Method for determining free amino acids in peanuts

Abstract

The invention relates to the technical field of detection, and particularly discloses a method for determining free amino acids in peanuts. The method comprises the following steps: S1, extracting free amino acids in a to-be-detected peanut sample by adopting an extraction solvent to obtain an extracting solution, and diluting the extracting solution to obtain a to-be-detected solution containing the free amino acids; s2, analyzing the to-be-detected solution by adopting ultra-high performance liquid chromatography-tandem mass spectrometry, and calculating to obtain the variety and content of free amino acids in the to-be-detected peanut sample according to the peak area and the standard curve of the amino acid mixed standard working solution. According to the determination method provided by the invention, a treatment mode for extracting the free amino acid in the peanut sample is simple, convenient and efficient, derivatization treatment is not needed, and the problems of complicated operation, impurity interference and the like caused by derivatization are avoided.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a method for determining free amino acids in peanuts. Background Technology

[0002] Peanuts are an important legume crop widely cultivated globally. Their kernels are rich in protein, unsaturated fatty acids, vitamin E, resveratrol, and various minerals, making them a recognized nutrient-dense food. Thanks to their unique nutritional value and wide range of applications, peanuts and their products (such as peanut oil, peanut butter, and snack foods) hold a solid position in the global agricultural market. In recent years, the global peanut industry has continued to expand, demonstrating strong growth momentum. Simultaneously, peanut product forms are deeply expanding from traditional raw materials and primary processed products towards high-end, functional, and personalized directions. For example, innovative products such as high-oleic peanut oil, low-fat peanut protein powder, and peanut beverages rich in specific amino acids are emerging to meet consumers' increasingly diversified health needs. With the growing health awareness of global consumers and increasingly fierce market competition, product quality has become a key barrier for enterprises to build core competitiveness. Against this backdrop, accurate detection and comprehensive control of key nutrients in peanuts (such as amino acid composition, fatty acid ratio, and vitamin content) and potential risk factors (such as aflatoxin) are not only the cornerstone of ensuring product safety and fulfilling nutritional claims, but also a core link in promoting high-quality development and value upgrading of the entire industry. This also presents unprecedentedly higher requirements for the improvement of component analysis technology and quality standards throughout the entire peanut industry chain.

[0003] Precise analysis of free amino acids in peanuts is the scientific cornerstone connecting their agricultural attributes with their function as high-value-added health foods. These trace components directly define the product's nutritional value (such as the balance of essential amino acids) and sensory experience (such as flavor precursors). Simultaneously, as a sensitive "metabolic window," they provide crucial data support for crop traceability and the digital optimization of processing techniques. Faced with the current trend of the industry expanding towards functional and personalized products, traditional detection methods struggle to effectively overcome the interference of high-fat matrices, making precise analysis of trace free amino acids difficult. This has become a core obstacle restricting peanut quality science from moving towards a precision era.

[0004] Currently, common methods for detecting free amino acids, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), and capillary electrophoresis, all face certain limitations in practical applications. HPLC is widely used, but its separation efficiency is limited, with a single analysis taking over 60 minutes, and its sensitivity is insufficient for detecting trace amino acids in complex samples. GC requires pre-derivatization of amino acids, a cumbersome and time-consuming process that may lead to target analyte loss. Capillary electrophoresis suffers from poor reproducibility and weak resistance to matrix interference, making it unsuitable for the rapid, batch detection requirements of industrial settings. As the peanut industry upgrades towards standardization and high quality, the industry urgently needs to develop a new detection solution that combines high sensitivity, high selectivity, rapid response, and accuracy to overcome the shortcomings of current technologies. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a method for determining free amino acids in peanuts.

[0006] The inventive concept of this invention is as follows:

[0007] This invention employs ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), which combines the rapid separation of UPLC with the precise identification of tandem mass spectrometry, offering advantages such as fast analysis speed, high resolution, and strong anti-interference capabilities. Based on this technology, this invention provides a method for determining free amino acids in peanuts without derivatization. This method is simple to operate, highly efficient, and exhibits good sensitivity and accuracy. It effectively solves the interference problem caused by a high-fat matrix and can effectively separate and detect multiple free amino acids in peanuts, including alanine, phenylalanine, and aspartic acid. This enables rapid and accurate detection of free amino acids in peanuts, providing a scientific and reliable detection technology support for the evaluation of peanut nutritional value, quality grading, and optimization of processing techniques.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for determining free amino acids in peanuts includes the following steps: S1: Free amino acids in the peanut sample to be tested are extracted using an extraction solvent to obtain an extract. The extract is then diluted to obtain a test solution containing free amino acids. S2: The test solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. Based on the peak area and the standard curve of the amino acid mixed standard working solution, the types and contents of free amino acids in the peanut sample were calculated.

