Method for determining various phenolic acid compounds in germinated triticale

Abstract

The invention relates to the technical field of agricultural product nutrition ingredient detection, in particular to a method for determining various phenolic acid compounds in germinated triticale, which specifically comprises the following steps: extracting a sample; drawing a standard curve; performing high performance liquid chromatography detection on the to-be-detected sample, calculating the concentration of each phenolic acid in the to-be-detected sample through an external standard method, and calculating the content of each phenolic acid compound in the to-be-detected sample according to a formula: # imgabs0 # omega is the content of the phenolic acid compound in the to-be-detected sample, and the unit is mu g / g; rho is the concentration of the detected component in the to-be-detected sample obtained from the standard curve, and the unit is mu g / mL; v is the volume of the added extraction solvent, and the unit is mL; m is the mass of the germinated triticale flour, and the unit is g. The method solves the problems that the contents of 11 phenolic acid compounds in the germinated triticale cannot be simultaneously and quantitatively detected and the used detection equipment is relatively expensive in the prior art, and has the advantages of simplicity, high efficiency, moderate price of the used detection equipment, low detection cost and the like.

Description

Technical Field

[0001] The invention relates to the technical field of detecting the nutritional components of agricultural products, in particular to a method for determining multiple phenolic acid compounds in germinated black wheat. Background Art

[0002] There are many kinds of active ingredients in black wheat and their content is rich. Common active ingredients mainly include phenolic acids, flavonoids, anthocyanins, dietary fiber, oligosaccharides, etc. These active ingredients are natural antioxidants in black wheat, which can effectively remove free radicals in the body, improve human immunity, lower blood lipids, prevent cancer, etc. Studies have shown that after black wheat germinates, the types of phenolic acid compounds contained in it will be further enriched and the content will change dynamically. Therefore, how to detect the content of these phenolic acid compounds sensitively, efficiently and quickly to achieve dynamic monitoring purposes will help accelerate and promote the development of functional black wheat research technology.

[0003] At present, the main methods for quantitative analysis of phenolic acid compounds in wheat include HPLC, UPLC, UPLC-MS, etc. For example, Chinese invention patent application No. 201711375184.X discloses a simple and efficient high-performance liquid chromatography tandem mass spectrometry detection method for phenolic acid in wheat, which fully dissolves the wheat sample that has completely passed through a 0.425 mm pore sieve in a 2 mol / L sodium hydroxide solution, and then extracts it with ethyl acetate after nitrogen hydrolysis under light-proof conditions, and takes the upper organic phase and rotary evaporates it to dryness; the residue is redissolved with 50% methanol, filtered through a 0.22 μm filter membrane, and stored at 4°C for testing; the sample to be tested is tested by high-performance liquid chromatography tandem mass spectrometry, and the standard curve is made with phenolic acid standards, and the test results are substituted into the standard curve for comparison to obtain the content of phenolic acid in the wheat sample; this detection method can be applied to the extraction and detection of 9 common phenolic acids in wheat. Compared with other detection methods, it has the characteristics of simple extraction, short time consumption, high sensitivity and good stability. The whole detection process takes 2 hours and 15 minutes (2 hours for hydrolysis and 15 minutes for instrument detection). It can be used for the quantitative detection of phenolic acids in large quantities of wheat samples.

[0004] However, the above detection method is not practical because the equipment used is relatively expensive. Moreover, it can only detect nine phenolic acids, namely gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, and sinapinic acid, while chlorogenic acid and salicylic acid contained in germinated black wheat cannot be detected.

[0005] At present, there is no public record of a method that can simultaneously and quantitatively detect the contents of 11 phenolic acid compounds, including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid, in germinated black wheat. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the present invention provides a method for determining multiple phenolic acid compounds in germinated black wheat to overcome the above-mentioned shortcomings of the prior art.

