High performance liquid chromatography method for simultaneously determining 10 polyphenol substances in fruits and vegetables

Abstract

The invention relates to a liquid chromatography method for simultaneously measuring 10 polyphenol substances in fruits and vegetables, which comprises the following steps: S1, weighing a sample to be measured, adding an ethanol aqueous solution containing hydrochloric acid, carrying out ultrasonic extraction, and centrifuging to obtain supernate; repeating once, combining the supernate, carrying out nitrogen blowing or rotary evaporation until the supernate is nearly dry, redissolving with a methanol aqueous solution, filtering, and standing for measurement on a machine; s2, preparing a standard solution of 10 polyphenol substances by taking methanol as a solvent; s3, determining the sample to be detected and the standard solution by using a high performance liquid chromatograph; and S4, calculating the content of the polyphenol substance according to the retention time, the peak area and the standard curve. The method provided by the invention can be used for determining the content of ten polyphenol substances including gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin, quercetin, luteolin and kaempferide in the fruits and vegetables, has the characteristics of rapidness, accuracy and high sensitivity, and provides timely and accurate technical and data support for determination and monitoring of phytochemicals in the fruits and vegetables.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis methods, and particularly relates to a high performance liquid chromatography method for simultaneously determining the contents of 10 polyphenols in fruits and vegetables. Background Art

[0002] Polyphenols in plant foods such as fruits, vegetables, and grains are an important class of non-nutritional phytochemicals. Their antioxidant activity plays anti-tumor, anti-infection, immunity-enhancing, and anti-aging roles in the human body, and can effectively reduce the risk of tumors, diabetes, cardiovascular diseases, and other diseases. Vegetables are the food with the largest daily recommended intake. Not only are there many types of vegetables, but polyphenols are unevenly distributed, and the composition and content of phenols in vegetables from different origins and varieties also vary greatly. In addition, the structure of polyphenols is mostly unstable and is easily oxidized by temperature, light, and other factors, which places higher requirements on the extraction, detection, and analysis of polyphenols.

[0003] The commonly used methods for detecting polyphenols in plant foods include spectrophotometry, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, etc. Among them, spectrophotometry measures the content of total phenols, total flavonoids or total anthocyanins, and cannot distinguish and measure the monomer content of polyphenols; the chromatography-mass spectrometry method has high sensitivity and can realize qualitative and quantitative analysis of polyphenols, but there are problems such as expensive equipment and high technical requirements for operators. Therefore, liquid chromatography is widely used in the macro-analysis and detection of polyphenols in plant foods. At present, most studies are on the use of liquid chromatography to determine specific polyphenol components in one or several vegetables, while there are relatively few liquid chromatography determination methods suitable for the simultaneous determination of polyphenols in different types of vegetables.

[0004] Therefore, it is necessary to establish a method for the simultaneous determination of polyphenols in different vegetables. Summary of the invention

[0005] In view of the problems existing in the prior art: currently, most studies are on the determination of specific polyphenol components in one or several vegetables by liquid chromatography, while the methods for the simultaneous determination of polyphenols in different types of vegetables are mostly high-precision instruments and equipment such as liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry or flight mass spectrometry, which are limited in popularization and promotion, and have high requirements for the matrix of the sample to be tested, and the pre-treatment steps are cumbersome and time-consuming, and also have high requirements for inspection technicians. The purpose of the present invention is to provide a high-performance liquid chromatography method for the simultaneous determination of 10 polyphenols in vegetables.

