A method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits

Abstract

The present invention provides a method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits, and belongs to the technical field of chemical analysis. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits provided by the present invention separates the pulp and peel of citrus fruits, extracts and prepares pulp sample solutions and peel sample solutions respectively, and adopts liquid chromatography-mass spectrometry for detection, so that 12 phenolic acids can be detected simultaneously in the end, with short detection time, low detection limit and high accuracy.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits. Background Art

[0002] Citrus is a general term for plants of the subfamily Citrus of the family Rutaceae, mainly including sweet oranges, broad-skinned citrus, grapefruits and pomeloes, lemons, hybrid citrus and kumquats, etc. Citrus fruits are rich in phenolic acids, which not only have important functions such as anti-inflammatory, antioxidant, anti-allergic, anti-cancer, anti-mutagenic and antibacterial, but their content and distribution are also important indicators for evaluating the quality and maturity of citrus fruits.

[0003] At present, the main methods for determining phenolic acid include spectral analysis, capillary electrophoresis, chromatography, electrochemical analysis, nuclear magnetic resonance, etc., but these methods have the disadvantages of long detection time, low efficiency, high detection limit, few types of phenolic acid that can be detected simultaneously, often requiring multiple batches of detection, cumbersome work, time-consuming and large amount of work. Therefore, establishing a rapid and accurate method for detecting phenolic acid substances in citrus and clarifying the composition, distribution and biological activity of phenolic acid compounds in citrus fruits is of great significance for the development and utilization of citrus fruits, breeding work, and citrus nutrition and health processing. Summary of the invention

[0004] In view of this, an object of the present invention is to provide a method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits provided by the present invention adopts liquid chromatography-mass spectrometry for detection, can simultaneously detect 12 phenolic acids, has short detection time, low detection limit and high accuracy.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits, comprising:

[0007] Step S1, preparation of sample solution: separating the peel and pulp of citrus fruit, and freezing them in a -80°C refrigerator for later use;

[0008] Taking an appropriate amount of pulp into a centrifuge tube, adding the extracting solution, mixing, ultrasonic extraction, and centrifuging to obtain a first supernatant and a first residue; adding the extracting solution to the residue again, mixing, ultrasonic extraction, and centrifuging to obtain a second supernatant and a second residue; combining the first supernatant and the second supernatant, fixing the volume, and filtering with an organic filter membrane to obtain a pulp sample solution;

[0009] Taking an appropriate amount of peel in a centrifuge tube, adding the extract, mixing, ultrasonic extraction, centrifuging, and obtaining a third supernatant and a third residue; adding the extract to the residue again, mixing, ultrasonic extraction, centrifuging, and obtaining a fourth supernatant and a fourth residue; combining the third supernatant and the fourth supernatant, fixing the volume, and filtering to obtain a peel sample solution;

[0010] Step S2, detection: Detection by liquid chromatography-mass spectrometry, accurately aspirate 1.0 μL of the peel sample solution, pulp sample solution and mixed standard solution, respectively, inject into liquid chromatography-mass spectrometry instrument, and determine the content of 12 phenolic acids therein, among which:

[0011] The liquid chromatography conditions are as follows: separation using a C18 chromatographic column, mobile phase A being a 2% to 5% by volume formic acid aqueous solution, mobile phase B being methanol; elution; injection volume 1.0 μL; detection time 4 min; column temperature 30° C. to 50° C.;

[0012] The mass spectrometry detection conditions were:

[0013] The negative ion detection mode was adopted in the scanning mode of multiple reaction monitoring, the ion source was ESI source, the ion spray voltage was -4500V, the ionization temperature was 550°C, the curtain gas was 35psi, the collision gas pressure was 9bar, the ion source gas was 150psi, the ion source gas was 255psi, the inlet potential was -10.0V, and the unit outlet potential was -12V. The mass spectrometry detection parameters for each compound to be tested were:

[0014] Gentisic acid: m / z 153→108, CE=-30V, DP=-60V, m / z 153→109, CE=-18V, DP=-58V;

[0015] Gallic acid: m / z169→125, CE=-20V, DP=-70V, m / z169→79, CE=-32V, DP=-63V;

[0016] p-Coumaric acid: m / z 163.1→119.1, CE=-21V, DP=-46V, m / z 163.1→90.9, CE=-42V, DP=-180V;

