A method for determining content of organic acid metabolites of fructus mume in colon tissue and application thereof

Abstract

The present invention discloses a method for determining the content of organic acid metabolites of black plum in colon tissue and its application. The determination method comprises the following steps: (1) adding sterile physiological saline to the colon tissue, grinding, adding methanol to continue grinding, centrifuging, collecting the supernatant, adding methanol and vortexing, centrifuging, and finally adding water and vortexing to obtain a sample solution to be tested; (2) using high performance liquid chromatography-mass spectrometry to draw standard curves of the three organic acids using citric acid, malic acid, and quinic acid standards, wherein citric acid is used in positive ion mode, and malic acid and quinic acid are used in negative ion mode; (3) using the same method to detect the sample to be tested, and the concentration or content of the three organic acid metabolites (citric acid, malic acid, and quinic acid) can be calculated based on the standard curve. The method of the present invention is simple and highly sensitive and can be used to study the mechanism of the effect of black plum on ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for determining the content of organic acid metabolites of ebony in colon tissue and an application thereof. Background Art

[0002] Wumei (Prunus mume) is the nearly ripe fruit of the Rosaceae plant, Prunus mume. Its use as a medicine was first documented in the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica). In modern clinical practice, Wumei is a commonly used remedy for diarrheal illnesses. It has a certain effect on relieving symptoms such as persistent diarrhea and loose stools caused by ulcerative colitis, helping to reduce bowel movements and improve diarrheal symptoms. Wumei is also a representative sour herb in traditional Chinese medicine. Organic acids are the primary active substances in Wumei and form the basis of these sour herbs. Twenty organic acids have been isolated from Wumei in free form, including citric acid, malic acid, succinic acid, and chlorogenic acid. Citric and malic acid are found in higher concentrations, while chlorogenic acid is converted to quinic acid. Organic acids can improve the intestinal microecology and help alleviate the symptoms of ulcerative colitis. Ulcerative colitis lesions are primarily located in the colon, but there have been no reports on measuring organic acids in colonic tissue to investigate the therapeutic mechanisms and dosage relationships of Wumei and guide clinical medication use.

[0003] The high sensitivity and specificity of high-performance liquid chromatography-mass spectrometry (HPLC-MS) have been widely used in drug analysis. It offers significant advantages for the qualitative and quantitative analysis of trace substances in complex samples, and HPLC-MS is often used for chemical composition analysis of biological samples. Because complex components in biological samples can interfere with the detection signal of the analytes, sample pretreatment techniques such as protein precipitation, liquid-liquid extraction, and solid-phase extraction columns are required to extract and purify the biological samples. Liquid chromatography is then used to separate the samples before they can be detected and analyzed by mass spectrometry. Therefore, sample pretreatment is a key technology.

[0004] Among current sample pretreatment techniques, protein precipitation involves adding several times the volume of an organic solvent (such as methanol or acetonitrile) to a biological sample to denature and precipitate the protein in the sample. After centrifugation, the supernatant is diluted and analyzed by liquid chromatography-mass spectrometry. The problems this technique faces with black plum metabolites are: (1) Blood samples can be directly precipitated with organic solvents, and the supernatant is then analyzed after centrifugation. However, colon tissue is a solid sample, unlike blood, and cannot be directly treated with organic solvents. Solid-liquid extraction cannot extract the target compound; (2) Metabolites are present in colon tissue, and sample processing requires extraction of the target compound in addition to protein precipitation. (3) There are no literature reports on how to process and extract colon tissue.

[0005] Liquid-liquid extraction involves extracting the analyte into an organic phase using a water-immiscible organic solvent and the biological sample. The organic solvent is then evaporated and reconstituted to yield a test solution. This technique faces a problem with ebony plum metabolites: the target metabolites are all water-soluble organic acids, making them unsuitable for liquid-liquid extraction.

[0006] The solid phase extraction cartridge method is to enrich, separate, and purify the sample by selective adsorption and selective elution. The biological sample passes through the solid phase extraction cartridge, and the interfering substances are washed away with the eluent. The target substance is adsorbed on the cartridge and eluted with the eluent. The eluted liquid nitrogen is collected and blown dry, redissolved, centrifuged, and the supernatant is measured. The problem faced by this technology for the metabolites of black plum is that the target substances to be measured in the metabolites of black plum are all water-soluble organic acids, which are easily soluble in water and methanol. During elution, the target substances are washed out together, and the target substances cannot be detected. Without suitable eluents and eluents, the solid phase extraction cartridge method is not suitable.

[0007] Citric acid, malic acid, and quinic acid can all be determined using high-performance liquid chromatography (HPLC). However, HPLC is less sensitive than mass spectrometry, and the low levels in biological samples and the high levels of interference can prevent detection. Therefore, LC-MS is often used. LC-MS can be used to determine the chemical components of black plums. For example, Chinese invention patent No. 202210297928.5 discloses a method for determining the content of chemical components in black plums. However, it does not disclose a method for simultaneously determining the three organic acids (citric acid, malic acid, and quinic acid) in colonic tissue. The metabolites of black plum organic acids are small-molecule organic acids, and these organic acids are also metabolites in the body. Since citric acid and quinic acid have essentially the same molecular weight, their parent ions and fragment ions share similarities. If these interferences are not distinguished during chromatographic-mass spectrometric separation, the accurate quantification of the target analyte will be affected. Therefore, the screening of chromatographic-mass spectrometric conditions is also a key technology. Summary of the Invention

[0008] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for determining the content of organic acid metabolites of black plum in colon tissue.

