Method for separating and determining aflatoxin B1 in traditional Chinese medicine powder based on liquid chromatography-mass spectrometry

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining aflatoxin B1 in traditional Chinese medicine powder based on liquid chromatography-mass spectrometry. The method comprises the following steps: 1) sample pretreatment: extracting AFB1 in traditional Chinese medicine powder by utilizing a restrictive supramolecular solvent; 2) separation: taking octadecylsilane chemically bonded silica as a chromatographic column stationary phase, taking a mixed solution of a formic acid solution and acetonitrile as a mobile phase, and separating AFB1 in the traditional Chinese medicine powder through isocratic elution; 3) detection: detecting in a detector to obtain a chromatogram and a mass spectrum; and carrying out qualitative and quantitative analysis on the AFB1 in the traditional Chinese medicine powder according to a detection result. The method is simple and convenient in sample pretreatment, short in time consumption and low in cost, and has relatively good sensitivity, specificity, accuracy and precision.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining aflatoxin B1 in traditional Chinese medicine powder by liquid chromatography-mass spectrometry. Background Art

[0002] During the processes of collection, preparation, storage, and transportation of traditional Chinese medicinal materials, due to improper storage conditions or dampness and mildew, they are very likely to be contaminated with aflatoxins. Aflatoxin B1 (AFB1) is a secondary metabolite produced by strains such as Aspergillus flavus and Aspergillus parasiticus, and is one of the most potent chemical carcinogens known so far. Its toxicity is far higher than that of cyanides, arsenides, and organic pesticides, causing obvious damage to the liver, and can cause the loss of immune function in humans or animals, inducing malformations and the occurrence of cancer. Therefore, the content of aflatoxins in traditional Chinese medicinal materials must be strictly controlled within a safe range.

[0003] Common detection methods for aflatoxins include thin-layer chromatography, high-performance liquid chromatography, enzyme-linked immunosorbent assay, liquid chromatography-tandem mass spectrometry, high-performance liquid chromatography-fluorescence detection method (HPLC-FLD), etc. However, due to the fact that traditional Chinese medicine powder contains a variety of complex and highly diverse matrices, the extraction of aflatoxins is easily interfered by matrix components such as polysaccharides, proteins, lipids, and pigments. For example, polysaccharide substances such as starch, cellulose, and pectin commonly found in traditional Chinese medicine may form colloids or precipitates during the extraction process, affecting the extraction efficiency of the target toxin; plant proteins or microbial proteins may bind to aflatoxins or form precipitates in highly polar solvents, affecting the extraction effect; fat-soluble components such as oils and waxes may compete with non-polar aflatoxins for extraction solvents, reducing the extraction efficiency; chlorophyll, carotenoids, flavonoid pigments, etc. may be simultaneously extracted during the extraction process, interfering with subsequent detection and analysis.

[0004] Therefore, it is necessary to conduct research on the extraction and determination methods of AFB1 in traditional Chinese medicine powder to eliminate interference and improve the detection effect. Summary of the Invention

[0005] In view of this, the present invention proposes a highly efficient, sensitive, widely applicable, and low-cost AFB1 detection method, which can be used for the separation and detection of AFB1 in traditional Chinese medicine powder. The present invention uses a restricted access supramolecular solvent (SUPRAS-RAM) composed of decanoic acid (capric acid) reverse micelles to extract AFB1 in traditional Chinese medicine powder, and uses liquid chromatography-mass spectrometry (LC-MS) for determination. This method is superior to the traditional methods in the pharmacopoeia in terms of sensitivity, selectivity, matrix effect, simplicity of sample pretreatment, and environmental friendliness, and is particularly suitable for the efficient and accurate detection of aflatoxins in complex matrices such as traditional Chinese medicine powder.

[0006] One of the objectives of the present invention is to provide a method for separating aflatoxin B1 from traditional Chinese medicine powder based on liquid chromatography - mass spectrometry (LC - MS), which can effectively separate aflatoxin B1 from traditional Chinese medicine powder in a relatively short time.