[0017] Salicylic acid: m / z 137→65.1, CE=-37V, DP=-56V, m / z 137→93, CE=-22V, DP=-50V;

[0018] 4-Hydroxybenzoic acid: m / z 137.1→93, CE=-19V, DP=-30V, m / z 137.1→119.1, CE=-16V, DP=-30V;

[0019] Caffeic acid: m / z 179→134, CE=-35V, DP=-56V, m / z 179→135, CE=-25V, DP=-56V;

[0020] Coumaric acid: m / z 139→51, CE=-15V, DP=-23V, m / z 139→95, CE=-14V, DP=-22V;

[0021] Ferulic acid: m / z 193.2→134, CE=-21V, DP=-50V, m / z 193.2→178.1, CE=-18V, DP=-72V;

[0022] Protocatechuic acid: m / z 153→90.9, CE=-35V, DP=-55V, m / z 153→109, CE=-22V, DP=-50V;

[0023] Sinapic acid: m / z 223.1→208, CE=-18V, DP=-70V, m / z 223.1→164.1, CE=-19V, DP=-70V;

[0024] Vanillic acid: m / z 167.1→152.1, CE=-16V, DP=-68V, m / z 167.1→108.1, CE=-30V, DP=-43V;

[0025] Cinnamic acid: m / z 147→103, CE=-17V, DP=-38V, m / z 147→77, CE=-30V, DP=-35V;

[0026] The data acquisition software is SCIEX OS 2.0 software.

[0027] Preferably, the method further comprises the preparation of a mixed standard solution, wherein the preparation steps of the mixed standard solution are as follows: accurately weighing each phenolic acid standard, dissolving them with chromatographically pure methanol and making up to volume, respectively preparing a standard stock solution with a mass concentration of 1.0 mg / mL; accurately measuring an appropriate amount of each standard stock solution and placing it in the same volumetric flask, diluting it with chromatographically pure methanol to prepare a series of mixed reference solutions, and storing them at 4°C in the dark.

[0028] Preferably, when the citrus is orange or sweet orange, the pulp sample needs to retain the husk and remove the seeds; when the citrus is pomelo, the pulp sample needs to remove the husk and seeds.

[0029] Preferably, the extract in step S1 is a mixed aqueous solution of methanol and formic acid, wherein the volume ratio of methanol, water and formic acid is 70:28:2, and the amount of the extract added is 2 to 5 times the weight of the pulp or the pulp.

[0030] Preferably, the frequency of the ultrasonic extraction is 40kHz, and the extraction time is 25min to 35min; the rotation speed of the centrifuge is 7000r / min to 9000r / min, and the centrifugation time is 13min to 18min; and the filtration is performed using a 0.22μm microporous organic filter membrane.

[0031] Preferably, the volume in step S1 is adjusted to 25 mL to 100 mL using chromatographically pure methanol.

[0032] Preferably, the elution flow rate in step S2 is 0.3000 mL / min.

[0033] Beneficial technical effects: The present invention provides a method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits. The pulp and peel of citrus fruits are separated, and pulp sample solutions and peel sample solutions are extracted and prepared respectively. Liquid chromatography-mass spectrometry is used for detection, and finally 12 phenolic acids can be detected simultaneously with short detection time, low detection limit and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A flow chart of the method for detecting the phenolic acid content in citrus fruits;

[0035] Figure 2 Flow chart of the method for detecting the phenolic acid content in different citrus fruits;

[0036] Figure 3 The following are the structural diagrams of 12 phenolic acids;

[0037] Figure 4 The following are representative extracted ion patterns of 12 phenolic acid monomer standards;

[0038] Figure 5 The following are representative extracted ion patterns of the mixed standard of 12 phenolic acids. DETAILED DESCRIPTION

[0039] The present invention provides a method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits, comprising:

[0040] Step S1, preparation of sample solution: separating the peel and pulp of citrus fruit, and freezing them in a -80°C refrigerator for later use;

[0041] In the present invention, the 12 phenolic acids are protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, coumaric acid, ferulic acid, sinapic acid, caffeic acid, p-coumaric acid, salicylic acid, gallic acid, gentisic acid and cinnamic acid; the structural diagrams of the 12 phenolic acids are shown in the attached Figure 3 .

