Method for determining γ-aminobutyric acid

Abstract

The present invention relates to a method for determining γ-aminobutyric acid. Specifically, the present invention provides a method for determining the content of γ-aminobutyric acid in a test sample, comprising the following steps: providing a liquid chromatograph, a charged aerosol detector and accessories thereof; providing liquid chromatography determination conditions; accurately weighing an appropriate amount of γ-aminobutyric acid standard, adding a solution solvent to dissolve, then adding a composite filler, vortex mixing and shaking, centrifuging, filtering, and using the filtrate as a standard solution to be measured; accurately weighing an appropriate amount of a test sample optionally containing γ-aminobutyric acid, and preparing a sample solution using the same preparation method of the standard solution; injecting the standard solution and the sample solution into the liquid chromatograph respectively, recording the chromatogram with a charged aerosol detector, and calculating the γ-aminobutyric acid in the test sample by an external standard method according to the peak area, to obtain. The method of the present invention has the advantages of simple operation, rapidity, strong specificity, and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of bioactive substances and relates to a method for detecting gamma-aminobutyric acid, including detecting foods containing gamma-aminobutyric acid, such as functional foods, detecting raw materials for preparing foods containing gamma-aminobutyric acid, etc. Background Art

[0002] γ-Aminobutyric acid (GABA) is a compound with the chemical formula C₄H₄NO₂ and a molecular weight of 103.1. It is an amino acid widely found in vertebrates, plants, and microorganisms. GABA appears as a white crystalline powder with no optical activity. Its melting point is 195-204°C (decomposition). It is miscible with water, slightly soluble in ethanol and acetone, and insoluble in benzene and ether. It decomposes by losing water to form pyrrolidone.

[0003] γ-Aminobutyric acid often exists in the form of zwitterions (negatively charged carboxyl groups and positively charged amino groups) in solution. Due to the electrostatic interaction between positive and negative charged groups, GABA can have both gaseous (folded) and solid (extended) molecular conformations in solution. The coexistence of multiple molecular conformations of GABA in solution enables it to bind to multiple receptor proteins and exert multiple important physiological functions.

[0004] Gamma-aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, possesses excellent water solubility and thermal stability. As a small, non-protein amino acid, GABA has been proven to be safe for consumption. GABA products, along with their functional foods and health supplements, have become a research hotspot in the food and pharmaceutical fields. Existing studies have shown that consuming a certain amount of GABA can improve sleep quality and lower blood pressure, among other physiological benefits.

[0005] In medicine, GABA has the effects of calming nerves, relieving anxiety, relieving pain, regulating blood pressure, eliminating fatigue, stabilizing blood sugar, improving brain activity, treating neurological diseases (anti-epileptic), auxiliary drugs for cancer treatment, increasing muscle growth, treating sequelae of stroke, promoting the ability of human sperm to penetrate eggs, promoting cell proliferation, and various types of hepatic coma.

[0006] While the European Food Safety Authority (EFSA) allows the addition of GABA to foods, with a dietary intake limit of 550 mg / day, its primary functional properties require rigorous human trials to substantiate its efficacy. Based on toxicology testing, the US Food and Drug Administration (FDA) has declared GABA safe for use in foods, including beverages, coffee, tea, and chewing gum. However, its addition to infant food, meat products, or products containing meat is prohibited. China's Ministry of Health, in its Announcement No. 12 of 2009, approved biologically produced GABA as a "new resource food," stipulating that its intake limit should not exceed 500 mg / day. Its use in beverages, cocoa products, chocolate and its beverages, candy, baked goods, and puffed foods is prohibited, but its addition to infant food is prohibited.

[0007] The chemical structure of GABA lacks significant absorption in the ultraviolet, visible, and fluorescent regions, making its detection quite difficult. Furthermore, the very low levels of GABA in typical functional foods and their ingredients, combined with the complex composition of these foods and their ingredients, further complicate its detection.

