution of analytical grade.

[0007] In the present invention, the crop seeds or tissues are dried before being crushed as appropriate.

[0008] The hydrolysis conditions are as follows: in an inert atmosphere, the hydrolysis temperature is 110°C ± 1°C, and the hydrolysis time is 22h ~ 24h; The pore size of the filter membrane may be 0.22 μm.

[0009] In the above method, the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometer is equipped with an electrospray ion source; The conditions of ultra-high performance liquid chromatography are as follows: Chromatographic column: ACQUITY UPLC BEH Amide column, 2.1×100 mm, 1.7 µm; Mobile phase A: 10 mmol / L ammonium acetate solution + 0.1% formic acid; Mobile phase B: acetonitrile + 10 mmol / L ammonium acetate solution + 0.1% formic acid; Flow rate: 0.40 mL / min; column temperature: 40 °C; injection volume: 1 μL; Elution gradient, based on the volume percentage of mobile phase A and B: 0 min, A 5%, B 95%; 0-4.5 min, A5%, B 95%; 4.5-7.0 min, A 5% to 40%, B 95% to 60%; 7.0-9.0 min, A 40% to 55%, B 60% to 45%; 9.0-10.0 min, A 55% to 55%, B 45% to 45%; 10.0-10.1 min, A 55% to 5%, B 45% to 95%; 10.1-15min, A 5% to 5%, B 95% to 95%.

[0010] In the above method, in the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, the mass spectrometry conditions are as follows: The electrospray ion source ESI positive ion (+) mode, multiple reaction monitoring mode, capillary voltage was 2.5 kV, ion source temperature was 150 °C, desolvation temperature was 500 °C, and desolvation flow rate was 1000 L / h.

[0011] In the above method, the amino acid mixed standard solution is a 0.1 mol / L hydrochloric acid solution prepared by mixing phenylalanine, leucine, isoleucine, methionine, tyrosine, valine, proline, alanine, threonine, glycine, serine, glutamine, aspartic acid, histidine, arginine and lysine.

[0012] The present invention has the following beneficial effects: This method is not subjected to derivatization treatment, and directly uses the LC-MS instrument configured with an Amide hydrophilic column for determination. After comparing reversed-phase columns such as C18 and T3 with hydrophilic columns such as HILIC and amide, this method found that compared with reversed-phase columns such as C18 and T3 and other hydrophilic columns such as HILIC, the amide column can separate isomers of leucine and isoleucine, and the chromatogram peak shape of the compound is also better, without double peaks or tailing. Therefore, the amide analysis column was selected, and the entire sample analysis time was 15 minutes. Compared with the fully automatic amino acid analyzer, its analysis time is about 50 minutes, which is greatly shortened. It can greatly save time costs for excellent resource screening of a large number of samples. In addition, since derivatization reagents such as ninhydrin are not used, which have serious chemical toxicity, it is environmentally friendly. In addition, due to the higher sensitivity and accuracy of LC-MS / MS, the reliability of the detection data is significantly improved, providing a reliable technical means for the accurate identification of excellent germplasms such as high sulfur amino acids in soybeans. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the ion diagram of each component of amino acid determined by the LC-MS / MS method of the present invention.

[0014] Figure 2 Amino acid component chromatogram determined by amino acid analyzer.

[0015] Figure 3 Effects of different flow ratios on histidine peak shape and separation performance.

[0016] Figure 4 The effect of different fluidity ratios on cystine peak shape and separation effect.

[0017] Figure 5 The effect of different flowability ratios on the peak shape and separation effect of leucine and isoleucine.

[0018] Figure 6 It is the amino acid component content in the soybean seeds in Example 1 of the present invention.

[0019] Figure 7 The figures show the addition levels and recovery rates of different amino acids in Example 2 of the present invention.