[0009] Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) combines the rapid separation of UPLC with the precise identification of tandem mass spectrometry, offering advantages such as fast analysis speed, high resolution, and strong anti-interference capabilities. Based on this technology, this invention provides a method for determining free amino acids in peanuts that requires no derivatization, is simple to operate, has high detection efficiency, and good sensitivity and accuracy, effectively solving the interference problem caused by a high-fat matrix. This invention can effectively separate and detect multiple free amino acids in peanuts, including alanine, phenylalanine, and aspartic acid, achieving rapid and accurate detection of free amino acids in peanuts. This provides scientific and reliable detection technology support for the evaluation of peanut nutritional value, quality grading, and optimization of processing techniques.

[0010] Preferably, in the method for determining free amino acids in peanuts, the free amino acids include at least one of alanine, arginine, asparagine, aspartic acid, cystine, γ-aminobutyric acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, theanine, threonine, tyrosine, and valine; more preferably, in the method for determining free amino acids in peanuts, the free amino acids include alanine, arginine, asparagine, aspartic acid, cystine, γ-aminobutyric acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, theanine, threonine, tyrosine, and valine.

[0011] Preferably, the extraction solvent is water; more preferably, the extraction solvent is ultrapure water. In this invention, using water as the extraction solvent effectively extracts free amino acids from peanuts while avoiding interference from matrix impurities such as sugars, acids, phenols, and fats. It also avoids the use of organic solvents, reducing detection costs while ensuring extraction efficiency and being environmentally friendly. Furthermore, using water as the extraction solvent avoids the damage to the instrument that can occur when using 10% sulfosalicylic acid as an extraction solvent.

[0012] Preferably, the extract is diluted with an acetonitrile aqueous solution at a mass percentage of 40% to 60%; more preferably, it is diluted with an acetonitrile aqueous solution at a mass percentage of 42% to 58%; even more preferably, it is diluted with an acetonitrile aqueous solution at a mass percentage of 45% to 55%; and even more preferably, it is diluted with an acetonitrile aqueous solution at a mass percentage of 48% to 52%. In this invention, diluting the extract with an acetonitrile aqueous solution reduces the solubility of free amino acids in the aqueous phase, and the diluted solution can reduce matrix interference.

[0013] Preferably, the mass-to-volume ratio of the peanut sample to the extraction solvent is 1:(10~50); more preferably, the mass-to-volume ratio of the peanut sample to the extraction solvent is 1:(15~45); even more preferably, the mass-to-volume ratio of the peanut sample to the extraction solvent is 1:(20~40); and even more preferably, the mass-to-volume ratio of the peanut sample to the extraction solvent is 1:(25~35). Wherein, the unit for the peanut sample is g, and the unit for the extraction solvent is mL.

[0014] Preferably, the peanut sample to be tested is in the form of peanut powder, which is obtained by crushing the peanut sample to be tested; more preferably, the particle size of the peanut powder is 0.2~0.5mm; even more preferably, the particle size of the peanut powder is 0.25~0.4mm; and even more preferably, the particle size of the peanut powder is 0.26~0.3mm.

[0015] Preferably, the extraction time is 15 min to 35 min; more preferably, the extraction time is 20 min to 35 min; even more preferably, the extraction time is 22 min to 30 min; and even more preferably, the extraction time is 24 min to 28 min.

[0016] In this invention, under the limited ratio of peanut sample to extraction solvent and extraction time, the extracted free amino acids can meet the analytical requirements, and excessive impurities will not be extracted, thus avoiding the impact of excessive impurities in the test solution on the accuracy of the detection.

[0017] Preferably, the extraction process is carried out under ultrasound. Ultrasound promotes the transfer of free amino acids from the solid phase to the aqueous phase.