[0006] This application optimizes sample extraction conditions, explores the selection of high performance liquid chromatography separation and detection conditions, uses retention time for qualitative analysis and external standard method for quantitative analysis, and invents a rapid, economical and accurate liquid chromatography determination method for the simultaneous detection of 10 polyphenols, including phenolic acids (gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid) and flavonoids (catechin, epicatechin, quercetin, luteolin, kaempferol), in fruits and vegetables, for the analysis and determination of phytochemicals in plant foods.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] One of the technical solutions of the present application provides a high performance liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables, comprising the following steps:

[0009] S1. Sample pretreatment: weigh the sample to be tested, add ethanol aqueous solution containing hydrochloric acid, extract by ultrasonication and centrifuge, and take the supernatant; repeat the extraction once, combine the supernatants, blow nitrogen or rotary evaporate to near dryness, re-dissolve in methanol aqueous solution, filter, and wait for the machine to measure;

[0010] S2. Preparation of standard solutions: using methanol as solvent, prepare standard solutions of 10 polyphenolic substances; the concentration gradient of the standard solutions is 0.1 to 100 μg / mL;

[0011] S3. The above-mentioned samples and standard solutions were measured using a high performance liquid chromatograph, and the measurement conditions were as follows: the liquid chromatograph consisted of a quaternary pump (for gradient separation), a column oven, a UV / visible detector and a workstation, and methanol / dilute phosphoric acid was used as the mobile phase to separate the samples / standard solutions; the chromatographic conditions were as follows:

[0012] The chromatographic column was a C18 column, 4.6 mm × 250 mm, 5 μm;

[0013] Mobile phase: Mobile phase A is methanol, mobile phase B is 0.025 mM phosphoric acid aqueous solution;

[0014] Flow rate: 1.0 mL / min;

[0015] Elution method: gradient elution according to volume ratio, 0-5.0min, 5.0%-18.0%; 5.0-15.0min, 18.0%-25.0%; 15.0-20.0min, 25.0%-42.5%; 20.0-30.0min, 42.5%-60.0%; 30.0-37.0min, 60.0%-95.0%; 37.0-38.0min, 95.0%-5.0%; 38.0-40.0%, 5.0%;

[0016] Detection wavelength: 280nm;

[0017] Column temperature: 30℃~35℃; the chromatogram is drawn at 35℃;

[0018] Injection volume: 5 μL to 20 μL; preferably 10 μL.

[0019] S4. Draw a standard curve based on the chromatographic results of the standard solution, and calculate the content of polyphenols based on the retention time, peak area and the standard curve.

[0020] Furthermore, in step S1, the relationship between the amount of the sample to be tested and the ethanol aqueous solution containing hydrochloric acid is 1 g: (2.5-20) mL, preferably 1 g: 10 mL;

[0021] Further, in step S1, the volume concentration of ethanol in the ethanol aqueous solution containing hydrochloric acid is 50% to 100%; the volume concentration of hydrochloric acid in the ethanol aqueous solution containing hydrochloric acid is 0.01% to 0.15%, preferably 0.015%;

[0022] Furthermore, in step S1, the temperature of the ultrasonic extraction is 25-65°C, and the time is 10-60 min; preferably 50°C, 10 min;

[0023] Furthermore, in step S1, the centrifugal speed is 5000-7000 rpm, and the time is 5-20 min;

[0024] Furthermore, in step S1, the temperature of the nitrogen blowing or rotary evaporation is 50°C.

[0025] Furthermore, in step S2, the 10 polyphenolic substances include gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin, quercetin, luteolin, and kaempferol.

[0026] The second technical solution of the present application provides an application of a liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables, wherein the method is used to determine the content of polyphenols in fruits and vegetables.

[0027] Furthermore, the fruits and vegetables include Solanaceae and Brassica fruits and vegetables.

[0028] Compared with the prior art, this application has at least the following advantages:

[0029] (1) The present invention provides a high performance liquid chromatography method for determining the contents of 10 polyphenols, including gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin, quercetin, luteolin and kaempferol, in fruits and vegetables. The method can detect the contents of the 10 polyphenols quickly, accurately and with high sensitivity, and provide timely and accurate technical and data support for the determination and monitoring of phytochemicals in fruits and vegetables.

[0030] (2) This detection method broadens the application of chromatographic detection in the analysis of substances such as polyphenols, and is also of great significance in other areas such as food nutrition research, agricultural production research, drug development and natural product research.