[0020] Figure 8 The results of the method detection limit, quantitative limit and stability test of each amino acid component in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0023] The present invention provides a method for determining the amino acid content in crops based on LC-MS / MS, comprising the following steps: 1. Sample hydrolysis Crop seeds or tissues (fresh tissues need to be properly dried) are mixed evenly and crushed through a 60-mesh sieve. According to the protein content (generally 10-20 mg), an appropriate amount of sample is accurately weighed and placed in a hydrolysis tube. Another appropriate amount of sample is taken to determine its moisture content. 10 mL of 6 mol / L hydrochloric acid is added to the hydrolysis tube. Connect the hydrolysis tube to the vacuum pump's exhaust pipe, evacuate and then fill with nitrogen, and tighten the screw cap while filling with nitrogen.

[0024] The sealed hydrolysis tube was placed in an electric heating blast drying oven at 110°C, and after hydrolysis for 22 h, it was taken out and cooled to room temperature. After filtering the hydrolyzate, 0.5 mL of the supernatant was diluted to 5 mL, and then filtered through a 0.22 μm filter membrane and transferred to a sample injection bottle for LC-MS / MS analysis.

[0025] 2 Chromatographic and mass spectrometry conditions Ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-XEVO TQ, Waters, USA) was equipped with an electrospray ion source. Chromatographic column: ACQUITY UPLC BEH Amide column (2.1×100 mm, 1.7 μm), or other equivalent; mobile phase A: 10 mmol / L ammonium acetate solution + 0.1% formic acid; mobile phase B: acetonitrile + 10 mmol / L ammonium acetate solution + 0.1% formic acid; flow rate: 0.40 mL / min; column temperature: 40 °C; injection volume: 1 μL; elution gradient is shown in Table 1. Electrospray ion source ESI (+) mode, multiple reaction monitoring mode, capillary voltage 2.5 kV, ion source temperature 150 °C, desolvation gas temperature 500 °C, desolvation gas flow rate 1000 L / h. Other mass spectrometry acquisition parameters are shown in Table 2.

[0026] Table 1 Liquid chromatography gradient elution program

[0027] Table 2 Mass spectrometry parameters of 16 amino acids

[0028] 3 Calculation of amino acid content Accurately pipette a certain amount of amino acid mixed standard solution to prepare the standard solution intermediate solution, then take an appropriate amount of the intermediate standard solution to prepare mixed standard working solutions of different mass concentration series, generate the standard curve of each amino acid component, inject the mixed amino acid standard working solution and the sample determination solution into LC-MS / MS at the same volume, and calculate the concentration of amino acids in the sample determination solution by peak area using the external standard method.

[0029] The calculation formula of amino acid content in the sample is: X i =

[0030] Where: X i -Amino acids in the sample i The content of is in g / 100g; c i -Amino acids in the sample solution i Content, nmol / ml; F-dilution factor; V-volume of sample hydrolyzate, ml; M-amino acids i Molar mass, g / mol; m-sample weight, g; w- moisture content of sample, %.

[0031] Example 1 The method for determining the amino acid content in crops based on LC-MS / MS specifically comprises the following steps: 1. Sample hydrolysis Weigh 0.050g of soybean seeds, mix them evenly, and crush them through a 60-mesh sieve. Accurately weigh an appropriate amount of sample according to its protein content and place it in a hydrolysis tube. Take another appropriate amount of sample to determine its moisture content. Add 10 mL of 6 mol / L hydrochloric acid to the hydrolysis tube. Connect the hydrolysis tube to the vacuum pump's exhaust pipe, evacuate and then fill it with nitrogen, and then tighten the screw cap while filling with nitrogen.

[0032] Place the sealed hydrolysis tube in an electric heating blast drying oven at 110℃±1℃, take it out after hydrolysis for 22h~24h, and cool it to room temperature. After filtering the hydrolyzate, take 0.5mL of the supernatant and dilute it to 5mL, pass it through a 0.22μm filter membrane, and transfer it to a sample injection bottle for LC-MS / MS analysis.

[0033] 2 Chromatographic and mass spectrometric conditions Ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-XEVO TQ, Waters, USA) was equipped with an electrospray ion source. Flow rate: 0.40 mL / min; column temperature: 40 °C; injection volume: 1 μL; elution gradient is shown in Table 1. Electrospray ion source ESI (+) mode, multiple reaction monitoring mode, capillary voltage 2.5 kV, ion source temperature 150 °C, desolvation gas temperature 500 °C, desolvation gas flow rate 1000 L / h.