[0018] Preferably, the ultrasonic power is 200W~600W; more preferably, the ultrasonic power is 250W~550W; even more preferably, the ultrasonic power is 300W~500W; and even more preferably, the ultrasonic power is 350W~450W.

[0019] Preferably, the temperature of the ultrasound is 20~30℃; more preferably, the temperature of the ultrasound is 22~28℃; and even more preferably, the temperature of the ultrasound is 22~26℃.

[0020] Preferably, the peanut powder is mixed with the extraction solvent before ultrasound; more preferably, the mixing method is vortex mixing; even more preferably, the vortex mixing time is 3-8 min; and even more preferably, the vortex mixing time is 4-6 min.

[0021] Preferably, the diluted extract in S1 is filtered through an organic filter membrane to obtain a test solution containing free amino acids; more preferably, the organic filter membrane is a polyvinylidene fluoride filter membrane; and even more preferably, the pore size of the polyvinylidene fluoride filter membrane is 0.22 μm.

[0022] Preferably, the free amino acids included in the amino acid mixed standard working solution in S2 are at least one of alanine, arginine, asparagine, aspartic acid, cystine, γ-aminobutyric acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, theanine, threonine, tyrosine, and valine.

[0023] Preferably, the amino acid mixed standard working solution is obtained by stepwise dilution of the amino acid mixed standard solution with acetonitrile aqueous solution.

[0024] Preferably, the mass concentration of amino acids in the amino acid mixed standard working solution in S2 is as follows: Alanine 11.1~223 ng / mL, Arginine 21.8~436 ng / mL, Asparagine 16.5~330 ng / mL, Aspartic acid 16.6~333 ng / mL, Cystine 30.0~601 ng / mL, Gamma-aminobutyric acid 12.9~258 ng / mL, Glutamine 18.3~365 ng / mL, Glutamic acid 18.4~368 ng / mL, Glycine 9.38~188 ng / mL, Histidine 19.4~388 ng / mL, Isoleucine 16. 4~328 ng / mL, leucine 16.4~328 ng / mL, lysine 18.3~365 ng / mL, methionine 18.7~373 ng / mL, phenylalanine 20.6~413 ng / mL, proline 14.4~288 ng / mL, serine 13.1~263 ng / mL, theanine 87.1~1742 ng / mL, threonine 14.9~298 ng / mL, tyrosine 22.6~453 ng / mL, valine 14.6~293 ng / mL.

[0025] Preferably, the detection conditions for ultra-high performance liquid chromatography separation in S2 include at least one of the following: (1) Column specifications: particle size 1.7μm~3.5μm, inner diameter 1.0mm~4.6mm, length 50mm~250mm; (2) Column temperature: 33℃~37℃; (3) Injection volume: 4.0 μL~6.0 μL; (4) Flow rate: 0.1 mL / min ~ 0.3 mL / min; (5) Mobile phase: Phase A is 0.1% ~ 0.3% formic acid aqueous solution, and Phase B is acetonitrile.

[0026] More preferably, the detection conditions for ultra-high performance liquid chromatography separation in S2 include at least one of the following: (1) Adopting ACQUITY UPLC BEH C 18 Chromatographic column, column specifications: 1.7μm, 2.1mm × 100mm; this column exhibits excellent retention and separation performance for free amino acids. (2) Column temperature: 35℃; (3) Injection volume: 5.0 μL; (4) Flow rate: 0.2 mL / min; (5) Mobile phase: Phase A is 0.2% formic acid aqueous solution, and Phase B is acetonitrile.

[0027] Preferably, the ultra-high performance liquid chromatography in S2 adopts an isocratic elution mode: the elution time is 0~5.0 min, and the elution process maintains 4%~6% B phase. More preferably, the ultra-high performance liquid chromatography in S2 adopts an isocratic elution mode: the elution time is 0~5.0 min, and the elution process maintains 5% B phase.

[0028] In this invention, the defined ultra-high performance liquid chromatography detection and elution conditions not only have short elution times, but also achieve effective separation of 21 free amino acids with symmetrical peak shapes.

