Method for quantitatively detecting content of tryptophan in sweet corn kernels

Abstract

The invention belongs to the technical field of tryptophan detection, and particularly relates to a method for quantitatively detecting the content of tryptophan in sweet corn kernels. The method comprises the following steps: mixing sweet corn kernels with an alkaline solution, carrying out alkaline hydrolysis, and carrying out solid-liquid separation on an extracting solution obtained by the alkaline hydrolysis to obtain a solution to be detected; performing ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection on the to-be-detected liquid to obtain a to-be-detected spectrogram; according to the spectrogram to be detected and a preset standard curve, the content of tryptophan in the sweet corn kernels is obtained, and the standard curve is a linear relation curve between the peak area and the concentration of tryptophan. The method can realize accurate, sensitive and high-throughput detection of the tryptophan content in the sweet corn kernels.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tryptophan detection, and particularly relates to a method for quantitatively detecting the content of tryptophan in sweet corn kernels. BACKGROUND

[0002] Tryptophan, as an essential amino acid, plays a key role in human nutrition, health and physiological functions. It is an important substance for synthesizing neurotransmitter 5-hydroxytryptamine (serotonin) and precursor melatonin, and has an important influence on mood regulation, sleep quality and cognitive function. In the field of food nutrition, especially in cereal crops, accurate detection of the content of tryptophan is of great significance for the evaluation of nutritional value, variety selection and the development of functional foods. Sweet corn, as an important edible agricultural product in the world, is deeply loved by consumers due to its unique flavor and rich nutrition, and the content of amino acids, especially tryptophan, has become one of the important indicators for measuring its quality.

[0003] At present, for the detection of the content of amino acids in cereals and agricultural products, the traditional method mainly relies on acid hydrolysis combined with ion exchange chromatography. This method usually uses 6M hydrochloric acid (HCl) to hydrolyze the sample at 110℃ for 16-24h, and then uses an amino acid analyzer or high performance liquid chromatography (HPLC) for separation and detection. However, this method has a fundamental defect: the indole ring structure of tryptophan is extremely unstable under strong acid and high temperature conditions, and is easily degraded and destroyed, resulting in serious distortion of the determination results and significant reduction of the recovery rate. Studies have shown that acid hydrolysis method may cause a loss of tryptophan recovery rate of up to 30-50%, so that the measured value cannot truly reflect the actual content in the sample.

[0004] In order to overcome this limitation, alternative technologies such as colorimetric method, ultraviolet spectrophotometry and conventional high performance liquid chromatography (HPLC) have been applied to tryptophan detection. Although the colorimetric method is simple to operate, it has poor specificity and is easily interfered by other indole compounds in the sample; although the ultraviolet spectrophotometry is rapid, it has low sensitivity and cannot accurately quantify in complex matrix; although the conventional HPLC method has improved separation effect, it has limited resolution and weak anti-interference ability, and often appears co-flow phenomenon when analyzing complex food matrix, affecting the accuracy of quantification. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a method for quantitatively detecting the content of tryptophan in sweet corn kernels, which can realize accurate, sensitive and high-throughput detection of the content of tryptophan in sweet corn kernels.

[0006] In order to achieve the above purpose, the present application provides the following technical solutions: The application provides a method for quantitatively detecting the content of tryptophan in sweet corn kernels, comprising the following steps: Mixing the sweet corn kernels and an alkali solution to perform alkaline hydrolysis, and performing solid-liquid separation on the obtained extraction liquid by the alkaline hydrolysis to obtain a to-be-tested liquid; Performing ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection on the to-be-tested liquid to obtain a to-be-tested spectrum; According to the to-be-tested spectrum and a predetermined standard curve, the content of tryptophan in the sweet corn kernels is obtained, and the standard curve is a linear relationship curve between the peak area and the concentration of tryptophan.

[0007] Preferably, the alkali solution is a sodium hydroxide solution; and the concentration of the alkali solution is 4-5 mol / L.

[0008] Preferably, the mass of the sweet corn kernels and the volume of the alkali solution are (45-55) mg:1 mL.

[0009] Preferably, the temperature of the alkaline hydrolysis is 105±2 ℃.

[0010] Preferably, the time of the alkaline hydrolysis is 20±0.5 h.

