UPLC-MS / MS high-throughput detection method for flavonoid compounds

Abstract

The invention discloses a UPLC-MS / MS high-throughput detection method for flavonoid compounds. The method comprises the following steps: after a sample is extracted, detecting a sample extracting solution and a standard solution by adopting an ultra-high performance liquid chromatography and mass spectrometry combined method; mobile phases adopted by the ultra-high performance liquid chromatography are as follows: a mobile phase A is an ultrapure water solution containing 0.1% of formic acid; the mobile phase B is an acetonitrile solution containing 0.1% of formic acid. According to the method, after the tissue sample is extracted and subjected to liquid chromatography separation, the flavonoid compounds are qualitatively and quantitatively detected in a targeted mode, and due to the high selectivity of the high-resolution mass spectrum, parameter optimization does not need to be conducted on each metabolite.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass spectrometry detection, and particularly relates to a method for high-throughput detection of flavonoid compounds based on UPLC-MS / MS and application. BACKGROUND

[0002] Flavonoids are a class of compounds existing in nature, which have a ketone hydroxyl group in the molecule, the first oxygen atom has alkalinity, and have the characteristics of C6-C3-C6, that is, a series of compounds formed by two aromatic rings A and B connected with each other through a central three-carbon chain. According to the connection mode of the benzene ring and the intermediate two-carbon part, the B ring connection position, the oxidation level of the three-carbon part, and the polymerization degree, etc., the natural flavonoids can be divided into multiple types, respectively, flavone, flavonol, dihydroflavone, dihydroflavonol, isoflavone, dihydroisoflavone, chalcone, dihydrochalcone, aurone, anthocyanidin, rutin, catechin, daidzein, etc. Most of the plant bodies contain flavonoids, and the types of metabolites are different in different plants. For example, the aleurone layer of colored wheat (blue, black, purple, red) contains a large amount of anthocyanin; flavonoids in citrus fruits generally exist in the form of glycosides; isoflavones are rich in soybeans; flavanols are abundant in apple, cherry, tea and other plants. Flavonoids play an important role in plant growth, development, flowering, fruiting, and antibacterial disease prevention. Most flavonoid metabolites have activity and health care function, so accurate quantification of flavonoids in vegetables, fruits, flowers, medicinal plants, food and other samples helps to understand the growth and development of plants, the resistance mechanism, and also provides a theoretical basis for the research on the influence of different processing methods on the nutritional value of food. Therefore, comprehensive, stable and high-throughput detection of flavonoids in biological bodies has important guiding significance for the research on biological metabolic pathways and plant growth and development. SUMMARY

[0003] To solve the above technical problems, the present application provides a method for detecting flavonoids based on UPLC-MS / MS and application. In the present application, after the tissue sample is extracted and separated by liquid chromatography, the flavonoids are detected qualitatively and quantitatively, which has the characteristics of simple pretreatment, high sensitivity, strong selectivity, good reproducibility, high throughput, and can detect more than 380 metabolites at a time.

[0004] To achieve this purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides a method for detecting flavonoid metabolites based on UPLC-MS / MS, the steps of the method comprising: after sample extraction, using the method of ultra-high performance liquid chromatography combined with mass spectrometry to detect the sample extraction liquid and the standard solution; The flavonoid metabolites include any one or at least two combinations of 11 chromones, 2 furanochromones, 46 isoflavones, 152 flavones, 7 dihydroisoflavones, 94 flavonols, 2 aurones, 9 chalcones, 31 dihydroflavones, 11 flavanones, 10 dihydroflavonols, 7 flavanols, 3 flavans, 1 pterocarpans, 9 anthocyanidins, 1 flavonolignan, 2 biflavones, and 45 others.

[0005] In the present application, more than 380 flavonoid metabolites can be simultaneously detected by the above detection method, the detection flux is extremely high, and the above flavonoid metabolites can be effectively separated.

[0006] Preferably, the sample extraction step includes: centrifuging the sample after the first extraction to obtain supernatant and precipitate, centrifuging the precipitate after the second extraction, collecting the supernatant after the second centrifugation for incubation, and performing the third centrifugation to obtain the sample extract after vacuum freeze-drying.

