Method for measuring content of organic acid in yellow tea by high performance liquid chromatography

Abstract

The invention discloses a method for determining the content of organic acids in yellow tea by high performance liquid chromatography, which comprises the following steps: carrying out gradient elution by using a C18 liquid chromatographic column AcclaimTM 120 at the column temperature of 30 DEG C and using methanol as a mobile phase A and 0.1 mol / L KH2PO4 as a mobile phase B; wherein the gradient elution procedure is as follows: 1% A is carried out in 0-12min; 12-15 min, 1%-50% of A is added; 15-25 min, 50% of A is determined; 25-27 min, 50%-1% of A is obtained; 27-30 min, 1% of A is set; a diode array detector is used for detecting the sample, the detection wavelength is 210 nm, the sample size is 30 [mu] L, and the initial flow velocity is 1.2 mL / min; the time is 0-12 minutes, and the flow velocity is 1.2-1.0 mL / min; the time is 12-30 minutes, and the flow velocity is 1.0 mL / min. According to the method, during gradient elution, the separation effect of all organic acids is good, components which are difficult to separate can be measured, the detection cost is saved, the detection efficiency is improved, and the method has important significance on quality research of yellow tea and establishment of a detection method standard of the organic acids in the tea leaves.

Description

Technical Field

[0001] The invention relates to the technical field of tea component detection, and in particular to a method for determining the content of organic acids in yellow tea by high performance liquid chromatography. Background Art

[0002] The different quality and flavor of tea are determined by the types, contents and different proportions of active ingredients contained in it. Organic acid is one of the important active ingredients in tea, accounting for about 3% of the total dry matter of tea. It is the precursor or intermediate product of some flavor and aroma components in tea, affecting the formation of tea quality, and has health benefits such as digestion and stomach conditioning, anti-oxidation, and antibacterial. Yellow tea is one of the six major types of tea in my country. It belongs to lightly fermented tea and is a unique type of tea in my country. It has a long history of drinking and a unique yellowing processing technology. It not only has a unique flavor, but also has significant health benefits. However, there is still a lack of systematic research on the components and contents of organic acids in tea, especially the research on organic acids in yellow tea has almost no reports. The main methods for determining organic acids in tea are potentiometric titration, capillary electrophoresis, ion chromatography, gas chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry and high performance liquid chromatography. Potentiometric titration uses the change in potential to determine the titration endpoint and is suitable for the determination of total organic acids. Capillary electrophoresis uses a quartz capillary as a separation channel and a high-voltage DC electric field as a driving force to achieve separation based on the differences in the components in the sample. It has the advantages of being simple, efficient, rapid, and low in reagent and sample consumption, but has poor reproducibility. Ion chromatography has high requirements for sample purification, and tea contains complex ingredients. It is particularly difficult to separate organic acids with similar structures, such as malic acid and succinic acid, using ion chromatography. In addition, when a large number of inorganic anions and organic acids coexist in the sample, the difficulty of detecting low molecular weight organic acids will increase. Gas chromatography is suitable for the determination of low molecular weight organic acids, but requires high-quality, low-volatile organic acids. Derivatization treatment is cumbersome; gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry have high separation of chromatography and high sensitivity of mass spectrometry, high accuracy and good specificity, but the instruments are expensive; high performance liquid chromatography has the characteristics of simple operation, high sensitivity and good separation effect, but it has high requirements for sample pretreatment, is easily affected by impurities, and is also greatly affected by pH and temperature; organic acids are easily ionized in water, so acidic mobile phases with low pH values ​​are generally used to inhibit the dissociation of organic acids, but long-term use of acidic mobile phases will affect the performance and service life of chromatographic columns; organic acids have basically no characteristic absorption in the ultraviolet band, so they are often selected for detection near 210nm, but the sensitivity of this band is relatively poor. The existing national standard GB 5009.157-2016 "National Food Safety Standard Determination of Organic Acids in Food" stipulates the method of using high performance liquid chromatography to determine tartaric acid, lactic acid, malic acid, citric acid, succinic acid, fumaric acid, and adipic acid in food, but this standard is not applicable to the determination of organic acids in tea.The internal components of tea are complex, especially for yellow tea, which undergoes a "yellowing" effect through the "yellowing" process, and its internal components will undergo complex changes. Therefore, it is necessary to provide a detection method for simply and accurately determining the content of organic acid components in yellow tea using high performance liquid chromatography, and continuously optimize sample pretreatment conditions and high performance liquid chromatography conditions to minimize the impact of interfering substances.

