A method for simultaneously detecting multiple mycotoxins in traditional Chinese medicines and preparations

Abstract

The present invention relates to a method and application for simultaneously detecting a variety of mycotoxins in Chinese medicinal materials and preparations, and belongs to the technical field of compound detection. The detection method of the present invention comprises the following steps: (1) Preparation of a sample solution to be tested: Mix the sample powder to be tested with an extract, oscillate, add sodium salt, magnesium salt and antioxidant to obtain a crude extract of the sample to be tested; After the crude extract of the sample to be tested is centrifuged, the supernatant is taken for solid phase extraction, eluted, and all effluents are collected; The effluent is dried, a re-solution is added, and the mixture is filtered to obtain a sample solution to be tested (2) Detection: A liquid chromatography-mass spectrometer is used for qualitative or quantitative analysis; The method of quantitative analysis is an external standard method. The method of the present invention can qualitatively and quantitatively analyze 33 kinds of mycotoxins in Chinese medicinal materials or Chinese medicinal preparations by simply extracting Chinese medicinal materials or Chinese medicinal preparations, and the method has good stability and specificity, and high accuracy and precision.

Description

Technical Field

[0001] The present invention relates to the technical field of compound detection, and in particular to a method and application for simultaneously detecting multiple mycotoxins in traditional Chinese medicines and preparations. Background Art

[0002] Traditional Chinese medicine is part of my country's excellent cultural heritage and has made outstanding contributions in disease prevention, treatment and health care. With the widespread use of traditional Chinese medicine, its safety issues have also attracted great attention. The mildew of traditional Chinese medicine caused by fungal contamination is one of the main problems affecting the quality and safety of traditional Chinese medicine. Most traditional Chinese medicines come from plants and animals, and the ingredients they contain are mostly organic substances such as fat, starch, protein, and sugar. These ingredients are easily infected by fungi under the influence of temperature, humidity, sunlight, air and other factors, causing fungal toxin contamination, thereby reducing their quality, affecting the treatment effect, and even endangering the health of patients.

[0003] Fungi are a type of eukaryotic microorganisms with a wide variety of species. The main hazards are pathogenic fungi and toxin-producing fungi. Toxin-producing fungi mainly include Aspergillus, Penicillium, Fusarium and other fungi, which can produce a variety of toxic secondary metabolites, such as aflatoxins, ochratoxins, trichothecenes, fumonisins, patulin, etc. A large number of studies have shown that these fungal toxins have harmful effects such as teratogenicity, carcinogenicity and nephrotoxicity.

[0004] At present, the existing analytical methods in my country either use immunoaffinity columns for purification, which is cumbersome, consumes a lot of organic solvents, and has low sensitivity; or use simple HLB universal small column purification. Due to the complex matrix of Chinese herbal medicines / herbal medicines or Chinese patent medicines, the use of a single small column for purification is not enough to remove the impurities of the sample. The final solution on the machine is still dark in color and has high viscosity, which is easy to contaminate the mass spectrometer and the detection limit is difficult to achieve. Therefore, there is an urgent need for a detection method that can make up for the shortcomings of the existing methods, which is both efficient and fast, and fully purifies the Chinese medicine samples. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method and application that is efficient, rapid, highly sensitive, accurate, stable and precise, and can simultaneously detect multiple mycotoxins in Chinese medicinal materials and preparations.

[0006] To achieve the above object, the technical solution adopted by the present invention is: the present invention provides a method for simultaneously detecting multiple mycotoxins in Chinese medicinal materials and preparations, comprising the following steps:

[0007] (1) Preparation of sample solution to be tested:

[0008] The powder of the sample to be tested is mixed with the extract, shaken, and sodium salt, magnesium salt and antioxidant are added to obtain a crude extract of the sample to be tested; the extract is a mixture of formic acid, acetonitrile, methanol and water; the antioxidant is citrate;

[0009] After the crude extract of the sample to be tested is centrifuged, the supernatant is taken for solid phase extraction, eluted, and all the effluent is collected;

[0010] The effluent is dried, a reconstitution solution is added, mixed and filtered to obtain a sample solution to be tested; the reconstitution solution is a mixture of formic acid, methanol, acetonitrile and water;

[0011] (2) Detection: qualitative or quantitative analysis is performed using liquid chromatography-mass spectrometry; the quantitative analysis method is an external standard method;

[0012] The fungal toxin is one or more of 15-acetyl-deoxynivalenol, 3-acetyl-deoxynivalenol, HT-2 toxin, T-2 toxin, α-zearalanol, α-zearalanol, β-zearalanol, β-zearalanol, fumonisin B1, fumonisin B2, fumonisin B3, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, gliotoxin, fusarium ketone-X, mycophenolic acid, penicillic acid, serpentin, deoxynivalenol, neosolani, nivalenol, fumonisin, verrucosporin, zearalanone, zearalenone, sterigmatotoxin, ochratoxin A, ochratoxin B, and ochratoxin C.

[0013] The present invention can detect the mycotoxins contained in Chinese medicinal materials and preparations by selecting a specific extraction method and specific detection conditions, and has the advantages of high efficiency, high sensitivity, accuracy, good stability and precision, and can detect 33 kinds of mycotoxins at the same time.

[0014] As a preferred embodiment of the method of the present invention, in step (1), the volume ratio of methanol, acetonitrile and water in the extract is methanol:acetonitrile:water=(3-4):(4-5):2, and the volume of formic acid in the extract is (1-2)% of the total volume of the extract.