[0007] To achieve the above objective, the present invention adopts the following technical solutions:

[0008] The method for separating aflatoxin B1 from traditional Chinese medicine powder based on liquid chromatography - mass spectrometry (LC - MS) includes the following steps:

[0009] (1) Sample pretreatment: Extract AFB1 from traditional Chinese medicine powder using a restricted access supramolecular solvent;

[0010] (2) Separation: Use octadecylsilane - bonded silica gel as the stationary phase of the chromatographic column, and a mixed solution of formic acid solution and acetonitrile as the mobile phase. Separate AFB1 in traditional Chinese medicine powder by isocratic elution; in the mobile phase, the volume ratio of formic acid solution to acetonitrile is 5–15:85–95.

[0011] The restricted access supramolecular solvent proposed by the present invention is spontaneously formed by adding water to a carboxylic acid solution in tetrahydrofuran. These SUPRAS are composed of reverse hexagonal aggregates of carboxylic acids, whose water cavities are surrounded by polar groups, while the hydrocarbon chains are dissolved in THF. The driving forces expected to extract AFB1 include the dispersive interaction between the carboxylic acid hydrocarbon chain and the aromatic structure of the mycotoxin, as well as the hydrogen - bond interaction between the donor and / or acceptor groups they carry. On the other hand, the size of the water cavity of the reverse hexagonal aggregate can be adjusted according to the environment of carboxylic acid self - assembly (such as the volume ratio of THF to water). Therefore, these SUPRAS can dissolve low - molecular - weight polar solutes while excluding the extraction of macromolecules. In addition, proteins precipitate in the presence of THF. Therefore, physical and chemical mechanisms work together to achieve the removal of matrix components, and the extraction and purification of analytes are completed in one step.

[0012] The restricted access supramolecular solvent is synthesized in situ from capric acid, tetrahydrofuran, and NaCl solution during the pretreatment of the sample.

[0013] Preferably, step (1) includes: successively adding capric acid, tetrahydrofuran, and NaCl solution to the traditional Chinese medicine powder containing AFB1, and reacting to obtain a supramolecular solvent sample solution extracting AFB1; the concentration of the tetrahydrofuran is 20% v / v–30% v / v; the concentration of the capric acid is 8–15 mg / mL; the concentration of the NaCl solution is 0.002–0.005 mM, and the pH is 2–4.

[0014] More preferably, the concentration of the tetrahydrofuran is 25% v / v; the concentration of the capric acid is 10 mg / mL; the concentration of the NaCl solution is 0.003 mM, and the pH is 3.

[0015] As a preferred solution, the sample pretreatment includes: weighing 1 g of the traditional Chinese medicine powder sample to be tested and placing it in a centrifuge tube, successively adding 100 mg of capric acid, 2.5 mL of THF, and 7.5 mL of an NaCl solution with a concentration of 0.003 mM and a pH of 3; vortexing for 20 min and centrifuging, taking out all the upper-layer supramolecular solvent, putting it into a centrifuge tube and volatilizing it to dryness in a fume hood, adding a certain volume of methanol to dissolve the residue, and filtering through a 0.22-μm organic filter membrane to obtain a sample solution.

[0016] As a preference, in the mobile phase, the concentration of the formic acid solution is 0.05%–0.2%, more preferably 0.1%.

[0017] As a more preference, in the mobile phase, the volume ratio of the formic acid solution to acetonitrile is 10:90.

[0018] As a preference, the flow rate of the mobile phase is 0.1–0.3 mL / min, more preferably 0.2 mL / min; the column temperature of the chromatographic column is 20–30 °C, more preferably 25 °C.

[0019] As a preference, the specification of the chromatographic column is 50×2.1 mm, 1.7 μm.

[0020] As a preference, the injection volume is 1 μL.

[0021] As a preference, the running time of the liquid chromatography is 1.5 min.

[0022] As a preference, the traditional Chinese medicine powder includes any one or more of notoginseng powder, Chinese prickly ash, fritillary bulb, dwarf lilyturf tuber, and polygonatum rhizome.

[0023] As a preference, the traditional Chinese medicine powder is notoginseng powder.

