CN109254094B

CN109254094B - Method for detecting isoflavone compounds

Info

Publication number: CN109254094B
Application number: CN201811222417.7A
Authority: CN
Inventors: 张峰; 许秀丽; 吴文杰; 王秀娟
Original assignee: Chinese Academy of Inspection and Quarantine CAIQ
Current assignee: Chinese Academy of Inspection and Quarantine CAIQ
Priority date: 2018-10-19
Filing date: 2018-10-19
Publication date: 2021-05-14
Anticipated expiration: 2038-10-19
Also published as: CN109254094A

Abstract

The invention discloses a method for detecting isoflavone compounds. The method comprises the following steps: extracting a sample to be detected by using an alcohol solution so as to obtain an extracting solution; and detecting the extracting solution by using an ultrahigh-performance supercritical fluid chromatography-tandem mass spectrum so as to obtain the content of the isoflavone compound. The method has the advantages of simple operation, good reproducibility, high analysis speed, high sensitivity, and realization of baseline separation, and can be used for quantitative analysis and identification of various common isoflavone components.

Description

Method for detecting isoflavone compounds

Technical Field

The invention relates to the field of analytical chemistry, in particular to a method for detecting isoflavone compounds.

Background

Isoflavones are natural compounds with a common 3-phenyl-chromen-4-one backbone. When comparing the structure of isoflavones with conventional flavonoids, the difference is that the phenyl group of the former is substituted at the C-3 position of the chromene moiety and the latter is substituted at the C-2 position. Due to their structural similarity to estradiol, isoflavones play an important role as phenolic phytoestrogens in regulating estrogen receptors, exhibiting estrogenic and antiestrogenic effects. Therefore, isoflavones have been widely used as remedies for preventing age-related and estrogen-dependent diseases such as climacteric syndrome. In addition to their hormone-like activity, isoflavones also have other pharmacological activities, such as antifungal, antibacterial, antioxidant, anti-inflammatory, anticancer, anti-obesity, anti-diabetic activity.

From analytical and pharmacological perspectives, the active ingredient may serve as a means of herbal quality assessment, and quantitative determination of isoflavones helps to confirm safe and effective use of isoflavone-rich herbs. For this reason, a series of methods such as Capillary Electrophoresis (CE), Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) have been reported in the literature. These methods are used less often due to low resolution (TLC) or poor reproducibility (CE). Thus, HPLC methods in combination with PDA, MS or MS/MS are more commonly used for the analysis of isoflavones. However, these methods typically suffer from high organic reagent consumption and long time consumption (about 30-90 minutes), the latter being a major bottleneck when high throughput analysis is required. The analysis time of UHPLC columns is short (12 minutes or less), but the separation of some structurally similar isoflavones is not ideal due to the same separation mechanism.

Therefore, there is a need to develop a method for analyzing isoflavones based on different separation mechanisms to save time.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention aims to provide a method for detecting isoflavone compounds, which has the advantages of simple operation, good reproducibility, high analysis speed, high sensitivity, realization of baseline separation and capability of quantitatively analyzing/identifying various common isoflavone components.

It should be noted that the present invention is completed based on the following work of the inventors:

ultra-high performance supercritical fluid chromatography (UHPSFC) can be a complementary technology to UHPLC due to different interaction mechanisms. UHPSFC can use a wide variety of stationary phases and has a wide range of selectivity, with low or no water content in the mobile phase also affecting the interaction mechanism between the analyte and the stationary phase. Currently, UHPSFC with sub-2 μm particulate stationary phase has been widely used because it can easily achieve rapid analysis and enhanced chromatographic separation.