[0007] In the present application, the solid sample is extracted twice, which significantly improves the concentration of the sample in high-performance liquid detection, maximizes the accuracy of the detection, and reduces the influence of the sample matrix on the detection; the liquid sample is extracted once.

[0008] Preferably, the mass-volume ratio of the sample to the solvent is 1: (2-100) g / mL, and the (2-100) can be 2, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100, etc.

[0009] Preferably, the first extraction reagent includes any one or at least two combinations of methanol, acetonitrile, or water.

[0010] Preferably, the first extraction reagent includes a combination of water and methanol.

[0011] Preferably, the volume ratio of water to methanol in the first extraction reagent is 1: (0.5-3). The (0.5-3) can be 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, or 3.0, etc.

[0012] Preferably, the first extraction time is 20-40 min, which can be 20 min, 25 min, 30 min, 35 min, or 40 min, etc.

[0013] Preferably, the first extraction temperature is 0-4℃, which can be 0℃, 1℃, 2℃, 3℃, or 4℃, etc.

[0014] Preferably, the centrifugation speed is 10000-14000 rpm, and the time is 5-15 min. The 10000-14000 rpm may be, for example, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, or 14000 rpm, etc. The 5-15 min may be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min, etc.

[0015] Preferably, the centrifugation temperature is 0-4℃, for example, 0℃, 1℃, 2℃, 3℃, or 4℃, etc.

[0016] Preferably, the second extraction reagent includes any one or a combination of at least two of methanol, acetonitrile, or water.

[0017] Preferably, the second extraction reagent includes a combination of water and methanol.

[0018] Preferably, the volume ratio of water to methanol in the second extraction reagent is 1:(0.5-3). The (1-3) may be, for example, 0.5, 1, 1.5, 2, 2.5, or 3, etc.

[0019] In the present application, the first extraction reagent and the second extraction reagent are the same, because for flavonoids, the extraction efficiency of methanol is better, and two extractions can ensure that the sample is extracted more fully, further improving the accuracy of content detection.

[0020] Preferably, the second extraction time is 15-40 min, for example, 15 min, 20 min, 25 min, 30 min, 35 min, or 40 min, etc.

[0021] Preferably, the second extraction temperature is 0-4℃, for example, 0℃, 1℃, 2℃, 3℃, or 4℃, etc.

[0022] Preferably, the second centrifugation speed is 10000-14000 rpm, and the time is 5-15 min. The 10000-14000 rpm may be, for example, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, or 14000 rpm, etc. The 5-15 min may be, for example, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min, etc.

[0023] Preferably, the temperature of the second centrifugation is 0-4℃, for example, it can be 0℃, 1℃, 2℃, 3℃ or 4℃, etc.

[0024] Preferably, the temperature of the second centrifugation is 0-4℃, for example, it can be 0℃, 1℃, 2℃, 3℃ or 4℃, etc.

[0025] Preferably, the temperature of the incubation is -15--25℃, for example, it can be -25℃, -24℃, -23℃, -22℃, -21℃, -20℃, -19℃, -18℃, -17℃, -16℃ or -15℃, etc.

[0026] Preferably, the incubation time is 0.5-2h, for example, it can be 0.5h, 1h, 1.5h or 2h, etc.

[0027] Preferably, the speed of the third centrifugation is 10000-14000rpm, and the time is 15-30min. The 10000-14000rpm, for example, can be 10000rpm, 11000rpm, 12000rpm, 13000rpm or 14000rpm, etc. The 15-30min, for example, can be 15min, 20min, 25min or 30min.

[0028] Preferably, the drying includes vacuum freeze-drying.

[0029] In the present application, vacuum freeze-drying is used to dry the sample, and the specific parameters are temperature: -50℃; time: 12-48h. The 12-48h, for example, can be 12h, 15h, 20h, 25h, 30h, 35h, 40h or 48h, etc.

[0030] Preferably, the preparation step of the standard solution includes: using methanol to dissolve the standard.

[0031] Preferably, the volume fraction of the methanol is 20-100%, for example, it can be 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.

[0032] Preferably, the concentration of the standard solution ranges from 1 to 500 nM, for example, it can be 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM or 500 nM, etc.

[0033] Preferably, before detection, the dried sample extract is reconstituted with methanol, and the supernatant is collected after centrifugation.