[0009] Another object of the present invention is to provide an application of the method for determining the content of organic acid metabolites of ebony in colon tissue.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A method for determining the content of organic acid metabolites of ebony in colon tissue comprises the following steps:

[0012] (1) Colon tissue pretreatment:

[0013] Sterile physiological saline (grinding agent) is added to the colon tissue, and the tissue is ground. Methanol (protein precipitant) is then added and ground again. The tissue is centrifuged (first centrifugation) and the supernatant is collected to obtain supernatant I. Methanol (extraction agent) is then added and vortexed, followed by centrifugation (second centrifugation) to obtain supernatant II. Finally, water (extraction agent) is added and vortexed to obtain the sample solution to be tested.

[0014] (2) Draw the standard curves of three organic acids:

[0015] Citric acid, malic acid, and quinic acid were prepared into concentration gradient standard curve working solutions using methanol-water solutions. High-performance liquid chromatography-mass spectrometry was then used for detection, with citric acid in positive ion mode and malic acid and quinic acid in negative ion mode. The chromatographic peak area of ​​each organic acid was obtained, and standard curves were plotted with the peak area as the ordinate (Y) and the mass concentration of the standard curve working solution as the abscissa (X). Linear equations for citric acid, malic acid, and quinic acid were obtained, respectively.

[0016] (3) Determination of the content of three organic acids:

[0017] The sample solution obtained in step (1) is detected by high performance liquid chromatography-mass spectrometry, wherein citric acid is detected in positive ion mode, and malic acid and quinic acid are detected in negative ion mode, to obtain the chromatographic peak area of ​​each organic acid, and then substitute it into the linear equation obtained in step (2) to calculate the concentration and / or content of citric acid, malic acid, and quinic acid, respectively.

[0018] The colon tissue described in step (1) is animal colon tissue; preferably rat colon tissue; further preferably rat colon tissue after treatment with black plum decoction for ulcerative colitis.

[0019] In step (1), the colon tissue can be frozen after separation and then taken out and placed at room temperature when needed.

[0020] The material-liquid ratio of the colon tissue and sterile saline in step (1) is 50 mg to 200 mg: 350 μl to 700 μl; preferably 100 mg: 700 μl.

[0021] The grinding described in step (1) is performed using a cryogenic grinding apparatus.

[0022] The grinding conditions described in step (1) are: using 2-4 mm zirconia grinding beads, intermittent grinding at 50-70 Hz and 4°C for 5-10 minutes; preferably: using 3 mm zirconia grinding beads, intermittent grinding at 70 Hz and 4°C for 10 minutes.

[0023] In step (1), the amount of methanol (protein precipitant) added for the first time is calculated based on 150 μl to 300 μl of methanol per 100 mg of colon tissue; preferably, it is calculated based on 200 to 300 μl of methanol per 100 mg of colon tissue.

[0024] In step (1), the conditions for the two centrifugations are: 4°C, 10,000 r / min to 12,000 r / min for 10 to 15 min; preferably: 4°C, 10,000 r / min for 15 min, or 4°C, 12,000 r / min for 10 min.

[0025] In step (1), the volume ratio of methanol (extraction agent) added for the second time to the supernatant I is (2-5):1; preferably 2:1.

[0026] The vortexing time in step (1) is 0.5 min to 2 min, preferably 1 min.

[0027] The dosage ratio of the supernatant II and water in step (1) can be adjusted according to the concentration of the analyte, and the volume ratio is preferably 1: (1 to 3); more preferably 1:1.

[0028] The concentration of the methanol aqueous solution in step (2) is 30 to 50% by volume, preferably 50% by volume.

[0029] In step (2), the standard curve working solution with a concentration gradient is prepared by preparing 5 to 9 standard curve working solutions containing citric acid, malic acid and quinic acid with different known concentrations.

[0030] In step (2), the concentration range of citric acid in the standard curve working solution is 500-60000 ng / mL, the concentration range of malic acid is 50-25000 ng / mL, and the concentration range of quinic acid is 40-5000 ng / mL.

[0031] The parameters of the high performance liquid chromatography described in steps (2) and (3) are as follows: chromatographic column Agilent ZORBAXRx-C18 (4.5×150mm, 5μm); mobile phase A is an aqueous solution containing 0.1% by volume of formic acid, mobile phase B is methanol, and the gradient elution is: 0-5min, 5%B; 5-7min, 5%-80%B; 7-8min, 80%-5%B; 9min, 5%B; column temperature: 35-40°C (preferably 40°C); flow rate: 0.2-0.4mL / min (preferably 0.3ml / min); injection volume: 1-3μL (preferably 1μl).