[0008] The main detection method found in the literature so far is thin layer chromatography [Falah, Fereshteh, Vasiee, Alireza, Tabatabaei Yazdi, Farideh, Moradi, Samira, Sabahi, Sahar. Optimizationof γ-aminobutyric acid (GABA) production by Lactobacillus spp. from agro-food waste[J]. Biomass Conversion and Biorefinery, 2022 (prepublish)], paper electrophoresis [Tang Chaqin, Zhang Ding, Chen Xuan, Xu Deliang, Li Xinghui, Xiao Runlin. Determination of γ-aminobutyric acid and glutamic acid in tea by paper electrophoresis], paper chromatography [Zhang Min, Xue Zhenglian, Yu Fei, Liu Yan, Wang Zhou. Determination of γ-aminobutyric acid in fermentation broth by paper chromatography-enzyme reader [J]. Food and Fermentation Industries, 2019, 45(15): 262-267. DOI: 10.13995 / j.cnki.11-1802 / ts.0208 44], Berthelot colorimetry [Tang Caiyun, Wang Tao, Tu Jie, Liu Guanhui, Li Peng, Zhao Jing. Comparison of colorimetry and HPLC for the determination of γ-aminobutyric acid in mulberry leaf tea [J]. Food Science, 2018, 39(24): 256-260], amino acid analyzer [Liu Xingyong, Fan Jianlin, Shi Jiang, Yin Benlin, Lin Tao, Li Qiwan, Wang Luxiang. Determination of γ-aminobutyric acid in maca and analysis of its content influence [J]. Modern Food Science], direct analysis in real time mass spectrometry (DART-MS) [ Li Wushuang, Lang Wencheng, Ma Lin, Li Long, Wu Guohua. Rapid determination of γ-aminobutyric acid content in mulberry leaves by direct analysis in real time mass spectrometry (DART-MS) [J]. Sericulture Science, 2018, 44(04): 567-572.DOI:10.13441 / j.cnki.cykx.2018.04.009], POD-UPLC detection method [Sun Xu. POD-UPLC detection of γ-aminobutyric acid content in solid sports nutrition products [J]. Food Science and Technology, 2020, 45(04): 286-290.DOI:10.13684 / j.cnki.spkj.2020.04.052], fluorescence detection method [Guo Xuguang, Yin Yuyun, Xu Xiaonan. Determination of γ-aminobutyric acid in health products by high performance liquid chromatography-post-column derivatization-fluorescence detection method [J]. Journal of Henan Preventive Medicine], UV detection method [Duan Zhihong, Huang Yongmei, Lv Yingnian, Liang Lizhong, Huang Yanxia, Ye Hua, Ye Shengquan. Determination of γ-aminobutyric acid in sea rice by HPLC-UV method with 2,4-dinitrofluorobenzene pre-column derivatization [J]. Food Industry Science and Technology, 2019, 40(02): 257-261+270.DOI:10.13386 / j.issn1002-0306.2019.02.044], high performance liquid chromatography tandem mass spectrometry [Zhan Xiuping, Li Jianyong, Chen Jianbo, et al. Improved QuEChERS pretreatment method combined with ultra performance liquid chromatography-tandem mass spectrometry for simultaneous determination of nine medium- and high-risk pesticide residues in typical leafy vegetables [J]. Pesticides, 2022, 61(11):833-839+849. DOI:10.16820 / j.nyzz.2022.2026]. Previously, no Quechers treatment method for γ-aminobutyric acid in functional foods and their raw materials was found.

[0009] The CAD detector is a charged aerosol detector, a new type of universal detector with high sensitivity and good reproducibility. Based on the principle of atomization detector, the eluent is atomized to form particles, which are dried in an evaporation tube and then collide with charged nitrogen, causing the surface of the analyte particles to be positively charged. The charge on the surface of the analyte particles is measured by an electrometer, so that the chromatographic peak area is related to the charge on the surface, which becomes the basis for determining the concentration of the substance. It can detect any non-volatile and semi-volatile substances. The CAD detector breaks through the limitations of other detector designs, achieves versatility, and provides consistent responsiveness for difficult-to-volatile compounds. The CAD detector has high sensitivity and low detection limits. When used in conjunction with a liquid chromatography separation system, it can accurately perform quantitative or semi-quantitative analysis of most non-volatile and semi-volatile organic compounds.

[0010] QuEChERS is a pretreatment method that has emerged and rapidly gained widespread adoption in recent years. Its name, a combination of the following words, stands for Quick, Easy, Cheap, Effective, Rugged, and Safe. Its principles are similar to those of high-performance liquid chromatography (HPLC) and solid-phase extraction (SPE), both utilizing the interaction of an adsorbent filler with impurities in the matrix to adsorb and purify them.

[0011] Therefore, those skilled in the art are eagerly looking forward to new methods for detecting γ-aminobutyric acid, such as a new method for detecting γ-aminobutyric acid in food, especially functional food and its raw materials using the QuEChERS model. Summary of the Invention

[0012] The present invention aims to provide a method for detecting γ-aminobutyric acid (GABA), particularly in foods containing GABA, such as functional foods, and in raw materials used to prepare GABA-containing foods. The present inventors have successfully developed a method for effectively detecting GABA in functional foods and their raw materials treated using the Quechers method using a charged aerosol detector. The present invention is based on this discovery.