[0029] Preferably, the mass spectrometry analysis conditions in S2 include at least one of the following: (1) Electrospray ionization (ESI) source, positive ion mode scanning; (2) Electrospray voltage: 2.5~5kV; (3) Ion source temperature: 400~600℃; (4) Atomizing gas pressure: 45~55psi; (5) Heating gas pressure: 45~55psi; (6) Air curtain pressure: 20~30psi; (7) Collision chamber inlet voltage: 8~15V; (8) Collision chamber outlet voltage: 8~15V; (9) The detection mode is multiple reaction monitoring (MRM); (10) Collision energy: ESI+: 20~100 V; ESI-: -20~-100 V; (11) De-clustering voltage: 4~5kV; (12) Solvent delay time: 2~5 min.

[0030] More preferably, the mass spectrometry analysis conditions in S2 include at least one of the following: (1) Electrospray ionization (ESI) source, positive ion mode scanning; (2) Electrospray voltage: 4.5kV; (3) Ion source temperature: 550℃; (4) Atomizing gas pressure: 50 psi; (5) Heating gas pressure: 50 psi; (6) Air curtain pressure: 25 psi; (7) Collision chamber inlet voltage: 10V; (8) Collision chamber outlet voltage: 10V; (9) The detection mode is multiple reaction monitoring (MRM); (10) Collision energy: ESI+: 20~100 V; ESI-: -20~-100 V; (11) De-clustering voltage: 4.5kV; (12) Solvent delay time: 3 min.

[0031] Preferably, the mass spectrometry analysis conditions in S2 also include quantitative ion pairs and qualitative ion pairs, and the parameters of the quantitative ion pairs and qualitative ion pairs are shown in Table 1.

[0032] In this invention, under the defined ultra-high performance liquid chromatography and mass spectrometry detection conditions, the standard curves obtained for different amino acids all showed correlation coefficients (r) greater than 0.99 within their respective mass concentration ranges, indicating good linearity. The detection method provided by this invention has high sensitivity and accuracy, strong anti-interference ability, low detection limit, and spiked recovery and precision that meet the detection requirements, enabling precise quantification of trace free amino acids in peanuts.

[0033] In some embodiments of the present invention, the detection limit for free amino acids is 0.05 µg / g to 1.5 µg / g; in some specific embodiments of the present invention, the detection limit for free amino acids is 0.16 µg / g to 1.41 µg / g.

[0034] In some embodiments of the present invention, the limit of quantification for free amino acids is 0.3 µg / g to 4.5 µg / g; in some specific embodiments of the present invention, the limit of quantification for free amino acids is 0.48 µg / g to 4.21 µg / g.

[0035] In some embodiments of the present invention, the relative standard deviation (RSD) of the method for determining free amino acids in peanuts is between 0.01 and 0.4; in some specific embodiments of the present invention, the relative standard deviation (RSD) of the method for determining free amino acids in peanuts is between 0.02 and 0.31.

[0036] In some embodiments of the present invention, the spiked recovery rate of the method for determining free amino acids in peanuts is 90% to 100%; in some specific embodiments of the present invention, the spiked recovery rate of the method for determining free amino acids in peanuts is 90.3% to 97.5%.

[0037] Preferably, the peanut sample includes at least one of the following: untreated raw sample, roasted peanut sample, fried peanut sample, and boiled peanut sample.

[0038] In some embodiments of the present invention, the peanut sample is a roasted peanut sample at a roasting temperature of 160~200℃; in some specific embodiments of the present invention, the peanut sample is a roasted peanut sample at a roasting temperature of 170~190℃; in some examples of the present invention, the peanut sample is a roasted peanut sample at a roasting temperature of 175~185℃.

[0039] In some embodiments of the present invention, the peanut sample is a roasted peanut sample, and the roasting time is 5-10 minutes; in some specific embodiments of the present invention, the peanut sample is a roasted peanut sample, and the roasting time is 6-9 minutes.

[0040] In some embodiments of the present invention, the peanut sample is a deep-fried peanut sample at a frying temperature of 120-160°C; in some specific embodiments of the present invention, the peanut sample is a deep-fried peanut sample at a frying temperature of 125-155°C; in some examples of the present invention, the peanut sample is a deep-fried peanut sample and the roasting temperature is 130-150°C.

[0041] In some embodiments of the present invention, the peanut sample is a deep-fried peanut sample, and the deep-frying time is 2-8 minutes; in some specific embodiments of the present invention, the deep-frying time is 3-5 minutes.