[0034] Preferably, the volume fraction of the methanol is 20-100%, for example, it can be 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%, etc.

[0035] Preferably, the speed of the centrifugation is 10000-14000 rpm, and the time is 10-30 min. The 10000-14000 rpm, for example, can be 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm or 14000 rpm, etc. The 10-30 min, for example, can be 10 min, 15 min, 20 min, 25 min or 30 min.

[0036] Preferably, the temperature of the centrifugation is 0-4℃, for example, it can be 0℃, 1℃, 2℃, 3℃ or 4℃, etc.

[0037] The mobile phase used in the ultra-high performance liquid chromatography is: the mobile phase A is an ultrapure water solution containing 0-0.1% formic acid. The 0-0.1% formic acid, for example, can be 0% formic acid, 0.04% formic acid, 0.06% formic acid, 0.1% formic acid, etc. The mobile phase B is a pure acetonitrile solution containing 0-0.1% formic acid. The 0-0.1% formic acid, for example, can be 0% formic acid, 0.04% formic acid, 0.06% formic acid, 0.1% formic acid, etc. By adding formic acid in the mobile phase A and B, the ionization efficiency of the metabolites can be improved, the peak shape of the metabolites can be improved, thereby reducing the detection limit of the metabolites and improving the stability of the detection system.

[0038] Preferably, the stationary phase of the chromatographic column used in the ultra-high performance liquid chromatography includes a trifunctional group bonded amide stationary phase.

[0039] Preferably, the column temperature of the chromatographic column is 25-45℃, for example, it can be 20℃, 25℃, 30℃, 35℃, 40℃, 41℃, 42℃, 43℃, 44℃ or 45℃, etc.

[0040] Preferably, the injection volume of the ultra-high performance liquid chromatography is 1-3 μL, for example, it can be 1 μL, 1.5 μL, 2 μL, 2.5 μL or 3 μL, etc.

[0041] Preferably, the elution procedure of the gradient elution of the ultra-high performance liquid chromatography is as follows: 0 min-1 min, the mobile phase B changes uniformly from 0% to 20%; 1 min-10 min, the mobile phase B changes uniformly from 20% to 80%; 10 min-14 min, the mobile phase B changes uniformly from 80% to 95%; 14min-16min, the mobile phase B remains 95% unchanged; 16.0min-16.1min, the mobile phase B changes from 95% to 0% at a constant rate; 16.1min-18min, the mobile phase B remains 0% unchanged.

[0042] The gradient elution is used in the present application, because the different types of flavonoids in the sample have large differences, and the above elution program can promote each flavonoid to reach a better separation effect according to its own appropriate capacity factor.

[0043] Preferably, the flow rate of the mobile phase is 0.1-0.5mL / min, for example, it can be 0.1mL / min, 0.2mL / min, 0.3mL / min, 0.4mL / min or 0.5mL / min, etc.

[0044] Preferably, the mass spectrometer used in the mass spectrum includes a quadrupole-electric field orbitrap high-resolution mass spectrometer.

[0045] Preferably, the scan mode of the mass spectrum includes full scan under positive ion condition.

[0046] Preferably, the sheath gas of the mass spectrum is 35-45arb, for example, it can be 35arb, 36arb, 37arb, 38arb, 39arb, 40arb, 41arb, 42arb, 43arb, 44arb or 45arb, etc.

[0047] Preferably, the auxiliary gas of the mass spectrum is 8-12arb, for example, it can be 8arb, 9arb, 10arb, 11arb or 12arb, etc.

[0048] Preferably, the ion spray voltage of the mass spectrum is +2.5kv to +3.5kv, for example, it can be +2.5kv, +2.6kv, +2.7kv, +2.8kv, +2.9kv, +3.0kv, +3.1kv, +3.2kv, +3.3kv, +3.4kv or +3.5kv, etc.

[0049] Preferably, the ion transmission tube temperature of the mass spectrum is 300-350℃, for example, it can be 300℃, 310℃, 320℃, 330℃, 340℃ or 350℃, etc.

[0050] Preferably, the first-order resolution of the mass spectrum is 70000, and the second-order resolution is 17500.

[0051] In a second aspect, the present application provides an application of the method for detecting flavonoids based on UPLC-MS / MS according to the first aspect in biological sample detection.