[0032] The mass spectrometry parameters in steps (2) and (3) are as follows: electrospray ionization (ESI), ion source temperature: 550°C, nebulizer pressure: 50 Pa; capillary voltage 4500 V; collision gas: high-purity nitrogen, collision gas pressure medium; ionization mode: electrospray, positive and negative ion mode, multiple reaction monitoring (MRM) mode, ion pairs: citric acid Q1 is 193.100, Q3 is 111.000, DP is 40 V, CE is 24 V, EP is 10 V, CXP is 11 V; malic acid Q1 is 133.000, Q3 is 115.000, DP is -50 V, CE is -15 V, EP is -10 V, CXP is -16 V; quinic acid Q1 is 191.100, Q3 is 93.000, DP is -90 V, CE is -29 V, EP is -10 V, CXP is -16 V.

[0033] In step (3), the retention time of quinic acid is 3.9 min, the retention time of malic acid is 4.2 min, and the retention time of citric acid is 4.8 min.

[0034] In step (3), the concentration C1 of each organic acid can be obtained based on the chromatographic peak area of ​​each organic acid in the sample solution to be tested and the standard curve of each organic acid, and then the content X of each component of the sample to be tested can be calculated according to the formula X = C1 × V / M (wherein, C1: concentration of organic acid; V: volume of the sample solution to be tested; M: sample weight).

[0035] The method for determining the content of organic acid metabolites of black plum in colon tissue is used in determining organic acids for purposes other than disease diagnosis and treatment.

[0036] The organic acid includes at least one of citric acid, malic acid and quinic acid; preferably citric acid and quinic acid, or citric acid, malic acid and quinic acid.

[0037] The present invention has the following advantages and effects compared to the prior art:

[0038] 1. The present invention provides a method for determining the content of organic acid metabolites of black plum in rat colon tissue, which adopts high performance liquid chromatography-mass spectrometry (LC-MS / MS) technology. By pre-treating the colon tissue, organic acids in the colon tissue are extracted, and appropriate chromatographic conditions and mass spectrometry conditions are selected, the content of citric acid, malic acid, and quinic acid metabolites of black plum in rat colon tissue (black plum decoction for the treatment of ulcerative colitis) can be simultaneously determined. The method has the advantages of simplicity, high sensitivity, and strong specificity, and can thus be used for studying the mechanism of black plum's effect on ulcerative colitis, dose relationship research, and guiding clinical medication.

[0039] 2. The present invention adopts a liquid chromatography-mass spectrometer to determine the chromatographic peak of each organic acid according to the characteristic ion peak of each organic acid in the mixed reference solution through mass spectrometry detection, and obtain the chromatographic peak area of ​​each organic acid; and establish a standard curve for each organic acid with the peak area of ​​each organic acid chromatographic peak as the ordinate and the concentration of each organic acid as the abscissa; then, under the same chromatographic conditions and mass spectrometry conditions, determine the chromatographic peak of each organic acid in the test sample according to the characteristic ion peak through mass spectrometry detection, obtain the chromatographic peak area of ​​each organic acid, and finally calculate the content of each organic acid in the sample according to the standard curve method; wherein, quinic acid uses a negative ion mode and citric acid uses a positive ion mode, so that the chromatographic peaks of citric acid and quinic acid can be completely separated, the interference peak is effectively avoided, and citric acid, malic acid, and quinic acid can be accurately analyzed at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 1 is the characteristic ion chromatogram of the mixed reference substance in Example 1; wherein A is the characteristic ion chromatogram of citric acid; B is the characteristic ion chromatogram of malic acid; and C is the characteristic ion chromatogram of quinic acid. DETAILED DESCRIPTION

[0041] The present invention will be described in further detail below in conjunction with the examples, but embodiments of the present invention are not limited thereto. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually based on conventional experimental conditions or the experimental conditions recommended by the manufacturer. Unless otherwise stated, the reagents and raw materials used in the present invention can be obtained commercially.

[0042] Example 1 Determination of the content of organic acid metabolites of black plum in rat colon tissue

[0043] This example provides a method for determining the content of organic acid metabolites of black plum in rat colon tissue, using high-performance liquid chromatography-mass spectrometry to determine the content of organic acids in black plum metabolites; the organic acids include citric acid, malic acid, and quinic acid. The specific steps are as follows:

[0044] 1. Liquid chromatography and mass spectrometry conditions

[0045] 1.1 Liquid chromatography conditions are as follows:

[0046] The chromatographic column was Agilent ZORBAX Rx-C18, 4.5×150 mm, 5 μm; the mobile phase A was water (0.1% (v / v) formic acid), the mobile phase B was methanol, and the gradient elution was (0–5 min, 5% B; 5–7 min, 5%–80% B; 7–8 min, 80%–5% B; 9 min, 5% B); the column temperature was 40°C; the flow rate was 0.3 ml / min; and the injection volume was 1 μl.

[0047] 1.2 Mass spectrometry conditions are as follows:

[0048] Electrospray ionization (ESI) was used in multiple reaction monitoring (MRM) scanning mode. Positive ionization mode was used for citric acid, and negative ionization mode was used for malic and quinic acid. The source temperature was 550°C; the nebulizer pressure was 50 Pa; and the capillary voltage was 4500 V. The collision gas was nitrogen; the collision gas pressure was medium. The ion pairs for citric acid were: Q1: 193.100, Q3: 111.000, DP: 40 V, CE: 24 V, EP: 10 V, CXP: 11 V; Q1: 133.000, Q3: 115.000, DP: -50 V, CE: -15 V, EP: -10 V, CXP: -16 V; and Q1: 191.100, Q3: 93.000, DP: -90 V, CE: -29 V, EP: -10 V, CXP: -16 V for malic acid.