[0015] Under this preferred ratio, the mycotoxins in Chinese medicinal materials and preparations can be effectively extracted, the obtained sample solution has fewer impurities, the extraction rate of mycotoxins is higher, and it can be quickly detected by liquid chromatography-mass spectrometry, further improving the detection efficiency of the method of the present invention. Adding a certain concentration of formic acid to the methanol-acetonitrile-water mixture can more effectively improve the extraction rate and recovery rate of the sample solution to be tested.

[0016] As a preferred embodiment of the method of the present invention, the volume ratio of methanol, acetonitrile and water in the extract is methanol:acetonitrile:water=3:5:2, and the volume of formic acid in the extract is 2% of the total volume of the extract.

[0017] Under this preferred ratio, fungal toxins in Chinese medicinal materials and preparations can be effectively extracted, the obtained test sample solution has few impurities, the extraction rate of fungal toxins is high, and it can be quickly detected by liquid chromatography-mass spectrometry, further improving the detection efficiency of the method of the present invention.

[0018] As a preferred embodiment of the method of the present invention, in step (1), the volume ratio of methanol, acetonitrile and water in the reconstitution solution is methanol:acetonitrile:water=(1-1.5):(3-4.5):(4-6), and the volume of formic acid in the reconstitution solution is (0.1-0.2)% of the total extract volume.

[0019] Under this preferred ratio, the recovery rate of mycotoxins can be effectively improved, and can be quickly detected by liquid chromatography-mass spectrometry, further improving the efficiency of the method of the present invention. Adding a certain concentration of formic acid to the methanol-acetonitrile-water mixture can more effectively improve the recovery rate of the sample solution to be tested.

[0020] As a preferred embodiment of the method of the present invention, the volume ratio of methanol, acetonitrile and water in the reconstitution solution is methanol:acetonitrile:water=1:3:6, and the volume of formic acid in the extract is 0.1% of the total extract volume.

[0021] Under this preferred ratio, the mycotoxins in the sample to be tested can be effectively increased, the recovery rate of the mycotoxins can be effectively improved, and the mycotoxins can be quickly detected by liquid chromatography-mass spectrometry, further improving the efficiency of the method of the present invention.

[0022] As a preferred embodiment of the method of the present invention, in the step (1), the mass ratio of sodium salt, magnesium salt and antioxidant is sodium salt: magnesium salt: antioxidant = 4:1:1.5; the sodium salt is sodium chloride; the magnesium salt is magnesium sulfate; the antioxidant is a mixture of sodium citrate and disodium citrate trihydrate, and the mass ratio of sodium citrate to disodium citrate trihydrate is sodium citrate: disodium citrate trihydrate = 2:1.

[0023] As a preferred embodiment of the method of the present invention, in step (1), the solid phase extraction method is: adding the supernatant to an HLB solid phase extraction column, waiting for the supernatant to drain, adding acetonitrile for elution, and collecting all the effluent.

[0024] The present invention firstly uses an extracting liquid to roughly extract the sample to be tested, and then uses an HLB solid phase extraction column for further purification. The obtained sample solution to be tested has less impurities and low viscosity, and can effectively reduce the influence of impurities on qualitative and quantitative analysis results.

[0025] As a preferred embodiment of the method of the present invention, in step (2), the liquid chromatography conditions of the liquid chromatography-mass spectrometer are as follows:

[0026] The chromatographic column is a C18 liquid chromatography column;

[0027] The mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.1-0.5 wt % formic acid aqueous solution, and the mobile phase B is a mixture of methanol and acetonitrile; the volume ratio of methanol to acetonitrile in the mobile phase B is methanol:acetonitrile=(3-4):(6-7); the mobile phase elution program is

[0028] Time / min Mobile phase A / % Mobile phase B / % 0 10 90 0.5 10 90 1 25 75 2 40 60 2.5 50 50 4.5 50 50 7 80 20 10 80 20 11 95 5 13 95 5 13.1 10 10 16 10 10

[0029] The column temperature was 35-40°C, the flow rate was 0.2-0.4 ml / min, and the injection volume was 5-10 μl.

[0030] Under the optimal parameter conditions, the use of aqueous mobile phase A and organic mobile phase B under the action of C18 column (reverse phase chromatography column) can better separate and ionize 33 kinds of mycotoxins, thereby achieving efficient and rapid qualitative and quantitative analysis of 33 kinds of mycotoxins. Adding 0.1-0.5% formic acid to mobile phase A can prevent chromatographic peak tailing, has the effect of modifying the peak shape to make the peak shape more symmetrical, and can improve the ionization efficiency of mycotoxins, improve the sensitivity of the method, and thus improve the qualitative and quantitative analysis efficiency of the method. The use of methanol-acetonitrile mixed solution as mobile phase B is based on the chemical properties and pH of 33 kinds of mycotoxins. The mobile phase system of methanol-acetonitrile mixed solution (4:6, v / v) was screened out through a large number of experiments, which can well adjust the polarity and viscosity of the mobile phase to achieve the effect of effectively separating 33 kinds of mycotoxins.

[0031] As a preferred embodiment of the method of the present invention, the liquid chromatography conditions of the liquid chromatography-mass spectrometry instrument are as follows:

[0032] The chromatographic column is a Waters CORTECES UPLC C18 chromatographic column, and the specifications of the chromatographic column are 1.6 μm, 2.1×100 mm;

[0033] Mobile phase A was 0.1 wt % formic acid in water, and the volume ratio of methanol to acetonitrile in mobile phase B was methanol:acetonitrile = 4:6;

[0034] The column temperature was 40°C, the flow rate was 0.25 ml / min, and the injection volume was 5 μl.