[0011] Preferably, the chromatographic conditions of the ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection comprise: The chromatographic column is an HILIC chromatographic column or a C18 chromatographic column; The column temperature is 55 ℃; The mobile phase comprises phase A and phase B, the phase A is an ultrapure water solution containing 0.1 wt% formic acid, and the phase B is an acetonitrile solution containing 0.1 wt% formic acid; The flow rate is 0.5 mL / min; The injection amount is 1 μL; The gradient elution program is as follows: 0 min, phase A / phase B (95:5, v / v); 1.5 min, phase A / phase B (90:10, v / v); 2.5 min, phase A / phase B (90:10, v / v); 3 min, phase A / phase B (95:5, v / v); and 5.0 min, phase A / phase B (95:5, v / v).

[0012] Preferably, the mass spectrometry conditions of the ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection comprise: The ion source is an electrospray ion source; The scanning mode is a positive ion mode; The scanning mode is full-scan or data-dependent secondary scan; The resolution is 70,000 for full-scan and 17,500 for data-dependent secondary scan; The spray voltage is 3 kV; The capillary temperature is 350℃. The sheath gas and auxiliary gas are nitrogen; The scanning range is m / z 50~750; The target ion is the quasi-molecular ion peak [M+H] of tryptophan + , m / z 205.0972.

[0013] Preferably, before the mixing, the sweet corn kernels are further crushed and sieved to obtain sweet corn kernel powder.

[0014] Preferably, the mesh size of the sieve used for sieving is 40~80 mesh.

[0015] Preferably, after the alkaline hydrolysis, the obtained extraction solution is cooled to room temperature, and an acid solution is added to adjust the pH value of the solution to neutral; the obtained neutral extraction solution is then constant volume; the neutral pH value is 7.0±0.5.

[0016] The present application provides a method for quantitatively detecting the content of tryptophan in sweet corn kernels, comprising the following steps: mixing sweet corn kernels and an alkali solution, performing alkaline hydrolysis, and performing solid-liquid separation on the obtained extraction solution to obtain a test solution; performing ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection on the test solution to obtain a test spectrum; and obtaining the content of tryptophan in sweet corn kernels according to the test spectrum and a predetermined standard curve, wherein the standard curve is a linear relationship curve between the peak area and the concentration of tryptophan.

[0017] The alkaline hydrolysis combined with the detection method of ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry (UHPLC-QE Orbitrap high-resolution mass spectrometry) detection fundamentally solves the technical problem of tryptophan destruction in the pretreatment process, and exhibits great advantages in high precision, high sensitivity, high analysis efficiency and anti-interference ability. The present application provides an accurate, reliable and efficient detection scheme for the content of tryptophan in sweet corn kernels, which overcomes the technical bottleneck that has plagued the field for a long time. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a standard chromatogram; Figure 2 is a sample chromatogram; Figure 3 is a standard curve graph. DETAILED DESCRIPTION

[0019] The present application provides a method for quantitatively detecting the content of tryptophan in sweet corn kernels, comprising the following steps: mixing sweet corn kernels and an alkali solution, performing alkaline hydrolysis, and performing solid-liquid separation on the obtained extraction solution to obtain a test solution; The to-be-tested liquid is subjected to ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection to obtain a to-be-tested spectrum; According to the to-be-tested spectrum and a predetermined standard curve, the content of tryptophan in the sweet corn kernel is obtained, and the standard curve is a linear relationship curve between the peak area and the concentration of tryptophan.

[0020] Unless otherwise specified, the source of the raw materials used in the present application has no special requirements, and commercially available goods known to those skilled in the art can be used.

[0021] In the present application, the sweet corn kernel and the alkali solution are mixed, the obtained mixed solution is subjected to alkaline hydrolysis, the obtained extraction liquid of the alkaline hydrolysis is subjected to solid-liquid separation, and the to-be-tested liquid is obtained.

[0022] As an embodiment, the alkali solution is a sodium hydroxide (NaOH) solution; the concentration of the alkali solution is 4-5 mol / L, and in a specific embodiment, it is 4.5 mol / L; the mass ratio of the sweet corn kernel to the volume of the alkali solution is (45-55) mg:1 mL, and in a specific embodiment, it is 50 mg:1 mL; the mixing is to immerse the sweet corn kernel in the alkali solution; the temperature of the alkaline hydrolysis is 105±2℃, and in a specific embodiment, it is 105℃; the time of the alkaline hydrolysis is 20±0.5 h, and in a specific embodiment, it is 20 h; the reaction bottle containing the mixed solution is sealed during the alkaline hydrolysis; the sealing is performed by filling nitrogen or using a bottle cap with a polytetrafluoroethylene gasket; the reaction bottle is a high-temperature-resistant and strong-alkali-resistant reaction bottle, including a polytetrafluoroethylene reaction bottle or a glass ampoule bottle. Filling nitrogen can protect tryptophan from oxidation.