[0052] Compared with the prior art, the present application has at least the following beneficial effects: 1. The flavonoid analysis method based on UPLC-MS / MS in the present application has the characteristics of simple pretreatment, high sensitivity, strong selectivity, low detection limit, good reproducibility, etc., and can detect more than 380 metabolites at one time.

[0053] 2. The present application uses high-resolution mass spectrometry for detection, uses primary mass spectrometry data for quantification, and secondary mass spectrometry fragments for qualitative analysis, which reduces false positives in detection results, and at the same time, the primary and secondary information of the compound can be obtained, and both the quantitative and qualitative results are greatly improved.

[0054] 3. The method of the present application can be widely used in the research fields of medicine, agronomy, microbiology, food, etc., for screening disease biomarkers, elucidating disease occurrence and treatment mechanisms, studying plant growth and development, stress resistance, pesticide residues and transgenic crops, food production and preservation, food nutrition identification, food safety monitoring, and studying metabolic pathways, phenotype identification and functional genomics. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 is the ion current extraction diagram of (-) -gallocatechin standard product.

[0056] Figure 2 is the ion current extraction diagram of cyanidin-3-glucoside standard product.

[0057] Figure 3 is the ion current extraction diagram of dihydromyricetin standard product.

[0058] Figure 4 is the ion current extraction diagram of myricitrin standard product.

[0059] Figure 5 is the ion current extraction diagram of prunusin standard product.

[0060] Figure 6 is the ion current extraction diagram of 5, 7-dihydroxychromone standard product.

[0061] Figure 7 is the ion current extraction diagram of dihydroxydihydroquercetin-7-O-β-D-glucoside standard product.

[0062] Figure 8 is the ion current extraction diagram of icariin A standard product.

[0063] Figure 9 is the ion current extraction diagram of theaflavin 3, 3'-digallate standard product.

[0064] Figure 10 is the ion current profile of naringenin standard.

[0065] Figure 11 is the ion current profile of furanocoumarin standard.

[0066] Figure 12 is the ion current profile of sophoranone standard.

[0067] Figure 13 is the ion current profile of biochanin A standard.

[0068] Figure 14 is the ion current profile of irilone standard.

[0069] Figure 15 is the ion current profile of 7-demethylbilobetin standard.

[0070] Figure 16 is the ion current profile of 17-alpha-hydroxyprogesterone standard.

[0071] Figure 17 is the total ion current profile actually detected in the sample.

[0072] From the results, the ion current profile of the standard and the total ion current profile of the actual sample both exhibit good peak shape, response, and resolution. Thus, it can be inferred that the method has the feasibility of being applied to high-throughput targeted detection of flavonoids. DETAILED DESCRIPTION

[0073] The technical solutions of the present application will be further described below by specific embodiments in combination with the accompanying drawings. However, the following examples are only simple examples of the present application, and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0074] The instrument manufacturers used in the following examples are as follows: Mass spectrometer: Q Exactive, Thermo Fisher Scientific, Waltham, MA, USA Example 1 The present embodiment provides a method for detecting flavonoids based on UPLC-MS / MS (1) Sample pretreatment After the rice sample is ground with liquid nitrogen, 100 mg of the sample is accurately weighed into a 2 mL centrifuge tube, 0.8 mL of 70% methanol solution is added, vortexed, ultrasonically extracted at 4°C for 30 min, centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant is taken to a new centrifuge tube. 0.8 mL of the extract, which is 70% methanol solution, is added to the precipitate, ultrasonically extracted at 4°C for 15 min, centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant is taken and combined with the supernatant of the first extraction in a new centrifuge tube, incubated at -20°C for 1 h, and centrifuged at 12000 rpm at 4°C for 10 min. The supernatant is taken and vacuum dried to obtain the sample extract.

[0075] (2) Preparation of standard solution The standard powder is weighed and dissolved in pure methanol to prepare the corresponding standard single stock solution. The same volume of each standard single stock solution is mixed, and pure methanol is added to prepare the standard mixed stock solution with a concentration of 10 μM.

[0076] The prepared standard solution is diluted with pure methanol to prepare standards with concentrations of 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM, or 500 nM.