[0024] The second object of the present invention is to provide a method for identifying aflatoxin B1 in traditional Chinese medicine powder, which can identify aflatoxin B1 in traditional Chinese medicine powder within a relatively short time.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] A method for identifying aflatoxin B1 in traditional Chinese medicine powder, comprising the following steps:

[0027] (1) Separation: Separating aflatoxin B1 in the traditional Chinese medicine powder by using the aforementioned separation method;

[0028] (2) Detection: Detecting by using a mass spectrometry detector to obtain a chromatogram and a mass spectrum;

[0029] (3) Identification: Comparing the chromatographic behavior of the test sample with that of the reference sample to judge whether aflatoxin B1 is contained in the test sample.

[0030] Preferably, in mass spectrometry detection, the ion source is ESI, the detection mode is multiple reaction monitoring, and the detection ion pair of aflatoxin B1 is 313.0 m / z → 285.0 m / z.

[0031] Preferably, QTRAP 4500 system is used for mass spectrometry analysis.

[0032] Preferably, in mass spectrometry detection, DP is 117; CE is 31.0; EP is 10.0; CXP is 7.8.

[0033] Preferably, the retention time is 0.75 ± 0.5 min, and it is determined as aflatoxin B1.

[0034] The third object of the present invention is to provide a method for detecting the content of aflatoxin B1 in traditional Chinese medicine powder, which can determine the content of aflatoxin B1 in traditional Chinese medicine powder within a relatively short time.

[0035] To achieve the above object, the present invention adopts the following technical solutions:

[0036] A method for detecting the content of aflatoxin B1 in traditional Chinese medicine powder, comprising the following steps:

[0037] (1) Separation and detection: Separate and detect aflatoxin B1 in traditional Chinese medicine powder by using the aforementioned identification method to obtain a chromatogram and a mass spectrum;

[0038] (2) Content determination: According to the spectra obtained in step (1), calculate the content of aflatoxin B1 by the external standard method.

[0039] Preferably, within the range of 1–25 ng / mL of aflatoxin B1, y = (431.91 ± 20.51)x + (1677.89 ± 252.41), R 2 = 0.9933; and / or y = (416.82 ± 20.51)x + (380.31 ± 218.17), R 2 = 0.9946; where y is the Y-axis, representing the peak area, x is the X-axis, representing the concentration, and R 2 is the linear coefficient.

[0040] Through content determination, we can further determine whether the content of aflatoxin B1 in traditional Chinese medicine powder is qualified. If the peak area of aflatoxin B1 in the test sample is greater than the peak area of aflatoxin B1 in the reference solution, it indicates that the content of aflatoxin B1 is unqualified; on the contrary, if the peak area of aflatoxin B1 in the test sample is not greater than the peak area of aflatoxin B1 in the reference solution, it indicates that the content of aflatoxin B1 is qualified.

[0041] The beneficial effects of the present invention are as follows:

[0042] 1. The present invention has studied and optimized the parameters affecting the extraction efficiency, including the type of surfactant, the proportion of THF, pH value, salt ion concentration, etc. The results show that when capric acid is used as the surfactant, with a THF volume fraction of 25%, a pH of 3, and an NaCl concentration of 0.003 mM, the extraction effect on AFB1 is the best.

[0043] 2. Compared with the high-performance liquid chromatography-fluorescence detection method commonly used in pharmacopoeias, the present method has significant advantages in terms of sensitivity, specificity, matrix effect, simplicity of sample pretreatment, and environmental friendliness. Under the method of the present invention, the detection limit of AFB1 is 0.086 μg / kg, and the lower limit of quantification is 0.28 μg / kg. The detection limit of this method is significantly lower than that of HPLC-FLD (usually 1–5 μg / kg), and it can detect a lower concentration of aflatoxin, especially suitable for trace analysis.

[0044] 3. For the LC-MS method for separating and determining AFB1 in traditional Chinese medicine powder proposed by the present invention, the matrix effect of AFB1 is 0.99, indicating no matrix effect.

[0045] 4. The LC-MS method for separating and determining AFB1 in traditional Chinese medicine powder proposed by the present invention has good accuracy and precision. The intra-day and inter-day spiked recoveries of AFB1 are respectively between 95.01%–102.88% and 102.35%–103.60%, and the relative standard deviation (RSD) is between 3.24%–11.53%.