[0035] Under the optimal parameter conditions, the Waters CORTECES UPLC C18 column has the best separation effect on 33 mycotoxins, with sharp peaks and high responses of each mycotoxin. The use of other C18 columns will reduce the separation of the 33 mycotoxins and may even fail to separate fumonisin-like mycotoxins. The use of 0.1% formic acid aqueous solution as mobile phase A and methanol-acetonitrile (4:6, v / v) mixed solution as mobile phase B can effectively separate 33 mycotoxins. If ultrapure water or sodium acetate aqueous solution is used as mobile phase A, fumonisin-like mycotoxins cannot be effectively separated, and the purpose of detecting 33 mycotoxins cannot be achieved.

[0036] As a preferred embodiment of the method of the present invention, in the step (2), the mass spectrometry conditions of the liquid chromatography-mass spectrometer are as follows: an electrospray ion source ESI is used, the scanning modes are positive ion and negative ion modes, the detection mode is multiple reaction ion monitoring, the curtain gas is 206.88 kPa, the collision gas is 5.376 kPa, the ionization temperature is 450°C, the auxiliary heating gas 1 is 275.8 kPa, and the auxiliary heating gas 2 is 413.7 kPa, wherein the ionization voltage of the positive ion mode is 5.5 kV, and the ionization voltage of the negative ion mode is -4.5 kV.

[0037] Under the optimal parameter conditions, each fungal toxin can be better ionized and the accuracy of the method can be improved.

[0038] As a preferred embodiment of the method of the present invention, the fungal toxin is 15-acetyl-deoxynivalenol, 3-acetyl-deoxynivalenol, HT-2 toxin, T-2 toxin, α-zearalanol, α-zearalanol, β-zearalanol, β-zearalanol, fumonisin B1, fumonisin B2, fumonisin B3, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, gliotoxin, fusarium ketone-X, mycophenolic acid, penicillic acid, serpentin, deoxynivalenol, neosolani fusarium ketone, nivalenol, fumonisin, verrucosporin, zearalanone, zearalenone, sterigmatotoxin, ochratoxin A, ochratoxin B, and ochratoxin C.

[0039] As a preferred embodiment of the method of the present invention, in step (1), the concentration of the standard solution in the external standard method is as follows:

[0040] Aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, sterigmatotoxin, ochratoxin A, ochratoxin B, ochratoxin C and the initial mobile phase solution were respectively prepared into a series of standard working solutions with concentrations of 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, and 50 μg / l;

[0041] A series of standard working solutions with concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, and 250 μg / l were prepared by respectively adding penicillinogen, zearalenone, zearalenone, mycophenolic acid, gliotoxin, α-zearalanol, α-zearalenol, β-zearalanol, and β-zearalenol to the initial mobile phase solution;

[0042] HT-2 toxin, T-2 toxin, fumonisin B1, fumonisin B2, fumonisin B3, penicillic acid, serpentin, and fusarium solani were respectively prepared with the initial mobile phase solution into a series of standard working solutions with concentrations of 1, 2, 4, 10, 20, 40, 100, 200, and 500 μg / l;

[0043] Fumagillin and the initial mobile phase solution were prepared into a series of standard working solutions with concentrations of 2.5, 5, 10, 25, 50, 100, 250, 500, and 1250 μg / l;

[0044] 15-acetyl-deoxynivalenol, 3-acetyl-deoxynivalenol, Fusarium-X, deoxynivalenol and the initial mobile phase solution were respectively prepared into a series of standard working solutions with concentrations of 5, 10, 20, 50, 100, 200, 500, 1000 and 2500 μg / l;

[0045] nivalenol and the initial mobile phase solution were prepared into a series of standard working solutions with concentrations of 10, 20, 40, 100, 200, 400, 1000, 2000, and 5000 μg / l;

[0046] The initial mobile phase solution is prepared by mixing mobile phase A and mobile phase B in a volume ratio of mobile phase A:mobile phase B=1:9.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The method of the present invention can perform qualitative or quantitative analysis on 33 kinds of mycotoxins in Chinese medicinal materials or Chinese medicinal preparations by simply extracting the Chinese medicinal materials or Chinese medicinal preparations. The 33 kinds of mycotoxins have a good linear relationship, the detection limit is 0.3-35 μg / kg, and the quantitative limit is 1-100 μg / kg. The method has good stability and specificity, high accuracy and precision, and can efficiently, quickly and accurately monitor and evaluate the quality and safety of Chinese medicinal materials and Chinese medicinal preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Liquid chromatography tandem mass spectra of aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, fumonisin B3, ochratoxin A, ochratoxin B, ochratoxin C, sterigmatocystin, T-2 toxin, HT-2 toxin, deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, fusarenone-X, neosolaniol, and penicillic acid standards determined by the method of Example 1 of the present invention;

[0050] Figure 2 It is a liquid chromatography tandem mass spectrometry of the gliotoxin, fumonisin, penicillinogen, aflatoxin M1, aflatoxin M2, serpentin, mycophenolic acid, zearalanone, zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, and nivalenol standard products determined by the method of Example 1 of the present invention;

[0051] Figure 3 This is the total ion current graph of 33 fungal toxin standards determined by the method of Example 2 of the present invention;

[0052] Figure 4 This is the total ion current diagram of the fungal toxins contained in Longze Xiongdan Capsules detected in Example 3 of the present invention;

[0053] Figure 5 The total ion current diagram of the negative sample in the method specificity test of Effect Example 1 of the present invention and the total ion current diagram of the negative sample with 33 kinds of fungal toxins added;

[0054] Figure 6 The total ion current diagram of chromatographic column A used in the selection of chromatographic column in Example 2 of the present invention;