[0013] Specifically, the first aspect of the present invention provides a method for determining the content of γ-aminobutyric acid in a test sample, which comprises the following steps:

[0014] (1) Provide liquid chromatograph, charged aerosol Detectors and their accessories;

[0015] (2) Provide liquid chromatography conditions, including column type, mobile phase and elution procedure, column temperature, charged aerosol Inspection Detector Drying tube atomization temperature, nitrogen pressure, data acquisition frequency, injection volume;

[0016] (3) Accurately weigh an appropriate amount of γ-aminobutyric acid standard, add the solution solvent to dissolve, then add the composite filler, vortex mix and shake, centrifuge, and remove the supernatant and filter it through a 0.22 μm filter membrane. The filtrate is used as the standard solution to be tested;

[0017] (4) Accurately weigh an appropriate amount of a test sample optionally containing γ-aminobutyric acid, dissolve it in a solution solvent, add the combined filler, vortex mix, centrifuge, and filter the supernatant through a microporous filter membrane. The filtrate is used as the sample solution to be tested;

[0018] (5) Inject the standard solution and sample solution into the liquid chromatograph respectively and use the charged atomizer The detector records the chromatogram, Calculate the concentration of the test sample by the external standard method according to the peak area γ-aminobutyric acid is obtained.

[0019] According to the method of the first aspect of the present invention, the test sample is labeled as a food containing γ-aminobutyric acid, such as a functional food or a γ-aminobutyric acid raw material.

[0020] According to the method of the first aspect of the present invention, the content of γ-aminobutyric acid in the test sample is 0-100%, and the preferred γ-aminobutyric acid content is greater than 0.1 mg / kg.

[0021] According to the method of the first aspect of the present invention, the liquid chromatograph, the charged aerosol Detector accessories include Nitrogen generator, air compressor, chromatography management software, high-speed refrigerated centrifuge, and / or chromatography columns.

[0022] According to the method of the first aspect of the present invention, the chromatographic column is an Agilent ZORBAX SB-Aq brand chromatographic column, for example, a chromatographic column with a specification of 4.6×250 mm and a diameter of 5 μm.

[0023] According to the method of the first aspect of the present invention, the liquid chromatography determination conditions are as follows: chromatographic column Agilent ZORBAX SB-Aq (4.6×250 mm, 5 μm); flow rate 0.8 ml / min; mobile phase A is acetonitrile, mobile phase B is 0.1% nonafluorovaleric acid, gradient elution: 0-5 min, 100% B; 5-25 min, 100%-85% B; 25-27 min, 85% B; 27-32 min, 85%-5% B; 32-32.5 min, 5%-100% B; column temperature 30°C; charged atomization Detector The drying tube nebulization temperature was 50°C, the nitrogen pressure was 60.1 psi, the acquisition frequency was 2 Hz, the filter was 3.6S, the power function was 1.2, and the injection volume was 5 μL.

[0024] According to the method of the first aspect of the present invention, the preparation solvent is water, or an aqueous solution containing 6% isopropyl alcohol.

[0025] According to the method of the first aspect of the present invention, the combined filler is a combination of C18 filler, PSA filler and NH2 filler.

[0026] According to the method of the first aspect of the present invention, the composite filler is a combination of C18 filler, PSA filler and NH2 filler in equal weight ratios.

[0027] According to the method of the first aspect of the present invention, the composite filler is added to the solution containing γ-aminobutyric acid, and the amount of the composite filler added per 5 ml of solution is 50-500 mg, such as 100-200 mg, for example 150 mg.

[0028] According to the method of the first aspect of the present invention, the centrifugation is performed at a speed of 5000-50000 r / min for 5-30 min, for example, at a speed of 5000-20000 r / min for 5-20 min, for example, at a speed of 6000-12000 r / min for 5-15 min, for example, at a speed of 9000 r / min for 10 min.

[0029] According to the method of the first aspect of the present invention, the microporous filter membrane is a 0.22 μm filter membrane.

[0030] According to the method of the first aspect of the present invention, the concentration of γ-aminobutyric acid in the standard solution to be tested and / or the sample solution to be tested is 0~1000µg / ml, for example, the concentration of γ-aminobutyric acid is 5~500µg / ml, for example, the concentration of γ-aminobutyric acid is 10~200µg / ml, for example, the concentration of γ-aminobutyric acid is 10~100µg / ml.

[0031] According to the method of the first aspect of the present invention, step (3) is to prepare the standard solution in the following manner: accurately weigh 10.0 mg of γ-aminobutyric acid standard and put it into a 100 ml volumetric flask, add an appropriate amount of liquid preparation solvent and ultrasonically vibrate until it is completely dissolved, add more liquid preparation solvent to make up to the scale, shake well, and use it as a standard preparation solution; accurately weigh 50 mg of C18 filler, 50 mg of PSA filler, and 50 mg of NH2 filler, place them in a centrifuge tube, add 5 ml of the above-mentioned standard preparation solution, vortex mix and shake well, centrifuge at 9000 r / min for 10 minutes, aspirate the supernatant and filter it through a 0.22 μm filter membrane, and use the filtrate as the standard solution to be tested.