[0046] 5. The present invention simplifies the sample pretreatment steps by using supramolecular solvent microextraction technology, reduces the usage amount of organic solvents, is simple and rapid to operate, and meets the requirements of green chemistry. While the pharmacopoeia method usually requires complex sample purification steps (such as immunoaffinity column purification), which is time-consuming and costly.

[0047] 6. The method of the present invention can effectively separate aflatoxin B1 in traditional Chinese medicine powder in a relatively short time and has a good separation effect. Description of the Drawings

[0048] Figure 1 It is a detection result diagram of the influence of three kinds of surfactants on the extraction efficiency of AFB1.

[0049] Figure 2 It is a detection result diagram of the influence of the dosage of capric acid on the extraction efficiency of AFB1.

[0050] Figure 3 It is a detection result diagram of the influence of pH on the extraction efficiency of AFB1.

[0051] Figure 4Detection result graph of the effect of NaCl concentration on the extraction efficiency of AFB1.

[0052] Figure 5 Graphs of the solvent-matched standard curve and matrix-matched standard curve of AFB1, with the peak area on the vertical axis and the AFB1 sample loading concentration on the horizontal axis.

[0053] Figure 6 LC-MS chromatogram of the extraction of aflatoxin B1 (AFB1) by supramolecular solvent; among them, Figure 6 -A is the LC-MS chromatogram of the methanol solution of AFB1 standard, with a concentration of 1 ppb; Figure 6 -B is the LC-MS chromatogram of the supramolecular solvent-extracted Chinese medicine powder spiked sample, with a concentration of 1 ppb; Figure 6 -C is the LC-MS chromatogram of the supramolecular solvent-extracted blank Chinese medicine powder sample.

[0054] Figure 7 Detection result graph of the extraction rate of lauric acid and myristic acid on AFB1, among which, Figure 7 -A is the detection result graph of the extraction rate of lauric acid on AFB1; Figure 7 -B is the detection result graph of the extraction rate of myristic acid on AFB1. Detailed implementation manners

[0055] The technical solutions of the present invention will be further described clearly and completely below in combination with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0056] In the embodiments of the present invention, the aflatoxin B1 standard (Aflatoxin B1, AFB1, concentration: 100 μg / mL, purity: ≥98%) was purchased from Tanmo Quality Inspection Technology Co., Ltd.; the Panax notoginseng Chinese medicine powder was purchased from Beijing Tongrentang Ginseng and Antler Chinese Medicine Products Co., Ltd.; chromatographic grade methanol (HPLC, purity: 99.90%), analytical grade tetrahydrofuran (THF, purity: ≥99.5%), decanoic acid (Decanoic acid, purity: 99%) and myristic acid (Tetradecanoic acid, purity: 98%) were all purchased from Titan Technology Co., Ltd. (Shanghai, China); lauric acid (Lauric acid, purity: ≥98%) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China); hydrochloric acid (HCl, concentration: 36–38%) and sodium chloride (purity: ≥99.5%) were purchased from Chengdu Kelong Chemical Co., Ltd. (China).

[0057] In the embodiments of the present invention, chromatographic analysis was performed using an ACQUITY UPLC system (Waters Corporation, Milford, Massachusetts, USA); mass spectrometry analysis was performed using a QTRAP 4500 system (AB SCIEX, Framingham, Massachusetts, USA); separation was performed using an ACQUITY UPLC BEH C18 chromatographic column (50×2.1 mm, 1.7 μm; Waters Corporation, Milford, Massachusetts, USA); a TGL-16M desktop refrigerated centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd., China); an HZK-FA120 electronic balance (Huazhi Electronic Technology Co., Ltd., China); and an XW-80A vortex mixer (Shanghai Chi Tang Electronics Co., Ltd.).

[0058] In the embodiment of the present invention, the standard solution is prepared by diluting a 100 μg / mL (100 ppm) AFB1 standard methanol solution with methanol to prepare a 1000 ng / mL (1000 ppb) stock solution, which is stored in a -20°C refrigerator away from light for future use.