[0077] (3) Mass spectrometry detection The sample extract is reconstituted with pure methanol, and the concentration of the reconstituted sample is 0.5 g / mL. The sample is centrifuged at 12000 rpm at 4°C for 20 min, and the supernatant is the sample extract. The sample extract and the standard solution are subjected to mass spectrometry detection.

[0078] Liquid chromatography conditions: The chromatographic system used a Vanquish ultra-high performance liquid chromatograph from Thermo. The chromatographic column was a Waters HSS T3 (100 x 2.1 mm, 1.8 μm), the mobile phase was: phase A was ultrapure water containing 0.1% formic acid, phase B was acetonitrile containing 0.1% formic acid; the flow rate was 0.3 mL / min; the column temperature was 40°C; the injection volume was 2 μL; the elution gradient was: 0 min-1 min, the mobile phase B changed from 0% to 20% at a constant rate; 1 min-10 min, the mobile phase B changed from 20% to 80% at a constant rate; 10 min-14 min, the mobile phase B changed from 80% to 95% at a constant rate; 14 min-16 min, the mobile phase B remained 95%; 16.0 min-16.1 min, the mobile phase B changed from 95% to 0% at a constant rate; 16.1 min-18 min, the mobile phase B remained 0%. During the entire analysis process, the sample was placed in a 4°C autosampler.

[0079] Mass spectrometry conditions: The mass spectrometry system used a Q Exactive Orbitrap mass spectrometer from Thermo. It was equipped with an electrospray ionization (ESI) source and operated in negative ion full scan (Full MS-DDMS2) mode. The scan conditions were as follows: sheath gas 40 arb; auxiliary gas 10 arb; ion spray voltage +3500 V; temperature 350°C; ion transfer tube temperature 320°C. The first scan range (scan m / z range) was 70-1000 Da, and the second scan range was 70-2000 Da. The first resolution was 70000, and the second resolution was 17500.

[0080] Example 2 The present example provides a method for detecting flavonoid compounds based on UPLC-MS / MS After the liquid sample was slowly thawed at 4°C, 0.8 mL of extraction solution was added, which was a 70% methanol solution. After ultrasonic-assisted extraction at 4°C for 30 min, centrifugation was performed at 12000 rpm for 10 min at 4°C, and the supernatant was transferred to a new 2 mL centrifuge tube. Incubation was performed at -20°C for 1 h, and centrifugation was performed at 12000 rpm for 10 min at 4°C. The supernatant was vacuum freeze-dried to obtain the sample extract.

[0081] (2) Preparation of standard solution The standard powder was weighed and dissolved in pure methanol to prepare the corresponding standard single standard mother liquor. The same volume of each standard single standard mother liquor was mixed uniformly, and pure methanol was added to make the concentration of the standard mixed standard mother liquor 10 μM.

[0082] The prepared standard solution was gradient diluted with pure methanol to 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM or 500 nM standard.

[0083] (3) Mass spectrometry detection The sample extract was reconstituted with pure methanol, and the sample concentration after reconstitution was 0.5 g / mL. The sample was centrifuged at 12000 rpm for 20 min at 4℃, and the supernatant was taken as the sample extract. The sample extract and the standard solution were subjected to mass spectrometry detection.

[0084] Liquid chromatography conditions: The chromatography system used was a Vanquish ultra-high performance liquid chromatograph from Thermo. The chromatography column was a Waters HSS T3 (100x2.1 mm, 1.8 μm), the mobile phase was A phase: 0.1% formic acid in ultrapure water, B phase: 0.1% formic acid in acetonitrile; flow rate was 0.3 mL / min; column temperature was 40℃; injection volume was 2 μL; elution gradient: 0 min-1 min, mobile phase B changed from 0% to 20% at a constant rate; 1 min-10 min, mobile phase B changed from 20% to 80% at a constant rate; 10 min-14 min, mobile phase B changed from 80% to 95% at a constant rate; 14 min-16 min, mobile phase B remained at 95%; 16.0 min-16.1 min, mobile phase B changed from 95% to 0% at a constant rate; 16.1 min-18 min, mobile phase B remained at 0%. During the entire analysis process, the sample was placed in a 4℃ automatic sampler.