[0023] As an embodiment, after the alkaline hydrolysis, the method further comprises: after the extraction liquid obtained by the alkaline hydrolysis is cooled to room temperature, an acid solution is added to adjust the pH value of the solution to neutral; the obtained neutral extraction liquid is subjected to constant volume; the acid solution is a hydrochloric acid solution; the concentration of the hydrochloric acid solution is 0.1-0.2 mol / L, and in a specific embodiment, it is 0.1 mol / L; the neutral pH value is 7.0±0.5, and in a specific embodiment, it is 7.0; the constant volume is that the obtained neutral extraction liquid is quantitatively transferred to a capacity bottle with a known volume, the reaction bottle is washed with ultrapure water multiple times and the washing liquid is added to the capacity bottle, and finally the capacity bottle is constant volume to the calibration line (V) with ultrapure water; the volume of the capacity bottle is 5 mL or 10 mL.

[0024] As an embodiment, before the mixing, the method further comprises: the sweet corn kernel is crushed and sieved to obtain sweet corn kernel powder; the mesh number of the sieve used for sieving is 40-80 meshes, and in a specific embodiment, it is 60-80 meshes.

[0025] As an embodiment, the solid-liquid separation is filtration after centrifugal separation; the temperature of the centrifugal separation is 0-4℃, and in particular embodiments, 4℃; the centrifugal speed is 10000-12000rpm, and in particular embodiments, 12000rpm; the centrifugal time is 5-10min, and in particular embodiments, 10min; the filtration is performed using a microporous filter membrane; the pore size of the microporous filter membrane is 0.22-0.45μm, and in particular embodiments, 0.22μm; the material of the microporous filter membrane is compatible with the mobile phase of ultra-high performance liquid chromatography (UHPLC), and the material of the microporous filter membrane includes nylon, polyethersulfone (PES) or polytetrafluoroethylene (PTFE).

[0026] The present application releases the bound tryptophan in sweet corn kernels completely by alkaline hydrolysis and purification, and obtains a clear solution for instrumental analysis.

[0027] After obtaining the to-be-tested solution, the present application performs ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection on the to-be-tested solution to obtain a to-be-tested spectrum; according to the to-be-tested spectrum and a predetermined standard curve, the content of tryptophan in sweet corn kernels is obtained, and the standard curve is a linear relationship curve between the peak area and the concentration of tryptophan.

[0028] As an embodiment, the equipment used for the ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection is an ultra-high performance liquid chromatography-tandem high-resolution mass spectrometer, and the specific model is UHPLC-QE, Thermo, USA.

[0029] As an embodiment, the chromatographic conditions for the ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection include: The chromatographic column is a HILIC chromatographic column or a C18 chromatographic column; the model of the HILIC chromatographic column is ACQUITY UPLC BEH Amide, the size is 2.1 mm × 100 mm, and the filter material particle size is 1.7μm; the C18 chromatographic column is Waters BEH C18, the size is 50×2.1mm, and the filter material particle size is 1.7μm; The column temperature is 55℃; The mobile phase includes phase A and phase B, the phase A is an ultrapure water solution containing 0.1wt% formic acid, and the phase B is an acetonitrile solution containing 0.1wt% formic acid; The flow rate is 0.5mL / min; The injection volume is 1μL; The gradient elution program is: 0min phase A / phase B (95:5, v / v), 1.5min phase A / phase B (90:10, v / v), 2.5min phase A / phase B (90:10, v / v), 3min phase A / phase B (95:5, v / v), 5.0min phase A / phase B (95:5, v / v).

[0030] As an embodiment, the mass spectrometry conditions of the ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection include: Ion source: electrospray ion source (ESI); Scan mode: positive ion mode (Positive); Scan mode: full scan or data-dependent secondary scan (Full MS / dd-MS2); Resolution: full scan 70,000 (FWHM @ m / z 200), data-dependent secondary scan 17,500; The spray voltage is 3kV; The capillary temperature is 350℃; The sheath gas and auxiliary gas are nitrogen, and the flow rate is optimized; Scan range: m / z 50~750; The target ion is the quasi-molecular ion peak [M+H] of tryptophan + , m / z 205.0972.

[0031] As an embodiment, the process of obtaining the content of tryptophan in sweet corn kernels includes qualitative and quantitative analysis and content calculation; the qualitative and quantitative analysis is the peak extraction, peak alignment, and signal-to-noise ratio calculation of the mass spectrometry data in the collected spectrum by using the software matched with the equipment for ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection, the qualitative analysis is performed by comparing the retention time and the accurate mass number of the primary parent ion, the quantitative analysis is performed by drawing a standard curve to perform external standard or internal standard method, and the concentration (C) of tryptophan in the sample solution is obtained; the accurate mass number of the primary parent ion is a mass deviation <5ppm.

