Method for detecting theanine in milk powder

Abstract

The invention provides a method for detecting theanine in milk powder. The method comprises the following steps: pretreating an object to be detected; the method specifically comprises the following steps: adding a first volume of boiling distilled water to dissolve a to-be-detected substance to obtain a to-be-detected substance solution, placing the to-be-detected substance solution in an environment at a first temperature, leaching for a first time, filtering, cooling, fixing the volume to the first volume, and filtering with a filter membrane to obtain a sample test solution; sampling the sample test solution to obtain an on-machine test solution; and detecting the loading test solution through a high performance liquid chromatography-tandem mass spectrometer, and determining the content of theanine. By adopting the high performance liquid chromatography, the content of theanine in the milk powder can be detected, the requirements of customers with the detection requirements are met, and meanwhile, the detection method provided by the invention has the advantages of excellent repeatability and reproducibility, simplicity and convenience in operation, high speed, easiness in obtaining used equipment and high generalizability.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and in particular to a method for detecting theanine in milk powder. Background Art

[0002] Theanine is a special amino acid contained in tea leaves. Since its discovery, it has not been found in other plants except for a mushroom and Camellia sasanqua. Since its discovery, theanine has been widely studied as the main quality component of tea leaves. In recent years, with the deepening of research, the physiological health benefits of theanine have attracted widespread attention. Because theanine has the effects of lowering blood pressure, calming the nerves, improving sleep, and promoting brain function, theanine has been used as a raw material for health foods and medicines.

[0003] Theanine can be used as a natural sedative to treat anxiety and insomnia. At present, there are many milk powders with tea theanine added on sale. The matrix of the detection method for theanine is limited to tea. At present, there is no detection method for theanine content in milk powder as the matrix in China. The existing detection method cannot determine whether theanine content in milk powder meets the standard. Summary of the invention

[0004] In view of the above problems, the present application is proposed to provide a method for detecting theanine in milk powder that overcomes the above problems or at least partially solves the above problems, comprising:

[0005] A method for detecting theanine in milk powder comprises the steps of:

[0006] Pre-treating the object to be tested; specifically, adding a first volume of boiling distilled water to dissolve the object to be tested to obtain an object to be tested solution, extracting the object to be tested solution under a first temperature environment for a first time, filtering and cooling, fixing the volume to the first volume, and filtering with a filter membrane to obtain a sample test solution;

[0007] Sampling the sample test solution to obtain an on-machine test solution;

[0008] The test solution is tested by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine.

[0009] Furthermore, the step of detecting the on-machine test solution by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine uses a chromatographic column model of Agilent AQ-SB 3um2.1*100mm.

[0010] Furthermore, the first volume is 100 ml / g.

[0011] Furthermore, the first temperature is 100°C.

[0012] Furthermore, the first duration is 30-40 minutes.

[0013] Furthermore, the membrane filtration is performed by filtering through a 0.45 μm aqueous phase membrane.

[0014] Furthermore, the step of detecting the content of theanine by high performance liquid chromatography tandem mass spectrometry is as follows:

[0015] The mass spectrometry conditions were as follows: parent ion 175 m / z; product ions 130 m / z, 158 m / z; cone voltage 30 V; collision voltage 25 V.

[0016] Furthermore, in the step of detecting the on-machine test solution by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine, the mobile phase is 0.1% formic acid aqueous solution and methanol solution.

[0017] Furthermore, in the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the elution gradient is:

[0018] 0-1 minute, formic acid aqueous solution 98%, methanol solution 2%; 1-2.5 minutes, formic acid aqueous solution 98%-5%, methanol solution 2%-98%; 2.5-4.0 minutes, formic acid aqueous solution 5%, formic acid acetonitrile solution 95%; 4.0-7 minutes, formic acid aqueous solution 5%-98%, formic acid acetonitrile solution 95%-2%.

[0019] Furthermore, it also includes performing a spiked recovery test on the analyte to determine a spiked recovery rate, and determining a test result based on the spiked recovery rate.

[0020] This application has the following advantages:

[0021] In the embodiments of the present application, there is no detection scheme for theanine content in milk powder in the prior art, and the blank of theanine content detection in matrices such as milk powder in the prior art is filled. The present application provides a detection method for theanine in milk powder, including the steps of: pre-treating the test object; specifically, adding a first volume of boiling distilled water to dissolve the test object to obtain a test object solution, placing the test object solution in a first temperature environment for extraction for a first time, filtering and cooling, fixing the volume to the first volume, and filtering with a filter membrane to obtain a sample test solution; sampling the sample test solution to obtain a machine test solution; detecting the machine test solution by a high performance liquid chromatography tandem mass spectrometer to determine the content of theanine. By adopting high performance liquid chromatography, the theanine content in milk powder can be detected, which meets the requirements of customers with this detection demand. At the same time, the detection method proposed in the present application has excellent repeatability and reproducibility, is easy to operate, fast, and the equipment used is easy to obtain, and is highly scalable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the description of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0023] Figure 1 This is a flow chart of the steps of a method for detecting theanine in milk powder provided in one embodiment of the present application;