[0055] Figure 7 This is the total ion current diagram of chromatographic column B used in the selection of chromatographic column in Effect Example 2 of the present invention;

[0056] Figure 8 This is the total ion current diagram of chromatographic column C used in the selection of chromatographic column in Effect Example 2 of the present invention;

[0057] Fig. 9 The effect of different concentrations of formic acid on the extraction rate of some fungal toxins in the selection of the extracting solution in Example 2 of the present invention;

[0058] Fig.10 The effect of different ratios of methanol-acetonitrile-water mixtures on the extraction rates of 33 fungal toxins in the selection of the extracting solution in Example 2 of the present invention;

[0059] Fig.11 This is the effect of different concentrations of formic acid on the recovery rates of 33 fungal toxins in the selection of the complex solution in Example 2 of the present invention. DETAILED DESCRIPTION

[0060] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0061] In the following examples and comparative examples, the sources of the standard products are shown in Table 1.

[0062] Table 1 Related information of various toxin standards

[0063]

[0064]

[0065] The HLB solid phase extraction cartridge is an Oasis PRiME HLB solid phase extraction cartridge with a specification of 3 ml 60 mg, provided by Waters;

[0066] Formic acid, methanol, and acetonitrile were all of chromatographic grade and provided by MacLean;

[0067] The water was ultrapure water;

[0068] Column A is a Waters CORTECS UPLC C18 column with specifications of 1.6 μm, 2.1 × 100 mm, provided by Waters;

[0069] Column B is a Waters ACQUITY UPLC BEH C18 column with specifications of 1.7 μm, 2.1 × 100 mm, provided by Waters;

[0070] Column C is an Agilent Poroshell 120EC-C18 column with specifications of 2.7 μm, 2.1 × 100 mm, provided by Agilent;

[0071] The remaining reagents and consumables, unless otherwise specified, can be obtained from commercial sources.

[0072] The preparation method of the standard solution in the following examples and comparative examples is as follows:

[0073] (a) Aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin B1 (AFB2), aflatoxin B2 (AFB2), ochratoxin A (OTA), ochratoxin B (OTB), ochratoxin C (OTC), fumonisin B1 (FB1), fumonisin B2 (FB2), fumonisin B3 (FB3), deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-A-DON), 15-acetyl-deoxynivalenol (DON), Fusarium enol (15-A-DON), sterigmatocystin (ST), T-2 toxin (T-2), HT-2 toxin (HT-2), zearalenone (ZAN), zearalenone (ZEN), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), fusarenolide-X (Fus-X), neo-orange fusarenolide (NEO), penicillic acid, gliotoxin, fumagillin, vcurve spore penicillin (VCG), aflatoxin M1 (AFM1), aflatoxin M2 (AFM2), nivalenol (NIV), dioscorea serovar Sinenol (DAS), mycophenolic acid (mycophenolic acid) were prepared with methanol to prepare a single standard stock solution of 100 μg / ml;

[0074] (b) preparing mixed standard intermediate solutions with methanol from single standard stock solutions of 15-A-DON, 3-A-DON, HT-2, T-2, α-ZAL, α-ZOL, β-ZAL, β-ZOL, FB1, FB2, FB3, AFB1, AFB2, AFG1, AFG2, AFM1, AFM2, gliotoxin, Fus-X, mycophenolic acid, penicillic acid, DAS, DON, NEO, NIV, fumagillin, VCG, ZAN, ZEN, ST, OTA, OTB, and OTC, respectively, according to Table 2;

[0075] Table 2 Mixed standard intermediate solutions of various mycotoxin standards

[0076]

[0077] (c) preparing a series of standard working solutions with concentrations of 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, and 50 μg / l by respectively mixing the mixed standard intermediate solutions of AFB1, AFB2, AFG1, AFG2, AFM1, AFM2, ST, OTA, OTB, and OTC with the initial mobile phase solution;

[0078] A series of standard working solutions with concentrations of 0.5, 1, 2, 5, 10, 20, 50, 100, and 250 μg / l were prepared by mixing the mixed standard intermediate solutions of VCG, ZAN, ZEN, mycophenolic acid, gliotoxin, α-ZAL, α-ZOL, β-ZAL, and β-ZOL with the initial mobile phase solution;

[0079] A series of standard working solutions with concentrations of 1, 2, 4, 10, 20, 40, 100, 200, and 500 μg / l were prepared by mixing the mixed standard intermediate solutions of HT-2, T-2, FB1, FB2, FB3, penicillic acid, DAS, and NEO with the initial mobile phase solution;

[0080] A series of standard working solutions with concentrations of 2.5, 5, 10, 25, 50, 100, 250, 500, and 1250 μg / l were prepared by mixing the mixed standard intermediate solution of fumigallin with the initial mobile phase solution;

[0081] A series of standard working solutions with concentrations of 5, 10, 20, 50, 100, 200, 500, 1000, and 2500 μg / l were prepared by mixing the mixed standard intermediate solutions of 15-A-DON, 3-A-DON, Fus-X, and DON with the initial mobile phase solution;

[0082] The mixed standard intermediate solution of NIV and the initial mobile phase solution were prepared into a series of standard working solutions with concentrations of 10, 20, 40, 100, 200, 400, 1000, 2000, and 5000 μg / l;

[0083] The initial mobile phase solution was prepared by mixing mobile phase A and mobile phase B in a volume ratio of mobile phase A:mobile phase B=1:9.

[0084] Example 1

[0085] An embodiment of the method for simultaneously detecting multiple mycotoxins in Chinese medicinal materials and preparations of the present invention comprises the following steps:

[0086] (1) Preparation of sample solution to be tested:

[0087] In this example, Chinese medicinal materials added with 33 kinds of mycotoxin standards were used as test samples. The amount of each mycotoxin added is shown in Table 6.