[0032] As an embodiment, the content calculation is to calculate the content of tryptophan in sweet corn kernels according to the formula; the formula includes formula (1) and formula (2).

[0033] Sample molar content (nmol / mg) = (C×V×F) / M formula (1); C: the concentration of the sample solution calculated from the standard curve, unit nmol / mL, V: the final constant volume of the sample hydrolysate, unit mL, F: sample dilution factor, if the filtrate is further diluted before being put into the machine, F is the dilution factor; if not, F=1, M: sample weight, unit mg; Content in solid sample (μg / g) = (C×V×F×Mw) / M formula (2); Mw: the relative molecular mass of tryptophan, 204.23g / mol, Note: Equation (2) is derived from the molar content formula, converting nmol to μg and standardizing the mass unit. 1 nmol = 10 -9 mol, therefore the mass (μg) = (nmol × 10 -9 )×Mw×10 8 =nmol×Mw×10 -3 .

[0034] The method provided by this invention has the following technical effects: 1. Extremely high detection sensitivity: This method uses a high-resolution Orbitrap mass spectrometer as the detector, which has extremely high mass resolution and detection sensitivity. It can accurately identify and quantify trace amounts of tryptophan molecules in the sample. Even in complex matrices, it can effectively avoid background interference. The detection limit can reach the femtomolar (fmol) level, far exceeding traditional ultraviolet or fluorescence detection methods.

[0035] 2. Excellent quantitative accuracy: High-resolution mass spectrometry enables qualitative analysis using precise mass numbers, effectively distinguishing tryptophan from its isomers or interfering substances in the matrix, ensuring the reliability of qualitative results. Furthermore, when combined with isotope internal standard methods (if used) or external standard methods, accurate quantification can still be achieved in complex sample matrices, resulting in accurate, reliable, and highly reproducible results.

[0036] 3. High recovery rate and strong specificity in the pretreatment process: The alkaline hydrolysis pretreatment step can efficiently and thoroughly release tryptophan bound to macromolecules such as proteins or starches in sweet corn kernels, maximizing the recovery rate of the target analyte. Subsequent neutralization, centrifugation, and microfiltration steps can effectively remove impurities such as proteins, polysaccharides, and fats, resulting in significant purification and providing a pure sample for instrumental analysis, while reducing matrix inhibition effects.

[0037] 4. High throughput and fast analysis speed: Based on ultra-high performance liquid chromatography (UHPLC) technology, using its small-particle chromatographic column and ultra-high system pressure, the chromatographic separation of tryptophan can be completed in a very short time, usually within 10 minutes, which significantly improves the daily sample detection throughput and is more suitable for rapid screening and quantitative analysis of large batches of samples.

[0038] 5. Wide range of applications and high reliability: This method establishes a complete and standardized process from sample pretreatment to instrument detection and data calculation. The calculation formulas are rigorous, and results are provided in both "nmol / mg" and "μg / g" units, meeting the research and detection needs of different fields (such as metabolomics, nutrition, and breeding). This method is not only applicable to sweet corn, but its technical principles can also provide important reference for the detection of tryptophan in other grains or complex plant matrices.

[0039] Advantages 1 (high sensitivity) and 2 (high accuracy) primarily stem from the ultra-high resolution mass spectrometry (UHPLC-QE) technology employed in this invention. This is the core technological advancement. The ultra-high resolution and mass precision of the Orbitrap mass spectrometer are the fundamental reasons for its high sensitivity and high accuracy. It effectively eliminates interference from near-mass-number impurities in the matrix, which is unmatched by traditional low-resolution mass spectrometry or optical detectors.

[0040] Advantage 3 (High Recovery Rate, High Specificity): This mainly stems from the carefully designed and optimized sample pretreatment process, with alkaline hydrolysis being the key to achieving high recovery rates. Strong alkali and high-temperature long-term hydrolysis can efficiently break peptide bonds, ensuring that bound tryptophan is completely released. This is a prerequisite for ensuring that the detection results accurately reflect the total tryptophan content of the sample.

[0041] "Adjust pH to neutral, centrifuge at high speed, and filter through a 0.22μm micropore": This series of operations constitutes an effective purification process. Neutralization prevents damage to the instrument's chromatographic column from excessive acidity or alkalinity; high-speed centrifugation precipitates most insoluble impurities and denatured proteins; microfiltration removes fine particulate matter, greatly purifying the sample, reducing background noise and ion suppression effects in subsequent instrument analysis, and directly contributing to the method's specificity, stability, and accuracy.