[0049] 2. Instruments, reagents, and materials used

[0050] 2.1 Required instruments and reagents

[0051] Instruments: Shimadzu ultra-high performance liquid chromatography-mass spectrometry instrument AB sciex5500 Qtrap triple quadrupole mass spectrometer (AB Company, USA); electronic balance, Seville cryogenic grinding apparatus (KZ-Ⅲ-F / FP); vortex mixer (VortexMixer XW-80A); refrigerated high-speed centrifuge (Xiangyi H2050R).

[0052] Reagents: Methanol was of mass spectrometry grade, formic acid was of chromatography grade, and water was ultrapure water.

[0053] 2.2 Required experimental materials

[0054] (1) Reference substances: Citric acid was purchased from the China Food and Drug Inspection Institute, batch number: 100396-202104, purity 99.7%; the malic acid used was L-malic acid, purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number: A271B213943, purity ≥98%; quinic acid was purchased from Chengdu Sodium Columbium Lithium Biotechnology Co., Ltd., batch number: nkl-00907241212, purity 99.3%.

[0055] (2) Black plum, purchased from Beijing Tong Ren Tang Pharmaceutical Co., Ltd.

[0056] (3) Experimental animals and groups:

[0057] Forty SPF SD male rats were randomly divided into blank control group, model group, high-dose black plum group, medium-dose black plum group, and low-dose black plum group, with 8 rats in each group.

[0058] The medicinal material of Prunus mume was identified as meeting the requirements of the first variety of the 2020 edition of the Chinese Pharmacopoeia. A decoction of Prunus mume flesh was prepared using a traditional decoction method: an appropriate amount of Prunus mume flesh was soaked for 30 minutes, extracted once with 8x water under reflux for 1 hour, and the filtrate was filtered. The extract was then refluxed with 5x water and extracted a second time for 30 minutes, and the filtrate was filtered. The two filtrates were combined and concentrated to the desired concentration (high dose 150 mg / ml, medium dose 75 mg / ml, low dose 37.5 mg / ml) to obtain the Prunus mume flesh decoction, which was stored at 4°C until ready for use.

[0059] Ulcerative colitis was induced using dextran sulfate sodium salt (DSS, MW: 36,000-50,000 Da) in free drinking water, in compliance with ethical animal experimentation guidelines (ethics number: 20231027003). Twenty-four hours after modeling, the doses administered to the treatment groups were calculated based on the human-animal equivalent dose ratio (BSA) of 6.3. The total daily dose of black plum pulp decoction was 1500 mg / kg for the high-dose group, 750 mg / kg for the medium-dose group, and 375 mg / kg for the low-dose group. Administration was intragastrically administered at a volume of 10 ml / kg rat body weight twice daily for 10 consecutive days. The model group and the blank control group were administered an equal volume of distilled water daily. After the last dose, the rats were fasted for 24 hours and anesthetized with 3% isoflurane inhalation. Distal colon tissue was harvested, washed with pre-chilled PBS, and cryopreserved for later use.

[0060] 3. Draw a standard curve

[0061] 3.1 Preparation of reference substance stock solutions: Accurately weigh appropriate amounts of citric acid, malic acid, and quinic acid reference substances, dissolve them in methanol, and prepare reference substance stock solutions. The concentrations of citric acid, malic acid, and quinic acid reference substance stock solutions are approximately 2 mg / ml, 1 mg / ml, and 0.5 mg / ml, respectively. Store in a refrigerator at 4°C until use.

[0062] 3.2 Preparation of standard curve working solution: Accurately measure appropriate amounts of citric acid, malic acid, and quinic acid reference stock solutions and place them in a 50ml volumetric flask. Dilute with 50% (v / v) methanol solution to prepare a mixed reference solution with a citric acid concentration of 60,000 ng / ml, a malic acid concentration of 25,000 ng / ml, and a quinic acid concentration of 5,000 ng / ml. Then, use 50% (v / v) methanol solution to prepare nine concentration gradients by doubling the solution. The concentration range of citric acid is 460-60,000 ng / ml, the concentration range of malic acid is 50-25,000 ng / ml, and the concentration range of quinic acid is 40-5,000 ng / ml.

[0063] 3.3 Drawing of the standard curve

[0064] 1) Under the same chromatographic and mass spectrometric conditions as in step 1 above, 1 μL of each mixed reference solution (the standard curve working solution with the concentration gradient prepared in step 3.2 above) was injected into the HPLC instrument. The chromatographic peaks of each organic acid were determined based on the characteristic ion peaks by mass spectrometry. Figure 1 ), and obtain the chromatographic peak area of ​​each organic acid;

[0065] 2) Draw a standard curve using the peak area as the ordinate (Y) and the mass concentration of the reference substance as the abscissa (X). Perform linear regression to establish a standard curve for each component and obtain the linear equation and correlation coefficient r for each organic acid. Gradually dilute the mixed reference solution and use the concentration of each reference substance at a signal-to-noise ratio (S / N) of 10 as the lower limit of quantification (LLOQ) for each organic acid.