[0024] Figure 2 This is an operation flow chart of a method for detecting theanine in milk powder provided in one embodiment of the present application;

[0025] Figure 3 This is a standard curve diagram of a method for detecting theanine in milk powder provided in one embodiment of the present application;

[0026] Figure 4 This is a chromatogram of a standard curve of a method for detecting theanine in milk powder provided in an embodiment of the present application at a concentration point of 0.1 mg / L;

[0027] Figure 5 This is a chromatogram of a standard curve of a method for detecting theanine in milk powder provided in an embodiment of the present application at a concentration point of 0.25 mg / L;

[0028] Figure 6 It is a chromatogram of a standard curve of a method for detecting theanine in milk powder provided in an embodiment of the present application at a concentration point of 0.50 mg / L;

[0029] Figure 7 This is a chromatogram of a standard curve of a method for detecting theanine in milk powder provided in an embodiment of the present application at a concentration point of 10 mg / L;

[0030] Figure 8 It is a graph of a method for detecting theanine in milk powder provided in an embodiment of the present application for determining the content of theanine in a spiked milk powder sample;

[0031] Fig. 9 The present invention provides a method for detecting theanine in milk powder provided in one embodiment of the present application, which is a graph showing the content of theanine in a milk powder sample. DETAILED DESCRIPTION

[0032] In order to make the objects, features and advantages of the present application more obvious and understandable, the present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0033] The inventors analyzed the prior art and found that theanine is an additive in milk powder. The existing detection schemes all detect the content of theanine in the tea matrix, which cannot determine the content of theanine in milk powder and cannot clarify whether the additives in milk powder comply with relevant regulations.

[0034] Reference Figure 1-2 , showing a step flow chart and an operation flow chart of a method for detecting theanine in milk powder provided in an embodiment of the present application:

[0035] The method comprises:

[0036] S110, pre-treating the test object; specifically, adding a first volume of boiling distilled water to dissolve the test object to obtain a test object solution, extracting the test object solution under a first temperature environment for a first time, filtering and cooling, fixing the volume to the first volume, and filtering with a filter membrane to obtain a sample test solution;

[0037] S120, sampling the sample test solution to obtain an on-machine test solution;

[0038] S130, testing the test solution by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine.

[0039] In the embodiments of the present application, there is no detection scheme for theanine content in milk powder in the prior art, and the blank of theanine content detection in matrices such as milk powder in the prior art is filled. The present application provides a detection method for theanine in milk powder, including the steps of: pre-treating the test object; specifically, adding a first volume of boiling distilled water to dissolve the test object to obtain a test object solution, placing the test object solution in a first temperature environment for extraction for a first time, filtering and cooling, fixing the volume to the first volume, and filtering with a filter membrane to obtain a sample test solution; sampling the sample test solution to obtain a machine test solution; detecting the machine test solution by a high performance liquid chromatography tandem mass spectrometer to determine the content of theanine. By adopting high performance liquid chromatography, the theanine content in milk powder can be detected, which meets the requirements of customers with this detection demand. At the same time, the detection method proposed in the present application has excellent repeatability and reproducibility, is easy to operate, fast, and the equipment used is easy to obtain, and is highly scalable.

[0040] Next, a method for detecting theanine in milk powder in this exemplary embodiment will be further described.

[0041] As described in step S110, the object to be tested is pre-treated; specifically, a first volume of boiling distilled water is added to dissolve the object to be tested to obtain an object to be tested solution, the object to be tested solution is placed in an environment of a first temperature for a first time, filtered and cooled, fixed to the first volume, and filtered through a filter membrane to obtain a sample test solution.

[0042] It should be noted that after filtering and cooling, 1 ml of sample can be taken, centrifuged at 13000 r / min for 10 min, and then subjected to liquid chromatography analysis.

[0043] In one embodiment of the present application, the first volume is 100 ml / g.

[0044] In an embodiment of the present application, the first temperature is 100° C.

[0045] In one embodiment of the present application, the first duration is 30-40 minutes.

[0046] In one embodiment of the present application, the membrane filtration is filtered through a 0.45 μm aqueous phase membrane.

[0047] In a specific implementation, 1 g of the milk powder sample is weighed and placed in a 200 ml beaker, 100 ml of boiling distilled water is added, and the mixture is stirred to dissolve. The beaker is placed in a constant temperature water bath at 100° C. and extracted for 30 min. The filtrate is filtered and transferred to a 100 ml volumetric flask. After cooling, the filtrate is fixed to volume, mixed, and then filtered with a 0.45 μm aqueous phase filter membrane to obtain a test solution for the machine.

[0048] As described in step S120, the sample test solution is sampled to obtain the machine test solution.

[0049] As described in step S130, the test solution is tested by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine.