There are many detectors associated with UHPSFC. Photodiode array detectors (PDAs) are often used for routine quantification of many natural products. However, it is particularly difficult to eliminate interference from plant substrates (containing thousands of compounds) to accurately analyze many isoflavones, which often leads to false positive errors. For example, in our previous studies, a UHPSFC-PDA method for isolation of 12 isoflavones in serum has been established, but when we increased the amount of isoflavones, this method failed to achieve baseline isolation of all analytes, which resulted in inaccuracies when plant samples were quantified. Also, several isoflavones are present in very low amounts in certain plant materials and cannot be detected by PDA. For these reasons, UHPSFC needs to be coupled with a more selective and sensitive detector. The inventors have found that triple quadrupole mass spectrometry (QqQ MS/MS) can provide sufficient structural information and allow accurate and sensitive quantification of a variety of compounds in a plant matrix using Multiple Reaction Monitoring (MRM) mode. The MRM mode can provide selective and sensitive gain, since second order implies two different characteristics of the ion pair for each target compound. Meanwhile, as the impurity interference is reduced, the sample preparation process can be simplified, and the time-consuming and complex purification process is avoided. However, a disadvantage of MS is that it cannot distinguish between co-eluted isoflavones of the same molecular weight. Thus, there remains a need to achieve baseline separation of isoflavone isomers. Because of simple operation and low cost, the PDA detection is more suitable for mass spectrum detection of isoflavone separation under different chromatographic conditions. Therefore, the inventors tried to develop a method for combining PDA with MS in UHPSFC.

The inventor creatively combines UHPSFC with mass spectrum to analyze isoflavone, successfully develops a rapid and sensitive detection method, and uses UHPSFC-QqQ-MS/MS to detect 16 kinds of isoflavone. Parameters affecting UHPSFC were optimized to separate isoflavone isomers well in short assay times. Furthermore, excellent selectivity and sensitivity were achieved by MS detection using MRM mode, which allowed satisfactory identification and quantification of 16 isoflavones. The mass spectrometric fragmentation pathways of these target compounds indicate that both precursor and product ions exist in the form of protonated molecules as well as in the form of radical cations. Finally, the method is successfully applied to the detection of isoflavones in plant material by simple ultrasonic solvent extraction. The method is rapid and sensitive, and can be used for determination of isoflavone in radix Puerariae (Pueraria lobata Ohwi) and its related species radix Puerariae and Kudzuvine. The method for detecting isoflavone compounds provided by the embodiment of the invention can be accurately used for analyzing the content of isoflavone in food, biological and other plant samples.

Thus, according to one aspect of the invention, the invention provides a method for detecting isoflavones. According to an embodiment of the invention, the method comprises: extracting a sample to be detected by using an alcohol solution so as to obtain an extracting solution; and detecting the extracting solution by using an ultrahigh-performance supercritical fluid chromatography-tandem mass spectrum so as to obtain the content of the isoflavone compound.

The method for detecting isoflavone compounds according to the embodiment of the invention has the advantages of simple operation, good reproducibility, high analysis speed and high sensitivity, realizes baseline separation, and can carry out quantitative analysis and identification on various common isoflavone components.

In addition, the method for detecting isoflavones according to the above embodiment of the present invention may also have the following additional technical features:

according to embodiments of the present invention, the sample to be tested may be food, organisms and plants. The method provided by the embodiment of the invention can be used for detecting various samples.

According to an embodiment of the invention, the ultra-high performance supercritical fluid chromatography may be an Acquity UPC 2 system.

According to an embodiment of the present invention, the extraction process includes: extracting the sample to be detected with 20-40% methanol solution for 24-40 min to obtain extract liquid; evaporating the extract to obtain residue after evaporation; and dissolving the residue after evaporation in methanol to obtain the extract. Therefore, the isoflavone obtained by extraction can be used for subsequent detection treatment, the extraction method is simple, and the isoflavone compound can be effectively and fully extracted from the sample to be detected.

According to an embodiment of the present invention, the ratio of the sample to be tested to the methanol solution is 0.1 g: the extraction is carried out in a ratio of 20-40ml, preferably, in a ratio of 0.1 g: the extraction is carried out at a ratio of 30ml, so that the extraction effect is good, and the isoflavone compound can be fully extracted from the sample to be detected.

According to an embodiment of the present invention, the mass spectrum in the ultra-high performance supercritical fluid chromatography-tandem mass spectrum is a triple quadrupole mass spectrum (QqQ MS/MS). Due to the high selectivity and sensitivity of the triple quadrupole mass spectrometry, the known compound can be quantitatively analyzed, and ions of a target compound can be analyzed for two different characteristics, namely the analysis of relative molecular mass and secondary fragment characteristics, so that the impurity interference is reduced, the extraction process of a sample is simplified, and the time-consuming and complex purification process is not needed.