[0059] In the embodiment of the present invention, the supramolecular solvent microextraction is optimized as follows:

[0060] (1) Add a certain amount of methanol solution containing AFB1 standard into a centrifuge tube and place it in a fume hood overnight to allow the methanol to evaporate completely.

[0061] (2) Accurately weigh a certain amount of capric acid (100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg and 400 mg) and place it in the test tube of step (1), add a certain volume of THF (2 mL, 2.5 mL, 3 mL, 3.5 mL and 4 mL), and after the capric acid is completely dissolved, add a certain amount of pre-prepared sodium chloride solution (concentrations of 0.001 mM, 0.003 mM, 0.005 mM, 0.007 mM, 0.01 mM and 0.03 mM, pH range of 3-7) to make the total volume 10 mL.

[0062] (3) Vortex the centrifuge tube in step (2) for 20 minutes and let it stand. A transparent and water-insoluble supramolecular solvent is formed on the upper layer of the solution. Take out all the supramolecular solvent in the upper layer, put it into a centrifuge tube and evaporate it to dryness in a fume hood, add a certain volume of methanol to dissolve the residue, filter it through a 0.22 μm organic filter membrane (Tianjin Linghang Experimental Equipment Co., Ltd.), and perform LC-MS analysis.

[0063] (4) Blank control: A blank control group without AFB1 standard was treated under the same conditions as above (three times in parallel), and the blank average value was deducted when analyzing the AFB1 content.

[0064] In the embodiment of the present invention, the actual Chinese medicine powder sample is extracted as follows:

[0065] 1) Weigh 1 g of Panax notoginseng traditional Chinese medicine powder and place it in a centrifuge tube. Add a certain amount of methanol solution of AFB1 standard product to submerge the traditional Chinese medicine powder. Open the tube and place it in a fume hood overnight. Wait until the methanol has completely evaporated to obtain a uniformly spiked traditional Chinese medicine powder sample.

[0066] 2) Accurately weigh 100 mg of capric acid and place it in the centrifuge tube in step 1). Add 2.5 mL of THF, and finally add 7.5 mL of sodium chloride solution (concentration is 0.003 mM, pH is 3).

[0067] 3) After vortexing the centrifuge tube in step 2) for 20 min, place it in a centrifuge to precipitate the traditional Chinese medicine powder at the bottom of the centrifuge tube. Take out all the upper-layer supramolecular solvent, place it in a centrifuge tube and let it evaporate to dryness in a fume hood. Add a certain volume of methanol to dissolve the residue, filter it through a 0.22 μm organic filter membrane (Tianjin Pilot Experimental Equipment Co., Ltd.), and perform LC-MS analysis.

[0068] 4) Blank control: The blank control group of the traditional Chinese medicine powder sample without AFB1 standard product is treated under the same conditions as above (in parallel three times), and the blank average value is deducted when analyzing the AFB1 content.

[0069] Example 1. Method for detecting aflatoxin B1 in traditional Chinese medicine powder based on LC-MS

[0070] (1) Preparation of the test solution

[0071] Sample solution: Weigh 1 g of Panax notoginseng traditional Chinese medicine powder and place it in a centrifuge tube. Add a certain amount of methanol solution of AFB1 standard product to submerge the traditional Chinese medicine powder. Open the tube and place it in a fume hood overnight. Wait until the methanol has completely evaporated to obtain a uniformly spiked traditional Chinese medicine powder sample. Accurately weigh 100 mg of capric acid and place it in the above centrifuge tube. Add 2.5 mL of THF, and finally add 7.5 mL of sodium chloride solution (concentration is 0.003 mM, pH is 3). After vortexing the centrifuge tube for 20 min, place it in a centrifuge to precipitate the traditional Chinese medicine powder at the bottom of the centrifuge tube. Take out all the upper-layer supramolecular solvent (the restricted access supramolecular solvent that has extracted AFB1), place it in a centrifuge tube and let it evaporate to dryness in a fume hood. Add a certain volume of methanol to dissolve the residue, filter it through a 0.22 μm organic filter membrane to obtain the sample solution.

[0072] (2) Chromatographic conditions and mass spectrometry parameters

[0073] The chromatographic conditions and mass spectrometry parameters are shown in Table 1.