[0042] In some embodiments of the present invention, the peanut sample is a boiled peanut sample at a boiling temperature of 80-100°C; in some specific embodiments of the present invention, the peanut sample is a boiled peanut sample at a boiling temperature of 90-100°C; in some examples of the present invention, the peanut sample is a boiled peanut sample at a boiling temperature of 95-100°C.

[0043] In some embodiments of the present invention, the peanut sample is a boiled peanut sample, and the boiling time is 25-30 minutes; in some specific embodiments of the present invention, the peanut sample is a boiled peanut sample, and the boiling time is 26-28 minutes.

[0044] The beneficial effects of this invention are: 1. The determination method provided by the present invention is simple and efficient in extracting free amino acids from peanut samples, without the need for derivatization, thus avoiding the problems of complex operation and impurity interference caused by derivatization.

[0045] 2. This invention adopts ACQUITY UPLC BEH C 18 The chromatographic column, combined with optimized isocratic elution conditions, enables the effective separation of various free amino acids, with symmetrical peak shapes, high resolution, and excellent separation effect. Employing tandem mass spectrometry in multiple reaction monitoring mode, it exhibits strong anti-interference capabilities, low detection limits, and spiked recovery and precision that meet detection requirements. It can accurately quantify trace free amino acids in peanuts, providing scientific and reliable detection technology support for the evaluation of peanut nutritional value, quality grading, and optimization of processing technology. Attached Figure Description

[0046] Figure 1 Chromatograms of serine, asparagine, aspartic acid, threonine, glycine, glutamic acid, alanine, leucine, proline, and phenylalanine obtained from a mixed standard working solution of amino acids; Figure 2 Chromatograms of tyrosine, isoleucine, valine, lysine, histidine, glutamine, cystine, arginine, γ-aminobutyric acid, theanine, and methionine obtained from a mixed standard working solution of amino acids. Figure 3 Images of four peanut samples; Figure 4 Chromatograms for detecting serine in peanut samples treated with different methods; Figure 5 Chromatograms for detecting asparagine in peanut samples treated with different methods; Figure 6 Chromatograms for detecting aspartic acid in peanut samples treated with different methods; Figure 7 Chromatograms for detecting threonine in peanut samples treated with different methods; Figure 8 Chromatograms for detecting glycine in peanut samples treated with different methods; Figure 9 Chromatograms for detecting glutamic acid in peanut samples treated with different methods; Figure 10 Chromatograms for detecting alanine in peanut samples treated with different methods; Figure 11 Chromatograms for detecting leucine in peanut samples treated with different methods; Figure 12 Chromatograms for detecting proline in peanut samples treated with different methods; Figure 13 Chromatograms for detecting phenylalanine in peanut samples treated with different methods; Figure 14 Chromatograms for detecting tyrosine in peanut samples treated with different methods; Figure 15 Chromatograms for detecting isoleucine in peanut samples treated with different methods; Figure 16 Chromatograms for detecting valine in peanut samples treated with different methods. Detailed Implementation

[0047] To enable those skilled in the art to more clearly understand this application, the present invention will be further described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced even without certain specific details. In some embodiments, materials, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the present invention.

[0048] It should be noted that, unless otherwise specified in the examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0049] The experimental equipment and samples used in this invention are as follows: 1. Sample: Peanuts from the same batch produced in Ding'an City, Hainan Province, containing plump peanut kernels; 2. Ultra-high performance liquid chromatography-mass spectrometry system: Brand: Agilent; 3. Liquid chromatography column: ACQUITY UPLC BEH C 18 Chromatographic column, 1.7μm, 2.1mm×100mm, brand: Agilent; The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. The essential features and significant effects of the present invention can be seen from the following embodiments. The described embodiments are some, but not all, embodiments of the present invention, and therefore do not limit the present invention in any way. Any non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are within the protection scope of the present invention.

[0050] Example 1 A method for determining free amino acids in peanuts includes the following steps: 1. Pretreatment of peanut samples to be tested: Peel the peanut sample and crush the peanut kernels to obtain peanut powder sample; weigh 1.00g of peanut powder sample into a 50mL centrifuge tube, then add 30mL of ultrapure water as the extraction solvent (ultrapure water is economical and environmentally friendly and can effectively extract free amino acids), vortex mix for 5min to ensure that the peanut powder and the extraction solvent are fully in contact; place the centrifuge tube in a 400W ultrasonic extractor and ultrasonically extract for 25min at room temperature to promote the transfer of free amino acids from the peanut powder solid phase to the aqueous phase; after extraction, make up to 50mL with ultrapure water, vortex mix, and then centrifuge at 8000r / min for 8min to remove peanut powder and other impurities; then take 1.0mL of supernatant and transfer it to a 10mL volumetric flask, add 50% acetonitrile aqueous solution to dilute and make up to the mark, vortex mix, filter the solution through a 0.22μm polyvinylidene fluoride organic filter membrane, and collect the filtrate as the test solution.