[0088] Take 1g of the sample powder to be tested that has passed through a No. 2 sieve, add 10ml of the extracting solution, shake and extract for 20min after the extracting solution fully soaks the sample powder to be tested, add 4g of magnesium sulfate, 1g of sodium chloride, 1g of sodium citrate and 0.5g of disodium citrate, vortex for 3min, and obtain a crude extract of the sample to be tested; the extracting solution comprises a methanol-acetonitrile-water mixture with a volume ratio of methanol: acetonitrile: water = 3:5:2 and formic acid accounting for 2% of the total volume of the extracting solution;

[0089] The crude extract of the sample to be tested was centrifuged at 10000r / min for 5min, 1ml of the supernatant was taken for solid phase extraction on an HLB solid phase extraction column, and after the supernatant was drained, 1ml of acetonitrile was added as the eluent, and all the effluent was collected;

[0090] The effluent was blown with nitrogen at 40°C until it was almost completely dry, 1 ml of the reconstitution solution was added, mixed and filtered through a 0.22 μm organic filter membrane to obtain a sample solution to be tested; the reconstitution solution contained a methanol-acetonitrile-water mixture with a volume ratio of methanol:acetonitrile:water = 1:3:6 and 0.1% formic acid in the total volume of the reconstitution solution.

[0091] (2) Detection: Qualitative and quantitative analysis was performed using a liquid chromatography-mass spectrometer (AB Sciex Triple Quad 5500); the quantitative analysis method was an external standard method;

[0092] The liquid chromatography conditions in the liquid chromatography-mass spectrometry instrument are as follows:

[0093] The chromatographic column is chromatographic column A (Waters CORTECES UPLC C18 chromatographic column);

[0094] The column temperature was 40 °C, the flow rate was 0.25 ml / min, and the injection volume was 5 μl;

[0095] The mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is a 0.1 wt% formic acid aqueous solution; mobile phase B is a mixture of methanol and acetonitrile, and the volume ratio of methanol to acetonitrile is methanol:acetonitrile=4:6; the elution degree of the mobile phase is shown in Table 3;

[0096] Table 3 Mobile phase elution program

[0097] Time / min Mobile phase A / % Mobile phase B / % 0 10 90 0.5 10 90 1 25 75 2 40 60 2.5 50 50 4.5 50 50 7 80 20 10 80 20 11 95 5 13 95 5 13.1 10 10 16 10 10

[0098] The mass spectrometry conditions in the liquid chromatography-mass spectrometry instrument are as follows:

[0099] An electrospray ion source ESI was used, the scanning modes were positive ion and negative ion modes, the detection mode was multiple reaction ion monitoring, the curtain gas was 206.88 kPa, the collision gas was 5.376 kPa, the ionization temperature was 450°C, the auxiliary heating gas 1 was 275.8 kPa, and the auxiliary heating gas 2 was 413.7 kPa. The ionization voltage in the positive ion mode was 5.5 kV, and the ionization voltage in the negative ion mode was -4.5 kV.

[0100] The retention time of the chromatographic peak of the fungal toxin detected in the sample solution is consistent with that of the standard. When the relative abundance of each qualifier ion is compared with the standard solution spectrum obtained under the same conditions with similar concentrations, the error does not exceed the range specified in Table 4, and the corresponding fungal toxin can be determined to exist in the sample. The liquid chromatography tandem mass spectra of each standard are shown in Figure 1-2 , mass spectrometry analysis parameters are shown in Table 5 (qualitative results).

[0101] Table 4 Range of relative abundance deviation of sample qualifier ions

[0102] Relative ion abundance ＞50 20-50 10-20 ＜10 Relative deviation range (%) ±20 ±25 ±30 ±50

[0103] Table 5 Standard mass spectrum analysis parameters

[0104]

[0105] The peak area of ​​the series of standard working solutions of the mycotoxin standard was used as the ordinate and the concentration of the standard working solution was used as the abscissa to draw the standard working curve. The mycotoxin in the sample solution to be tested was quantified using the standard curve. The content of mycotoxin in the sample to be tested was calculated according to the formula (the blank value should be deducted from the calculation result). The results are shown in Table 6 (quantitative results). The formula is as follows:

[0106] X=(C×V) / m

[0107] Wherein, X is the mycotoxin content in the sample to be tested, in μg / kg;

[0108] C is the concentration of mycotoxins in the sample solution to be tested, in ng / ml;

[0109] V is the fixed volume of the sample solution to be tested, in ml;

[0110] m is the weight of the sample to be tested, in g.

[0111] Table 6 Results of 33 kinds of mycotoxins

[0112]

[0113] Example 2

[0114] An embodiment of the method for simultaneously detecting multiple mycotoxins in Chinese medicinal materials and preparations of the present invention is similar to the detection method of embodiment 1, except that in step (2), mobile phase A is a 0.5wt% formic acid aqueous solution, the volume ratio of methanol to acetonitrile in mobile phase B is 3:7, the column temperature is 35°C, the flow rate is 0.4ml / min, and the injection volume is 10μl; the remaining steps and their parameter conditions remain unchanged. Qualitative results are shown in Figure 3 , the quantitative results are shown in Table 7.

[0115] Table 7 Results of 33 kinds of mycotoxins

[0116]

[0117] Example 3

[0118] An embodiment of the method for simultaneously detecting multiple mycotoxins in Chinese medicinal materials and preparations of the present invention is similar to the detection method of Example 1, except that:

[0119] In step (2), Longze Xiongdan Capsules were used instead of the licorice herbs with 33 kinds of mycotoxin standards as the test sample; the remaining steps and their parameters remained unchanged. The qualitative results of the mycotoxin detected are shown in Figure 4 , the quantitative results are shown in Table 8.