[0066] 3) The linear regression equation for citric acid was Y=473X-214987, r=0.9996, with a concentration range of 460-60,000 ng / ml and a lower limit of quantification (LLOQ) of 460 ng / ml. The linear regression equation for malic acid was Y=4686X+605790, r=0.9995, with a concentration range of 50-25,000 ng / ml and a lower limit of quantification (LLOQ) of 50 ng / ml. The linear regression equation for quinine acid was Y=369X-976, r=0.9999, with a concentration range of 40-5,000 ng / ml and a lower limit of quantification (LLOQ) of 40 ng / ml.

[0067] 4. Prepare each test sample

[0068] 4.1 Preparation of quality control samples

[0069] Take the citric acid, malic acid, and quinic acid reference substance stock solutions prepared in step 3.1 above, add methanol to prepare a mixed reference substance solution containing 1,000,000 ng / ml of citric acid, 500,000 ng / ml of malic acid, and 10,000 ng / ml of quinic acid to obtain quality control sample mixed solution A; then dilute the quality control sample mixed solution A 2- and 4-fold with methanol, respectively, to obtain quality control sample mixed solutions B and C; then dilute the quality control sample mixed solution A 3-, 6-, and 12-fold with methanol, respectively, to obtain quality control sample mixed solutions E, F, and G.

[0070] 4.2 Preparation of recovery sample solution

[0071] (1) Take out the frozen blank rat colon tissue, cool it to room temperature, and weigh 100 mg; add 100 μl of quality control sample mixed solution A, B, and C respectively;

[0072] (2) Add 700 μl of sterile saline and 3 mm zirconium oxide grinding beads and grind intermittently at 70 Hz and 4°C for 10 min;

[0073] (3) Add 200 μl of methanol and continue grinding intermittently at 70 Hz and 4°C for 5 min;

[0074] (4) Centrifuge at 10,000 rpm for 15 min at 4°C;

[0075] (5) Take 200 μl of supernatant and add 400 μl of methanol, and vortex for 1 min;

[0076] (6) Centrifuge at 4°C, 10,000 rpm for 15 min;

[0077] (7) Add water to the supernatant at a volume ratio of 1:1 and vortex for 1 min to obtain the spiked test solution;

[0078] (8) Repeat the above steps (1) to (7) to prepare three spiked test solutions, which are named quality control recovery sample solutions 1-3.

[0079] 4.3 Preparation of recovered reference solution

[0080] (1) Take 100 μl of 50% (v / v) methanol solution and add 100 μl of quality control sample mixed solution A, B, and C respectively;

[0081] (2) Add 700 μl of sterile saline and 3 mm zirconium oxide grinding beads and grind intermittently at 70 Hz and 4°C for 10 minutes;

[0082] (3) Add 200 μl of methanol and continue grinding intermittently at 70 Hz and 4°C for 5 min;

[0083] (4) Centrifugation at 4°C, 10,000 rpm for 15 min;

[0084] (5) Take 200 μl of supernatant and add 400 μl of methanol, and vortex for 1 min;

[0085] (6) Centrifuge at 4°C, 10,000 rpm for 15 min;

[0086] (7) Take the supernatant and add water in a volume ratio of 1:1, vortex and mix for 1 min, and name it as the recovery reference control solution 4-6.

[0087] 4.4 Sample preparation

[0088] (1) Take out the frozen rat blank group colon tissue sample, cool it to room temperature, and weigh 100 mg;

[0089] (2) Add 700 μl of sterile saline and 3 mm zirconium oxide grinding beads and grind intermittently at 70 Hz and 4°C for 10 minutes;

[0090] (3) Add 300 μl of methanol and continue grinding intermittently at 70 Hz and 4°C for 5 min;

[0091] (4) Centrifugation at 4°C, 10,000 rpm for 15 min;

[0092] (5) Take 200 μl of supernatant and add 400 μl of methanol, and vortex for 1 min;

[0093] (6) Centrifuge at 4°C, 10,000 rpm for 15 min;

[0094] (7) Add water to the supernatant at a volume ratio of 1:1 and vortex for 1 min to mix thoroughly to obtain the test solution (blank group background).

[0095] 4.5 Preparation of Matrix Effect Samples

[0096] 4.5.1 Preparation of Matrix Quality Control Samples

[0097] Take 50 μl of the quality control sample mixed solutions E, F, and G prepared in step 4.1 above, add them to 450 μl of the test solution prepared in step 4.4 above, and mix well. Prepare three aliquots for each concentration to obtain matrix quality control sample solutions 7-9.

[0098] 4.5.2 Preparation of Matrix Reference Control Solution

[0099] Take 50 μl of the quality control sample mixed solutions E, F, and G prepared in step 4.1 above, add them to 450 μl of 50% (v / v) methanol solution and mix well to obtain matrix reference control solutions 10-12.

[0100] 4.6 Determination

[0101] According to the parameter settings in 1.1 and 1.2, measure and record the peak area of ​​the chromatogram.

[0102] 5. Methodological Validation

[0103] 5.1 Precision

[0104] 5.1.1 Intra-day Precision: Take 10 ml of the matrix reference solution prepared in step 4.5.2 above, inject it six times in a row, record the chromatogram, and calculate the mean peak area and RSD (relative standard deviation) of the reference substance. The results showed that the RSDs for citric acid, malic acid, and quinic acid were 2.11%, 1.76%, and 1.26%, respectively.