[0007] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0008] The method for determining multiple phenolic acid compounds in germinated black wheat specifically comprises the following steps:

[0009] (1) Sample extraction: Weigh a certain amount of germinated black wheat flour into a centrifuge tube, add an extractant to perform vortex extraction, vortex extract, condense by rotary evaporation, and filter through a 0.22 μm filter membrane to obtain a sample to be tested;

[0010] (2) Drawing a standard curve: Weigh a certain amount of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid standards, dissolve them in chromatographic methanol and prepare a standard stock solution with a mass concentration of 1 mg / mL, take an appropriate amount of the standard stock solution and dilute it with chromatographic methanol to prepare a mixed gradient dilution solution with mass concentrations of 20 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL of 11 phenolic acids, respectively, and filter through a 0.22 μm filter membrane, then perform high performance liquid chromatography detection, and then draw a standard curve with the mass concentration of the standard as the horizontal axis and the chromatographic peak area y of the standard as the vertical axis;

[0011] (3) The sample obtained in step (1) is subjected to high performance liquid chromatography detection, and the detection result is substituted into the standard curve obtained in step (2) for comparison, and the concentrations of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid in the sample are determined by external standard method, and then the content of each phenolic acid compound in the sample is calculated according to formula (1):

[0012]

[0013] Where:

[0014] ω is the content of phenolic acid compounds in the sample to be tested, in μg / g;

[0015] ρ is the concentration of the component to be tested in the sample obtained from the standard curve, in μg / mL;

[0016] v is the volume of the added extraction solvent, in mL;

[0017] m is the mass of germinated black wheat flour, in g.

[0018] Further, the HPLC detection conditions described in step (2) and step (3) are the same, both using Shimadzu LC-20AT HPLC, Sigma-C18 chromatographic column (4.6mm×250mm, 5μm), mobile phase A is a chromatographic methanol solution of 1% by volume of acetic acid, and mobile phase B is a 1% by volume acetic acid solution; the injection volume is 10μL, the flow rate is 1mL / min, the detection wavelength is 280nm, the column temperature is 25°C, and gradient elution is adopted, and the specific gradient elution program is: 0-10min, 8%A→10%A, 92%B→90%B; 10-23min, 10%A→30%A, 90%B→70%B; 23-38min, 30%A→33%A, 70%B→67%B; 38-45min, 33%A→50%A, 67%B→50%B.

[0019] Furthermore, in step (2), the specific preparation operation of the mixed gradient dilution is as follows: accurately weigh 50 mg of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid standards, dissolve them with chromatographic methanol and make up to volume in a 50 mL volumetric flask to prepare a mixed standard stock solution with a mass concentration of each phenolic acid of 1 mg / mL, then take 5 mL, 10 mL, 12.5 mL, 25 mL and 50 mL of the standard stock solution into 5 250 mL volumetric flasks, respectively, make up to volume with chromatographic methanol to prepare 11 kinds of phenolic acid mass concentrations of 20 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL mixed gradient dilutions.

[0020] Furthermore, in step (1), during the vortex extraction, the extractant is a methanol aqueous solution with a volume percentage of 80%, the solid-liquid ratio of germinated black wheat flour to the extractant is 1 g:30 mL, and the vortex extraction time is not less than 6 minutes.

[0021] Furthermore, in step (1), the temperature of rotary evaporation concentration is 60°C.

[0022] Furthermore, in step (1), the germinated black wheat flour is the germinated black wheat flour obtained by freeze-drying, crushing and passing through a 60-mesh sieve.

[0023] Furthermore, in step (1), the sample to be tested is subjected to high performance liquid chromatography within 24 hours after preparation.

[0024] Furthermore, ω is expressed as the average of three measurement results, and the result is rounded to two significant figures.

[0025] Compared with the prior art, the present invention has at least the following beneficial effects:

[0026] 1. For the first time, a method for simultaneous and quantitative detection of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid in germinated black wheat was proposed. This method can be extended to the testing of other germinated grains.

[0027] 2. The test results are accurate and reliable. Through linear equation fitting and evaluation of precision, repeatability and stability, the results show that when the concentrations of these 11 phenolic acid compounds are in the range of 20-200 μg / mL, they have a good linear relationship with the peak area (R 2 ≥0.9994, n=6), the peak area RSD values ​​of precision, repeatability and stability were all less than 2%, and the spiked recovery was between 98.02% and 101.12%.

[0028] 3. The test method is simple and efficient. Both the sample extraction and the detection after extraction are simple and fast. The sample extraction time is about 35 minutes, and the detection time after extraction is about 45 minutes. The entire test cycle can be completed in only about 80 minutes.