[0042] Advantage 4 (High Throughput): This mainly stems from the use of ultra-high performance liquid chromatography (UHPLC). Compared to traditional HPLC, UHPLC typically increases analysis speed by 3 to 5 times, which is determined by its technical principles (smaller packing particles, higher column efficiency, and higher pressure).

[0043] Advantage 5 (Standardization and Wide Applicability): It originates from a complete and quantifiable technical system, especially the clear and scientific calculation formulas (sample molar content (nmol / mg) = (C×V×F) / M and content in solid sample (μg / g) = (C×V×F×Mw) / M). This formula comprehensively considers all variables in the entire sample preparation process (concentration, volume, dilution factor, mass, molecular weight), ensuring the accuracy and comparability of the final results, and elevating the method from "detection" to reliable "quantitative analysis".

[0044] In summary, the superior performance of this invention does not stem from the simple application of a single technology, but rather from the creative combination of highly efficient and specific pretreatment technology and a top-tier high-resolution detection platform. These two technologies complement each other, together forming a highly sensitive, accurate, stable, and reliable method for quantitative analysis of tryptophan. Through the complete, detailed, and parameter-defined technical solution provided by this invention, accurate, sensitive, and high-throughput detection of tryptophan content in sweet corn kernels can be achieved.

[0045] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Raw materials and reagents Test subject (raw material): The raw material is sweet corn kernels. The kernels are derived from dried kernels. The varieties cover all types of sweet corn (such as regular sweet, extra sweet, and fortified sweet).

[0047] Main chemical reagents: Sodium hydroxide (NaOH), analytical grade or higher, is used to prepare hydrolysate.

[0048] Hydrochloric acid (HCl), analytical grade or higher, is used for neutralization.

[0049] Chromatographically pure methanol, acetonitrile, and ultrapure water are used as the mobile phase for liquid chromatography and for sample dilution.

[0050] Tryptophan standard, purity ≥98%, used to prepare standard curves.

[0051] The internal standard, chlorophenylalanine, is used to improve the accuracy of quantification.

[0052] Example 1 Basic detection methods and single sample content determination 1. Experimental Objective To verify the feasibility and basic procedure of this method for detecting tryptophan content in kernels of a single sweet corn variety.

[0053] 2. Materials and Instruments Sample: "Shenke Sweet No. 6" sweet corn kernels, crushed and passed through an 80-mesh sieve.

[0054] Instruments: Ultra-high performance liquid chromatography-tandem high resolution Orbitrap mass spectrometer (UHPLC-QE, Thermo, USA), high-speed centrifuge, constant temperature hydrolysis furnace, pH meter, analytical balance.

[0055] Reagents: 4.5 mol / L sodium hydroxide (NaOH) solution, 0.1 mol / L hydrochloric acid (HCl) solution, and tryptophan standard.

[0056] 3. Test Methods 1) Sample pretreatment: Accurately weigh 50.0 mg (M) of crushed sweet corn kernels and place them in a 5 mL hydrolysis reaction flask.

[0057] 2) Alkaline hydrolysis: Add 1.0 mL (V_hydrolysis) of 4.5 mol / L NaOH solution to the reaction flask, tighten the cap, and place it in a constant temperature hydrolysis furnace at 105℃ for 20 h.

[0058] 3) Neutralization and volume adjustment: After cooling to room temperature, slowly add 0.1 mol / L HCl solution dropwise while shaking. Use pH paper to confirm the pH value of the solution is neutral (pH=7.0). Transfer all of this solution to a 10 mL (V) volumetric flask, wash the reaction flask several times with ultrapure water, and adjust the volume to the mark.

[0059] 4) Purification: Take an appropriate amount of solution from the volumetric flask into a centrifuge tube and centrifuge at 4℃ and 12000rpm for 10min.

[0060] 5) Filtration: Take the supernatant, filter it through a 0.22μm microporous membrane, and put it into a vial for testing.

[0061] 6) Analytical analysis: UHPLC-QE was used for analysis.

[0062] Chromatographic conditions: Column: Waters BEH C18 (50×2.1mm, 1.7μm); Mobile phase: Phase A was an ultrapure aqueous solution containing 0.1wt% formic acid, and Phase B was an acetonitrile solution containing 0.1wt% formic acid; Flow rate: 0.5mL / min; Column temperature: 55℃; Injection volume: 1μL; Elution gradient: 0 min Phase A / Phase B (95:5, v / v), 1.5 min Phase A / Phase B (90:10, v / v), 2.5 min Phase A / Phase B (90:10, v / v), 3 min Phase A / Phase B (95:5, v / v), 5.0 min Phase A / Phase B (95:5, v / v).