[0032] According to the method of the first aspect of the present invention, step (3) is to prepare the sample solution as follows: accurately weigh an appropriate amount of a test sample (such as a functional food or raw material) expected to contain 10.0 mg of γ-aminobutyric acid into a 100 ml volumetric flask, add an appropriate amount of a preparation solvent, and ultrasonically vibrate until completely dissolved, add more preparation solvent to the scale, and shake well to serve as a sample preparation solution; accurately weigh 50 mg of C18 filler, 50 mg of PSA filler, and 50 mg of NH2 filler, place them in a centrifuge tube, add 5 ml of the above-mentioned sample preparation solution, vortex mix and shake well, centrifuge at 9000 r / min for 10 min, aspirate the supernatant, filter through a 0.22 μm filter membrane, and use the filtrate as the sample solution to be tested.

[0033] Furthermore, the second aspect of the present invention provides a method for determining the content of γ-aminobutyric acid in a sample using a composite filler, wherein the composite filler is a combination of C18 filler, PSA filler, and NH2 filler.

[0034] According to the use of the second aspect of the present invention, the composite filler is a combination of C18 filler, PSA filler and NH2 filler in equal weight ratios.

[0035] According to the use of the second aspect of the present invention, the method for determining the content of γ-aminobutyric acid in the test sample is as described in any scheme of the first aspect of the present invention.

[0036] As described in detail herein, the method of the present invention has the advantages of being simple to operate, rapid, highly specific, and highly sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Chromatogram of γ-aminobutyric acid reference solution.

[0038] Figure 2 : Chromatogram of γ-aminobutyric acid solid beverage sample solution.

[0039] Figure 3 : Chromatogram of GABA sample solution.

[0040] Figure 4 : Chromatogram of γ-aminobutyric acid sample solution. Implementation Method

[0041] The present application can be further described by the following examples, however, the scope of the present application is not limited to the following examples. Those skilled in the art will appreciate that, without departing from the spirit and scope of the present application, various changes and modifications can be made to the present application. The present application provides a general and / or specific description of the materials and test methods used in the test. Although many materials and operating methods used to achieve the purpose of the present application are well known in the art, the present application is still described in as much detail as possible. The following examples further illustrate the present application, rather than limiting the present application. The steps of the preparation or operation below are all for the purpose of illustration, and based on the comparability of each example, certain specific descriptions have been made. Those skilled in the art can fully summarize the products or methods involved in the present application based on existing knowledge. Some instruments, equipment, reagents, and reagents used in the present invention are commonly used in the art and can be easily obtained from commercial sources.

[0042] Example 1: Determination of γ-aminobutyric acid in functional foods and raw materials

[0043] 1. Instruments and reagents

[0044] Liquid chromatography (Vanquish, Thermo Fisher Scientific, USA), nitrogen generator (Corona1010, Peak Scientific), air compressor (Peak Scientific), charged aerosol Detector ( Chromatographic data were collected using a CAD detector (VH-D20-A), Chromeleon chromatography management software 7.2.10 ES, an ultrasonic cleaner SB-25-12D, a high-speed refrigerated centrifuge (ST 40R, Thermo Fisher Scientific, USA), a circulating water multi-purpose vacuum pump SHB-Ⅲ, and an Agilent ZORBAX SB-Aq (4.6 × 250 mm, 5-Micron PN880975-914) chromatographic column.

[0045] Nonafluoropentanoic acid (NFPA, Sigma-Aldrich), mass spectrometry-grade acetonitrile (Cleman), GABA reference substance (purity > 99%, Beijing Manhag Biotechnology Co., Ltd.), C18 packing (octadecylsilane bonded silica column packing, particle size 50 μm, pore size 60 Å, Shanghai Guchen), PSA packing (N-propylethylenediamine bonded packing, particle size 50 μm, pore size 60 Å, Amicrom), NH2 packing (NNH2 amino solid-phase extraction column packing, particle size 40 μm, pore size 60 Å, Lianqiao Bio) are all easily purchased through commercial channels.

[0046] C18 filler, namely octadecylsilane bonded silica filler, is a reverse phase C18 filler with silica gel as the matrix. It has a high bonding density and its specific surface area can reach 400m2 / g.

[0047] PSA filler, namely N-propylethylenediamine bonded filler, is a polar adsorbent with N-propylethylenediamine (-Si(CH2)3NH(CH2)2NH2) bonded to a high-purity silica gel matrix. Its retention mechanism is weak anion exchange, polar adsorption, chelation, etc. Since PSA has two amino groups (primary amine and secondary amine), and the pKa values of the two amino groups are higher, PSA has a higher exchange capacity.

[0048] NH2 filler, also known as amino solid phase extraction filler, is a polar adsorbent with a polar bonded phase aminopropyl (-Si(CH2)3NH2) bonded to a high-purity silica gel matrix. It has both hydrogen bonding and anion exchange mechanisms, and is end-capped. Its specific surface area can reach over 300m2 / g.