[0051] 2. Prepare a mixed standard working solution of amino acids. Accurately pipette 1 mL of the mixed amino acid standard solution (all amino acid concentrations 1 mmol / L), dissolve it in 50% acetonitrile aqueous solution, and dilute to 10 mL to prepare a free amino acid mixed stock solution (all amino acid concentrations 0.1 mmol / L). Transfer 1 mL of this stock solution and serially dilute it with 50% acetonitrile aqueous solution to prepare six series of amino acid mixed standard working solutions with concentrations of 0.1 μmol / L, 0.5 μmol / L, 1.0 μmol / L, 1.5 μmol / L, 2.0 μmol / L, and 2.5 μmol / L.

[0052] 3. Set the ultra-high performance liquid chromatography conditions: (1) Adopting ACQUITY UPLC BEH C 18 Chromatographic column, column specifications: 1.7μm, 2.1mm × 100mm; (2) Column temperature: 35℃; (3) Injection volume: 5.0 μL; (4) Flow rate 0.2 mL / min; (5) Mobile phase: Phase A: 0.2% formic acid aqueous solution by mass; Phase B: acetonitrile; (6) Elution mode: Isocratic elution mode: elution time is 0~5.0 min, and 5% B phase is maintained during the elution process.

[0053] ACQUITY UPLC BEH C 18The chromatographic column has good retention and separation effects on polar compounds. The above-mentioned ultra-high performance liquid chromatography conditions can effectively separate 21 free amino acids with symmetrical peak shapes.

[0054] 4. Set mass spectrometry conditions: (1) Electrospray ionization (ESI) source, positive ion mode scanning; (2) Electrospray voltage: 4.5kV; (3) Ion source temperature: 550℃; (4) Atomizing gas pressure: 50 psi; (5) Heating gas pressure: 50 psi; (6) Air curtain pressure: 25 psi; (7) Collision chamber inlet voltage: 10V; (8) Collision chamber outlet voltage: 10V; (9) The detection mode is multiple reaction monitoring (MRM); (10) Collision energy: ESI+: 20~100 V; ESI-: -20~-100 V; (11) De-clustering voltage: 4.5kV; (12) Solvent delay time: 3 min.

[0055] The mass spectrometry analysis conditions for qualitative and quantitative ion pairs of each free amino acid are shown in Table 1.

[0056]

[0057] 5. Qualitative and quantitative analysis Qualitative analysis: using high performance liquid chromatography Tandem mass spectrometry (TMS) is used for qualitative determination of peanut samples. Under the same chromatographic and mass spectrometric conditions, the test solution exhibits chromatographic peaks for quantitative and qualitative ion pairs. The detection results of the test solution are compared with the retention time and characteristic ion pairs of the amino acid standard working solution. If the retention time deviation is ≤0.05 min and the relative abundance ratio of the characteristic ion pairs is consistent with that of the amino acid standard, the presence of the free amino acid in the sample can be determined.

[0058] Quantitative analysis: A standard curve was plotted with the concentration of each free amino acid standard working solution as the abscissa (X) and the corresponding quantitative ion pair chromatographic peak area as the ordinate (Y) to obtain the linear regression equation; the quantitative ion pair chromatographic peak area of ​​each free amino acid in the test solution was substituted into the regression equation to calculate the content of each free amino acid in the sample.

[0059] Methodological Validation 1. Standard curve, limit of detection, and limit of quantitation Accurately measure 1 mL of each concentration of the mixed standard working solution of amino acids from step 2, and detect them under the chromatographic and mass spectrometric conditions of steps 3 and 4 to obtain qualitative and quantitative ion-pair chromatograms. Plot a standard curve with the peak area of ​​the quantitative ion-pair chromatogram of each amino acid standard as the ordinate and the mass concentration as the abscissa to obtain a linear regression equation. Calculate the limit of detection (LOD) of each standard in the sample using a signal-to-noise ratio of 3 and the limit of quantitation (LOQ) using a signal-to-noise ratio of 10. The relevant data for the standard curve are shown in Table 2.