[0074] Table 1. Table of chromatographic conditions and mass spectrometry parameters

[0075]

[0076] (3) Detection

[0077] Precisely measure 1 μL of the sample solution to be tested and inject it directly. Conduct the detection according to the aforementioned chromatographic and mass spectrometric conditions to obtain the chromatogram and mass spectrum. Then, use the external standard method (that is, by measuring the standard solution with a known concentration, establishing a standard curve of signal intensity (such as peak area) versus concentration, and then calculating the AFB1 content based on the sample signal) to calculate the AFB1 content.

[0078] Example 2. Preparation and extraction optimization of supramolecules

[0079] The supramolecular solvent formed based on long-chain alkyl acids is a reverse micelle with an ordered structure formed by mixing long-chain alkyl acids, water, and an organic solvent (such as THF) in an appropriate ratio through spontaneous self-assembly. When the ratio of alkyl acid to THF is different, the pore size of the formed reverse micelle is different, which will have different degrees of purification effects on the sample matrix. Different types of long-chain alkyl acids will affect the composition and size of the supramolecule, thereby affecting the extraction efficiency. In this experiment, capric acid, lauric acid, and myristic acid (C8, C 12 、C 14 ) were selected for the optimization of the type and dosage of alkyl acids; and after selecting the optimal surfactant, the optimization of the ratio of THF, solution pH, NaCl salt ion strength, equilibrium solution, and powder mass was carried out. Panax notoginseng powder was selected as the sample, and AFB1 was used as the target analyte for the standard addition recovery experiment, and the extraction efficiency was evaluated by the AFB1 peak area after the final extraction.

[0080] (1) Influence of surfactant type and different THF percentages on extraction effect

[0081] Fix the mass of three long-chain alkyl acids (capric acid, lauric acid, and myristic acid; C8, C 12 and C 14 ) at 200 mg each, add different volumes of tetrahydrofuran (2 mL, 2.5 mL, 3 mL, 3.5 mL, and 4 mL) to dissolve them, and then add deionized water (pH = 3) to a total volume of 10 mL, that is, the volume fractions of THF are 20%, 25%, 30%, 35%, and 40% (v / v), respectively. All three alkyl acids can form a water-insoluble supramolecular solvent on the upper layer of the solution and effectively extract aflatoxin AFB1. As Figure 1 shown, the AFB1 peak area extracted by the supramolecular solvent prepared with capric acid is significantly larger than that of the supramolecular solvents prepared with lauric acid and myristic acid. Therefore, capric acid was finally selected as the surfactant for this experiment.

[0082] (2) Optimization of surfactant dosage

[0083] Weigh different masses of capric acid (100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, and 400 mg) respectively, and add 2.5 mL of THF to dissolve it. Subsequently, add 7.5 mL of deionized water (pH = 3) to the solution to ensure that the THF concentration is fixed at 25% (v / v). As Figure 2 shown, when the amount of capric acid used is 100 mg, the formed supramolecular solvent has the best extraction effect on AFB1.

[0084] (3) Optimization of the pH of the aqueous solution

[0085] Weigh 100 mg of capric acid and add 2.5 mL of THF to completely dissolve it. Add 7.5 mL of deionized water with the pH adjusted by hydrochloric acid (pH values are 3, 4, 5, 6, 7) respectively. As Figure 3 shown, when the pH is 3, the formed supramolecular solvent has the best extraction effect on AFB1.

[0086] (4) Optimization of salt ions

[0087] Weigh 100 mg of capric acid and add 2.5 mL of THF to completely dissolve it. Add NaCl solutions with concentrations of 0.001 mM, 0.003 mM, 0.005 mM, 0.007 mM, 0.01 mM, and 0.03 mM (pH = 3) respectively. As Figure 4 shown, when the NaCl concentration is 0.003 mM, the formed supramolecular solvent has the best extraction effect on AFB1.

[0088] Example 2. Method verification

[0089] Under the optimized best experimental conditions of Example 2, the sensitivity, accuracy, and precision of the detection method for aflatoxin B1 in the Panax notoginseng traditional Chinese medicine powder of the present invention are evaluated respectively, and parameters such as the linear range, detection limit, quantification limit, spiked recovery rate, and relative standard deviation of the method are given.