[0029] 4. The test cost and difficulty are low. The sample extraction device only involves conventional small devices such as vortex mixers, rotary evaporators and centrifuges. The extraction reagent is inexpensive. The test device after extraction only requires a high performance liquid chromatograph. Therefore, it is very practical and suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments 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 creative work.

[0031] Figure 1 For mixed standard ( Figure 1 A) and the sample to be tested ( Figure 1 B) chromatogram, wherein: 1. gallic acid, 2. protocatechuic acid, 3. p-hydroxybenzoic acid, 4. chlorogenic acid, 5. vanillic acid, 6. caffeic acid, 7. syringic acid, 8. p-coumaric acid, 9. ferulic acid, 10. sinapic acid, 11. salicylic acid.

[0032] Figure 2 This is a test chart of the effect of methanol volume fraction on the content of 11 phenolic acids.

[0033] Figure 3 This is a test chart of the effect of vortex extraction time on the content of 11 phenolic acids.

[0034] Figure 4This is a test chart showing the effect of rotary evaporation concentration temperature on the contents of 11 phenolic acids.

[0035] Figure 5 This is a test chart showing the effect of vortex material-liquid ratio on the contents of 11 phenolic acids. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0037] The method for obtaining the raw material germinated black wheat used in the following examples is as follows: accurately weigh 30g of black wheat seeds (Yunhei 161 black wheat, provided by the Cotton Research Institute of Shanxi Agricultural University), rinse with tap water 5 times, and soak the black wheat seeds in tap water for 8h after draining. The mass volume ratio of black wheat seeds to tap water is 1:6. After soaking, drain the black wheat seeds and place them in a germination box with holes at the bottom in an incubator with a constant temperature of 25°C and a constant humidity of 85% to germinate in the dark. During this period, water once every 8h, 100mL each time. Samples were taken on the 1st, 3rd, 5th, and 7th days after germination, and the black wheat grains without any treatment were used as the non-germinated control group. After the samples were collected, they were placed in an ultra-low temperature (-80°C) refrigerator for 12h, and then dried in a vacuum freeze dryer. After grinding, they were sieved through a 60-mesh sieve for later use.

[0038] The phenolic acid standards in the following examples - gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, salicylic acid were all purchased from Sigma-Aldrich, USA, chromatographic methanol was purchased from Xilong Science Co., Ltd., acetic acid was purchased from Tianjin Ruijinte Chemical Co., Ltd., and all the water used was ultrapure water.

[0039] In the following examples, the manufacturer information of each device is as follows: high performance liquid chromatograph, Shimadzu LC-20AT, Shimadzu Corporation, Japan; constant temperature and humidity incubator, HWS-160, Ningbo Jiangnan Instrument Factory; high-speed refrigerated centrifuge, GL-20B, Shanghai Anting Scientific Instrument Co., Ltd.; vacuum freeze dryer, Ningbo Xinzhi Technology Co., Ltd.; ultraviolet visible spectrophotometer, UV9000S, Shanghai Yuanxi Instrument Co., Ltd.; rotary evaporator, Shanghai Yarong Biochemical Instrument Factory.

[0040] Other raw materials, reagents, instruments, equipment, etc. not described in detail in the following examples can be purchased from the market or prepared by existing methods. Unless otherwise specified, they are common commercial products.

[0041] The data processing involved in the following examples was performed using Excel 2019 and IMB SPSS statistics 25, and Origin 2021 software was used for drawing.

[0042] 1. Determination of chromatographic analysis conditions

[0043] 1) Determination of detection wavelength

[0044] Accurately weigh 50 mg of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid standards respectively, dissolve them in chromatographic methanol and make up to volume in a 50 mL volumetric flask to prepare a standard solution with a mass concentration of 1 mg / mL.

[0045] Each standard solution was scanned at full wavelength in a UV spectrophotometer with chromatographic methanol as the blank solvent. The scanning results showed that all 11 phenolic acids had strong UV absorption at 248nm and 280nm. However, when 248nm was used as the detection wavelength, the test peak appeared inverted and the baseline was unstable, so 280nm was used as the detection wavelength.