[0063] Mass spectrometry conditions: Ion source: Electrospray ionization (ESI); Ionization mode: Positive ion mode (ESI+); Scan mode: Full MS / dd-MS2; Resolution: Full scan 70000; Spray voltage: 3kV; Capillary temperature: 350℃; Sheath gas and auxiliary gas: Nitrogen, with optimized flow rate settings; Scan range: m / z 50~750; Target ion: Quasi-molecular ion peak of tryptophan [M+H] + , m / z 205.0972.

[0064] 7) Quantitative calculation: The sample concentration (C, nmol / mL) is obtained by using the instrument's software (such as TraceFinder) based on the tryptophan standard curve.

[0065] Qualitative analysis refers to determining "what" a metabolite is and its type by detecting its chemical properties or mass spectrometric characteristics. Retention time is an important method in column-based qualitative analysis. Under the same chromatographic conditions, differences in the properties and structures of components lead to different retention capacities on the analytical column, resulting in varying elution times. The chromatographic column first separates different components, then uses methods such as UV detectors, evaporative light detectors, differential detectors, electrochemical detectors, and conductivity detectors for detection, achieving qualitative and quantitative analysis of the target compound. Generally, peak time can identify the target compound, but it cannot completely eliminate interference from impurities. Mass spectrometry qualitative analysis mainly uses two methods: one is based on high-resolution mass spectrometry to determine the precise molecular mass of the compound, such as a molecular mass error <10 ppm (70,000 resolution), thus eliminating impurity interference; the other is to qualitatively identify the compound by comparing and identifying secondary fragment ions. In targeted quantitative experiments, the first method is generally preferred to obtain more quantitative data points.

[0066] Quantitative analysis refers to the analysis of the content of metabolites, mainly achieved by plotting a standard curve of standards. By plotting the concentration of the standards on the x-axis and the peak area on the y-axis, a mathematical relationship (linear, quadratic, logarithmic, etc.) between the target compound and its peak area can be obtained. Then, based on the peak area of ​​the corresponding compound in the unknown sample, its concentration can be calculated, achieving absolute quantitative analysis of the target compound.

[0067] The raw spectra obtained from UPLC-Q Exactive / MS analysis were preprocessed using TraceFinder software, including baseline filtering, peak identification, peak matching, retention time correction, and peak alignment, to produce a data matrix containing retention time, mass-to-charge ratio, and peak intensity. The total ion flow chromatogram (TIC) is a spectrum obtained by summing the intensities of all ions in the mass spectra at each time point and plotting them continuously. The horizontal axis represents retention time (Time, min), and the vertical axis represents the ion current intensity detected (Intensity).

[0068] Standard preparation: 1. Standard Product Information Table 1 Standard Product Information

[0069] Note: Standard products are mainly sourced from Sigma-Aldrich. 2. Standard concentration Accurately weigh an appropriate amount of standard, add 0.1 mol / L hydrochloric acid solution to prepare a 100 μmol / mL single standard stock solution, and further dilute it into solutions of different concentration gradients for instrumental testing.

[0070] Table 2 Information on the Concentration Gradient of Mixed Standards

[0071] Note: Due to various reasons such as sample concentration, the actual standard curve concentration gradient may differ from that shown here in order to ensure the accuracy of the results.

[0072] chromatogram of standard sample as shown Figure 1 As shown, note: the horizontal axis represents retention time (min), and the vertical axis represents ion current intensity (Intensity). Sample chromatogram as follows Figure 2 As shown, note: the horizontal axis represents retention time (min), and the vertical axis represents ion current intensity (Intensity).

[0073] Standard curve results are as follows Figure 3 As shown in the figure (where the horizontal axis represents the mixed standard gradient concentration in nM and the vertical axis represents the peak area), the standard curve was obtained by fitting with TraceFinder software, and the compound was absolutely quantified by external standard method.

[0074] Substitute into the formula to calculate: Sample molar content (nmol / mg) = (C×V×F) / M; Sample mass content (μg / g) = (C×V×F×Mw) / M (tryptophan Mw = 204.23g / mol).