[0105] 5.1.2 Inter-Day Precision: Take the recovered reference solution 4 prepared in step 4.3 above and inject it at intervals of 24, 48, and 72 hours. Record the chromatograms and calculate the mean peak area and RSD of the reference solution. The results show that the RSDs for citric acid, malic acid, and quinic acid are 3.36%, 1.53%, and 2.16%, respectively.

[0106] The above results show that the method of this application has good precision.

[0107] 5.2 Repeatability

[0108] (1) Take out the frozen high-dose group colon tissue samples, cool them to room temperature, and weigh 6 portions, each weighing 100 mg;

[0109] (2) Add 700 μl of sterile saline and 3 mm zirconium oxide grinding beads, grind intermittently at 70 Hz and 4°C for 10 min;

[0110] (3) Add 300 μl of methanol and continue grinding intermittently at 70 Hz and 4°C for 5 min;

[0111] (4) Centrifuge at 4°C and 10,000 rpm for 15 min;

[0112] (5) Take 200 μl of supernatant and add 400 μl of methanol, and vortex for 1 min;

[0113] (6) Centrifuge at 4°C and 10,000 rpm for 15 min;

[0114] (7) Take the supernatant and add water in a volume ratio of 1:1, vortex and mix for 1 min to obtain the test solution;

[0115] (8) The test solution was analyzed by liquid chromatography-mass spectrometry, the chromatogram was recorded, and the mean and relative standard deviation (RSD) of the peak areas were calculated. The results showed that the RSDs of citric acid, malic acid, and quinic acid in colon tissue were 3.91%, 6.50%, and 5.50%, respectively. This indicates that the method of this application has good reproducibility.

[0116] 5.3 Stability

[0117] The quality control recovery sample solutions 1-3 prepared in step 4.2 above were stored at 23°C for 8 hours. The samples were assayed, the chromatograms recorded, and the average peak areas and RSDs calculated to test their stability. The results showed that the RSDs for citric acid, malic acid, and quinic acid were 3.58%, 5.72%, and 8.51%, respectively.

[0118] Take the quality control recovery sample solutions 1-3 prepared in step 4.2 above, incubate at 4°C for 72 hours, measure the concentrations, record the chromatograms, and calculate the mean peak area and RSD to test their stability. The results showed that the RSDs for citric acid, malic acid, and quinic acid were 2.65%, 3.78%, and 6.74%, respectively.

[0119] 5.4 Sample recovery

[0120] Take quality control recovery sample solutions 1-3, recovery reference control solutions 4-6, and blank group samples, measure them, obtain the peak area, and calculate the recovery rate according to the following formula.

[0121] Recovery rate (%) = (mean peak area of ​​quality control sample solution - background peak area) ÷ mean peak area of ​​reference control solution × 100%; Average recovery rate (%) = mean value of quality control samples at each concentration.

[0122] The results are shown in Table 1. The average recoveries of citric acid, malic acid, and quinic acid were all greater than 80%.

[0123] Table 1 Recovery results

[0124]

[0125] 5.5 Matrix Effects

[0126] Take 7-9 of the matrix quality control sample solution, 10-12 of the matrix reference control solution, and the blank group sample, measure them, obtain the peak area, and calculate the matrix effect according to the following formula.

[0127] Matrix effect % = (mean peak area of ​​matrix quality control sample - background peak area) ÷ mean peak area of ​​matrix reference control solution × 100%.

[0128] The results are shown in Table 2. The RSDs of citric acid, malic acid, and quinic acid in tissues were 85%-102%, 80%-95%, and 82%-89%, respectively, meeting the requirements for biological samples.

[0129] Table 2 Matrix effect results

[0130]

[0131] Example 2 Sample determination

[0132] This example provides a method for determining the contents of citric acid, malic acid, and quinic acid in rat colon tissue, which specifically comprises the following steps:

[0133] 1. Liquid chromatography and mass spectrometry conditions: same as in Example 1.

[0134] 2. Instruments, reagents, and materials used: Same as in Example 1.

[0135] 3. Draw a standard curve: same as in Example 1.

[0136] 4. Determination of citric acid, malic acid and quinic acid content

[0137] (1) Take out the frozen colon tissues of the high, medium, low dose groups, model group, and blank group, cool them to room temperature, and weigh 100 mg of each;

[0138] (2) Add 700 μl of sterile saline and 3 mm zirconium oxide grinding beads and grind intermittently at 70 Hz and 4°C for 10 min;

[0139] (3) Add 300 μl of methanol and continue grinding intermittently at 70 Hz and 4°C for 5 min;

[0140] (4) Centrifugation at 4°C, 10,000 rpm for 15 min;

[0141] (5) Take 200 μl of supernatant and add 400 μl of methanol, and vortex for 1 min;

[0142] (6) Centrifuge at 4°C, 10,000 rpm for 15 min;

[0143] (7) Add water to the supernatant at a volume ratio of 1:1 and vortex for 1 min to obtain the test solution;

[0144] (8) Analyze the test solution using liquid chromatography-mass spectrometry to obtain the response result.

[0145] (9) Substitute the obtained response results into the standard curve for calculation.

[0146] In this example, the measurement results of colon tissue samples of the high-, medium-, and low-dose groups of black plum, the model group, and the blank group are shown in Table 3. The results are expressed as mean ± SD, unit: ug / g.