[0049] 2. Preparation of standard solution

[0050] Accurately weigh 10.0 mg of γ-aminobutyric acid standard into a 100 ml volumetric flask. Add an appropriate amount of dosing solvent and ultrasonically shake until completely dissolved. Add more dosing solvent to the mark and shake well to prepare the standard stock solution. Accurately weigh 50 mg of C18 filler, 50 mg of PSA filler, and 50 mg of NH2 filler into a centrifuge tube. Add 5 ml of the above standard stock solution, vortex to mix and shake well. Centrifuge at 9000 r / min for 10 min. Aspirate the supernatant and filter through a 0.22 μm filter membrane. The filtrate is used as the standard solution to be tested (100 μg / ml) for further testing. Unless otherwise specified, the dosing solvent refers to an aqueous solution containing 6% isopropanol.

[0051] 3. Sample solution preparation

[0052] Accurately weigh an appropriate amount of a functional food or raw material expected to contain 10.0 mg of γ-aminobutyric acid into a 100 ml volumetric flask, add an appropriate amount of the preparation solvent, and ultrasonically shake until completely dissolved. Add more preparation solvent to the mark and shake well to prepare the sample preparation solution. Accurately weigh 50 mg of C18 filler, 50 mg of PSA filler, and 50 mg of NH2 filler, place them in a centrifuge tube, add 5 ml of the above sample preparation solution, vortex mix and shake well, centrifuge at 9000 r / min for 10 minutes, aspirate the supernatant and filter it through a 0.22 μm filter membrane. The filtrate is used as the sample solution to be tested (100 μg / ml) for testing. In order to make the sample solution and the target substance content in the standard solution equivalent, and to make functional foods with ultra-low target substance content measurable, for functional foods with a γ-aminobutyric acid content of less than 1%, prepare the sample solution based on the amount of γ-aminobutyric acid expected to be 1%.

[0053] 4. Chromatographic conditions

[0054] The chromatographic conditions used in this example are as follows:

[0055] The chromatographic column was an Agilent ZORBAX SB-Aq (4.6×250 mm, 5-Micron, PN880975-914); the flow rate was 0.8 ml / min; the mobile phase A was acetonitrile (mass spectrometry grade), and the mobile phase B was 0.1% nonafluorovaleric acid; the gradient elution was: 0–5 min, 100% B; 5–25 min, 100%–85% B; 25–27 min, 85% B; 27–32 min, 85%–5% B; 32–32.5 min, 5%–100% B; the column temperature was 30°C; the flow rate was 0.8 ml / min; the mobile phase A was acetonitrile (mass spectrometry grade), and the mobile phase B was 0.1% nonafluorovaleric acid. Detector The drying tube atomization temperature was 50°C, the nitrogen pressure was 60.1 psi (1 psi ≈ 6.895 kPa), the acquisition frequency was 2 Hz, the filter was 3.6 s, the power function was 1.2, and 5 μL of each test solution was injected during the test.

[0056] Some methodological investigations of this embodiment are carried out below.

[0057] 5. Linear relationship

[0058] Accurately pipette 1 ml, 2 ml, 4 ml, 6 ml, and 8 ml of the standard solution obtained in step 2 above, add water to 10 ml, and prepare solutions of 10 μg / ml, 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, and 100 μg / ml, respectively. Sample analysis is performed and the peak area is recorded. The standard curve is plotted with the standard concentration (μg / ml) as the abscissa and the peak area (pA*min) as the ordinate to obtain a regression analysis equation between the concentrations of 10 μg / ml and 100 μg / ml (the regression equation obtained in this embodiment is y=0.007762x-0.01027), and the correlation coefficient is calculated, which should be greater than 0.999 (the correlation coefficient obtained in this embodiment is 0.999867, indicating a good linear relationship).

[0059] 6. Precision

[0060] The GABA standard solution obtained in step 2 above was diluted with water to 40 μg / ml, and the sample was injected six times continuously, with 5 μL injected each time. The precision was calculated based on the peak area (in this example, the average peak area was 0.3013 pA*min, and the RSD was 0.37%, indicating that this method has high measurement precision).

[0061] 7. Spike recovery rate

[0062] Accurately weigh 9 portions (10 mg) of the powder of Sample No. 2 in Table 2 and place them in 9 100 ml volumetric flasks. Dissolve and dilute to the mark with the preparation solvent and mix well to prepare the sample solution.

[0063] Accurately weigh an appropriate amount of γ-aminobutyric acid and dissolve it in a preparation solvent to prepare a solution with a concentration of 1 mg / ml as the standard solution;

[0064] Accurately pipette 2.5 ml of the sample solution into nine 10 ml volumetric flasks. In groups of three, accurately add 0.125 ml, 0.25 ml, and 0.375 ml of the standard solution, respectively. Dose each of the nine volumetric flasks to 10 ml with the preparation solvent to serve as the recovery preparatory solution for later use.