[0046] 2) Determination of mobile phase

[0047] 2 mL of the 1 mg / mL standard solution was pipetted into a 50 mL volumetric flask and the volume was fixed with chromatographic methanol to prepare a mixed standard solution with a concentration of 40 μg / mL for the 11 phenolic acid compounds tested. The peaks and separation of the phenolic acid compounds were compared when the mobile phases were: methanol-water (the aqueous phase contained 0.1% by volume acetic acid), acetonitrile-water (the aqueous phase contained 0.1% by volume acetic acid), methanol-water (the aqueous phase contained 0.1% by volume phosphoric acid), and acetonitrile-water (the aqueous phase contained 0.1% by volume phosphoric acid). The test results show that when acetonitrile-water (the aqueous phase contains 0.1% acetic acid by volume) and acetonitrile-water (the aqueous phase contains 0.1% phosphoric acid by volume) are used as the mobile phase, the separation of vanillic acid, caffeic acid and syringic acid is low; when methanol-water (the aqueous phase contains 0.1% phosphoric acid by volume) is used as the mobile phase, the chromatographic peaks of sinapinic acid and ferulic acid overlap; when methanol-water (the aqueous phase contains 0.1% acetic acid by volume) is used as the mobile phase, the chromatographic peaks corresponding to each phenolic acid have a good separation. On this basis, the content of acetic acid in the aqueous phase is increased, and the results show that when the volume fraction of acetic acid in the aqueous phase reaches 1%, the chromatographic peaks can achieve baseline separation; in order to further optimize the peak shape, the present invention attempts to add acetic acid to the organic phase, and the results show that when the volume fraction of acetic acid in the organic phase and the aqueous phase is 1%, the separation of each component is good, and the peak shape is symmetrical and has no tailing phenomenon. Therefore, it is determined that the mobile phase is methanol-water, and 1% acetic acid by volume is added to both the methanol phase and the aqueous phase.

[0048] 3) Determination of chromatographic test conditions

[0049] Based on the determination of the mobile phase, a gradient elution program was adopted: 0-10 min, 8% A→10% A, 92% B→90% B; 10-23 min, 10% A→30% A, 90% B→70% B; 23-38 min, 30% A→33% A, 70% B→67% B; 38-45 min, 33% A→50% A, 67% B→50% B. The instrument and other important technical parameters were as follows: Shimadzu LC-20AT high performance liquid chromatograph, Sigma-C18 chromatographic column (4.6 mm×250 mm, 5 μm), mobile phase A was a chromatographic methanol solution of 1% by volume of acetic acid, and mobile phase B was a 1% by volume acetic acid solution; the injection volume was 10 μL, the flow rate was 1 mL / min, the detection wavelength was 280 nm, and the column temperature was 25°C.

[0050] 2. Determination of sample extraction method

[0051] 1) Determination of extraction solvent

[0052] The present invention compares the effects of different volume fractions of methanol-water solutions on the extraction rates of 11 phenolic acid compounds. Figure 2 As shown: when the volume fraction of methanol is 80%, the total content of the 11 phenolic acids measured is the highest, so it is finally determined to use 80% methanol aqueous solution as the extraction solvent.

[0053] 2) Determination of vortex extraction time

[0054] The present invention investigates the effect of vortex extraction time on the total content of 11 phenolic acids. Figure 3 As shown in the figure, the total contents of 11 phenolic acids increased with the extension of vortex extraction time within 2-6 minutes, but there was no significant increase after 6 minutes. Therefore, the vortex extraction time should not be less than 6 minutes. Considering the extraction efficiency and extraction effect comprehensively, 6 minutes is the most suitable vortex extraction time.

[0055] 3) Determination of rotary evaporation concentration temperature

[0056] On the basis of determining the vortex extraction time, the present invention further studies the effect of the rotary evaporation concentration temperature on the total content of the 11 phenolic acid compounds measured, such as Figure 4 As shown in the figure: when the rotary evaporation concentration temperature is 60℃, the total content of phenolic acid compounds measured in germinated black wheat is the highest, so 60℃ is the appropriate rotary evaporation concentration temperature.