[0147] Table 3 Sample measurement results

[0148]

[0149] Example 3 Optimization of colon tissue extraction method

[0150] Referring to the method of Example 2, different pretreatment methods were used to treat the rat colon tissue, and the remaining steps were the same as in Example 2. The specific grouping is as follows:

[0151] Method 1: Conventional extraction method 1: 100 mg of rat colon tissue was ground with 700 μl of normal saline, then 350 μl of methanol was added (to precipitate protein), centrifuged, and the supernatant was measured;

[0152] Method 2: Conventional extraction method 2: 100 mg of rat colon tissue was ground with 700 μl of normal saline, centrifuged, and 100 μl of supernatant was taken. 400 μl of methanol (to precipitate protein) was added and vortexed for 1 min. The supernatant was centrifuged and assayed.

[0153] Method 3: Using methanol as the grinding agent and extraction agent: 100 mg of rat colon tissue was ground with 700 μl of methanol, extracted by shaking, centrifuged, and the supernatant was measured;

[0154] Method 4: Pretreatment method of the present invention: 100 mg of rat colon tissue was ground with 700 μl of physiological saline, then ground again with 300 μl of methanol, shaken, and centrifuged. 200 μl of supernatant was extracted with 400 μl of methanol, vortexed for 1 min, and centrifuged. The supernatant was added with water in a volume ratio of 1:1 and vortexed for 1 min. The liquid was taken for measurement.

[0155] The pretreatment method (Method 4) of the present invention primarily comprises three steps: Step 1: Adding a grinding agent (physiological saline) to the colonic tissue and grinding with grinding beads to convert the colonic tissue into a homogenous liquid; Step 2: Precipitating proteins using methanol; Step 3: Adding extractants: methanol and water. Specifically, the grinding agent in the present invention is physiological saline, the protein precipitant is methanol, and the analyte extraction solvents are methanol and water. Unlike conventional single-step treatment, where the supernatant is collected for analysis, the present method continues grinding after adding the grinding agent to precipitate proteins. The first centrifugation is performed, and the supernatant is extracted by vortexing. A second centrifugation is performed, and the supernatant is extracted with water to obtain the test solution.

[0156] The measurement results show that: taking malic acid data as an example, the content results are: Method 4 > Method 2 > Method 1 > Method 3; the content measured by the pretreatment method of the present invention is the highest, and the result is set to 1; the result of Method 3 is 0.4 of that of the pretreatment method of the present invention, the result of Method 1 is 0.65 of that of the pretreatment method of the present invention; and the result of Method 2 is 0.80 of that of the pretreatment method of the present invention.

[0157] Compared to conventional methods, a single protein precipitation only removes proteinaceous substances from the sample, while other components that may interfere with the quantification of the analyte are not purified. Furthermore, the tissue composition is complex and matrix interference is significant. The pretreatment process can only continuously dilute the sample, thereby reducing the sensitivity of the detection method. The pretreatment method of the present invention, however, uses secondary centrifugation to further remove proteinaceous substances and other interferences, allowing for better extraction of organic acids. After removing interference, extraction with water results in high extraction recovery, minimal matrix interference, and high content determination results, meeting the requirements of biological sample determination.

[0158] Example 4 Selection of Detection Modes for Citric Acid and Quinic Acid by Mass Spectrometry

[0159] The negative ion mode has a better response for the routine determination of citric acid. However, since the molecular weights of quinic acid and citric acid are basically the same, both are 192.0 (the molecular formula of citric acid is C6H8O7, the precise molecular weight is 192.0196; the molecular formula of quinic acid is C7H 12 O6, accurate molecular weight 192.0560). In negative ion mode, the parent ions Q1 of citric acid and quinic acid are identical at 191.0, and the fragment ions Q3 have multiple identical ions: 85.0, 87.0, 111.0, and 173.0. Therefore, if citric acid is measured in negative ion mode, interfering peaks will be present, affecting the results.

[0160] In the present invention, by optimizing chromatographic conditions, quinic acid was analyzed in negative ion mode, with a parent ion Q1 of 191.0 and a quantitative fragment ion Q3 of 93.0. Citric acid was analyzed in positive ion mode, with a parent ion Q1 of 193.0 and a quantitative fragment ion Q3 of 111.0. Fragment ions of other compounds are also present in the tissue extract, which produce interfering peaks with citric acid in negative ion mode but do not interfere in positive ion mode.

[0161] The test results showed that after optimizing the chromatographic conditions, the retention time of citric acid was 4.8 minutes, and the retention time of quinic acid was 3.9 minutes. The chromatographic peaks of citric acid and quinic acid were completely separated, and there was no interference between citric acid and quinic acid. In other words, the positive ion mode selected for the detection of citric acid in the present invention can effectively avoid interfering peaks and ensure accurate and reliable measurement results.