[0042] In the present invention, before separating the peel and pulp of citrus fruits, it is necessary to gently wipe off the attachments on the surface of the citrus samples with clean gauze; the peel and pulp of citrus fruits are separated by using a diagonal segmentation method, taking the diagonal part to separate the peel and the pulp; when the citrus fruits are broad-skinned mandarin oranges and sweet oranges, the pulp samples need to retain the husk and remove the seeds; when the citrus fruits are pomelo, the husk and seeds need to be removed from the pulp samples.

[0043] Taking an appropriate amount of pulp into a centrifuge tube, adding the extracting solution, mixing, ultrasonic extraction, and centrifuging to obtain a first supernatant and a first residue; adding the extracting solution to the residue again, mixing, ultrasonic extraction, and centrifuging to obtain a second supernatant and a second residue; combining the first supernatant and the second supernatant, fixing the volume, and filtering with an organic filter membrane to obtain a pulp sample solution;

[0044] Taking an appropriate amount of peel in a centrifuge tube, adding the extract, mixing, ultrasonic extraction, centrifuging, and obtaining a third supernatant and a third residue; adding the extract to the residue again, mixing, ultrasonic extraction, centrifuging, and obtaining a fourth supernatant and a fourth residue; combining the third supernatant and the fourth supernatant, fixing the volume, and filtering to obtain a peel sample solution;

[0045] In the present invention, the extract in step S1 is preferably a mixed aqueous solution of methanol and formic acid, wherein the volume ratio of methanol, water and formic acid is preferably 70:28:2, and the amount of the extract added is preferably 2 to 5 times the weight of the pulp or pulp, and more preferably 3 to 4 times the weight of the pulp or pulp; the frequency of the ultrasonic extraction is preferably 40kHz, and the extraction time is preferably 25min to 35min, and more preferably 30min; the speed of the centrifugation is preferably 7000r / min to 9000r / min, and more preferably 8000r / min, and the centrifugation time is preferably 13min to 18min, and more preferably 15min; the filtration is preferably performed by filtration with a 0.22μm microporous organic filter membrane; the constant volume in step S1 is preferably constant volume with chromatographic pure methanol, and the constant volume is preferably selected according to the volume after the supernatant is combined.

[0046] The present invention also includes the preparation of a mixed standard solution: accurately weigh each phenolic acid standard, dissolve them in chromatographically pure methanol and make up to volume, respectively, to prepare a standard stock solution with a mass concentration of 1.0 mg / mL; accurately measure an appropriate amount of each standard stock solution and place it in the same volumetric flask, dilute it with chromatographically pure methanol, prepare a series of mixed reference solutions, and store them at 4°C in the dark.

[0047] Step S2, detection: Detection by liquid chromatography-mass spectrometry, accurately aspirate 1.0 μL of the peel sample solution, pulp sample solution and mixed standard solution, respectively, inject into liquid chromatography-mass spectrometry instrument, and determine the content of 12 phenolic acids therein, among which:

[0048] The liquid chromatography conditions are as follows: separation using a C18 chromatographic column, mobile phase A is a 2% to 5% by volume formic acid aqueous solution, mobile phase B is methanol; gradient elution; injection volume 1.0 μL; detection time 4 min; column temperature 30° C. to 50° C.; flow rate 0.3000 mL / min;

[0049] In the present invention, the C18 chromatographic column is preferably Kinetex C18 (3.0×150 mm, 2.6 μm), phenomenex, USA; the elution conditions in step S2 are shown in Table 1.

[0050] Table 1 Gradient elution parameter settings

[0051] time Flow rate Mobile phase A Mobile phase B 1 0.00 0.3000 50.0 50.0 2 4.00 0.3000 50.0 50.0

[0052] The mass spectrometry detection conditions are as follows: negative ion detection mode under the scanning mode of multiple reaction monitoring, the ion source is ESI source; the ion spray voltage is -4500 V; the ionization temperature is 550 ° C; the curtain gas is 35 psi; the collision gas pressure is 9 bar; the ion source gas is 150 psi; the ion source gas is 255 psi; the inlet potential is -10.0 V; the unit outlet potential is -12 V; the mass spectrometry detection parameters for each compound to be tested are shown in Table 2; the data acquisition software is SCIEX OS 2.0 software.