[0050] In one embodiment of the present invention, the specific process of "detecting the on-machine test solution by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine" in step S130 can be further explained in combination with the following description.

[0051] The instrument parameters are:

[0052] Instrument: HPLC / MSMS

[0053] Chromatographic column: Agilent AQ-SB 3um 2.1*100mm, or equivalent.

[0054] Mobile phase: A is 0.1% formic acid aqueous solution, B is methanol solution.

[0055] Flow rate: 0.60mL / min.

[0056] Column temperature: 30℃.

[0057] Injection volume: 2 μL.

[0058] The flow rate gradient elution program is shown in Table 1:

[0059]

[0060]

[0061] Table 1

[0062] Mass spectrometry reference conditions:

[0063] Ion source: ESI;

[0064] Detection method: multiple reaction monitoring (MRM);

[0065] Scanning mode: positive ion mode;

[0066] Ion source temperature: 120°C;

[0067] Desolventizing gas flow rate: 1000L / H;

[0068] Desolventization temperature: 300℃;

[0069] Capillary voltage: 3.0kV;

[0070] Collision gas flow: 0.15L / H;

[0071] Cone hole gas flow: 150L / H;

[0072] Dwell time: 0.02S.

[0073] The ionization mode, parent ion, daughter ion, declustering voltage, and collision energy are shown in Table 2:

[0074]

[0075] Table 2

[0076] refer to Figure 3-7 , which is the chromatogram of each concentration point of theanine standard curve and the linearity of theanine measured by high performance liquid chromatography tandem mass spectrometry using the treatment scheme of the present application, the linear range of the standard curve is: 0.1-10 mg / L.

[0077] Fig. 9The spectral data of theanine measured by HPLC-MS / MS for a certain milk powder sample. Figure 8 The spectrum data of theanine measured by HPLC-MS / MS after adding the spiked milk powder sample is shown in the figure. The method can achieve high efficiency separation and stable baseline. The recovery rate is within the satisfactory range of 60%-120% through the spiked recovery test of the sample. It has good repeatability and reproducibility and accurate quantification.

[0078] This application can detect theanine in milk powder by pre-treating the sample and testing the sample by high performance liquid chromatography tandem mass spectrometry under set conditions, filling the market gap and meeting the requirements of customers with such testing needs. The high performance liquid chromatography method has excellent repeatability and reproducibility, is easy to operate, fast, and the equipment used is easy to obtain, which is highly scalable.

[0079] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0080] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or terminal device including the elements.

[0081] The above is a detailed introduction to the detection method of theanine in milk powder provided by the present application. Specific examples are used in this article to illustrate the principle and implementation method of the present application. The description of the above embodiments is only used to help understand the method and its core idea of ​​the present application. At the same time, for those skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A method for detecting theanine in milk powder, characterized in that: Includes steps: Pre-treating the object to be tested; specifically, adding a first volume of boiling distilled water to dissolve the object to be tested to obtain an object to be tested solution, extracting the object to be tested solution under a first temperature environment for a first time, filtering and cooling, fixing the volume to the first volume, and filtering with a filter membrane to obtain a sample test solution; Sampling the sample test solution to obtain an on-machine test solution; The test solution is tested by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine.

2. The detection method according to claim 1, characterized in that: The step of detecting the on-machine test solution by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine uses a chromatographic column model of Agilent AQ-SB 3um 2.1*100mm.

3. The detection method according to claim 1, characterized in that: The first volume is 100 ml / g.

4. The detection method according to claim 1, characterized in that: The first temperature is 100°C.

5. The detection method according to claim 1, characterized in that: The first duration is 30-40 minutes.

6. The detection method according to claim 1, characterized in that: The membrane filtration was carried out through a 0.45 μm aqueous phase filter.

7. The detection method according to claim 1, characterized in that: The step of testing the test solution by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine, The mass spectrometry conditions were as follows: parent ion 175 m / z; product ions 130 m / z, 158 m / z; cone voltage 30 V; collision voltage 25 V.

8. The detection method according to claim 1, characterized in that: In the step of detecting the on-machine test solution by high performance liquid chromatography tandem mass spectrometry to determine the content of theanine, the mobile phase is 0.1% formic acid aqueous solution and methanol solution.

9. The detection method according to claim 8, characterized in that: In the step of detecting the test solution on the machine by liquid chromatography-tandem mass spectrometry, the elution gradient is: 0-1 minute, formic acid aqueous solution 98%, methanol solution 2%; 1-2.5 minutes, formic acid aqueous solution 98%-5%, methanol solution 2%-98%; 2.5-4.0 minutes, formic acid aqueous solution 5%, formic acid acetonitrile solution 95%; 4.0-7 minutes, formic acid aqueous solution 5%-98%, formic acid acetonitrile solution 95%-2%.

10. The detection method according to claim 1, characterized in that: The method further includes performing a spike recovery test on the analyte to determine a spike recovery rate, and determining a test result based on the spike recovery rate.

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