[0057] 4) Determination of material-liquid ratio

[0058] On the basis of determining the appropriate extraction solvent, vortex extraction time and rotary evaporation concentration temperature, the present invention further studied the effect of the solid-liquid ratio on the total content of 11 phenolic acid compounds in germinated black wheat, such as Figure 5 As shown: when the solid-liquid ratio of germinated black wheat flour to extractant is 1g:30mL, the total content of 11 phenolic acid compounds extracted is the highest, so 1g:30mL is determined to be the appropriate solid-liquid ratio.

[0059] 3. Specific test applications

[0060] 1) Standard curve drawing

[0061] Accurately weigh 50 mg of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid standards, dissolve them with chromatographic methanol and make up to volume in a 50 mL volumetric flask to prepare a mixed standard stock solution with a mass concentration of each phenolic acid of 1 mg / mL, then take 5 mL, 10 mL, 12.5 mL, 25 mL and 50 mL of the standard stock solution into 5 250 mL volumetric flasks, respectively, make up to volume with chromatographic methanol to prepare mixed gradient dilutions with mass concentrations of 11 phenolic acids of 20 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL, respectively, pass through a 0.22 μm filter membrane, and then perform high performance liquid chromatography detection. The test conditions are as shown in step 3) in 1, and then draw a standard curve with the mass concentration of the standard as the abscissa and the chromatographic peak area y of the standard as the ordinate. The chromatogram of the standard is as follows Figure 1 As shown in A.

[0062] 2) Prepare sample solution

[0063] Accurately weigh 1 g of germinated black wheat flour to be tested into a 50 mL centrifuge tube, first add 15 mL of chromatographic methanol, vortex for 6 min, centrifuge at 5000 r / min for 5 min, take the supernatant and filter it, add 15 mL of chromatographic methanol to the precipitate and repeat the above steps, combine the supernatants after two filtrations, concentrate at 60 ° C to 2 mL, pass through a 0.22 μm filter membrane, and perform high performance liquid chromatography test within 24 h. The test conditions are as shown in step 3) in 1.

[0064] In order to investigate the effect of germination time on the content of 11 phenolic acid compounds, the germinated black wheat flour with germination time of 0, 1, 3, 5, and 7 days was used as the test sample to measure the content of 11 phenolic acids. Taking black wheat on the fifth day of germination as an example, the chromatogram of the determination of 11 phenolic acid compounds is shown in the figure below. Figure 1 As shown in B.

[0065] 3) Content calculation

[0066] The test results of the samples were substituted into the standard curve for comparison. The concentrations of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid in the samples were determined by the external standard method. The content of each phenolic acid compound in the samples was calculated according to formula (1):

[0067]

[0068] Where:

[0069] ω is the content of phenolic acid compounds in the sample to be tested, in μg / g;

[0070] ρ is the concentration of the component to be tested in the sample obtained from the standard curve, in μg / mL;

[0071] v is the volume of the added extraction solvent, in mL;

[0072] m is the mass of germinated black wheat flour, in g.

[0073] Note: ω is expressed as the average value of 3 test results, and the result is retained to two significant figures. The final test results are shown in Table 1.

[0074] Table 1 Effect of germination time on the contents of 11 phenolic acids in black wheat

[0075]

[0076] Note: 0d means the black wheat grain control group without any treatment, ND means not detected, different lowercase letters in the same row represent the difference in the effects of different germination times on the contents of various phenolic acid compounds, and the significant level of the difference is P<0.05.

[0077] As shown in Table 1: Gallic acid was not detected in black wheat without any treatment. Within 1-3 days of germination, the gallic acid content increased significantly (p < 0.05), reaching a peak of 113.82 μg / g on the third day. As the germination time increased, the gallic acid content showed a downward trend; as the germination time increased, the protocatechuic acid, caffeic acid and syringic acid contents all showed an increasing trend; the vanillic acid, p-coumaric acid and ferulic acid contents increased within the first day of germination. The highest contents were on the 5th day of germination, which were 180.00μg / g, 67.63μg / g and 105.22μg / g respectively; the contents of p-hydroxybenzoic acid, sinapic acid and salicylic acid were the highest on the 5th day of germination, which were 469.07μg / g, 352.72μg / g and 160.66μg / g respectively; within 1-5 days of germination, the total content of 11 phenolic acids increased significantly (p<0.05), and then showed a downward trend on the 7th day of germination. In summary, germination treatment can significantly change the content and composition of these 11 phenolic acid compounds in black wheat.