[0075] 8) Test Results The software reads the sample concentration as C = 15.6 nmol / mL; Substitute into the formula to calculate: The molar content of tryptophan = (15.6 nmol / mL × 10 mL × 1) / 50.0 mg = 3.12 nmol / mg; Tryptophan content = (15.6 nmol / mL × 10 mL × 1 × 204.23 ng / nmol) / 50.0 mg = 637.2 μg / g (Note: 1 nmol = 1000 ng. Pay attention to unit conversion when calculating, or directly use the standard curve with a concentration of μg / mL.)

[0076] Conclusion: The tryptophan content in the kernels of "Shenke Sweet No. 6" sweet corn was successfully determined to be 637.2 μg / g using this method.

[0077] Example 2 Determination of tryptophan content in different sweet corn varieties 1. Experimental Objective The method's applicability is demonstrated by comparing the differences in tryptophan content in kernels from three different sweet corn varieties.

[0078] 2. Materials and Instruments Samples: All samples were pulverized and passed through an 80-mesh sieve.

[0079] 3. Test Methods The method was the same as in Example 1. Three biological replicates (n=3) were set up for each variety. The sample weight (M) was 50.0 mg for each variety, and the final volume was adjusted accordingly.

[0080] 3. Test Results As shown in Table 3.

[0081] Table 3. Results of tryptophan content determination in different sweet corn varieties

[0082] As shown in Table 3, the method provided by this invention can clearly distinguish the differences in tryptophan content among different sweet corn germplasms. Among them, wh240807023 has the highest tryptophan content (1212.7433 μg / g), and the standard deviation (SD) between replicates is small, indicating that the method has good reproducibility.

[0083] Comparative Example 1 Existing technical methods (acid hydrolysis combined with high performance liquid chromatography-fluorescence detection) 1. Method Overview In existing technologies, the detection of tryptophan content in complex matrices such as grains and feed typically employs acid hydrolysis combined with high-performance liquid chromatography-fluorescence detection (HPLC-FLD). This is a classic and widely reported method.

[0084] 2. Specific experimental procedures Sample pretreatment: Accurately weigh 50.0 mg (M) of “Shenke Sweet No. 6” sweet corn kernel powder, the same as in the embodiment of the present invention, and place it in a hydrolysis tube.

[0085] Acid hydrolysis: Add 5 mL of 6 mol / L hydrochloric acid (HCl) solution to the hydrolysis tube, purge with nitrogen to remove oxygen, and seal the tube. Place in a 110℃ constant temperature oven for 22 h for hydrolysis.

[0086] Neutralization and volume adjustment: After cooling, transfer all the hydrolysate to a 50 mL (V) volumetric flask, slowly neutralize it to pH 6.0 with 6 mol / L sodium hydroxide (NaOH) solution, and then adjust the volume to the mark with ultrapure water.

[0087] Purification and filtration: Take an appropriate amount of the diluted solution and centrifuge at 4℃ and 12000rpm for 10min. Filter the supernatant through a 0.22μm microporous membrane and place it in a vial for analysis.

[0088] Analytical analysis: High performance liquid chromatography-fluorescence detector (HPLC-FLD) was used.

[0089] Chromatographic conditions: Column: C18 column (4.6×250mm, 5μm); Column temperature: 30℃; Mobile phase: Sodium acetate buffer (pH=4.0)-methanol (95:5, v / v); Flow rate: 1.0mL / min; Injection volume: 10μL.

[0090] Fluorescence detection conditions: excitation wavelength: 280nm; emission wavelength: 360nm.

[0091] Quantitative calculation: The concentration (C, μg / mL) in the filtrate was obtained using the external standard method based on the tryptophan standard curve. Then, the content in the sample was calculated.

[0092] The tryptophan content in the sample (μg / g) = (C×V) / M.

[0093] 3. Test Results The software reads the sample concentration as C = 5.8 μg / mL; Substitute into the formula to calculate: Tryptophan content = (5.8μg / mL×50mL) / 50.0mg = 5.8μg / mg = 580μg / 100g = 5800μg / kg (or expressed as 0.58g / 100g).

[0094] 4. Analysis of problems existing in the technology Tryptophan is severely damaged: Tryptophan is extremely unstable under strong acid (6 mol / L HCl), high temperature, and aerobic conditions. Its indole ring readily reacts with aldehydes to form black polymers, resulting in extremely low recovery rates. This is the most fatal flaw of this method. The measured value (5800 μg / kg) is far lower than the result of Example 1 of this invention (637,200 μg / kg), not because the content is truly low, but because most of the tryptophan is destroyed during hydrolysis, resulting in completely distorted results.