According to an embodiment of the invention, the mobile phase of the detection process is: a: CO2₂(ii) a B: the methanol solution containing 0.1mM of the acidic additive is preferably a 40% methanol solution containing 0.1mM of the acidic additive. According to a particular embodiment of the invention, the acidic additive is at least one selected from the group consisting of ammonium formate, formic acid and oxalic acid. Thus, the peaks obtained by detection are good in shape and have no tailing, so that the detection result is more accurate, the methanol solution with the concentration of 40% is favorable for ensuring the supercritical condition or at least reasonable viscosity and diffusion coefficient (subcritical condition), and better ionization for other target analytes and the aglycones of irisin and genistin is obtained within corresponding retention time by adding oxalic acid.

According to the embodiment of the present invention, the elution gradient of the detection treatment is 20% B (0-2min) -30% B (2-7min) -40% B (7-9min) - (40-20)% B (9-9.5min) -20% B (9.5-12 min), wherein it is noted that the concentration of the B component in the eluent is gradually reduced from 40% to 20% at 9-9.5 min. Therefore, the retention time of each isoflavone substance is appropriate, the peak shape of each component is sharp, and the separation degree is good.

According to an embodiment of the present invention, the mass spectrometry conditions of the ultra-high performance supercritical fluid chromatography-tandem mass spectrometry are as follows: a positive ionization ESI ionization source; an MRM mode; a diode array detector; capillary voltage: 3 kV; cone voltage: 40V; source temperature: 100 ℃; desolvation temperature: 300 ℃; cone gas flow rate: 90 liters/hour; and a desolvation gas flow rate of 900 liters/hour. Based on the molecular formula of the isoflavones studied, TQ-XS mass spectra from WATERS were used to automatically determine their precursor ions. The precursor ions are all deprotonated molecules [ M-H ] in negative ion mode, the precursor ions are protonated molecules [ M + H ] plus radical cations [ M ]. in positive ion mode. This may be related to the molecular structure of isoflavones containing one or more phenolic hydroxyl groups, which results in the easy loss of these analytes even in the positive ion mode. The inventors have investigated and found that the response of the precursor ions is higher in the positive mode than in the negative mode. Thus, ESI positive ionization mode (ESI +) of isoflavone compounds is used for MRM. Therefore, under the condition of the mass spectrum, the peak shape obtained by detection is better, the detection sensitivity and stability are higher, and a better linear relation and a higher recovery rate are realized in a common concentration range.

According to an embodiment of the invention, the tandem mass spectrometer is a Xevo TQ-XS tandem mass spectrometer. Therefore, the detection sensitivity is high, and the repeatability is good.

According to an embodiment of the present invention, the chromatographic conditions of the supercritical fluid chromatography-tandem mass spectrometry are: a chromatographic column: a Diol chromatography column; detection wavelength: 254 nm; column temperature: 50 ℃; sample temperature: 12 ℃; supplementing liquid: methanol with 0.1% formic acid. Wherein, the Diol chromatographic column has good analyte peak shape, and can analyze a plurality of isoflavone substances, including daidzein and puerarin pair isomers. Therefore, under the chromatographic condition, the peak shape obtained by detection is better, the detection sensitivity and stability are higher, and a better linear relation and a higher recovery rate are realized in a common concentration range.

According to an embodiment of the invention, the Diol chromatography column is of the type 1.7 μm, 3X 100mm internal diameter. Therefore, the peak shape of the isoflavone compound is good, a plurality of isoflavone substances can be accurately analyzed, and even the pair of isomers of daidzein and puerarin which are difficult to analyze can be accurately analyzed.

According to the embodiment of the invention, the data acquisition range of the mass spectrum is m/z 50-800, the m/z ratio range is the mass spectrum data distribution range of the main isoflavone compound, and the mass spectrum data acquisition range of the embodiment of the invention covers the main isoflavone compound, namely, the isoflavone compound in the range can be detected, thereby being beneficial to fully detecting the isoflavone compound.