[0078] 4. Methodological Validation

[0079] 1) Precision test

[0080] A mixed standard solution was prepared according to the method shown in step 1) of step 3, and standard solutions of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid with a mass concentration of 100 μg / mL were respectively injected and measured under the chromatographic conditions shown in step 3) of step 1, the injection volume was set to 10 μL, the injection was repeated 6 times, and the relative standard deviation (RSD) value was calculated based on the chromatographic peak area.

[0081] The results showed that the RSD values ​​(n=6) of the chromatographic peak areas of 11 phenolic acid compounds, including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid, were 0.74%, 0.63%, 0.63%, 0.49%, 0.59%, 0.42%, 0.68%, 0.58%, 0.63%, 0.70% and 1.44%, respectively, all lower than 2.00%, indicating that the instrument had good precision.

[0082] 2) Repeatability test

[0083] Weigh 6 portions of black wheat flour from the same batch on the 5th day of germination, 1 g each, and prepare sample solutions according to the method shown in step 3, step 2). Then, determine gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapinic acid and salicylic acid according to the above method, and calculate the RSD value based on the chromatographic peak area corresponding to each phenolic acid compound.

[0084] The test results showed that the peak area RSDs (n=6) of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid were 1.88%, 1.81%, 1.03%, 1.09%, 1.89%, 1.58%, 1.59%, 1.88%, 1.73%, 1.73% and 1.89%, respectively, all lower than 2.00%, indicating that the instrument had good repeatability.

[0085] 3) Stability test

[0086] Weigh 3 portions of black wheat flour from the same batch on the 5th day of germination and prepare sample solutions according to the method shown in 3 step 2), and inject the prepared sample solutions every 4 hours within 24 hours. Calculate the RSD value based on the chromatographic peak areas corresponding to 11 phenolic acid compounds in the samples, including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapinic acid and salicylic acid.

[0087] The test results showed that the RSD (n=7) values ​​of the peak areas of 11 phenolic acids, including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid, were 1.34%, 1.74%, 1.71%, 0.93%, 1.04%, 1.96%, 1.14%, 1.95%, 1.86% and 1.20%, respectively, all lower than 2.00%, indicating that the sample solution had good stability within 24 hours.

[0088] 4) Spike recovery test

[0089] Weigh 1g of each of 3 portions of black wheat flour on the 5th day of germination, prepare samples and determine their content. Weigh 1g of 9 portions of germinated black wheat flour, and divide 3 portions into 1 group. Add standard solution into three levels: high, medium and low. Prepare samples and determine their content from low concentration to high concentration. Analyze the data and calculate the average spike recovery. The results of sample content and spike recovery are shown in Table 2.

[0090] Table 2 Sample content determination and spike recovery test (n = 3)

[0091]

[0092]

[0093] As shown in Table 2, the average spiked recoveries of 11 phenolic acids, including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid, were between 98.02% and 101.12%, and the RSD values ​​were all lower than 2.00%, indicating that the extraction and determination methods used in this experiment had good accuracy.

[0094] In summary, the present invention proposes a method for determining 11 phenolic acids in germinated black wheat: using Shimadzu LC-20AT high performance liquid chromatograph and Sigma-C18 chromatographic column (4.6mm×250mm, 5μm) as the stationary phase, methanol (containing 1% acetic acid by volume)-water (containing 1% acetic acid by volume) as the mobile phase, gradient elution, flow rate 1mL / min, detection wavelength 280nm, column temperature 25°C. The determination results show that 11 phenolic acid compounds such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid have a good linear relationship between concentration and peak area in the range of 20-200μg / mL (R 2 ≥0.9994, n=6), the peak area RSD values ​​of precision, repeatability and stability were all less than 2%, and the recovery rate of spiked addition was between 98.02% and 101.12%, indicating that the determination method proposed by the present invention is fast and the results are accurate and reliable. The determination method is used to determine the contents of 11 phenolic acid compounds in germinated black wheat, which can lay a foundation for the study of functional active ingredients in germinated black wheat and provide a reference for the quality control and evaluation of its products.

[0095] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace and deform the above embodiments within the scope of the present invention. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples without contradiction.