[0031] (3) The present invention optimizes sample pretreatment conditions through single factor and response surface design, explores the detection method established after selecting the mobile phase composition, gradient elution ratio, detection wavelength, etc. of chromatographic separation, and constructs a reasonable detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The HPLC chromatogram of 10 polyphenols; the marks in the figure are in the order of retention time: 1 gallic acid, 2 catechin, 3 chlorogenic acid, 4 caffeic acid, 5 epicatechin, 6 p-coumaric acid, 7 ferulic acid, 8 quercetin, 9 luteolin, 10 kaempferol;

[0033] Figure 2 is the standard curve of gallic acid;

[0034] Figure 3 is the standard curve of catechins;

[0035] Figure 4 is the standard curve of chlorogenic acid;

[0036] Figure 5 is the standard curve of caffeic acid;

[0037] Figure 6 is the standard curve of epicatechin;

[0038] Figure 7 is the standard curve of p-coumaric acid;

[0039] Figure 8 is the standard curve of ferulic acid;

[0040] Fig. 9 is the standard curve of quercetin;

[0041] Fig.10 is the standard curve of luteolin;

[0042] Fig.11 is the standard curve of kaempferol;

[0043] Fig.12 The HPLC spiked recoveries and relative standard deviations of 10 polyphenols are shown;

[0044] Fig.13 is the content of 10 polyphenols in 5 vegetable samples;

[0045] Fig.14 The effect of the proportion of organic phase in the extraction solvent on the extraction efficiency;

[0046] Fig.15 The effect of hydrochloric acid concentration in the extraction solvent on the extraction efficiency;

[0047] Fig.16 The effect of solid-liquid ratio on extraction efficiency;

[0048] Fig.17 The effect of ultrasonic extraction time on extraction efficiency;

[0049] Fig.18 The effect of ultrasonic extraction temperature on extraction efficiency;

[0050] Fig.19 3D diagram of response surface design optimization of ethanol proportion (%) and hydrochloric acid concentration (%) of sample extraction conditions;

[0051] Fig. 20 3D diagram of response surface design optimization of ultrasonic temperature (℃) and ethanol proportion (%) for sample extraction conditions;

[0052] Fig.21 3D diagram of response surface design optimization of ultrasonic temperature (℃) and hydrochloric acid concentration (%) for sample extraction conditions. DETAILED DESCRIPTION

[0053] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0054] All raw materials of the present invention have no particular limitation on their sources. If not otherwise specified, they can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0055] The materials, instruments, etc. involved in the implementation are as follows:

[0056] (1) Instrument

[0057] Waters e2695 liquid chromatograph-2489 UV / Vis detector; analytical balance, ultrasonic cleaning machine, ultrapure water instrument, any commercially available one that can achieve the corresponding functions can be used.

[0058] (2) Reagents and materials

[0059] Methanol (chromatographic grade) and ethanol (analytical grade) are both commercially available.

[0060] Standard substances: 10 kinds of polyphenol standard substances: gallic acid (CAS149-91-7), chlorogenic acid (CAS 327-97-9), caffeic acid (CAS 331-39-5), p-coumaric acid (CAS 501-98-4), ferulic acid (CAS 1135-24-6), catechin (CAS 99-50-3), epicatechin (CAS 490-46-0), quercetin (CAS 117-39-5), luteolin (CAS 491-70-3), kaempferol (CAS 491-54-3) standard reference substances.

[0061] Example 1 Fruit and vegetable sample collection

[0062] Collect 5 kinds of vegetable samples from two categories, namely, Solanaceae (purple eggplant, tomato) and Brassica (cauliflower, cauliflower, broccoli) in the circulation process. After proper washing and drying, take the edible part and divide the sample into quarters and homogenize it, then pack and freeze it for later use. The specific operation is as follows:

[0063] Chop the vegetable sample into pieces of about 0.2-0.5 cm in size, mix evenly and spread them into a circle, divide them into four equal parts with a cross through the center of the circle, take two opposite parts, mix them and divide them equally again, until the vegetable sample is about 100 g, put it into a food processor and grind it thoroughly to achieve homogenization.