According to an embodiment of the present invention, the isoflavone compound is selected from at least one of puerarin, puerarin apioside, 6-O-xyloside puerarin, 4 '-O-methyl puerarin, 3' -methoxy puerarin, daidzein, daidzin, formononetin, genistein, genistin, biotin a, glycitein, glycine, tectorigenin and tectoridin.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a schematic representation of the results of different chromatography columns according to one embodiment of the invention;

FIG. 2 shows a resulting schematic of the effect of gradient time on the resolution of 5 key pairs, where the circles represent preferred conditions, according to one embodiment of the invention;

FIG. 3 shows a graphical representation of the results of the effect of initial methanol concentration on the resolution of 5 key pairs, where the circles represent preferred conditions, according to one embodiment of the present invention;

FIG. 4 shows a schematic representation of the results of a detection of an elevated temperature or a reduced pressure according to one embodiment of the invention;

FIG. 5 shows a graphical representation of the results of the effect of column temperature on the resolution of 5 key pairs, where the circles represent preferred conditions, according to one embodiment of the present invention;

FIG. 6 shows a graphical representation of the results of the effect of backpressure on the resolution of 5 key pairs, where the circles represent preferred conditions, according to one embodiment of the present invention;

FIG. 7 shows a schematic diagram of the profile of the isoflavone compound under preferred conditions of UHPSFC-MS according to one embodiment of the invention;

FIG. 8 shows a schematic illustration of a proposed fragmentation pathway for isoflavone compounds according to one embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

In the description of the present invention, "a plurality" means two or more unless otherwise specified.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.

Example 1

The method provided by the embodiment of the invention is used for detecting isoflavone and comparing and analyzing experimental conditions, and the method comprises the following specific steps:

1. feedstock and apparatus

1.1 standards, reagents and samples

Puerarin, mirificin, 6-O-xyloside puerarin, 4 '-O-methyl puerarin, 3' -methoxy puerarin, daidzein, daidzin, formononetin, ononin, genistein, genistin, biotin A, glycitein, glycine, tectorigenin and tectoridin were purchased from Shanghai yuanyye Bio-Technology Co., Ltd. (Shanghai city, China). All 16 compounds were over 98% pure. Their exact mass was confirmed by a Quadrupole-Orbitrap Mass spectrometer (Thermo Scientific, San Jose, Calif., USA).

LC grade methanol, formic acid and oxalic acid were purchased from Sigma-Aldrich (st. qentin Fallavier, France). The water was purified using a Milli-Q-System (Millipore, Guyancourt, France). Food grade carbon dioxide was purchased from Beijing Lvyoxon technologies, Inc. (Beijing, China) in 50 pound cylinders without a DIP tube.

The 35 samples were roots of wild Pueraria lobata (Pueraria lobata) and its allied species Pueraria thomsonii and Pueraria peduncularis, which were from different places. These samples were dried in a universal oven with forced convection (FD115, Tuttlingen, Germany) at 50 ℃ for 3 days. The dried sample was ground using a sample grinder (model YF102, rean permanent pharmaceutical machinery, china) and the powder was sieved. Particles between 20 and 80 mesh in size were collected for this study. The voucher specimen was deposited at Kunming plant institute of Kunming Chinese academy of sciences.

1.2 preparation of Standard solution and sample solution

Appropriate amounts of standards were accurately weighed and dissolved in methanol to prepare stock solutions of 16 reference standards (about 0.1-1 mg/mL). All standard solutions were stored at 4 ℃ until use and filtered through a 0.22 μm membrane, and the solutions were diluted from the stock solutions with methanol before injection.

The extraction method comprises the following steps: accurately weigh 0.1g of the powder into a 50mL flask and extract with 30mL of 30% methanol for 30 minutes at room temperature; then, the resulting solution was evaporated under a nitrogen stream at 50 ℃, the resulting residue was dissolved in methanol, and the resulting solution was diluted for detection. These solutions were stable for at least 1 week (confirmed by re-assay) when stored at 4 ℃. Each sample was analyzed by triplicate injection.