[0003] Although yellow tea is a niche tea product among the six major tea categories, it has a long production history and has health benefits such as antibacterial, anti-oxidation, and regulating gastrointestinal function. Organic acids are important active ingredients in tea. The basic processing technology of yellow tea includes killing green, steaming yellow and drying. Steaming yellow is a unique process in yellow tea processing and is the key process to form its yellow soup, yellow leaves, mellow and sweet quality. According to the steaming yellowing method, it can be divided into dry steaming yellowing and wet steaming yellowing. During the steaming yellowing process, the killed green leaves are piled up while hot, so that the tea leaves undergo thermochemical changes under wet or dry heat conditions, and the metabolism of tea leaves is accelerated, laying the foundation for the formation of coloring substances, flavoring substances and aromatic substances in yellow tea. Among them, organic acids are intermediate products of the metabolism of various substances such as sugars and fats, and their content generally increases with the acceleration of metabolism. At present, there are few studies on organic acids in tea at home and abroad, and there are almost no reports on the research on organic acids in yellow tea. In the existing research on organic acids in tea using high-performance liquid chromatography, the types of organic acids that can be detected simultaneously by the established high-performance liquid chromatography detection method are mostly 10 or less. Tea contains a lot of low molecular weight organic acids, and the elution times of some low molecular weight organic acid chromatographic peaks are close, so it is difficult to separate them. Summary of the invention

[0004] The invention provides a method for determining the content of organic acids in yellow tea by high performance liquid chromatography, and aims to solve the technical problem of how to optimize the extraction method and detection conditions of main organic acids in yellow tea so as to accurately determine the content of multiple organic acids in yellow tea, so as to lay a foundation for in-depth research on the composition and content of organic acids in yellow tea, provide guidance for the improvement of yellow tea processing technology, the enhancement of flavor quality and the material basis research of health care function, and provide reference for the formulation of standards for detection methods of organic acid components in tea.

[0005] In view of the above problems in the prior art, according to one aspect disclosed in the present invention, the present invention adopts the following technical solution:

[0006] A method for determining the content of organic acids in yellow tea by high performance liquid chromatography, comprising the following detection conditions:

[0007] The C 18 Liquid chromatography column Acclaim TM120, column temperature was 30°C, gradient elution was performed with methanol as mobile phase A and 0.1 mol / L KH2PO4 as mobile phase B; wherein the gradient elution program was: 0-12 min, 1% A; 12-15 min, 1%-50% A; 15-25 min, 50% A; 25-27 min, 50%-1% A; 27-30 min, 1% A; samples were detected by diode array detector, the detection wavelength was 210 nm, the injection volume was 30 μL, the initial flow rate was 1.2 mL / min; 0-12 min, the flow rate was 1.2-1.0 mL / min; 12-30 min, the flow rate was 1.0 mL / min.

[0008] In order to better realize the present invention, a further technical scheme is: the preparation method of the 0.1 mol / L KH2PO4 is: weigh 13.6 g KH2PO4 into a beaker, add nearly 1 L of pure water to dissolve it, adjust the pH to 2.7 with H3PO4, transfer it to a 1 L volumetric flask, make up to volume with pure water and shake it well, filter it into a mobile phase bottle with a 0.45 μm water filter membrane, and ultrasonicate it for 10 minutes.

[0009] Furthermore, the sample pretreatment conditions are as follows: 3 g of ground sample is weighed and placed in a 500 mL conical flask, 450 mL of boiling pure water is added, and the sample is transferred into a boiling water bath and extracted for 45 min.

[0010] Furthermore, after the extraction is completed, the product is filtered under reduced pressure while hot, and the filtrate is transferred to a 500 mL volumetric flask. After cooling, the product is diluted to the mark with pure water, shaken well, filtered with a 0.22 μm water filter membrane, and then subjected to high performance liquid chromatography for determination. The product is qualitatively determined by retention time and quantitatively determined by external standard method.

[0011] Compared with the prior art, one of the beneficial effects of the present invention is:

[0012] The invention discloses a method for determining the content of organic acids in yellow tea by using high performance liquid chromatography. The method uses methanol and 0.1 mol / L KH2PO4 with a pH value of 2.7 as mobile phases, and has good separation effect of various organic acids during gradient elution. By optimizing high performance liquid chromatography conditions such as column temperature, flow rate, gradient elution program, etc., tartaric acid, quinic acid, formic acid, pyruvic acid and the like with similar elution times can be better separated, and malic acid and succinic acid which are difficult to be separated by using ion chromatography can also be separated and determined. The sample pretreatment method adopted avoids interference of impurity peaks on components to be determined, and the pretreatment method adopted can also be applied to determining the content of free amino acids and caffeine in tea leaves. The invention saves detection cost and improves detection efficiency, and has important significance for quality research of yellow tea and formulation of standards for detection methods of organic acids in tea leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present application document or the technical solutions in the prior art, the drawings required for use in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are only references to some embodiments in the present application document. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0014] Figure 1 The figure is a high performance liquid chromatogram of 12 organic acid standard substances according to one embodiment of the present invention.