[0085] Mass spectrometry conditions: The mass spectrometry system used was a Q Exactive Orbitrap high-resolution mass spectrometer from Thermo. It was equipped with an electrospray ionization (ESI) source, and the scanning mode was full scan (Full MS-DDMS2) mode under negative ion conditions. The scanning conditions were as follows: sheath gas 40 arb; auxiliary gas 10 arb; ion spray voltage +3500V; temperature 350℃; ion transfer tube temperature 320℃. The first scan range (scan m / z range) was 70-1000 Da, and the second scan range was 70-2000 Da. The first resolution was 70000, and the second resolution was 17500.

[0086] Example 3 The present embodiment provides a method for detecting flavonoids based on UPLC-MS / MS (1) Sample pretreatment After the rice sample was ground with liquid nitrogen, 100 mg of the sample was accurately weighed into a 2 mL centrifuge tube, 1.6 mL of 70% methanol solution was added, vortexed, ultrasonically extracted at 4°C for 30 min, centrifuged at 12000 rpm at 4°C for 10 min, the supernatant was transferred to a new centrifuge tube, incubated at -20°C for 1 h, centrifuged at 12000 rpm at 4°C for 10 min, and the supernatant was vacuum freeze-dried to obtain the sample extract.

[0087] (2) Preparation of standard solution The standard powder was weighed and dissolved in pure methanol to prepare the corresponding standard single stock solution. The same volume of each standard single stock solution was mixed and pure methanol was added to prepare the standard mixed stock solution with a concentration of 10 μM.

[0088] The prepared standard solution was gradiently diluted with pure methanol to prepare standards with concentrations of 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM or 500 nM.

[0089] (3) Mass spectrometry detection The sample extract was reconstituted with pure methanol, and the sample concentration after reconstitution was 0.5 g / mL. The sample was centrifuged at 12000 rpm at 4°C for 20 min, and the supernatant was used as the sample extract. The sample extract and the standard solution were subjected to mass spectrometry detection.

[0090] Liquid chromatography conditions: The chromatography system used was a Vanquish ultra-high performance liquid chromatograph from Thermo. The chromatography column was a Waters HSS T3 (100 x 2.1 mm, 1.8 μm), the mobile phase was A phase: 0.1% formic acid in ultrapure water, B phase: 0.1% formic acid in acetonitrile; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 2 μL; elution gradient: 0 min-1 min, mobile phase B from 0% to 20%; 1 min-10 min, mobile phase B from 20% to 80%; 10 min-14 min, mobile phase B from 80% to 95%; 14 min-16 min, mobile phase B remained at 95%; 16.0 min-16.1 min, mobile phase B from 95% to 0%; 16.1 min-18 min, mobile phase B remained at 0%. During the entire analysis process, the sample was placed in a 4°C autosampler.

[0091] Mass spectrometry conditions: The mass spectrometry system uses a Q Exactive Orbitrap mass spectrometer of Thermo. It is equipped with an electrospray ionization (ESI) source and operates in negative ion full scan (Full MS-DDMS2) mode. The scan conditions are as follows: sheath gas 40 arb; auxiliary gas 10 arb; ion spray voltage +3500 V; temperature 350℃; ion transfer tube temperature 320℃. The first scan range (scan m / z range) is 70-1000 Da, and the second scan range is 70-2000 Da. The first resolution is 70000, and the second resolution is 17500.

[0092] Example 4 This example provides a method for detecting flavonoids based on UPLC-MS / MS. The difference from Example 1 is only that the extraction reagent is 70% ethanol.

[0093] The rest is consistent with Application Example 1.

[0094] Example 5 This example provides a method for detecting flavonoids based on UPLC-MS / MS. The difference from Example 1 is only that the elution program is: 0min-1min, mobile phase B changes from 0% to 10% at a constant rate; 1min-10min, mobile phase B changes from 10% to 50% at a constant rate; 10min-14min, mobile phase B changes from 50% to 90% at a constant rate; 14min-16min, mobile phase B remains 90%; 16.0min-16.1min, mobile phase B changes from 90% to 0% at a constant rate; 16.1min-18min, mobile phase B remains 0%.

[0095] The rest is consistent with Example 1.