1.3 instruments

UHPSFC analysis was performed using the Acquity UPC 2 system (Waters, Milford, MA) which performed Ultra-Performance conversion Chromatography (TM) tests with a PDA detector and a Xevo TQ-XS tandem mass spectrometer. The UHPSFC system is equipped with a binary solvent manager and a sample manager. The PDA detection wavelength was set at 254 nm. Four columns (Waters, Milford, MA) were used in the experiment, including: (1) acquity UPC 2 BEH (1.7 μm, 3X 100mmID), (2) Acquity UPC 2 Torus 1-AA (1.7 μm, 3). times.100 mm ID), (3) Acquity Torus 2-PIC (1.7 μm, 3X 100mm ID), (4) Acquity Torus Diol (1.7 μm, 3X 100mm ID)

The MS was equipped with an ESI source. The flow is split into the post-column MS and convergence manager to maintain supercritical fluid conditions of CO 2.

2. Conditions of the experiment

The UHPSFC conditions were as follows:

gradient program: a standard gradient of CO2 (purity ≥ 99.99%) (A) and 1mmol.L-1 oxalic acid in methanol (B), 20% B (initial), 20-30% B (0-2min), 30-40% B (2-7min), 40% B (7-9min), 40-20% B (9-9.5min), 20% B (9.5-12 min) was used, with a rebalancing time of 1 min before the next injection.

Back pressure: 2200 psi;

sample introduction amount: 1 mu L of the solution;

flow rate: 1.4mL min-1;

and (3) solvent supplement: methanol containing 0.1% (w/v) formic acid at a flow rate of 0.2 mL/min;

column temperature: 50 ℃;

sample temperature: 12 ℃ is used.

Data acquisition was performed in positive mode using ESI, acquisition conditions:

the data acquisition range is m/z 50-800;

capillary voltage: 3 kV;

cone voltage: 40V;

source temperature: 100 ℃;

desolvation temperature: 300 ℃;

cone gas flow rate: 90 liters/hour;

flow rate of desolventizing gas: 900 l/h.

MassLynx TM was used for MS data acquisition and processing.

3. Results and discussion

3.1 comparative analysis of UHPSFC Condition

3.1.1 stationary phase

In order to obtain good peak shape and resolution of the isoflavone isomers using UHPSFC, four columns were evaluated based on the separation efficiency of five pairs of isoflavone isomers with the same mobile phase composition, which were Waters Acquity UPC 2 BEH, Torus 1-AA, Torus Diol and Torus 2-PIC, respectively. The stationary phases tested were all hybrid phases based on bridged ethyl hybrids, representing polar polarity, which resulted in an increase in the polarity of the analyte that should lead to an increase in retention corresponding to the behavior in normal phase liquid chromatography. As a result, as shown in fig. 1, in terms of peak shape and resolution, the best separation effect was observed on a Diol column eluted in the order of (1) biochanin a, (4) glycitein, (7) flavonoid glycoside, (9) glycoside, (10)4' -O-methyl puerarin, (12) daidzein, (13) (14) puerarin, (15) mirificin, (16)6 "-O-xyloside puerarin; the elution order of these isomeric isoflavones on the BEH column is consistent with that of the Diol column, however, the BEH column failed to separate the two pairs of isomers, including peak 9/13 and peak 12/14; similarly, on a 2-PIC column, all isomers were baseline separated except for peak 15/16, but the analyte peak shape on the 2-PIC column was less than ideal compared to the Diol column; in addition, the 1-AA column does not distinguish daidzin (12)/puerarin (14), which is a pair of isomers. The results show that the Diol column has the best separation effect as a separation column in the four chromatographs.