[0034] The chromatographic column was optimized using soybean seed amino acid hydrolyzate: The C18 and T3 reversed phase columns, as well as HILIC and Amide hydrophilic columns were compared. It was found that the separation and retention effects of C18 and T3 reversed phase columns were poor, the chromatographic peaks were separated within 1-3 minutes, and the matrix interference was large; the hydrophilic column HILIC had a double peak shoulder phenomenon for histidine and glutamic acid, and the separation effect of isomers such as leucine and isoleucine was poor. Therefore, this method selected the Amide hydrophilic column for amino acid analysis. The Amide column had the best separation effect, and the peak shape of 17 amino acids was good. Therefore, the ACQUITY UPLC BEH Amide column (100 mm×2.1 mm, 1.7 μm) of Waters was selected.

[0035] The LC-MS / MS of the present invention only takes 15 minutes, and its chromatogram is as follows Figure 1 As shown. The time taken by the amino acid analyzer is about 50 minutes (such as Figure 2 As shown), it is obvious that the present invention can shorten the detection time.

[0036] Mobile phase optimization: Using acetonitrile and water as the flowability A and B respectively, the effects of the following buffer salts and acids on the ionization degree and peak shape of the compounds were compared (see Table 3, Figure 3-Figure 5 ).

[0037] Table 3 Effects of different buffer salts and acids on amino acid separation

[0038] Based on the above experimental results, the optimal conditions are as follows: Chromatographic column: ACQUITY UPLC BEH Amide column (2.1×100 mm, 1.7 μm), or other equivalent; mobile phase A: 10 mmol / L ammonium acetate solution + 0.1% formic acid; mobile phase B: acetonitrile + 10 mmol / L ammonium acetate solution + 0.1% formic acid; flow rate: 0.40 mL / min; column temperature: 40 °C; injection volume: 1 μL; elution gradient is shown in Table 1. Electrospray ion source ESI (+) mode, multiple reaction monitoring mode, capillary voltage 2.5 kV, ion source temperature 150 °C, desolvation gas temperature 500 °C, desolvation gas flow rate 1000 L / h. Other mass spectrometry acquisition parameters are shown in Table 2.

[0039] 3 Calculation of amino acid content Accurately pipette a certain amount of 2.5 mmol / ml of amino acid mixed standard solution to prepare a 500 nmol / ml standard solution intermediate solution, then take an appropriate amount of the intermediate standard solution to prepare mixed standard working solutions of different mass concentration series (10, 50, 100, 200, 300 nmol / ml) to generate a standard curve for each amino acid component. The mixed amino acid standard working solution and the sample determination solution are injected into LC-MS / MS at the same volume, and the concentration of amino acids in the sample determination solution is calculated by peak area using the external standard method.

[0040] The calculation formula of amino acid content in the sample is: X i =

[0041] Where: X i -Amino acids in the sample i The content of is in g / 100g; c i -Amino acids in the sample solution i Content, nmol / ml; F-dilution factor; V-volume of sample hydrolyzate, ml; M-amino acids i Molar mass, g / mol; m-sample weight, g; w- moisture content of sample, %.

[0042] Weigh 50 mg of soybean seed powder and measure its moisture content to be 10.5%. The dilution factor is 10. According to the measured values ​​of each amino acid component given by the instrument, c i Calculate the content of each amino acid component X in soybeans i， The content of each component is shown in Figure 6 shown.

[0043] Example 2: Accuracy Analysis In order to verify the accuracy of this method in matrices such as soybeans, 0.050g of soybean seed samples were weighed, and low, medium and high levels of aspartic acid, leucine, isoleucine, cystine, proline, glycine and other amino acids were added to the soybean samples according to the background content of amino acids in soybeans. First, methionine and cystine, two amino acid standards with low content in soybeans, were prepared into a 10mg / ml standard solution with 0.1mol / l, and the other types of amino acid standards were prepared into 50mg / ml standard solutions. 0.050g of soybean samples were weighed, and then appropriate volumes of amino acid standard solutions were measured to make the added amino acid components at three different levels of low, medium and high. See details for details. Figure 7 The soybean samples after adding amino acid standard were hydrolyzed according to the sample hydrolysis steps and then measured on the machine. The recovery rate of addition was calculated based on the measured value. Figure 7 .