[0065] Accurately weigh 9 portions of 50 mg C18 filler, 50 mg PSA filler, and 50 mg NH2 filler, place them in centrifuge tubes, add 5 ml of the above recovery preparatory solution to each, vortex mix and shake, centrifuge at 9000 r / min for 10 min, aspirate the supernatant and filter through a 0.22 μm filter membrane. The filtrate is used as the test solution for the spiked recovery rate at low, medium, and high levels of the test sample for testing;

[0066] The above chromatographic conditions were used for determination according to the law, and the recovery rate and the corresponding RSD value were calculated. The results are shown in Table 1 below. The results of the spiked recovery rate indicate that the method of this embodiment has excellent accuracy.

[0067] Table 1: Recovery results of spiked γ-aminobutyric acid sample 4 (n=9)

[0068]

[0069] Note: The measured amount of 356.3 μg in the table refers to the actual amount of γ-aminobutyric acid measured in 10 ml of the recovered preparatory solution obtained from the "Low Concentration-1" test sample.

[0070] 8. Detection limit

[0071] Accurately measure 3.5 ml of the 0.1 mg / ml GABA standard solution obtained in step 2 and dilute to 100 ml. Prepare the test solution according to the method of this example. Under the selected chromatographic conditions, the GABA content corresponding to the peak height 3 times the signal-to-noise ratio is used as the detection limit, and the GABA content corresponding to the peak height 10 times the signal-to-noise ratio is used as the quantification limit, which are 0.035 mg / kg and 0.1 mg / kg, respectively.

[0072] 9. Stability

[0073] The sample solution at a concentration of -1, as shown in Table 2 for spiked recovery, was injected and analyzed at time points of 0, 6, 12, 20, and 24 hours. The peak area was measured (averaging six injections at each time point) and compared. The RSD for the five time points was 1.36%, indicating that the sample solution was stable over the 24 hours studied.

[0074] 10. Sample determination

[0075] Ten commercially available functional foods and GABA raw materials containing GABA were purchased as test samples. Detailed information is shown in Table 2. The GABA content (%) of these ten materials was determined using the conditions and procedures of Steps 1 to 4 of Example 1. The GABA content of the test samples was calculated using the peak area using the external standard method. The results are reported in Table 2.

[0076] Table 2: Information and test results of 10 GABA functional foods and GABA raw materials

[0077]

[0078] The above results show that the γ-aminobutyric acid content measured using the method of the present invention is consistent with the content in these materials.

[0079] The HPLC retention time of γ-aminobutyric acid is about 10.0 min. For some typical samples, the chromatogram of γ-aminobutyric acid reference solution is as follows: Figure 1The chromatogram of the γ-aminobutyric acid solid beverage sample solution of No. 1 in Table 2 is as shown in FIG. Figure 2 As shown in Table 2, the chromatogram of the GABA sample solution No. 2 is as follows Figure 3 As shown in Table 2, the chromatogram of the γ-aminobutyric acid sample solution No. 3 is as follows Figure 4 As shown, each figure shows that GABA has an excellent peak shape and no interfering peaks; each chromatogram provided is only recorded up to about 14 minutes, and the subsequent elution time is not recorded.