[0015] Figure 2 The figure is a high performance liquid chromatogram of organic acids in a yellow tea sample according to an embodiment of the present invention. A total of 10 organic acids were detected in the yellow tea sample, which are oxalic acid (1), quinic acid (3), formic acid (4), pyruvic acid (5), L-malic acid (6), acetic acid (8), citric acid (9), fumaric acid (10), succinic acid (11) and salicylic acid (12) in the order of peak elution time. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0017] A method for determining the content of organic acids in yellow tea by high performance liquid chromatography, using C 18 Liquid chromatography column Acclaim TM120 (5μm, 250mm×4.6mm), column temperature 30℃, gradient elution with methanol (mobile phase A) and 0.1mol / L KH2PO4 (mobile phase B) as mobile phases, preparation of 0.1mol / L KH2PO4 is as follows: weigh 13.6g KH2PO4 in a beaker, add nearly 1L pure water to dissolve, adjust pH to 2.7 with H3PO4, transfer to a 1L volumetric flask, make up to volume with pure water and shake well, filter to the mobile phase bottle with a 0.45μm water filter, and ultrasonicate for 10min. The role of methanol is to adjust the retention time of each component so that each chromatographic peak can be completely separated. The gradient elution program was: 0-12 min, 1% A, 12-15 min, 1%-50% A, 15-25 min, 50% A, 25-27 min, 50%-1% A, 27-30 min, 1% A. Samples were detected by a diode array detector with a detection wavelength of 210 nm, an injection volume of 30 μL, an initial flow rate of 1.2 mL / min, 0-12 min, a flow rate of 1.2-1.0 mL / min, and 12-30 min, a flow rate of 1.0 mL / min. The optimal pretreatment conditions of the samples were established as follows: 3 g (accurate to 0.001 g) of ground sample was weighed into a 500 mL conical flask, 450 mL of boiling pure water was added, and it was immediately transferred into a boiling water bath and extracted for 45 min (shaken once every 10 min). After the extraction is completed, the product is immediately filtered under reduced pressure while hot, and the filtrate is transferred to a 500-mL volumetric flask. After cooling, the product is diluted to the mark with pure water, shaken, filtered with a 0.22 μm water filter membrane, and then subjected to high performance liquid chromatography for determination. The product is qualitatively determined by retention time and quantitatively determined by external standard method. This method can be used to simultaneously and rapidly determine the contents of oxalic acid, tartaric acid, quinic acid, formic acid, pyruvic acid, L-malic acid, lactic acid, acetic acid, citric acid, fumaric acid, succinic acid and salicylic acid in yellow tea. In one embodiment, the method quantitative limits of the 12 organic acids measured are: oxalic acid (0.20 μg / mL), tartaric acid (1.50 μg / mL), quinic acid (4.00 μg / mL), formic acid (3.00 μg / mL), pyruvic acid (0.70 μg / mL), L-malic acid (4.00 μg / mL), lactic acid (6.00 μg / mL), acetic acid (5.00 μg / mL), citric acid (3.00 μg / mL), fumaric acid (0.02 μg / mL), succinic acid (5.00 μg / mL) and salicylic acid (0.02 μg / mL). See Figure 1 As shown, Figure 1This is a chromatogram of 12 kinds of organic acid standard substances, among which the retention times of chromatographic peaks 1-12 are: oxalic acid (2.187min), tartaric acid (2.554min), quinic acid (2.705min), formic acid (2.898min), pyruvic acid (3.081min), L-malic acid (3.349min), lactic acid (4.486min), acetic acid (4.915min), citric acid (6.287min), fumaric acid (6.886min), succinic acid (8.187min), and salicylic acid (22.906min).