[0044] Example 3 The detection limit, quantification limit and stability test of the method were carried out for each amino acid component. The detection limit and quantification limit of the method were obtained according to the signal-to-noise ratio of 3 times and 10 times of the instrument baseline, respectively. In order to verify the stability of the method, the above soybean samples were measured in parallel 6 times, and the RSD of each amino acid component was calculated. The experimental data are shown in Figure 8 shown.

[0045] Depend on Figure 8 It can be seen from the results that the method and technology of the present invention is used to determine and analyze the amino acid components in soybeans with extremely high sensitivity and accuracy. Compared with the current standard "GB5009.124-2016 Food Safety Standard Determination of Amino Acids in Foods" which uses amino acid analysis to determine the detection limit and quantification limit of amino acids in solid or semi-solid samples, the sensitivity of different amino acid components is improved by 10-500 times, and it has good stability. The determination time is also greatly shortened compared with the determination time of the amino acid analyzer. Therefore, this method is of great significance in screening excellent germplasm, exploring excellent varieties and cultivating new varieties in crops such as soybeans.

Claims

1. A method for determining the amino acid content in crops based on LC-MS / MS, comprising the following steps: 1) crushing and sieving the crop seeds or tissues, mixing with an acidic solution for hydrolysis, filtering the obtained hydrolyzate, taking the supernatant and filtering through a filter membrane to obtain a sample to be tested; 2) The sample to be tested is subjected to ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, and the amino acid mixed standard solution is detected by the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry under the same conditions, and the concentration of amino acids in the sample to be tested is calculated by peak area using the external standard method to obtain the amino acid content in the crop.

2. The method according to claim 1, characterized in that The crops include at least one of soybean, corn, wheat, rice, oats and buckwheat.

3. The method according to claim 1 or 2, characterized in that The crop seeds or tissues are crushed and then passed through a 60-mesh sieve; the acidic solution is a 6 mol / L hydrochloric acid solution; The hydrolysis conditions are as follows: in an inert atmosphere, the hydrolysis temperature is 110°C ± 1°C, and the hydrolysis time is 22h ~ 24h; The pore size of the filter membrane is 0.22 μm.

4. The method according to any one of claims 1 to 3, characterized in that The ultra-high performance liquid chromatography-tandem quadrupole mass spectrometer is equipped with an electrospray ion source; The conditions of ultra-high performance liquid chromatography are as follows: Chromatographic column: ACQUITY UPLC BEH Amide column, 2.1×100 mm, 1.7 µm; Mobile phase A: 10 mmol / L ammonium acetate solution + 0.1% formic acid; Mobile phase B: acetonitrile + 10 mmol / L ammonium acetate solution + 0.1% formic acid; Flow rate: 0.40 mL / min; Column temperature: 40 °C; injection volume: 1 μL; Elution gradient, based on the volume percentage of mobile phase A and B: 0 min, A 5%, B 95%; 0-4.5 min, A 5%, B 95%; 4.5-7.0 min, A 5% to 40%, B 95% to 60%; 7.0-9.0 min, A 40% to 55%, B 60% to 45%; 9.0-10.0 min, A 55% to 55%, B 45% to 45%; 10.0-10.1 min, A 55% to 5%, B 45% to 95%; 10.1-15min, A 5% to 5%, B 95% to 95%.

5. The method according to any one of claims 1 to 4, characterized in that In the ultra-high performance liquid chromatography-tandem quadrupole mass spectrometry, the mass spectrometry conditions are as follows: The electrospray ion source (ESI) was in positive ion mode and multiple reaction monitoring mode. The capillary voltage was 2.5 kV, the ion source temperature was 150 °C, the desolvation temperature was 500 °C, and the desolvation flow rate was 1000 L / h.

6. The method according to any one of claims 1 to 5, characterized in that The amino acid mixed standard solution is a 0.1 mol / L hydrochloric acid solution prepared by mixing phenylalanine, leucine, isoleucine, methionine, tyrosine, valine, proline, alanine, threonine, glycine, serine, glutamine, aspartic acid, histidine, arginine and lysine.

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