[0120] Table 8 Results of mycotoxin content in Longze Xiongdan Capsules

[0121]

[0122]

[0123] Effect Example 1 Methodological Verification

[0124] The following methodologies were established based on experiments in Example 1.

[0125] 1. Linear relationship, detection limit and quantification limit

[0126] The peak area of ​​the series of mixed standard working solutions of the 33 kinds of mycotoxin standards was used as the ordinate and the corresponding mass concentration was used as the abscissa to draw the standard working curve. The concentration was calculated based on the peak area of ​​each standard at a 3-fold signal-to-noise ratio (S / N≥3) to determine the method detection limit (LOD); the concentration was calculated based on the peak area of ​​each standard at a 10-fold signal-to-noise ratio (S / N≥3) to determine the method quantification limit (LOQ). The linear regression equation, linear range, correlation coefficient, detection limit, and quantification limit of each standard are shown in Table 9.

[0127] As shown in Table 9, the standard working curves of the 33 fungal toxin standards obtained by the method of the present invention had good linear relationships, with correlation coefficients all greater than 0.996, LODs of 0.3-35 μg / kg, and LOQs of 1-100 μg / kg.

[0128] Table 9 Linearity, detection limit and quantification limit of 33 mycotoxin standards

[0129]

[0130]

[0131] 2. Method accuracy

[0132] The licorice herbs without any mycotoxin detected were taken as negative samples.

[0133] Mixed standard intermediate solutions of 33 mycotoxin standards at three different concentration levels, low, medium and high, were added to the negative samples to prepare test samples containing mycotoxins at different concentration levels. The samples were treated according to step (2) of Example 1, injected into a liquid chromatography-mass spectrometer, and the average recovery rate and relative standard deviation (RSD) of different standards were calculated. Six parallel tests were performed on the standard solutions at each concentration level. The results are shown in Table 10.

[0134] As shown in Table 10, the recoveries of 33 fungal toxins were stable in the range of 78.11-119.42%, and the relative standard deviations were in the range of 0.41-9.66%, indicating that the detection method of the present invention has good accuracy.

[0135] Table 10 Recovery and relative standard deviation of 33 mycotoxin standards

[0136]

[0137]

[0138] 3. Method precision

[0139] The licorice herbs without any mycotoxin detected were taken as negative samples.

[0140] The negative sample was prepared into a test sample solution according to the method of step (2) of Example 1, and standard solutions of fungal toxins of different concentrations were added to the test sample solutions. The samples were injected six times continuously, the peak areas were measured, and the relative standard deviation (RSD) was calculated. The results are shown in Table 11.

[0141] The concentrations of the above mycotoxin standard solutions are as follows:

[0142] The concentrations of AFB1, AFB2, AFG1, AFG2, AFM1, AFM2, OTA, OTB, OTC, and ST were 2 μg / l;

[0143] The concentration of ZAN, ZEN, α-ZOL, β-ZOL, α-ZAL, β-ZAL, gliotoxin, VCG, and mycophenolic acid is 10 μg / l;

[0144] The concentration of FB1, FB2, FB3, T-2, HT-2, NEO, DAS, and penicillic acid was 20 μg / l;

[0145] The concentration of fumagillin was 50 μg / l;

[0146] The concentrations of DON, 3-A-DON, 15-A-DON, and Fus-X were 100 μg / l;

[0147] The concentration of NIV was 200 μg / l.

[0148] Table 11 Precision results of 33 mycotoxins

[0149]

[0150]

[0151] As shown in Table 11, the relative standard deviations of the 33 fungal toxins were all less than 8.75%, indicating that the method of the present invention had good precision.

[0152] 4. Method stability

[0153] The licorice herbs without any mycotoxin detected were taken as negative samples.

[0154] The negative sample was prepared into a test sample solution according to the method of step (2) of Example 1, and standard solutions of fungal toxins of different concentrations were added to the test sample solutions. The samples were injected at different time intervals within 48 hours, the peak areas were measured, and the relative standard deviation (RSD) was calculated. The results are shown in Table 12.

[0155] The concentrations of the above mycotoxin standard solutions are as follows:

[0156] The concentrations of AFB1, AFB2, AFG1, AFG2, AFM1, AFM2, OTA, OTB, OTC, and ST were 4 μg / l;

[0157] The concentration of ZAN, ZEN, α-ZOL, β-ZOL, α-ZAL, β-ZAL, gliotoxin, VCG, and mycophenolic acid is 20 μg / l;

[0158] The concentration of FB1, FB2, FB3, T-2, HT-2, NEO, DAS, and penicillic acid was 40 μg / l;

[0159] The concentration of fumagillin was 100 μg / l;

[0160] The concentration of DON, 3-A-DON, 15-A-DON, and Fus-X was 200 μg / l;

[0161] The concentration of NIV was 400 μg / l.

[0162] Table 12 Stability results of 33 fungal toxins

[0163]

[0164]

[0165] As shown in Table 9, 33 fungal toxins can be detected at different times within 48 hours, and the relative standard deviations are all less than 8.53%, indicating that the method of the present invention has good stability.

[0166] 5. Method Specificity

[0167] The total ion current of the negative sample and the total ion current of the negative sample with 33 kinds of mycotoxins added are shown in Figure 5 .Depend on Figure 5 It can be seen that the negative sample has no interference with the determination, indicating that the method of the present invention has good specificity.