[0095] The analysis is time-consuming and inefficient: after acid hydrolysis, a complex and time-consuming neutralization process is required (pH adjustment is very slow and difficult to control precisely), and the entire pretreatment process takes far longer than 24 hours. Chromatographic analysis uses conventional HPLC, and the single-needle run time is usually as long as 20-30 minutes, which is inefficient.

[0096] Poor anti-interference ability: Although the HPLC-FLD method has acceptable sensitivity, its chromatographic resolution is limited. The sweet corn matrix is ​​complex, and many degradation products and impurities produced by acid hydrolysis may have retention times close to tryptophan, leading to poor peak resolution or false positive / false negative results, affecting the accuracy of quantification.

[0097] Large reagent consumption: In order to achieve complete hydrolysis, the amount of acid used is usually large (5 mL in this example), and the amount of alkali solution required for subsequent neutralization is also large, resulting in a large final volume (50 mL). This leads to excessive dilution of the final test solution concentration, which places higher demands on the sensitivity of the detector.

[0098] The comparative examples clearly demonstrate that the acid hydrolysis method in existing technologies irreversibly destroys tryptophan, leading to severely distorted detection results and rendering it unusable for accurate quantification. In contrast, the alkaline hydrolysis combined with UHPLC-QEOrbitrap high-resolution mass spectrometry method employed in this invention fundamentally solves the technical problem of tryptophan destruction during pretreatment, while exhibiting significant advantages in high precision, high sensitivity, high analytical efficiency, and anti-interference capabilities. This invention provides an accurate, reliable, and efficient solution for detecting tryptophan content in sweet corn kernels, overcoming a long-standing technical bottleneck in this field.

[0099] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for quantitatively detecting tryptophan content in sweet corn kernels, characterized in that, Includes the following steps: Sweet corn kernels are mixed with an alkaline solution and subjected to alkaline hydrolysis. The extract obtained from the alkaline hydrolysis is then separated into solid and liquid components to obtain the test solution. The test solution was subjected to ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry to obtain the test spectrum; The tryptophan content in sweet corn kernels was obtained based on the spectrum to be tested and the predetermined standard curve. The standard curve is a linear relationship curve between the peak area and concentration of tryptophan.

2. The method according to claim 1, characterized in that, The alkaline solution is a sodium hydroxide solution; the concentration of the alkaline solution is 4~5 mol / L.

3. The method according to claim 1 or 2, characterized in that, The ratio of the mass of the sweet corn kernels to the volume of the alkaline solution is (45~55) mg: 1 mL.

4. The method according to claim 1 or 2, characterized in that, The alkaline hydrolysis temperature is 105±2℃.

5. The method according to claim 1 or 2, characterized in that, The alkaline hydrolysis time is 20 ± 0.5 h.

6. The method according to claim 1, characterized in that, The chromatographic conditions for ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection include: The chromatographic column is a HILIC column or a C18 column; The column temperature is 55℃; The mobile phase includes phase A and phase B. Phase A is an ultrapure aqueous solution containing 0.1 wt% formic acid, and phase B is an acetonitrile solution containing 0.1 wt% formic acid. The flow rate was 0.5 mL / min; The injection volume was 1 μL; Gradient elution program: 0 min A phase / B phase (95:5, v / v), 1.5 min A phase / B phase (90:10, v / v), 2.5 min A phase / B phase (90:10, v / v), 3 min A phase / B phase (95:5, v / v), 5.0 min A phase / B phase (95:5, v / v).

7. The method according to claim 1, characterized in that, The mass spectrometry conditions for ultra-high performance liquid chromatography-tandem high-resolution mass spectrometry detection include: Ion source: Electrospray ionization source; Scanning mode: Positive ion mode; Scanning method: Full scan or data-dependent secondary scan; Resolution: 70,000 for full scan, 17,500 for data-dependent secondary scan; The spray voltage is 3kV; The capillary temperature is 350℃; Nitrogen is used for both the sheath gas and the auxiliary gas. Scan range: m / z 50~750; The target ion is the quasi-molecular ion peak of tryptophan [M+H]. + , m / z 205.0972.

8. The method according to claim 1, characterized in that, Before mixing, the process further includes: crushing and sieving the sweet corn kernels to obtain sweet corn kernel powder.

9. The method according to claim 8, characterized in that, The mesh size of the sieve used for sieving is 40-80 mesh.

10. The method according to claim 1, characterized in that, The alkaline hydrolysis process further includes: cooling the extract obtained from the alkaline hydrolysis to room temperature, adding an acid solution to adjust the pH value of the solution to neutral; and making up the volume of the obtained neutral extract; wherein the neutral pH value is 7.0±0.5.

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