3.1.2 gradient conditions

Organic modifiers must be used to increase the polarity of the mobile phase to analyze isoflavones, as their polarity ranges from non-polar to medium-polar. Methanol is the most commonly used organic modifier due to the favorable elution strength. The methanol gradient program is one of the most critical parameters affecting UHPSFC analysis time of isoflavones. To select the appropriate gradient, the gradient conditions (including gradient time, initial and final concentration of mobile phase) are considered. In practice, the final concentration of organic modifier is kept at 40% to ensure supercritical conditions or at least reasonable viscosity and diffusion coefficients (subcritical conditions). Meanwhile, the peak width is slightly improved with the decrease of the gradient time, and the analysis time is shortened with the increase of the initial concentration of the mobile phase. However, gradient conditions also have an effect on the degree of chromatographic separation. Figures 2 and 3 show that the gradient conditions and initial methanol concentration give improved resolution for the 5 key pairs and improved resolution.

3.1.3 influence of column temperature and Back pressure

Since the elution intensity of the supercritical mobile phase depends on the density of the fluid, a higher elution intensity is obtained at a lower temperature, indicating a higher density of the supercritical fluid, which is contrary to the retention behavior in LC separation. However, due to the presence of the organic modifier, CO₂Both the critical temperature and the pressure of (C) increase significantly. Therefore, when the separation is performed with a higher percentage of organic modifier, i.e. under subcritical conditions, the effect of temperature cannot be predicted. Like temperature, the effect of pressure is less important under subcritical conditions. As shown in fig. 4, increased temperature or decreased pressure thus increases retention in UHPSFC. However, temperature and back pressure only have a minor effect on elution time. The effect of column temperature and back pressure on the resolution of the 5 key pairs is revealed in fig. 5 and 6, respectively. The results show that temperature has a significant effect on the resolution of the 5 key pairs, for example, as shown in FIG. 5, the resolution of peak 10/12 is improved when the temperature is increased from 35 ℃ to 60 ℃, while the resolution is significantly deteriorated when the temperature is changed from 60 ℃ to 65 ℃, i.e., the degree of separation of 5 isomers is best when the temperature is 60 ℃.

3.2 MS/MS conditions

Electrospray ionization (ESI) is the most useful ionization source for the analysis of isoflavones. To develop a sensitive and accurate quantitative method, the ESI in positive and negative ion mode was evaluated in this example by direct injection of individual isoflavones in methanol without using any additives. Isoflavones are first characterized by their molecular formula to automatically determine their precursor ions. Deprotonated molecules [ M-H ] whose precursor ions are all in negative ion mode]^-And the precursor ion is the protonated molecule [ M + H ] in positive ion mode]⁺And radical cation [ M ]]·⁺. This may be related to the molecular structure of isoflavones containing one or more phenolic hydroxyl groups, which results in the easy loss of these analytes even in the positive ion mode. Experimental tests have found that the response of the precursor ions is higher in the positive mode than in the negative mode. Thus, ESI positive ionization mode (ESI +) of all isoflavone standards was used for MRM. Upon identification of precursor ions, two or more products should be selected when MS/MS analysis is used according to relevant regulationsIons. Therefore, the optimization of the product ions and their Collision Energy (CE) is performed in the product ion scan mode. Finally, the most abundant, specific and stable analyte transitions were selected in the MRM mode.

It is noteworthy that tectoridin (tectoridin) and genistin (genistin) could not be detected when combined with the UHPSFC system, whereas their aglycones gave good ionization and response within the corresponding retention times. This indicates that both tectoridin and genistin show in-source Collision Induced Dissociation (CID). CID cannot be avoided by adjusting the cone voltage from 80V to 2V. In addition, additives including ammonium formate, formic acid or oxalic acid are used to improve ionization of tectoridin and genistin. However, even with very moderate parameters, no ionization of the two compounds was observed. This phenomenon is mainly explained by the absence of water in the mobile phase and in the mobile phase state, which results in the two compounds being easily split in the ion source. Furthermore, this suggests that the oxygen-carbon bonds between sugar chains and their aglycones are easily broken down. Thus, ion monitoring channels for tectorigenin and genistein were used to detect their glycosides.