[0080] The above Example 1 established the method of the present invention for determining the content of γ-aminobutyric acid in functional foods containing γ-aminobutyric acid and their raw materials, and used this method to successfully determine some actual commercial samples. However, in fact, the inventors have found in some studies that in order to obtain excellent recovery rates, it is necessary to use a combination of a specific dosing solvent and a composite filler, otherwise the recovery rate will be significantly reduced. These studies are provided in the form of the following supplementary examples. Supplementary Example 1: With reference to the various operations and conditions of the above Example 1, the only difference is that the dosing solvent used is water without the addition of isopropanol. In the spiked recovery test, the recoveries measured for the three low-concentration samples were in the range of 90.3~92.1% (average 91.04%), the recoveries measured for the three medium-concentration samples were in the range of 88.8~91.4% (average 90.13%), and the recoveries measured for the three high-concentration samples were in the range of 89.6~93.2% (average 91.51%); Supplementary Example 2: With reference to the various operations and conditions of the above Example 1 , the only difference is that instead of using the combination of three fillers, an equal amount of 150 mg of C18 filler is used. In the spiked recovery test, the recoveries of the three low-concentration samples are in the range of 92.2-93.6% (mean 91.83%), the recoveries of the three medium-concentration samples are in the range of 90.5-91.9% (mean 91.43%), and the recoveries of the three high-concentration samples are in the range of 88.4-92.7% (mean 89.53%). Supplementary Example 3: Referring to the various operations and conditions of Example 1 above, the only difference is that instead of using the combination of three fillers, an equal amount of 150 mg of C18 filler is used. In the spiked recovery test, the recoveries of the three low-concentration samples are in the range of 92.2-93.6% (mean 91.83%), the recoveries of the three medium-concentration samples are in the range of 90.5-91.9% (mean 91.43%), and the recoveries of the three high-concentration samples are in the range of 88.4-92.7% (mean 89.53%). Combination, but instead of using the same amount of PSA filler 150 mg, in the spiked recovery test, the recoveries measured for 9 concentration samples were in the range of 86.4-90.3% (average 88.76%); Supplementary Example 4: Referring to the various operations and conditions of Example 1 above, the only difference is that instead of using the combination of three fillers, an equal amount of NH2 filler 150 mg was used. In the spiked recovery test, the recoveries measured for 9 concentration samples were in the range of 87.6-91.4% (average 89.23%); Supplementary Example 5: Referring to the various operations and conditions of Example 1 above, The conditions are as follows, the only difference being that instead of using a combination of three fillers, 75 mg each of C18 filler and PSA filler, or 75 mg each of C18 filler and NH2 filler, or 75 mg each of PSA filler and NH2 filler are used. In the spiked recovery test, the recoveries of all samples at different concentrations of the three filler combinations are within the range of 85.7-93.2% (average 88.47%). The results of these supplementary examples show that the recovery rate obtained by changing the dosing solvent is significantly reduced, and the combination of three fillers is also extremely important for obtaining a high recovery rate. The present invention provides a method for detecting γ-aminobutyric acid, and in particular, provides a method for detecting foods containing γ-aminobutyric acid, such as functional foods, and detecting raw materials for preparing foods containing γ-aminobutyric acid.The present inventors have successfully developed a method for detecting the content of gamma-aminobutyric acid (GABA) in functional foods and their raw materials using the Quechers method using a charged aerosol detector (CAD). This method demonstrates excellent methodological performance. For example, the method utilizes an Agilent ZORBAX SB-Aq (4.6×250mm, 5-Micron PN880975-914) column for separation, with a mobile phase consisting of a gradient elution of acetonitrile and 0.1% nonafluorovaleric acid at a flow rate of 0.8 mL / min. The CAD was operated at a nebulization temperature of 50°C and a nitrogen pressure of 60.1 psi (1 psi ≈ 6.895 kPa). Peak area was determined using an external standard. Results showed excellent linearity for GABA over a concentration range of 10.0 to 100.0 μg / mL, with a correlation coefficient exceeding 0.999. The detection limit was as low as 0.035 mg / kg and the quantification limit was as low as 0.1 mg / kg. Furthermore, the method exhibited excellent spike recovery and precision. These results show that the method of the present invention has the advantages of simple operation, rapidity, strong specificity and high sensitivity.

[0081] Any embodiment of any aspect of the present invention may be combined with other embodiments, as long as they do not conflict. In addition, any technical feature in any embodiment of any aspect of the present invention may be applicable to the same technical feature in other embodiments, as long as they do not conflict. The present invention is further described below.

[0082] All documents cited herein are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with those of the present invention, the present invention shall prevail. In addition, various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still intends to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the generally known meanings, the meanings expressed in the present invention shall prevail.

[0083] The above-described embodiments are merely preferred embodiments for the purpose of fully illustrating the present application, and the scope of protection of the present application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art based on the present application are within the scope of protection of the present application. The scope of protection of the present application shall be subject to the claims.