[0096] Example 6 This example provides a method for detecting flavonoids based on UPLC-MS / MS. The difference from Example 1 is only that the elution program is: 0min-1min, mobile phase B changes from 0% to 40% at a constant rate; 1min-10min, mobile phase B changes from 40% to 80% at a constant rate; 10min-14min, mobile phase B changes from 80% to 95% at a constant rate; 14 min-16 min, mobile phase B remains 95% unchanged; 16.0 min-16.1 min, mobile phase B changes uniformly from 95% to 0%; 16.1 min-18 min, mobile phase B remains 0% unchanged.

[0097] The rest is consistent with example 1.

[0098] Example 7 The difference between this example and example 1 is that the first resolution is 60000 and the second resolution is 15000. The rest is consistent with example 1.

[0099] Comparative example 1 The difference between this example and example 1 is that the mobile phase B of ultra-high performance liquid chromatography is a methanol solution containing 0.1% formic acid. The rest is consistent with example 1.

[0100] Comparative example 2 The difference between this example and example 1 is that the mobile phase A and B of ultra-high performance liquid chromatography do not contain 0.1% formic acid. The rest is consistent with example 1.

[0101] Test example The detection results of this example are further analyzed to verify the feasibility of the method provided by the present application: the peak area obtained by the first mass spectrum data is quantified, and the fragment data of the second mass spectrum is qualitatively analyzed. The raw spectrum obtained by the high-efficiency mass spectrometer used in the present application is processed by baseline filtering, peak identification, peak matching, retention time correction, and peak alignment through the peak accumulation software, so as to obtain a data matrix containing retention time, mass-to-charge ratio, and peak intensity (peak area), which is used for subsequent calculation of detection results.

[0102] The external standard method is used for quantification, the concentration of the standard is taken as the abscissa, and the peak area value of the standard is taken as the ordinate to establish a standard curve. The standard curve results of some compounds are shown in Table 1, and the extracted ion flow chart is shown in Figures 1-16 . Figure 17 The total ion flow chart of the sample in example 1.

[0103] Table 1 Standard curve information of some flavonoids 380 kinds of flavonoids can present good linear relationship in their respective linear concentration ranges, and R 2≥0.99, fully meet the quantitative requirements. The peak area of flavonoids in rice leaves is substituted into the standard curve, and the absolute content of flavonoids in rice leaves can be accurately calculated with high throughput and high sensitivity. The calculation formula is: the product content of flavonoids in rice sample (ng / mg) = (C x V x F x Mw) / M, wherein C is the sample concentration, unit nmol / L; V is the sample extraction liquid volume, unit L; F is the sample dilution multiple; M is the sample weighing mass, unit mg.

[0104] The contents of some flavonoids in Examples 1-4 are shown in Table 2: Table 2 Contents of some flavonoids In Example 1, 396 flavonoids were detected, with a content range of 0.8916-6948.6123 ng / g, and the contents of some compounds are shown in Table 2, which shows that this method is suitable for the determination of more than 380 flavonoids at one time, and the detection limit is in the ng level. In Example 2, 396 flavonoids were also detected, with a content range of 0.6754-1069.2279 ng / mL, which shows that this method is also suitable for the determination of more than 380 flavonoids in liquid samples at one time. In Example 3, 256 flavonoids were detected, and the content was lower than that in Example 1, which shows that when the ultrasonic extraction time is only 30 min, the extraction is not sufficient, resulting in that the flavonoids with low content cannot be detected, so the experimental conditions in Example 1 are more suitable. In Example 4, 342 flavonoids were detected, and the content was lower than that in Example 1, which shows that the extraction efficiency of 70% ethanol solution is lower than that of 70% methanol solution.

[0105] The contents of some flavonoids in Examples 5-7 are shown in Table 3: Table 3 Contents of some flavonoids The results of Examples 5 and 6 are similar. When the elution gradient is adjusted, it is found that 68.16% and 71.03% of the compounds will peak in the 2-8 min time period, the 5-8 isomers are not well separated, and some compounds appear "bifurcation" and "tailing", so we optimize the elution gradient. The elution gradient in Example 1 not only makes the peak time of metabolites more uniform, but also makes the multiple isomers well separated, reduces the phenomenon of "bifurcation" and "tailing" of metabolite peak type, so the experimental conditions in Example 1 are more suitable. There is no significant difference between Example 7 and Example 1, which shows that adjusting the resolution has little effect on the detection system.