[0053] Table 2 Mass spectrometry detection parameters of 12 phenolic acids

[0054]

[0055]

[0056] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0057] Example 1

[0058] (1) Preparation of sample solution:

[0059] Samples and sources: Six kinds of citrus fruits were collected at the mature stage, among which sample 1 was Miyagawa fruit, collected on October 25, 2021; sample 2 was Ponkan fruit, collected on November 15, 2021; sample 3 was Yura Wenzhou mandarin fruit, collected on October 10, 2021; sample 4 was Red Beauty fruit, collected on November 15, 2021; sample 5 was Oita fruit, collected on September 21, 2021; sample 6 was local early fruit, collected on November 15, 2021. The above samples were all collected in the orange orchard of Zhejiang Citrus Research Institute, and the sampling time was the mature and market period of each variety.

[0060] Sample collection method: Select contiguous orange trees and use the diagonal method to arrange sampling points. Draw a diagonal line from one corner of the sampling area to the other corner, and arrange sampling points at equal distances on this line. Set a total of 5 sampling points. When sampling, evenly pick about 1kg of fruit from the top, middle, bottom, inside, outside and fruit bearing directions (east, south, west and north) of the orange tree. Mix the fruits picked at each point thoroughly, reduce them by the quartering method, and finally take the required number of portions, each about 1kg, put them into bags, stick labels, and tie the bags tightly. The sampled orange trees are similar in age, growth, and number of fruits.

[0061] Sample pretreatment: The pretreatment methods of the above 6 different varieties of citrus samples were the same. Use clean gauze to gently wipe off the attachments on the surface of the citrus fruit. Use the diagonal segmentation method to take the diagonal part, separate the peel and the pulp, and freeze them in a -80℃ refrigerator for later use.

[0062] Preparation of pulp sample solution: accurately weigh 1.0 g of pulp sample into a 10 mL centrifuge tube, add 2.0 mL of extracting solution (V methanol: V water: V formic acid = 70: 28: 2) under light-proof conditions, mix thoroughly, perform ultrasonic extraction for 30 min, centrifuge at 4 ° C and 8000 r / min for 15 min, take out the centrifuge tube, and collect the supernatant; add 2.0 mL of extracting solution to the residue, repeat ultrasonic extraction once, combine the supernatants of the two extractions, make the volume to 10 mL with methanol, and filter through a 0.22 μm microporous organic filter membrane to obtain the solution.

[0063] Preparation of peel sample solution: accurately weigh 0.5 g of sample into a 10 mL centrifuge tube, add 2.0 mL of extracting solution (V methanol: V water: V formic acid = 70: 28: 2) under light-proof conditions, mix thoroughly, perform ultrasonic extraction for 30 min, centrifuge at 4 ° C and 8000 r / min for 15 min, take out the centrifuge tube, and collect the supernatant; add 2.0 mL of extracting solution to the residue, repeat ultrasonic extraction once, combine the supernatants of the two extractions, make the volume to 10 mL with methanol, and filter through a 0.22 μm microporous organic filter membrane to obtain the result.

[0064] (2) Preparation of standard substances: Accurately weigh each phenolic acid standard substance, dissolve it in chromatographic grade methanol and make up to volume to prepare a standard stock solution with a mass concentration of 1.0 mg / mL; accurately measure an appropriate amount of the stock solution and mix it in a volumetric flask, dilute it with methanol to prepare a series of mixed reference substance solutions, and store them at 4°C in the dark.

[0065] (3) Detection conditions: Liquid chromatography conditions: Kinetex C18 (3.0×150mm, 2.6μm) chromatographic column was used for separation, mobile phase A was 2% to 5% formic acid aqueous solution by volume, and mobile phase B was methanol; the gradient elution parameter settings are shown in Table 1; injection volume 1.0μL; detection time 4min; column temperature 30℃~50℃; flow rate 0.3000mL / min;

[0066] The mass spectrometry detection conditions were as follows: negative ion detection mode under the scanning mode of multiple reaction monitoring, the ion source was ESI source; the ion spray voltage was -4500 V; the ionization temperature was 550 °C; the curtain gas was 35 psi; the collision gas pressure was 9 bar; the ion source gas was 150 psi; the ion source gas was 255 psi; the inlet potential was -10.0 V; the unit outlet potential was -12 V; the mass spectrometry detection parameter settings for each compound to be tested are shown in Table 2; the data acquisition software was SCIEX OS 2.0 software.