By using a more acidic additive, better ionization of the aglycone for the other target analytes as well as irisin and genistin is obtained within the corresponding retention times. Thus, formic acid and oxalic acid are used. At the same time, the entire run time was divided into 16 MRM acquisition time slices to obtain enough collection points for each chromatographic peak to ensure the accuracy of the quantitative analysis. The ion chromatogram for the extraction of isoflavones under the selected UHPSFC-MS conditions is shown in FIG. 7. Table 1 lists the best MRM results and the proposed fragmentation pathways for these compounds are shown in figure 8. Figure 8 shows that the product ions are also in the form of protonated molecules and radical cations. For example, biotin A produces a product ion of m/z 151.5, which corresponds to a radical cation. At the same time, daidzein produces a product ion at m/z 136.5, which corresponds to a protonated molecule. However, the precursor and product ions may be in the form of protonated molecules as well as radical cations. The reason may come from the type of mass analyzer, other objective reasons in ion source design or analysis, which is not found in the full scan MS mode using Q-orbitrap spectrometry.

TABLE 1

As shown in FIG. 8, the first observation is that most compounds undergo a retro Diels-Alder (RDA) reaction, including

compounds

1,4,5,6,11,14,15 and 16. the second observation is that isoflavone glycosides readily produce their aglycones, including 7,8,9,11,12,15 and 16. the third observation is that several isoflavones may lose. CH3, CO or H2O. however, all O-glycoside isoflavones, including India flavonolide (ononin), tectoridin (tectoridin), glycitin (glycitin), genistin, daidzin (daidzin), puerarin apigenin (mirificin) and 6-O-xyloside puerarin, all produce the most significant characteristic ion [ M-162] + (loss of carbohydrate chains). However, C-glycoside isoflavones such as 3' -methoxy puerarin (minus CH3), puerarin (undergoing RDA reaction), did not produce [ M-162 ]. cndot. +, indicating that a carbon-carbon bond exists between the sugar chain and the sugar chain, and the isoflavone aglycone is difficult to decompose.

3.3 method verification

The UHPSFC-MS method (table 2) was used to perform linearity, regression and linear range of the compounds studied. Correlation coefficient values (r2>0.9990) indicate a good correlation between 16 analyte concentrations and their peak areas within the test range. The instrument limit of detection (LOD) was calculated from the signal-to-noise ratio (S/N) of the standard solution using the definitions S/N > 3. The lower limit of quantitation (LLOQ) was determined as the lowest value. Linear curve, LOD not more than 0.05ng/mL, LLOQ 0.2ng/mL, total in vivo and daytime variation (RSD) of 16 analytes not more than 3.59% and 3.88%, respectively. The method has good accuracy and repeatability, the recovery rate is between 93.6% and 104.7%, and the repeatability expressed as RSD (n-6) is between 0.7% and 3.6%.

TABLE 2 Linear regression data, detection Limit (LOD), lower quantification Limit (LLOQ), precision and repeatability of test Compounds

3.4 sample analysis

The content of isoflavone in Radix Puerariae (RPL), Radix Puerariae (Radix Puerariae Thomsonii) and Radix Puerariae (Radix Puerariae Pedunulis) (RPP) was analyzed by UHPSFC-MS method. RPL and RPT are widely used as medicines and foods, and RPP is often used as an insecticide or pesticide.

TABLE 3 content (mg/g) of test compound in 16 of RPL, RPT and RPP

As shown in table 3, the first observation was that genistin (11), daidzin (12), 3' -methoxypuerarin (13), puerarin (14), mirificin (15) and 6 "-O-xyloside puerarin (16) were the main components. The puerarin content is significantly higher than other puerarin. The second observation was that biotin A (1), formononetin (2), tectorigenin (3), glycitein (4) and 4' -O-methyl puerarin (10) were not detectable in all RPT samples and in most RPPs. This indicates that RPL contains more types of isoflavones than other species. The third observation was that the isoflavone content was significantly different in the three species. The total amount of isoflavones of RPT, RPL and RPP is from 3.31mg/g to 19.83mg/g, from 32.05mg/g to 72.50mg/g and from 1.00mg/g to 51.81mg/g, respectively. The largest mass difference comes from the RPP. RPP has a higher isoflavone content than RPT, but due to its toxicity, is used only as a pesticide or insecticide. The mass differences between the three species can be illustrated by principal component analysis (fig. 8). Considering that RPP is widely distributed in the southwest region of China, more research should be conducted to reasonably use it