[0064] Example 2

[0065] (1) Preparation of standard solution

[0066] Accurately weigh gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin, quercetin, luteolin, and kaempferol standard reference substances and dissolve them in methanol to make a standard stock solution with a concentration of 2000 μg / mL. Prepare the standard stock solutions of 10 polyphenols into a mixed standard application solution with a concentration of 100.0 μg / mL. Further dilute before use to prepare a mixed standard series solution with concentrations of 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 μg / mL. Mix the single standard application solutions of the 10 polyphenols and the mixed series standard application solutions (the measurement results are as shown in Figure 1 The standard liquid chromatograms of the 10 polyphenols were determined by using the concentration of the series of standard solutions and the corresponding peak areas to establish the standard curves of each polyphenol (as shown in Figure 10). Figures 2 to 11 shown).

[0067] (2) Sample pretreatment

[0068] Accurately weigh 0.5g of sample into a centrifuge tube, add 5.0mL of ethanol aqueous solution containing 0.015% hydrochloric acid (4:1, V / V), ultrasonically extract at 50℃ for 10min, centrifuge at 6000rpm for 10min, and take the supernatant into another centrifuge tube. Repeat the extraction once, combine the supernatants, blow nitrogen at 50℃ to dryness, re-dissolve in methanol solution, filter with organic phase nylon needle filter (13mm×0.45μm, Shanghai Anpu Laboratory Technology Co., Ltd., SCAA-103), and wait for determination.

[0069] Example 3 Liquid chromatography determination

[0070] (1) The liquid chromatograph was a Waters e2695 liquid chromatograph equipped with a 2489 UV / Vis detector. The conditions for sample separation and determination were as follows: the liquid chromatographic column was a Waters Symmetry column (4.6 mm×250 mm, 5 μm), the column temperature was 35° C., the injection volume was 10 μL, the flow rate was 1.0 mL / min, methanol was used as the mobile phase A, and 0.025 mM phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed according to the volume ratio:

[0071] 0~5.0min, A 5.0%~18.0%;

[0072] 5.0~15.0min, A 18.0%~25.0%;

[0073] 15.0~20.0min, A 25.0%~42.5%;

[0074] 20.0~30.0min, A 42.5%~60.0%;

[0075] 30.0~37.0min, A 60.0%~95.0%;

[0076] 37.0~38.0min, A 95.0%~5.0%;

[0077] 38.0~40.0min, 5.0%.

[0078] Detection wavelength: 280nm.

[0079] (2) Accuracy and stability

[0080] Under the above chromatographic separation conditions, the samples were measured three times in parallel for three consecutive days to calculate the precision of the method. The spike recovery rate and relative standard deviation (RSD) of the HPLC method were calculated by the three levels of spike recovery of high (100 μg / g), medium (50 μg / g) and low (20 μg / g) samples. Fig.12 shown).

[0081] (3) Measurement results

[0082] The results of the determination of 5 vegetable samples of two categories, namely, Solanaceae (purple eggplant, tomato) and Brassica (cauliflower, cauliflower, broccoli), were analyzed (e.g. Fig.13 As shown in the data, the total content of 10 polyphenols is from high to low: broccoli > cauliflower > cauliflower > purple eggplant > tomato.

[0083] Effects of the organic phase ratio in the extraction solvent, the hydrochloric acid concentration in the extraction solvent, the solid-liquid ratio, the ultrasonic extraction time, and the ultrasonic extraction temperature on the extraction efficiency of Examples 4 to 8

[0084] The initial extraction conditions were ethanol aqueous solution (80:20, V / V) containing 0.03% hydrochloric acid, a solid-liquid ratio of 1:5, an ultrasonic temperature of 25°C, and an ultrasonic time of 10 min. Different levels of adjustment were made for each of the five factors, namely, hydrochloric acid concentration, ethanol-water ratio, solid-liquid ratio, ultrasonic temperature, and ultrasonic time. The effects of the above factors on the extraction efficiency were investigated while the other four factors remained unchanged. Figures 14 to 18 ).