[0067] (4) Detection: 1.0 μL of the peel sample solution, pulp sample solution and mixed standard solution were accurately aspirated and injected into a liquid chromatography-mass spectrometer to determine the contents of the 12 phenolic acids therein. The test results are shown in Tables 3 to 5.

[0068] Table 3 Solvent standard curve and matrix calibration curve of phenolic acids

[0069]

[0070]

[0071]

[0072] Table 4 Determination results of phenolic acid content in the pulp of different citrus samples (mg / kg·FW)

[0073]

[0074] Table 5 Determination results of phenolic acid content in peels of different citrus samples

[0075]

[0076]

[0077] (5) Method accuracy (recovery rate) test:

[0078] Matrix solution extraction: (a) Preparation of pulp matrix solution: Accurately weigh 1.0 g of citrus pulp sample into a 10 mL centrifuge tube, add 2.0 mL of extracting solution (V methanol: V water: V formic acid = 70:28:2) under light-proof conditions, mix thoroughly, extract by ultrasonication for 30 min, centrifuge at 4°C, 8000 r / min for 15 min, remove the centrifuge tube, and collect the supernatant; add 2.0 mL of extracting solution to the residue, repeat the ultrasonic extraction once, combine the supernatants of the two extractions, make up to 10 mL with methanol, and filter through a 0.22 μm microporous organic filter membrane to obtain the matrix solution. (b) Preparation of peel matrix solution: Accurately weigh 0.5 g of citrus peel sample into a 10 mL centrifuge tube, add 2.0 mL of extract solution (V methanol: V water: V formic acid = 70: 28: 2) under light-proof conditions, mix thoroughly, perform ultrasonic extraction for 30 min, centrifuge at 4°C and 8000 r / min for 15 min, remove the centrifuge tube, and collect the supernatant; add 2.0 mL of extract solution to the residue, repeat ultrasonic extraction once, combine the supernatants of the two extractions, make the volume up to 10 mL with methanol, and filter through a 0.22 μm microporous organic filter membrane to obtain the matrix solution.

[0079] Add standard solution: Add different levels of 12 phenolic acid standard solutions to the blank citrus matrix (peel, pulp) and determine the recovery rate at each addition level. The addition levels were 0.5, 2.5, 50, and 250 μg / kg, and each level was repeated 5 times. Add to the two matrices respectively, mix well, let stand for 1 hour, and test on the machine. The test results are shown in Table 6.

[0080] Table 6 Recovery of phenolic acids added to citrus peel and pulp

[0081]

[0082]