[0168] Effect Example 2 Optimization of Chromatographic Conditions

[0169] 1. Selection of chromatographic column

[0170] The mixed standard intermediates of 33 kinds of mycotoxins obtained in Example 1 were mixed into standard solutions, and the separation effects of chromatographic columns A, B and C on the 33 kinds of mycotoxins were investigated under the same liquid chromatography conditions. The results are shown in FIG. Figure 6-8 .

[0171] like Figure 6-8As shown, when column A is used, the peaks of various fungal toxins are sharp and the separation is high; when column B is used, the responses of mycotoxins ZAN, fumarillin and β-ZAL are high, but the responses of other fungal toxins are lower than those of column A, among which the response of fumonisin fungal toxins is the worst and the separation is low; when column C is used, the responses of mycotoxins OTA, OTC, ST, T-2 and VCG are high, and the responses of other fungal toxins are lower than those of columns A and B. Therefore, the present invention selects column A Waters CORTECS UPLC C18 column (1.6 μm, 2.1×100 mm) as the liquid chromatography column of the liquid chromatography-mass spectrometry instrument.

[0172] 2. Selection of mobile phase

[0173] Since the composition of the mobile phase affects the chromatographic behavior and ionization efficiency of the target components, the mobile phase combinations were investigated.

[0174] Five groups of organic reagents with different combinations were set as mobile phase B, namely 0.1% formic acid-acetonitrile mixture, 0.1% formic acid-methanol mixture, methanol-acetonitrile (1:1, v / v) mixture, methanol-acetonitrile (4:6, v / v) mixture, and methanol-acetonitrile (6:4, v / v) mixture. The results showed that acetonitrile was better than methanol in response and separation of various mycotoxins. Since the 33 mycotoxins involved many types, each mycotoxins had different chemical properties and different acid-base, adding formic acid could not take into account the response of all compounds, so the methanol-acetonitrile system was selected as the mobile phase. On this basis, methanol and acetonitrile were mixed according to different volume ratios, and it was found that when the volume ratio of methanol to acetonitrile was 4:6, the response of each mycotoxin was the highest, and its peak shape was sharper.

[0175] Six different combinations of reagents were set as mobile phase A, namely 0.1% formic acid water, ultrapure water, 0.01% formic acid water, 2mmol / l ammonium acetate, 5mmol / l ammonium acetate, and 10mmol / l ammonium acetate. The results showed that when ultrapure water was used as mobile phase A, fumonisin fungal toxins were not retained, and the separation of other fungal toxins was low; when 0.01% formic acid water was used as mobile phase A, fumonisin B1 was not retained, and the separation of a few fungal toxins was low; when different concentrations of ammonium acetate were used as mobile phase A, the separation of fumonisin fungal toxins was low, and the ionization efficiency of the target component in negative ion mode was low; when 0.1% formic acid water was used as mobile phase A, the ionization efficiency of the target component was the highest, and the sensitivity was increased.

[0176] In summary, the present invention selects 0.1% formic acid water as mobile phase A and a methanol-acetonitrile (4:6, v / v) mixed solution as mobile phase B.

[0177] 3. Selection of extraction solution

[0178] In order to take into account the extraction effects of multiple mycotoxins, three groups of mixed solutions were set as extraction solutions, namely, methanol-water (8:2, v / v) mixed solution, acetonitrile-water (8:2, v / v) mixed solution, and methanol-acetonitrile-water (4:4:2, v / v / v) mixed solution. The above three groups of extraction solutions replaced the extraction solution in step (2) of Example 1, and the remaining steps were determined in the same way as in Example 1, and the extraction rate was calculated based on the mycotoxin content contained in the sample to be tested. The results showed that the methanol-acetonitrile-water mixed solution had a better extraction efficiency for mycotoxins in the sample to be tested.

[0179] On this basis, 0, 1, and 2% formic acid were added to the methanol-acetonitrile-water mixture, and the extraction rates of some fungal toxins were shown in Fig. 9 .

[0180] like Fig. 9 As shown, adding formic acid to the methanol-acetonitrile-water mixture can effectively improve the extraction effect of the extract, and adding 2% formic acid has the best effect.

[0181] In order to further improve the extraction rate of mycotoxins in the test samples, the ratio of methanol, acetonitrile and water was optimized on the basis of the above, and three groups of extraction solutions were set up, namely methanol:acetonitrile:water=4:4:2, methanol:acetonitrile:water=3:5:2, methanol:acetonitrile:water=5:3:2. The extraction rate results are shown in Fig.10 .

[0182] like Fig.10 As shown, when the methanol:acetonitrile:water=4:4:2 and methanol:acetonitrile:water=3:5:2 in the methanol-acetonitrile-water mixture, the extraction rate of fungal toxins in the test sample is higher, among which the extraction rate of fungal toxins in methanol:acetonitrile:water=3:5:2 is the highest.

[0183] In summary, the selection of a methanol-acetonitrile-water (3:5:2, v / v / v) mixture containing 2% formic acid as the extraction solution has the best extraction effect on fungal toxins in the test samples.

[0184] 4. Selection of reconstitution solution

[0185] In order to improve the recovery rate of fungal toxins, three groups of methanol-acetonitrile-water mixed solutions were set as re-solution solutions, namely methanol:acetonitrile:water=0.5:1.5:8, methanol:acetonitrile:water=1.5:4.5:4, and methanol:acetonitrile:water=1:3:6. The test steps are similar to the selection of extraction solution in item 3 of this effect example. The difference is that this experiment only needs to calculate the recovery rate of fungal toxins, not the extraction rate.