[0060]

[0061] As shown in Table 2, the correlation coefficients (r) of the 21 amino acid standards in the mixed standard working solution were all greater than 0.99 within their respective mass concentration ranges, indicating a good linear relationship.

[0062] The qualitative and quantitative ion-pair chromatograms of the mixed standard working solution of amino acids were obtained by detection, as shown in the figure below. Figures 1-2 As shown. Figure 1 and Figure 2 The left side of the spectrum for a pair of different amino acids is a qualitative ion-pair chromatogram, and the right side is a quantitative ion-pair chromatogram. Figure 1 The chromatograms are for serine (SER), asparagine (ASN), aspartic acid (ASP), threonine (THR), glycine (GLY), glutamic acid (GLU), alanine (ALA), leucine (LEU), proline (PRO), and phenylalanine (PHE). Figure 2 The spectrum shows the chromatograms of tyrosine (TYR), isoleucine (ILE), valine (VAL), lysine (LYS), histidine (HIS), glutamine (GLN), cysteine ​​(CYS), arginine (ARG), γ-aminobutyric acid (GABA), theanine (THE), and methionine (MET). Figure 1 and Figure 2 It can be seen that the determination method of the present invention can effectively separate 21 free amino acids with high separation degree.

[0063] 2. Repeatability test Take the same peanut sample (untreated raw sample, CK) and set up 3 replicate groups (CK-1, CK-2, CK-3). Determine the content according to the above steps and calculate the content by regression equation. The results are shown in Table 3.

[0064]

[0065] As shown in Table 3, there were no significant differences in the detection data of different replicate groups of the same peanut sample, and the RSD was between 0.02 and 0.31, indicating that the present invention has good repeatability.

[0066] 3. Spike recovery rate Select a peanut sample (untreated raw sample) and prepare the test solution according to step 1, dividing it into 3 groups. Add amino acid mixed standard working solutions at three concentration levels (low (1 µmol / mL), medium (10 µmol / mL), and high (25 µmol / mL)) to the test solutions respectively, and then perform the determination as described above. Each concentration level is measured in triplicate, and the spiked recovery rate and the relative standard deviation (RSD) of the measured values ​​are calculated.

[0067] The equation for calculating the spiked recovery rate is as follows: Recovery rate (%) = .

[0068] The test results are shown in Table 4.

[0069] Table 4 Spike Recovery Results

[0070] As shown in Table 4, the recovery rate of peanut spiked by the present invention is 90.3%~97.5%, and the relative standard deviation is ≤6.9%, which meets the requirements of mass spectrometry determination.

[0071] Example 2 The method for determining free amino acids in peanuts in Example 1 was used to test different peanut samples.

[0072] Peanuts were collected from Ding'an City, Hainan Province for this study. Peanuts from the same batch with plump kernels were selected. After peeling, the peanut kernels were air-dried, weighed, and randomly divided into four groups. One group served as the raw sample (CK) without any treatment. The other three groups were treated by roasting (T1), frying (T2), and boiling (T3), respectively. Each group had three replicates. Images of the four peanut samples are shown below. Figure 3 As shown.

[0073] All samples were whole and unpeeled before processing. Baking was performed at 180°C for 8 minutes; frying was performed at 140°C for 4 minutes; and boiling was performed in boiling water (100°C) for 27 minutes.

[0074] Peanut samples obtained by different treatment methods were analyzed using the detection method of Example 1. The chromatograms of different amino acids in peanut samples obtained by different treatment methods are shown below. Figures 4-16 As shown. Figures 4-16Chromatograms of serine (SER), asparagine (ASN), aspartic acid (ASP), threonine (THR), glycine (GLY), glutamic acid (GLU), alanine (ALA), leucine (LEU), proline (PRO), phenylalanine (PHE), tyrosine (TYR), isoleucine (ILE), and valine (VAL) in peanut samples (CK, T1, T2, T3) under different treatments are shown below. (For samples with the same treatment, the left side of a pair of chromatograms is the qualitative ion-pair chromatogram, and the right side is the quantitative ion-pair chromatogram.) Figure 4 For example, Figure 4 The images show the chromatograms of serine in peanut samples (CK, T1, T2, T3) after different treatments. For the CK sample, the left side is the qualitative ion-pair chromatogram, and the right side is the quantitative ion-pair chromatogram.