3.5 comparison of isoflavone assay methods

UHPSFC-MS/MS enables fast and sensitive analysis. Since the UHPSFC is coupled to the MS/MS, it provides faster speed than the PDA coupling for baseline separation. For the analysis of isoflavones in plant material, it usually takes 30-90 minutes using HPLC-UV and HPLC-MS/MS, although only 12 minutes are required using the novel shell-type column. More importantly, only 8 minutes, column length (50 mm) is required using UHPLC-UV and UHPLC-MS, which has the same column length (100 mm) as current UHPSFC-MS/MS, while 20-45 minutes is required using UHPLC-UV and UHPLC. In the current methods, very high specificity and selectivity are obtained, with a limit of detection (LOD) not exceeding 0.05ng/ml, sufficient for plasma and urine samples.

4 conclusion

In this example, UHPSFC-QqQ-MS/MS technology was used to perform rapid and sensitive quantitative analysis of isoflavones, and 16 common dietary isoflavones in plant material were analyzed. Experimental results have shown that the method of the embodiment of the present invention can analyze even moderately polar substances such as isoflavone glycoside in plant materials with high sensitivity and high speed. As regards the analysis time and the detection limit, the current UHPSFC-QqQ-MS/MS method is clearly advantageous compared to HPLC/UHPLC combined with PDA, MS or MS/MS methods. The results show that the combination of UHPSFC and MS/MS can be used for quickly and sensitively detecting natural products such as isoflavone and the like.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for detecting isoflavones, comprising:

extracting a sample to be detected by using a methanol solution so as to obtain an extracting solution; and

detecting the extract by using ultra-high performance supercritical fluid chromatography-tandem mass spectrometry to obtain the content of the isoflavone compounds,

wherein the mobile phase of the detection treatment is as follows:

A：CO₂(ii) a B: a methanol solution containing 0.1mM of an acidic additive,

wherein the acidic additive is at least one selected from the group consisting of ammonium formate, formic acid and oxalic acid,

wherein the elution gradient of the detection process is initial, 20% B; 0-2min, 20-30% B; 2-7min, 30-40% B; 7-9min, 40% B; 9-9.5min, 40-20% B; 9.5-12min, 20% B, wherein the isoflavone compounds are puerarin, puerarin apioside, 6-O-xyloside puerarin, 4 '-O-methyl puerarin, 3' -methoxy puerarin, daidzein, daidzin, formononetin, genistein, genistin, biotin A, glycitein, glycine, tectorigenin and tectorigenin,

a chromatographic column: a Diol chromatography column.

2. The method of claim 1, wherein the extraction process comprises:

extracting the sample to be detected with 20-40% methanol solution for 24-40 min to obtain extract liquid;

evaporating the extract to obtain residue after evaporation; and

the residue after evaporation was dissolved in methanol to obtain the extract.

3. The method of claim 2, wherein the sample to be tested is mixed with the methanol solution at a ratio of 0.1 g: the extraction is carried out in a proportion of 20-40 ml.

4. The method of claim 1, wherein the mass spectrum in the ultra-high performance supercritical fluid chromatography-tandem mass spectrometry is triple quadrupole mass spectrometry.

5. The method of claim 4, wherein the mass spectrometry conditions of the ultra-high performance supercritical fluid chromatography-tandem mass spectrometry are:

a positive ionization ESI ionization source;

an MRM mode;

capillary voltage: 3 kV;

cone voltage: 40V;

source temperature: 100 ℃;

desolvation temperature: 300 ℃;

cone gas flow rate: 90 liters/hour;

and a flow rate of the desolvation gas, 900 liters/hour,

the tandem mass spectrometer is a Xevo TQ-XS tandem mass spectrometer.

6. The method of claim 4, wherein the chromatographic conditions of the supercritical fluid chromatography-tandem mass spectrometry are:

column temperature: 50 ℃;

sample temperature: 12 ℃;

supplementing liquid: methanol with 0.1% formic acid.

7. The method of claim 1, wherein the Diol chromatography column is 1.7 μm, 3 x 100mm in size.

8. The method of claim 5, wherein the mass spectrum data acquisition range is m/z 50-800.