Claims

1. A method for determining multiple phenolic acid compounds in germinated black wheat, characterized in that: The specific steps include: (1) Sample extraction: Weigh a certain amount of germinated black wheat flour into a centrifuge tube, add an extractant for vortex extraction, condense by rotary evaporation and filter through a 0.22 μm filter membrane to obtain a sample to be tested; (2) Drawing a standard curve: Weigh a certain amount of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid standards, dissolve them in chromatographic methanol and prepare a standard stock solution with a mass concentration of 1 mg / mL, take an appropriate amount of the standard stock solution and dilute it with chromatographic methanol to prepare a mixed gradient dilution solution with mass concentrations of 20 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL of 11 phenolic acids, respectively, and filter through a 0.22 μm filter membrane, then perform high performance liquid chromatography detection, and then draw a standard curve with the mass concentration of the standard as the horizontal axis and the chromatographic peak area y of the standard as the vertical axis; (3) Performing high performance liquid chromatography on the sample obtained in step (1), substituting the test result into the standard curve obtained in step (2) for comparison, calculating the concentrations of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid in the sample by external standard method, and then calculating the content of each phenolic acid compound in the sample according to formula (1): Where: ω is the content of phenolic acid compounds in the sample to be tested, in μg / g; ρ is the concentration of the component to be tested in the sample obtained from the standard curve, in μg / mL; v is the volume of the added extraction solvent, in mL; m is the mass of germinated black wheat flour, in g.

2. The method for determining multiple phenolic acid compounds in germinated black wheat according to claim 1, characterized in that: The high performance liquid chromatography detection conditions described in step (2) and step (3) are the same, both using Shimadzu LC-20AT high performance liquid chromatograph, Sigma-C18 chromatographic column (4.6mm×250mm, 5μm), mobile phase A is a chromatographic methanol solution of 1% by volume acetic acid, and mobile phase B is a 1% by volume acetic acid solution; the injection volume is 10μL, the flow rate is 1mL / min, the detection wavelength is 280nm, the column temperature is 25°C, and gradient elution is adopted. The specific gradient elution program is: 0-10min, 8%A→10%A, 92%B→90%B; 10-23min, 10%A→30%A, 90%B→70%B; 23-38min, 30%A→33%A, 70%B→67%B; 38-45min, 33%A→50%A, 67%B→50%B.

3. The method for determining multiple phenolic acid compounds in germinated black wheat according to claim 1, characterized in that: In step (2), the specific preparation operation of the mixed gradient dilution is as follows: accurately weigh 50 mg of gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid and salicylic acid standards, dissolve them with chromatographic methanol and make up to volume in a 50 mL volumetric flask to prepare a mixed standard stock solution with a mass concentration of each phenolic acid of 1 mg / mL, then respectively absorb 5 mL, 10 mL, 12.5 mL, 25 mL, and 50 mL of the standard stock solution into 5 250 mL volumetric flasks, make up to volume with chromatographic methanol to prepare 11 kinds of phenolic acid mass concentrations of 20 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL mixed gradient dilutions.

4. The method for determining multiple phenolic acid compounds in germinated black wheat according to claim 1, characterized in that: In step (1), during the vortex extraction, the extractant is a methanol aqueous solution with a volume percentage of 80%, the solid-liquid ratio of germinated black wheat flour to the extractant is 1 g:30 mL, and the vortex extraction time is not less than 6 minutes.

5. The method for determining multiple phenolic acid compounds in germinated black wheat according to claim 1, characterized in that: In step (1), the temperature of rotary evaporation concentration is 60°C.

6. The method for determining multiple phenolic acid compounds in germinated black wheat according to claim 1, characterized in that: In step (1), the germinated black wheat flour is the germinated black wheat flour obtained by freeze-drying, crushing and passing through a 60-mesh sieve.

7. The method for determining multiple phenolic acid compounds in germinated black wheat according to claim 1, characterized in that: In step (1), the sample to be tested is subjected to high performance liquid chromatography within 24 hours after being prepared.

8. The method for determining multiple phenolic acid compounds in germinated black wheat according to any one of claims 1 to 7, characterized in that: ω is expressed as the average of three measurement results, and the result is rounded to two significant figures.

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