Claims

1. A method for determining the content of γ-aminobutyric acid in a test sample, comprising the following steps: (1) Provide liquid chromatograph, charged aerosol Detector and accessories thereof, the accessories including Nitrogen generator, air compressor, chromatography management software, high-speed refrigerated centrifuge and chromatography column, wherein the chromatography column is an Agilent ZORBAX SB-Aq brand chromatography column ； (2) Provide liquid chromatography conditions, including mobile phase and elution program, column temperature, charged aerosol Detector Drying tube atomization temperature, nitrogen pressure, data acquisition frequency and injection volume; the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is acetonitrile, mobile phase B is 0.1% nonafluorovaleric acid, the mobile phase elution program is the following gradient elution: 100% B during 0-5 min, 100%-85% B during 5-25 min, 85% B during 25-27 min, 85%-5% B during 27-32 min, 5%-100% B during 32-32.5 min, the flow rate of the mobile phase is 0.8 ml / min; column temperature is 30 ° C; charged spray Detector The drying tube nebulization temperature was 50°C, the nitrogen pressure was 60.1 psi, the acquisition frequency was 2 Hz, the filter was 3.6 s, the power function was 1.2, and the injection volume was 5 μL. (3) Accurately weigh an appropriate amount of γ-aminobutyric acid standard, dissolve it in the preparation solvent, add the composite filler, vortex mix, centrifuge, and filter the supernatant through a 0.22 μm filter membrane. The filtrate is used as the standard solution to be tested; the concentration of γ-aminobutyric acid in the standard solution to be tested is 0-1000 μg / ml; (4) Accurately weigh an appropriate amount of a test sample optionally containing γ-aminobutyric acid, dissolve it in a solution solvent, add a composite filler, vortex mix, centrifuge, and filter the supernatant through a microporous filter membrane. The filtrate is used as the sample solution to be tested; the test sample is a γ-aminobutyric acid raw material or a food labeled as containing γ-aminobutyric acid, and the content of γ-aminobutyric acid in the food is 0-100%; the concentration of γ-aminobutyric acid in the sample solution to be tested is 0-1000 μg / ml; (5) Inject the standard solution and sample solution into the liquid chromatograph respectively and use the charged atomizer The detector records the chromatogram, peak by peak The area of the test sample was calculated by external standard method. γ-aminobutyric acid, that is; In steps (3) and (4), the preparation solvent is an aqueous solution containing 6% isopropanol; the composite filler is a combination of C18 filler, PSA filler, and NH2 filler in an equal weight ratio; the composite filler is added to the solution containing γ-aminobutyric acid, and the amount of composite filler added per 5 ml of solution is 50-500 mg. The method according to claim 1 , wherein the food is a functional food.

3. The method according to claim 2, wherein the content of γ-aminobutyric acid in the food is greater than 0.1 mg / kg.

4. The method according to claim 1, wherein the chromatographic column is a chromatographic column with a specification of 4.6×250 mm and a diameter of 5 μm.

5. The method according to claim 1, wherein the composite filler is added to the solution containing γ-aminobutyric acid, and the amount of the composite filler added is 100-200 mg per 5 ml of solution.

6. The method according to claim 1, wherein the composite filler is added to the solution containing gamma-aminobutyric acid, and the amount of the composite filler added per 5 ml of solution is 150 mg.

7. The method according to claim 1, wherein the centrifugation is performed at a speed of 5000-50000 r / min for 5-30 min.

8. The method according to claim 1, wherein the centrifugation is performed at a speed of 5000-20000 r / min for 5-20 min.

9. The method according to claim 1, wherein the centrifugation is performed at a speed of 6000-12000 r / min for 5-15 min.

10. The method according to claim 1, wherein the centrifugation is performed at a speed of 9000 r / min for 10 min.

11. The method according to claim 1, wherein the concentration of γ-aminobutyric acid in the standard solution to be tested and / or the sample solution to be tested is 5-500 μg / ml.

12. The method according to claim 1, wherein the concentration of γ-aminobutyric acid in the standard solution to be tested and / or the sample solution to be tested is 10-200 μg / ml.

13. The method according to claim 1, wherein the concentration of γ-aminobutyric acid in the standard solution to be tested and / or the sample solution to be tested is 10-100 μg / ml.

14. The method according to claim 1, wherein step (3) is to prepare the standard solution as follows: accurately weigh 10.0 mg of γ-aminobutyric acid standard and place it in a 100 ml volumetric flask, add an appropriate amount of the preparation solvent, and ultrasonically vibrate until it is completely dissolved, add more preparation solvent to the scale, and shake well to prepare the standard solution; accurately weigh 50 mg of C18 filler, 50 mg of PSA filler, and 50 mg of NH2 filler, place them in a centrifuge tube, add 5 ml of the above standard solution, vortex mix and shake well, centrifuge at 9000 r / min for 10 min, aspirate the supernatant and filter it through a 0.22 μm filter membrane, and the filtrate is used as the standard solution to be tested.

15. The method according to claim 1, wherein step (4) is to prepare the sample solution as follows: accurately weigh an appropriate amount of a test sample expected to contain 10.0 mg of γ-aminobutyric acid and place it in a 100 ml volumetric flask, add an appropriate amount of a preparation solvent, and ultrasonically vibrate until completely dissolved, add more preparation solvent to the scale, and shake well to prepare the sample preparatory solution; accurately weigh 50 mg of C18 filler, 50 mg of PSA filler, and 50 mg of NH2 filler, place them in a centrifuge tube, add 5 ml of the above sample preparatory solution, vortex mix and shake well, centrifuge at 9000 r / min for 10 min, aspirate the supernatant and filter it through a 0.22 μm filter membrane, and use the filtrate as the sample solution to be tested.

16. Use of a composite filler in a method for determining the content of γ-aminobutyric acid in a sample, wherein the composite filler is a combination of a C18 filler, a PSA filler, and an NH2 filler in an equal weight ratio, and the method for determining the content of γ-aminobutyric acid in a sample is as described in any one of claims 1 to 15.

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