[0106] The content of the partial flavonoids in Comparative Example 1-2 is shown in Table 4: Table 4 Content of partial flavonoids In Comparative Example 1, when the mobile phase is adjusted to pure methanol, it is found that the peak order and time of 68.16% of the metabolites change, the peak time of some compounds is too late, and the peak shape of many compounds appears "splitting" phenomenon, and the content is lower than that of Example 1; In Comparative Example 2, when the mobile phase does not contain 0.1% formic acid solution, it will cause the compounds with low content to be undetected, and the peak shape of some compounds is poor, which shows that formic acid can not only improve the peak shape, but also improve the resolution of the instrument and reduce the detection limit of the substance.

[0107] In summary, after the biological sample is extracted and separated by liquid chromatography, and then detected by mass spectrometry, qualitative and quantitative analysis is carried out, which has the characteristics of simple pretreatment, high sensitivity, strong selectivity, low detection limit, good reproducibility and high throughput, and more than 380 flavonoid metabolites can be detected at one time.

[0108] The above merely illustrates the specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the present application can be easily thought out by those skilled in the art, and all fall within the protection scope and disclosure scope of the present application.

Claims

1. A method for high-throughput detection of flavonoids by UPLC-MS / MS, characterized in that, The method comprises: after sample extraction, using a method of ultra-high performance liquid chromatography combined with mass spectrometry to detect the sample extraction solution and the standard solution; After the first extraction of the sample, the supernatant and the precipitate are obtained, the precipitate is subjected to the second extraction, and then subjected to the second centrifugation, the supernatant after the two centrifugations is collected for incubation, subjected to the third centrifugation, and dried to obtain the sample extract; The mass spectrometer used in the mass spectrometry comprises a quadrupole-electric field orbitrap high-resolution mass spectrometer; The scanning mode of the mass spectrometry comprises full scanning under positive ion condition; the sheath gas of the mass spectrometry is 35-45 arb, the auxiliary gas is 8-12 arb; the ion spray voltage is +2.5kv to +3.5kv; the ion transmission tube temperature is 300-350℃; the first-order resolution of the mass spectrometry is 70000, and the second-order resolution is 17500.

2. The method of claim 1, wherein, The flavonoids include any one or at least two combinations of 11 chromones, 2 furan chromones, 46 isoflavones, 152 flavones, 7 dihydroisoflavones, 94 flavonols, 2 aurones, 9 chalcones, 31 dihydroflavones, 11 flavanones, 10 dihydroflavonols, 7 flavanols, 3 flavans, 1 pterocarpans, 9 anthocyanidins, 1 flavonol xanthohumol, 2 biflavones, and 45 other types.

3. The method of claim 2, wherein, The sample extraction comprises twice solid sample extraction and once liquid sample extraction; The extraction reagent comprises any one or at least two combinations of methanol, acetonitrile or water; The extraction time is 20-40min, and the extraction temperature is 0-4℃; The centrifugation speed is 10000-14000rpm, the time is 5-15min, and the temperature is 0-4℃; The drying comprises vacuum freeze drying.

4. The method of claim 3, wherein, The mobile phase used in the ultra-high performance liquid chromatography comprises: the mobile phase A is an ultrapure water solution containing 0-0.1% formic acid; the mobile phase B is a pure acetonitrile solution containing 0-0.1% formic acid; and the stationary phase of the chromatographic column used in the ultra-high performance liquid chromatography comprises a trifunctional group bonded amide stationary phase; The column temperature of the chromatographic column is 35-45℃; The injection amount of the ultra-high performance liquid chromatography is 1-3μL.

5. The method of claim 4, wherein, The elution procedure of the gradient elution of the ultra-high performance liquid chromatography is as follows: 0min-1min, the mobile phase B changes from 0% to 20% at a constant speed; 1min-10min, the mobile phase B changes from 20% to 80% at a constant speed; 10min-14min, the mobile phase B changes from 80% to 95% at a constant speed; 14min-16min, the mobile phase B remains unchanged at 95%; 16.0min-16.1min, the mobile phase B changes from 95% to 0% at a constant speed; 16.1min-18min, the mobile phase B remains unchanged at 0%; The flow rate of the mobile phase is 0.1-0.5mL / min.

6. Application of the UPLC-MS / MS high-throughput detection method of the flavonoids according to any one of claims 1-5 in in vivo detection.

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