[0075] The test results are shown in Table 5.

[0076]

[0077] In Table 5, each value represents the mean ± standard deviation (n = 3); a, b, c, and d represent significant differences between the means (p < 0.05).

[0078] As shown in Table 5, the detection method in Example 1 can effectively detect the types and contents of free amino acids in peanut samples treated with different methods. Furthermore, the types of amino acids in peanuts vary depending on the peanut growing region and variety.

[0079] This invention also tested free amino acids in "Yueyou 271" and "Zhanyou 75" peanuts. Both types of peanuts showed the detection of 16 amino acids: aspartic acid, asparagine, serine, arginine, threonine, glycine, glutamic acid, tyrosine, valine, proline, alanine, leucine, isoleucine, tyrosine, phenylalanine, and methionine. Therefore, this invention can detect a wider variety of free amino acids in peanut samples containing more types of amino acids.

[0080] In summary, the detection method provided in this embodiment of the invention uses direct ultrasonic extraction of free amino acids from peanut samples with ultrapure water for determination. This eliminates the need for derivatization, avoiding the operational complexity and interference from impurities associated with derivatization. Furthermore, the extraction solvent is environmentally friendly and economical, and the extraction efficiency is high. The detection method in this embodiment of the invention uses ACQUITY UPLC BEH C. 18The chromatographic column, combined with optimized isocratic elution conditions, enables the effective separation of various free amino acids, with symmetrical peak shapes, high resolution, and excellent separation effect. Employing tandem mass spectrometry in multiple reaction monitoring mode, it exhibits strong anti-interference capabilities, low detection limits, and spiked recovery and precision that meet detection requirements. It can accurately quantify trace free amino acids in peanuts, providing scientific and reliable detection technology support for the evaluation of peanut nutritional value, quality grading, and optimization of processing technology.

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for determining free amino acids in peanuts, characterized by, The method comprises the following steps: S1: extracting free amino acids in a peanut sample to be tested by using an extraction solvent to obtain an extract, and diluting the extract to obtain a solution to be tested containing free amino acids; the extraction solvent is water; S2: analyzing the solution to be tested by using ultra-high performance liquid chromatography-tandem mass spectrometry, and calculating the types and contents of free amino acids in the peanut sample to be tested according to peak areas and a standard curve of an amino acid mixed standard working solution; The detection conditions of the ultra-high performance liquid chromatography in S2 at least include the following one: (1) column specifications: a particle size of 1.7 μm to 3.5 μm, an inner diameter of 1.0 mm to 4.6 mm, and a length of 50 mm to 250 mm; (2) column temperature: 33 ℃ to 37 ℃; (3) injection volume: 4.0 μL to 6.0 μL; (4) flow rate: 0.1 mL / min to 0.3 mL / min; (5) mobile phase: A phase is 0.1% to 0.3% formic acid aqueous solution, and B phase is acetonitrile; An isocratic elution mode is adopted: an elution time is 0 to 5.0 min, and a elution process is kept at 4% to 6% B phase; The mass spectrometry analysis conditions in S2 at least include the following one: (1) collision energy: ESI+: 20 V to 100 V; ESI-: -20 V to -100 V; (2) declustering voltage: 4 kV to 5 kV; (3) solvent delay time: 2 min to 5 min.

2. The method of determining free amino acids in peanuts according to claim 1, wherein, The extract is diluted by using an acetonitrile aqueous solution with a mass percentage of 40% to 60%.

3. The method of determining free amino acids in peanuts of claim 1, wherein, The mass-volume ratio of the peanut sample to be tested to the extraction solvent is 1 g to (10 to 50) mL.

4. The method of determining free amino acids in peanuts of claim 1, wherein, The peanut sample to be tested is in the form of peanut powder.

5. The method of determining free amino acids in peanuts of claim 1, wherein, The extraction process is performed under ultrasonic.

6. The method of determining free amino acids in peanuts of claim 1, wherein, The types of the free amino acids include at least one of alanine, arginine, asparagine, aspartic acid, cystine, γ-aminobutyric acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, theanine, threonine, tyrosine and valine.

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