[0085] Example 9 Response surface design optimization of sample extraction conditions

[0086] In order to further establish the optimal extraction conditions, the Box-Behnken method was used to fit the above factors that affect the extraction efficiency at three levels. The hydrochloric acid concentration (A), ethanol concentration (B), and ultrasonic temperature (C) were used as independent variables, and the catechin content was used as the response value (Y). The optimal extraction conditions were determined through multiple regression fitting. The relationship between the hydrochloric acid concentration (A), ethanol concentration (B), and catechin content (Y) is shown in the figure. Fig.19 As shown; the relationship between ethanol concentration (B), ultrasonic temperature (C), and catechin content (Y) is as follows Fig. 20 As shown; the relationship between hydrochloric acid concentration (A), ultrasonic temperature (C), and catechin content (Y) is as follows Fig.21 shown.

[0087] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables, characterized in that: The following steps are involved: S1. Sample pretreatment: weigh the sample to be tested, add ethanol aqueous solution containing hydrochloric acid, extract by ultrasonication and centrifuge, and take the supernatant; repeat the extraction once, combine the supernatants, blow nitrogen or rotary evaporate to near dryness, re-dissolve in methanol aqueous solution, filter, and wait for the machine to measure; S2. Preparation of standard solutions: using methanol as solvent, prepare standard solutions of 10 polyphenolic substances; the concentration gradient of the standard solutions is 0.1 to 100 μg / mL; S3. The above-mentioned samples and standard solutions were measured using a high performance liquid chromatograph, and the measurement conditions were as follows: the liquid chromatograph consisted of a quaternary pump (for gradient separation), a column oven, a UV / visible detector and a workstation, and methanol / dilute phosphoric acid was used as the mobile phase to separate the samples / standard solutions; the chromatographic conditions were as follows: The chromatographic column was a C18 column, 4.6 mm × 250 mm, 5 μm; Mobile phase: Mobile phase A is methanol, mobile phase B is 0.025 mM phosphoric acid aqueous solution; Flow rate: 1.0 mL / min; Elution method: gradient elution according to volume ratio, 0-5.0min, 5.0%-18.0%; 5.0-15.0min, 18.0%-25.0%; 15.0-20.0min, 25.0%-42.5%; 20.0-30.0min, 42.5%-60.0%; 30.0-37.0min, 60.0%-95.0%; 37.0-38.0min, 95.0%-5.0%; 38.0-40.0%, 5.0%; Detection wavelength: 280nm; Column temperature: 30℃~35℃; Injection volume: 5μL~20μL; S4. Draw a standard curve based on the chromatographic results of the standard solution, and calculate the content of polyphenols based on the retention time, peak area and the standard curve.

2. A liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 1, characterized in that: In step S1, the dosage of the sample to be tested and the ethanol aqueous solution containing hydrochloric acid is in the ratio of 1 g: (2.5-20) mL.

3. The liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 1, characterized in that: In step S1, the volume concentration of ethanol in the ethanol aqueous solution containing hydrochloric acid is 50% to 100%.

4. The liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 1, characterized in that: In step S1, the volume concentration of hydrochloric acid in the ethanol aqueous solution containing hydrochloric acid is 0.01% to 0.15%.

5. The liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 1, characterized in that: In step S1, the temperature of the ultrasonic extraction is 25-65°C and the time is 10-60 minutes.

6. The liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 1, characterized in that: In step S1, the centrifugal speed is 5000-7000 rpm and the time is 5-20 min.

7. The liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 1, characterized in that: In step S1, the temperature of the nitrogen blowing or rotary evaporation is 50°C.

8. The liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 1, characterized in that: In step S2, the 10 polyphenolic substances include gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, catechin, epicatechin, quercetin, luteolin, and kaempferol.

9. The use of a liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables as claimed in claim 1, characterized in that: The method is used to determine the content of polyphenols in fruits and vegetables.

10. The use of a liquid chromatography method for simultaneously determining 10 polyphenols in fruits and vegetables according to claim 9, characterized in that: The fruits and vegetables include solanaceae and brassica fruits and vegetables.

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