[0018] The principle of the above determination scheme is further illustrated by the following examples: oxalic acid, tartaric acid, quinic acid, formic acid, pyruvic acid, L-malic acid, lactic acid, acetic acid, citric acid, fumaric acid, succinic acid and salicylic acid in yellow tea were simultaneously determined, with methanol or acetonitrile as mobile phase A, and phosphoric acid aqueous solutions with different pH values ​​(2.5, 2.6, 2.7, 2.8), different concentrations (0.05%, 0.1%), and different concentrations (0.01, 0.0 5, 0.1mol / L) NH4H2PO4, KH2PO4 or NaH2PO4 aqueous solution were used as mobile phase B. The results showed that the separation effect of organic acids was best when methanol was used as mobile phase A and 0.1mol / L KH2PO4 with a pH value of 2.7 was used as mobile phase B for gradient elution. Different column temperatures (25, 30, 35, 40℃), flow rates (0.8, 0.9, 1.0, 1.1, 1.2mL / min), injection volume The results showed that the column temperature was 30℃, the initial flow rate was 1.2mL / min, the flow rate was 1.2-1.0mL / min from 0 to 12min, the flow rate was 1.0mL / min from 12 to 30min, and the injection volume was 30μL. The tartaric acid, quinic acid, formic acid, pyruvic acid and other acids with similar elution times can be better separated, and the ion chromatography can also separate the acids that are difficult to separate. Malic acid and succinic acid were separated and determined by the high performance liquid chromatography analysis method established by the present invention. When the methanol content was 1%, 11 organic acids except salicylic acid all eluted within 10 minutes, but salicylic acid was difficult to elute when the methanol content was 1%. Therefore, gradient elution was adopted. When the retention time was between 12 minutes and 15 minutes, the methanol content increased from 1% to 50%. From 15 minutes to 25 minutes, the volume ratio of methanol was maintained at 50%. Under this condition, salicylic acid eluted at about 23 minutes. The present invention tests two pretreatment methods, water bath extraction and ultrasonic extraction, and tests different extraction temperatures, material-liquid ratios and extraction times, and finally selects the sample pretreatment method in GB / T8314-2013 "Determination of the total amount of free amino acids in tea", which is also the sample pretreatment method for determining the caffeine content by ultraviolet spectrophotometry in GB / T 8312-2013 "Determination of caffeine in tea". By adopting this method, only one sample pretreatment is needed to determine the free amino acids and caffeine content in yellow tea while determining the organic acid content in yellow tea, which can save detection costs and improve detection efficiency. At the same time, by adopting this pretreatment method, when the injection volume is 30 μL, each organic acid component has a good response, and the extract only needs to be filtered with a 0.22 μm water filter membrane and then be measured by a high performance liquid chromatograph, and the impurity peak will not interfere with the components to be measured. The invention is of great significance to the quality research of yellow tea and the formulation of the standard for the detection method of organic acids in tea leaves.

[0019] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0020] References to "one embodiment", "another embodiment", "embodiment", etc. in this specification refer to specific features, structures or characteristics described in conjunction with the embodiment as included in at least one embodiment generally described in this application. The appearance of the same expression in multiple places in the specification does not necessarily refer to the same embodiment. Further, when a specific feature, structure or characteristic is described in conjunction with any embodiment, it is claimed that the realization of such feature, structure or characteristic in conjunction with other embodiments also falls within the scope of the present invention.

[0021] Although the present invention is described herein with reference to a plurality of illustrative embodiments of the present invention, it should be understood that those skilled in the art may devise many other modifications and implementations that fall within the scope and spirit of the principles disclosed herein. More specifically, within the scope of the present disclosure and claims, a variety of variations and improvements may be made to the components and / or layout of the subject combination layout. In addition to variations and improvements made to the components and / or layout, other uses will also be apparent to those skilled in the art.

Claims

1. A method for determining the content of organic acids in yellow tea by high performance liquid chromatography, characterized in that: The following testing conditions are included: Choose C 18 Liquid chromatography columns Acclaim TM 120, column temperature was 30°C, gradient elution was performed with methanol as mobile phase A and 0.1 mol / L KH2PO4 as mobile phase B; wherein the gradient elution program was: 0-12 min, 1% A; 12-15 min, 1%-50% A; 15-25 min, 50% A; 25-27 min, 50%-1% A; 27-30 min, 1% A; samples were detected by diode array detector, the detection wavelength was 210 nm, the injection volume was 30 μL, the initial flow rate was 1.2 mL / min; 0-12 min, the flow rate was 1.2-1.0 mL / min; 12-30 min, the flow rate was 1.0 mL / min.

2. The method for determining the content of organic acids in yellow tea by high performance liquid chromatography according to claim 1, characterized in that: The preparation method of 0.1 mol / L KH2PO4 is as follows: weigh 13.6 g KH2PO4 into a beaker, add nearly 1 L of pure water to dissolve it, adjust the pH to 2.7 with H3PO4, transfer it to a 1 L volumetric flask, make up to volume with pure water and shake it well, filter it into a mobile phase bottle with a 0.45 μm water filter membrane, and ultrasonicate it for 10 minutes.

3. The method for determining the content of organic acids in yellow tea by high performance liquid chromatography according to claim 1, characterized in that: The sample pretreatment conditions are as follows: weigh 3 g of ground sample and place it in a 500 mL conical flask, add 450 mL of boiling pure water, move it into a boiling water bath, soak for 45 minutes, and shake it once every 10 minutes.

4. The method for determining the content of organic acids in yellow tea by high performance liquid chromatography according to claim 3, characterized in that: After the extraction is completed, filter under reduced pressure while hot, transfer the filtrate into a 500mL volumetric flask, cool it and dilute it to the scale with pure water, shake it well, filter it with a 0.22μm water filter membrane and then conduct high performance liquid chromatography determination. Qualitative analysis is performed by retention time and quantitative analysis is performed by external standard method.

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