[0186] The results showed that when the methanol:acetonitrile:water in the methanol-acetonitrile-water solution was 0.5:1.5:8, the isomers of ZAN, α-ZOL, β-ZOL and α-ZAL, β-ZAL could not be effectively separated; when the methanol:acetonitrile:water in the methanol-acetonitrile-water solution was 1.5:4.5:4, the recovery rate of 33 fungal toxins was 55.6-146.7%; when the methanol:acetonitrile:water in the methanol-acetonitrile-water solution was 1:3:6, the recovery rate of 33 fungal toxins was 70-130%. Therefore, methanol:acetonitrile:water=1:3:6 is the optimal combination, followed by methanol:acetonitrile:water=1.5:4.5:4.

[0187] On this basis, in order to further improve the recovery rate of mycotoxins, 0.1, 0.2, and 2% formic acid were added to the methanol-acetonitrile-water (1:3:6, v / v / v) mixture, respectively. The recovery results are shown in Fig.11 .

[0188] like Fig.11 As shown, adding 0.1% and 0.2% formic acid to the methanol-acetonitrile-water mixture can effectively improve the recovery rate of mycotoxins, among which the recovery rate and response are the highest when 0.1% formic acid is added.

[0189] In summary, the selection of methanol-acetonitrile-water (1:3:6, v / v / v) mixed solution containing 0.1% formic acid as the re-solution had the best recovery rate and the highest response for the fungal toxins in the test samples.

[0190] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for simultaneously detecting multiple mycotoxins in Chinese medicinal materials and preparations, characterized in that: The following steps are involved: (1) Preparation of sample solution: The powder of the sample to be tested is mixed with the extract, shaken, and sodium salt, magnesium salt and antioxidant are added to obtain a crude extract of the sample to be tested; the extract is a mixture of formic acid, acetonitrile, methanol and water; the antioxidant is citrate; After the crude extract of the sample to be tested is centrifuged, the supernatant is taken for solid phase extraction, eluted, and all the effluent is collected; The effluent is dried, a reconstitution solution is added, mixed and filtered to obtain a sample solution to be tested; the reconstitution solution is a mixture of formic acid, methanol, acetonitrile and water; (2) Detection: qualitative or quantitative analysis is performed using liquid chromatography-mass spectrometry; the quantitative analysis method is the external standard method; The mycotoxins are 15-acetyl-deoxynivalenol, 3-acetyl-deoxynivalenol, HT-2 toxin, T-2 toxin, α-zearalanol, α-zearalanol, β-zearalanol, β-zearalanol, fumonisin B1, fumonisin B2, fumonisin B3, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, gliotoxin, fusarium enone-X, mycophenolic acid, penicillic acid, serpentin, deoxynivalenol, neosolani, nivalenol, fumonisin, verrucosporin, zearalanone, zearalanenone, sterigmatotoxin, ochratoxin A, ochratoxin B, ochratoxin C; In step (1), the volume ratio of methanol, acetonitrile and water in the extract is methanol:acetonitrile:water = (3-4):(4-5):2, and the volume of formic acid in the extract is (1-2)% of the total volume of the extract; In the step (1), the solid phase extraction method is: adding the supernatant to an HLB solid phase extraction column, adding acetonitrile for elution until the supernatant is drained, and collecting all the effluent; In the step (2), the liquid chromatography conditions of the liquid chromatography-mass spectrometer are as follows: The chromatographic column is a C18 liquid chromatography column; The mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is a 0.1-0.5 wt% formic acid aqueous solution, and the mobile phase B is a mixture of methanol and acetonitrile; the volume ratio of methanol to acetonitrile in the mobile phase B is methanol:acetonitrile = (3-4): (6-7); the mobile phase elution program is The column temperature was 35-40°C, the flow rate was 0.25-0.4 ml / min, and the injection volume was 5-10 μl; In the step (2), the mass spectrometry conditions of the liquid chromatography-mass spectrometry are as follows: an electrospray ion source ESI is used, the scanning modes are positive ion and negative ion modes, the detection mode is multiple reaction ion monitoring, the curtain gas is 206.88 kPa, the collision gas is 5.376 kPa, the ionization temperature is 450°C, the auxiliary heating gas 1 is 275.8 kPa, and the auxiliary heating gas 2 is 413.7 kPa, wherein the ionization voltage in the positive ion mode is 5.5 kV, and the ionization voltage in the negative ion mode is -4.5 kV.

2. The method according to claim 1, characterized in that The volume ratio of methanol, acetonitrile and water in the extract is methanol:acetonitrile:water=3:5:2, and the volume of formic acid in the extract is 2% of the total volume of the extract.

3. The method according to claim 1, characterized in that In the step (1), the volume ratio of methanol, acetonitrile and water in the reconstitution solution is methanol:acetonitrile:water=(1-1.5):(3-4.5):(4-6), and the volume of formic acid in the reconstitution solution is (0.1-0.2)% of the total volume of the extract.

4. The method according to claim 3, characterized in that The volume ratio of methanol, acetonitrile and water in the reconstituted solution is methanol:acetonitrile:water=1:3:6, and the volume of formic acid in the extract is 0.1% of the total extract volume.

5. The method according to claim 1, characterized in that The liquid chromatography conditions of the liquid chromatography-mass spectrometry instrument are as follows: The chromatographic column is a Waters CORTECES UPLC C18 chromatographic column, and the specifications of the chromatographic column are 1.6 μm, 2.1×100 mm; Mobile phase A was 0.1 wt% formic acid in water, and the volume ratio of methanol to acetonitrile in mobile phase B was methanol:acetonitrile = 4:6; The column temperature was 40°C, the flow rate was 0.25 ml / min, and the injection volume was 5 μl.

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