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits, characterized in that: include: Step S1, preparation of sample solution: separating the peel and pulp of citrus fruit, and freezing them in a -80°C refrigerator for later use; Taking an appropriate amount of pulp into a centrifuge tube, adding the extracting solution, mixing, ultrasonic extraction, and centrifuging to obtain a first supernatant and a first residue; adding the extracting solution to the residue again, mixing, ultrasonic extraction, and centrifuging to obtain a second supernatant and a second residue; combining the first supernatant and the second supernatant, fixing the volume, and filtering with an organic filter membrane to obtain a pulp sample solution; Taking an appropriate amount of peel in a centrifuge tube, adding the extract, mixing, ultrasonic extraction, centrifuging, and obtaining a third supernatant and a third residue; adding the extract to the residue again, mixing, ultrasonic extraction, centrifuging, and obtaining a fourth supernatant and a fourth residue; combining the third supernatant and the fourth supernatant, fixing the volume, and filtering to obtain a peel sample solution; Step S2, detection: liquid chromatography-mass spectrometry is used for detection, 1.0 μL of the peel sample solution, pulp sample solution and mixed standard solution are respectively accurately aspirated, injected into a liquid chromatography-mass spectrometer, and the contents of 12 phenolic acids therein are determined, wherein the 12 phenolic acids are protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, coumaric acid, ferulic acid, sinapic acid, caffeic acid, p-coumaric acid, salicylic acid, gallic acid, gentisic acid and cinnamic acid; wherein: The liquid chromatography conditions are as follows: using a C18 chromatographic column for separation, mobile phase A is a 2% to 5% by volume formic acid aqueous solution, and mobile phase B is methanol; elution; injection volume 1.0 μL; detection time 4 min; column temperature 30°C to 50°C; the extract is a mixed aqueous solution of methanol and formic acid, wherein the volume ratio of methanol, water, and formic acid is 70:28:2; the chromatographic elution conditions are: 0-4 min, and the ratio of mobile phase A to mobile phase B is 50:50; The mass spectrometry detection conditions were as follows: negative ion detection mode under the scanning mode of multiple reaction monitoring, the ion source was ESI source; the ion spray voltage was -4500 V; the ionization temperature was 550 °C; the curtain gas was 35 psi; The collision gas pressure was 9 bar; the ion source gas was 150 psi; the ion source gas was 255 psi; the inlet potential was -10.0 V; the cell outlet potential was -12 V; the mass spectrometry detection parameters for each compound to be tested were: Gentisic acid: m / z 153→108, CE=-30V, DP=-60V, m / z 153→109, CE=-18V, DP=-58V; Gallic acid: m / z169→125, CE=-20V, DP=-70V, m / z169→79, CE=-32V, DP=-63V; p-Coumaric acid: m / z 163.1→119.1, CE=-21V, DP=-46V, m / z 163.1→90.9, CE=-42V, DP=-180V; Salicylic acid: m / z 137→65.1, CE=-37V, DP=-56V, m / z 137→93, CE=-22V, DP=-50V; 4-Hydroxybenzoic acid: m / z 137.1→93, CE=-19V, DP=-30V, m / z 137.1→119.1, CE=-16V, DP=-30V; Caffeic acid: m / z 179→134, CE=-35V, DP=-56V, m / z 179→135, CE=-25V, DP=-56V; Coumaric acid: m / z 139→51, CE=-15V, DP=-23V, m / z 139→95, CE=-14V, DP=-22V; Ferulic acid: m / z 193.2→134, CE=-21V, DP=-50V, m / z 193.2→178.1, CE=-18V, DP=-72V; Protocatechuic acid: m / z 153→90.9, CE=-35V, DP=-55V, m / z 153→109, CE=-22V, DP=-50V; Sinapic acid: m / z 223.1→208, CE=-18V, DP=-70V, m / z 223.1→164.1, CE=-19V, DP=-70V; Vanillic acid: m / z 167.1→152.1, CE=-16V, DP=-68V, m / z 167.1→108.1, CE=-30V, DP=-43V; Cinnamic acid: m / z 147→103, CE=-17V, DP=-38V, m / z 147→77, CE=-30V, DP=-35V; The data acquisition software is SCIEX OS2.0 software.

2. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits according to claim 1, characterized in that: The method also includes the preparation of a mixed standard solution, wherein the preparation steps of the mixed standard solution are: Accurately weigh each phenolic acid standard, dissolve it in chromatographic grade methanol and make up to volume to prepare a standard stock solution with a mass concentration of 1.0 mg / mL; Accurately measure an appropriate amount of each standard stock solution and place it in the same volumetric flask, dilute it with chromatographically pure methanol to prepare a series of mixed reference solutions, and store them at 4°C away from light.

3. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits according to claim 1, characterized in that: When the citrus is tangerine or sweet orange, the pulp sample needs to retain the husk and remove the seeds; when the citrus is pomelo, the pulp sample needs to remove the husk and seeds.

4. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits according to any one of claims 1 to 3, characterized in that: The amount of the extract added in step S1 is 2 to 10 times the weight of the peel or pulp.

5. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits according to claim 4, characterized in that: The frequency of the ultrasonic extraction is 40kHz, and the extraction time is 25min-35min; the rotation speed of the centrifuge is 7000r / min-9000r / min, and the centrifugation time is 13min-18min; the filtration is performed by filtering with a 0.22μm microporous organic filter membrane.

6. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits according to claim 1, characterized in that: In step S1, the volume is adjusted to 25 mL to 100 mL using chromatographically pure methanol.

7. The method for simultaneously detecting the contents of 12 phenolic acids in citrus fruits according to claim 1, characterized in that: The flow rate of the mobile phase in step S2 is 0.3000 mL / min.

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