[0162] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for determining the content of organic acid metabolites of ebony in colon tissue, characterized in that: The steps include: (1) Colon tissue pretreatment: Sterile physiological saline was added to the colon tissue, and the tissue was ground. Methanol was then added and the grinding was continued. The tissue was centrifuged and the supernatant was collected to obtain supernatant I. Methanol was then added and the tissue was vortexed and centrifuged to obtain supernatant II. Finally, water was added and the tissue was vortexed to obtain the sample solution to be tested. (2) Draw the standard curves of three organic acids: Citric acid, malic acid, and quinic acid were prepared into standard curve working solutions with a concentration gradient using methanol-water solution, and then detected by high performance liquid chromatography-mass spectrometry, wherein citric acid was analyzed in positive ion mode and malic acid and quinic acid were analyzed in negative ion mode. The chromatographic peak area of ​​each organic acid was obtained, and then standard curves were plotted with the peak area as the ordinate (Y) and the mass concentration of the standard curve working solution as the abscissa (X), to obtain linear equations for citric acid, malic acid, and quinic acid, respectively. (3) Determination of the content of three organic acids: The sample solution obtained in step (1) is detected by high performance liquid chromatography-mass spectrometry, wherein citric acid is detected in positive ion mode, and malic acid and quinic acid are detected in negative ion mode, to obtain the chromatographic peak area of ​​each organic acid, and then substitute it into the linear equation obtained in step (2) to calculate the concentration and / or content of citric acid, malic acid, and quinic acid respectively; The parameters of the high performance liquid chromatography described in steps (2) and (3) are as follows: chromatographic column Agilent ZORBAX Rx-C18, 4.5×150 mm, 5 μm; mobile phase A is an aqueous solution containing 0.1% by volume of formic acid, mobile phase B is methanol, and the gradient elution is: 0-5 min, 5% B; 5-7 min, 5%-80% B; 7-8 min, 80%-5% B; 9 min, 5% B; column temperature: 35-40°C; flow rate: 0.2-0.4 mL / min; injection volume: 1-3 μL; The mass spectrometry parameters in steps (2) and (3) are as follows: electrospray ionization source ESI, ion source temperature: 550 ° C, nebulizer pressure: 50 Pa; capillary voltage 4500 V; collision gas: high-purity nitrogen, collision gas pressure medium; ionization mode: electrospray, positive and negative ion mode, multiple reaction monitoring (MRM) mode, ion pairs: citric acid Q1 is 193.100, Q3 is 111.000, DP is 40 V, CE is 24 V, EP is 10 V, and CXP is 11 V; malic acid Q1 is 133.000, Q3 is 115.000, DP is -50 V, CE is -15 V, EP is -10 V, and CXP is -16 V; quinic acid Q1 is 191.100, Q3 is 93.000, DP is -90 V, CE is -29 V, EP is -10 V, and CXP is -16 V.

2. The method according to claim 1, wherein: The material-liquid ratio of the colon tissue and sterile saline described in step (1) is 50-200 mg:350-700 μl; In step (1), the amount of methanol added for the first time is calculated based on 150 μl to 300 μl of methanol per 100 mg of colon tissue; In step (1), the volume ratio of the methanol added for the second time to the supernatant I is 2 to 5:1; The volume ratio of the supernatant II and water in step (1) is 1:1-3.

3. The method according to claim 2, wherein: The material-liquid ratio of the colon tissue and sterile saline described in step (1) is 100 mg:700 μl; In step (1), the amount of methanol added for the first time is calculated based on 200-300 μl of methanol per 100 mg of colon tissue; In step (1), the volume ratio of methanol added for the second time to supernatant I is 2:1; The volume ratio of the supernatant II and water described in step (1) is 1:

1.

4. The method according to claim 1, wherein: The grinding conditions described in step (1) are: using 2-4 mm zirconium oxide grinding beads, intermittent grinding at 50-70 Hz and 4°C for 5-10 minutes; In step (1), the centrifugation conditions for both times are: 4°C, 10,000 rpm to 12,000 rpm for 10 to 15 min; The vortexing time in step (1) is 0.5 min to 2 min.

5. The method according to claim 4, characterized in that: The grinding conditions described in step (1) are as follows: using 3 mm zirconium oxide grinding beads, intermittent grinding at 70 Hz and 4°C for 10 minutes; In step (1), the centrifugation conditions for both times were: 4°C, 10,000 r / min for 15 min, or 4°C, 12,000 r / min for 10 min; The vortexing time in step (1) was 1 min.

6. The method according to claim 1, wherein: The concentration of the methanol aqueous solution in step (2) is 30 to 50% by volume; In step (2), the standard curve working solution with a concentration gradient is prepared by preparing 5 to 9 standard curve working solutions containing citric acid, malic acid and quinic acid with different known concentrations; In step (2), the concentration range of citric acid in the standard curve working solution is 500-60000 ng / mL, the concentration range of malic acid is 50-25000 ng / mL, and the concentration range of quinic acid is 40-5000 ng / mL.

7. The method according to claim 1, wherein: The colon tissue described in step (1) is animal colon tissue.

8. The method according to claim 7, wherein: The colon tissue described in step (1) is rat colon tissue.

9. The method according to claim 8, characterized in that: The colon tissue described in step (1) is the colon tissue of rats after ulcerative colitis was treated with black plum decoction.

10. Use of the method for determining the content of organic acid metabolites of ebony in colon tissue according to any one of claims 1 to 9 in determining organic acids for non-disease diagnosis and treatment purposes, characterized in that: The organic acid includes at least one of citric acid, malic acid and quinic acid.

11. The use according to claim 10, characterized in that: The organic acid is citric acid and quinic acid, or citric acid, malic acid and quinic acid.

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