[0090] (1) Linearity and matrix effect

[0091] Weigh 1 g of the traditional Chinese medicine powder sample and add a series of methanol solutions of AFB1 standard products respectively. Under the optimal extraction conditions, operate according to the "supramolecular solvent microextraction" and "extraction of actual traditional Chinese medicine powder sample" parts described above to make the final loading mass concentration of AFB1 be 1, 2.5, 5, 10, 25 ng / mL. Establish a matrix-matched standard curve with the concentration of each analyte as the abscissa and its corresponding peak intensity (peak area) as the ordinate. Repeat the experiment three times for each concentration. Under the same operating conditions, the solvent-matched standard curve is without the influence of the traditional Chinese medicine powder sample.

[0092] From Figure 5It can be seen that within the range of 1–25 ng / mL, both the matrix-matched and solvent-matched standard curves of AFB1 showed good linearity, and the linear determination coefficient R 2 > 0.99. The equation of the matrix-matched standard curve is: y = (431.91 ± 20.51)x + (1677.89 ± 252.41), and the linear coefficient R 2 = 0.9933. Under the same optimal conditions, after the supramolecular solvent extracted AFB1 and was detected by the instrument, the equation of the solvent-matched standard curve obtained was: y = (416.82 ± 20.51)x + (380.31 ± 218.17), and the linear coefficient R 2 = 0.9946.

[0093] The matrix effect is represented by the ratio of the slopes of the two standard curves. It is considered that the matrix effect can be divided into 7 types: strong enhancement effect (> 1.5), medium enhancement effect (1.2–1.5), slight enhancement effect (1.1–1.2), no matrix effect (0.9–1.1), slight inhibition effect (0.8–0.9), medium inhibition effect (0.5–0.8) and strong inhibition effect (< 0.5). The results showed that the matrix effect of AFB1 in this experiment was 0.99, which was a no matrix effect and met the requirements of the quantitative analysis method.

[0094] (2) Method sensitivity

[0095] Sensitivity is often expressed by the limit of detection (LOD) and / or the lower limit of quantitation (LLOQ). In instrumental analysis, it is commonly determined by the ratio of the signal value to the noise, that is, the signal-to-noise ratio (S / N). Generally, the mass concentration of the analyte when S / N is 10 is used as the LLOQ, and the mass concentration of the analyte when S / N is 3 is used as the LOD.

[0096] The limit of detection (LOD) for detecting aflatoxin B1 in Panax notoginseng powder by the aforementioned method was 0.086 μg / kg; the lower limit of quantitation (LLOQ) was 0.28 μg / kg.

[0097] (3) Recovery rate and precision

[0098] AFB1 standard methanol solution was added to the blank TCM powder sample to make the final mass concentration of AFB1 in the TCM powder 1μg / kg. The content of the target compound in the spiked TCM powder sample was detected according to the optimized analytical method, and three parallel samples were set up. In addition, the intra-day precision experiment was measured three times on the same day (once every 3 hours); the inter-day precision experiment was measured three times on different 3 days (once a day). The spike recovery of AFB1 was calculated using the accompanying calibration curve, and the relative standard deviation (RSD%) was used to represent the precision. The results are shown in Table 2. The average spike recoveries of AFB1 in the three intra-days were 101.34, 95.01 and 102.88%, respectively; the RSD was 3.56–11.53%; the spike recoveries between days were 102.35 and 103.60%, respectively; the RSD was 3.24–9.32%.

[0099] Table 2. Intra-day and inter-day recoveries and standard deviations

[0100]

[0101] (4) Method specificity

[0102] Comparison of the chromatograms of blank samples and spiked samples can verify the specificity of the method. If the blank sample has no interfering peaks at the peak position of the target substance, while the spiked sample has obvious target peaks, it means that the method can specifically detect the target substance and is not interfered by the matrix. Figure 6 -C shows that the blank sample of Chinese medicine powder has no interfering peak at the peak position of the target substance; Figure 6 -A. Figure 6 -B shows that AFB1 after supramolecular solvent extraction has the same good peak shape and the same retention time as the AFB1 standard solution. The retention time of aflatoxin B1 is 0.75min, which meets the requirements of quantitative analysis. The peak signal of the target in the spiked sample is significantly higher than the noise level in the blank sample, indicating that this method has high sensitivity and low detection limit.

[0103] Comparative Example 1

[0104] The extraction rate of AFB1 by the supramolecular solvent formed by capric acid and THF under the optimal conditions (capric acid: 100 mg; THF: 25%; pH: 3; NaCl: 0.003 mM) was compared with the extraction rate of AFB1 by the supramolecular solvent formed by lauric acid and myristic acid before optimization. Figure 7 As shown, the extraction efficiency of AFB1 by the supramolecular solvent formed by lauric acid and myristic acid is much lower than the extraction efficiency of AFB1 by the supramolecular solvent formed by capric acid and THF under the optimal conditions (Table 2).

Claims

1. A method for separating aflatoxin B1 from traditional Chinese medicine powder based on liquid chromatography-mass spectrometry, characterized in that: The steps include: (1) Sample pretreatment: AFB1 was extracted from Chinese herbal medicine powder using restricted access supramolecular solvent; (2) Separation: Octadecylsilane bonded silica gel was used as the stationary phase of the chromatographic column, and a mixed solution of formic acid solution and acetonitrile was used as the mobile phase. AFB1 in the traditional Chinese medicine powder was separated by isocratic elution; in the mobile phase, the volume ratio of formic acid solution to acetonitrile was 5–15:85–95.

2. The method according to claim 1, characterized in that: Step (1) comprises: adding capric acid, tetrahydrofuran and NaCl solution to the Chinese medicine powder to be tested in sequence, reacting to obtain a supramolecular solvent that extracts AFB1; the concentration of the tetrahydrofuran is 20% v / v-30% v / v; the concentration of the capric acid is 8-15 mg / mL; the concentration of the NaCl solution is 0.002-0.005 mM, and the pH is 2-4.

3. The method according to claim 1, characterized in that In the mobile phase, the concentration of formic acid solution is 0.05%-0.2%.

4. The method according to claim 1, characterized in that: The mobile phase flow rate was 0.1–0.3 mL / min, and the column temperature was 20–30 °C.

5. The method according to claim 1, characterized in that The Chinese medicine powder includes any one or more of Panax notoginseng powder, Zanthoxylum bungeanum, Fritillaria cirrhosa, Ophiopogon japonicus and Polygonatum sibiricum.

6. A method for identifying aflatoxin B1 in traditional Chinese medicine powder, characterized in that: The steps include: (1) Separation: Separating aflatoxin B1 from traditional Chinese medicine powder using the method described in any one of claims 1 to 5; (2) Detection: Detection is performed using a mass spectrometer to obtain a chromatogram and a mass spectrum; (3) Identification: Compare the chromatographic behavior of the test sample with that of the reference sample to determine whether the test sample contains aflatoxin B1.

7. The method according to claim 6, characterized in that In mass spectrometry detection, the ion source is ESI, the detection mode is multiple reaction monitoring, and the detection ion pair of aflatoxin B1 is 313.0 m / z→285.0 m / z.

8. The method according to claim 6, characterized in that The retention time was 0.75±0.5min, and it was determined to be aflatoxin B1.

9. A method for detecting the content of aflatoxin B1 in traditional Chinese medicine powder, characterized in that: The steps include: (1) Separation and detection: Separating and detecting aflatoxin B1 in the traditional Chinese medicine powder by the method described in any one of claims 6 to 8 to obtain a chromatogram and a mass spectrum; (2) Content determination: Based on the spectrum obtained in step (1), the content of aflatoxin B1 was calculated using the external standard method.

10. The method according to claim 9, characterized in that The aflatoxin B1 is in the range of 1–25 ng / mL. y=(431.91±20.51)x+(1677.89±252.41), R 2 =0.9933; and / or y = (416.82 ± 20.51) x + (380.31±218.17), R 2 =0.9946; where y is the Y axis, representing the peak area, x is the X axis, representing the concentration, R 2 is the linear coefficient.

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