Method for detecting ochratoxin A in Xiaoyao pills

Abstract

The invention discloses a method for detecting ochratoxin A in Xiaoyao pills. The method comprises the following steps: 1, preparing an ochratoxin A standard solution with mixed matrixes; 2, constructing a standard curve; 3, purifying and preparing a test solution by using an HLB (Hydrophile-Lipophile Balance) universal small column; and 4, detecting the test solution by adopting high performance liquid chromatography-tandem mass spectrometry, and quantitatively calculating quantitative ions by using a standard curve to obtain the concentration of the ochratoxin A in the test solution. According to the method, a to-be-detected sample is purified by adopting an HLB (Hydrophile Lipophile Balance) universal small column, and detection is performed by adopting a liquid chromatography-tandem mass spectrometry method, so that the pretreatment time and detection time of the sample can be effectively saved; the ochratoxin A standard solution mixed with the matrix is adopted for quantitative determination, and the influence of the matrix on the accuracy of the detection result can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for detecting ochratoxin A in Xiaoyao pills. Background Art

[0002] Traditional Chinese medicine (TCM) is part of my country's outstanding cultural heritage and has made outstanding contributions to disease prevention, treatment, and healthcare. Most TCMs are derived from plants and animals and contain organic substances such as fat, starch, protein, and sugar. These ingredients are susceptible to fungal infection and mycotoxin contamination under the influence of various factors such as temperature, humidity, sunlight, and air. This can reduce their quality, affect therapeutic efficacy, and even endanger patients' health. With the development of production technology, traditional TCM decoctions have changed their dosage form to granules, capsules, pills, and other forms for easier administration and portability. Although these preparations have changed in dosage form, due to the characteristics of mycotoxins, fungi in the medicinal materials can be introduced into TCM patent medicines, which still pose a threat to patients' health.

[0003] Ochratoxin A (OTA) is a mycotoxin primarily produced by Penicillium verrucosum, Aspergillus ochraceus, and Aspergillus carbonarius. Toxicological studies have shown that OTA is a highly toxic compound with side effects including nephrotoxicity, hepatotoxicity, teratogenicity, carcinogenicity, and mutagenicity. Its primary human harm is kidney damage. It is now classified as a Group 2B possible carcinogen by the International Agency for Research on Cancer (IARC).

[0004] At present, foreign research on ochratoxin A mainly focuses on cereals, dried nuts, condiments, alcoholic beverages, coffee, and infant foods. Only the EU food standards have set limits for ochratoxin A in licorice and its extracts. The detection range of ochratoxin A in relevant domestic standards and literature mainly applies to cereals, oils and their products, alcoholic beverages, soy sauce, vinegar, sauce and sauce products, raisins, peppercorns / powder, chili peppers and their products, raw coffee, and cooked coffee. As the national legal technical standard for drugs, the 2020 edition of the "Chinese Pharmacopoeia" does not set limit standards for ochratoxin A under the various varieties in Part I and the general rules in Part IV. The existing methods for detecting mycotoxins proposed in the "Chinese Pharmacopoeia" include thin layer chromatography, enzyme-linked immunosorbent assay, colloidal gold immunochromatography, high performance liquid chromatography, and liquid chromatography-mass spectrometry. Thin-layer chromatography is primarily used for initial screening. It's simple to operate and inexpensive, but the results aren't accurate enough to accurately determine the ochratoxin A content. Immunoaffinity chromatography offers the advantages of high specificity and sensitivity, but it's cumbersome to operate and expensive, making it both time-consuming and costly for large-scale screening. Colloidal gold immunochromatography is suitable for on-site, single or small sample testing, but it's inadequate for large-scale screening. High-performance liquid chromatography offers strong specificity and good reproducibility, but it takes a long time to test, is susceptible to matrix interference, and can be difficult to accurately measure when the content is low.

[0005] At present, the detection of mycotoxins is mainly concentrated on the determination of medicinal materials and decoction pieces. Except for the varieties specified in the 2020 edition of the Chinese Pharmacopoeia, there are no standard regulations for the detection of mycotoxins in other varieties of medicinal materials and decoction pieces. The types of mycotoxins detected under the 2020 edition of the Chinese Pharmacopoeia are mainly aflatoxins and zearalenone. Among them, aflatoxins involve 24 varieties, zearalenone involves 1 variety, and ochratoxin A only has a determination method that does not involve specific medicinal materials and decoction pieces. Chinese patent medicines are Chinese medicinal materials as raw materials. Under the guidance of traditional Chinese medicine theory, they are processed into a certain dosage form according to the prescribed prescription and preparation process. They are a type of approved commercialized Chinese medicine preparations. Xiaoyao Pills (concentrated pills) are a Chinese medicine preparation. Its prescription is composed of seven herbs: Bupleurum, Angelica, White Peony Root, Stir-fried Atractylodes, Poria, Roasted Licorice, and Mint. The licorice in the prescription is very easy to produce the fungal toxin ochratoxin A during the harvesting, processing, transportation and storage. In the preparation process for Xiaoyao Pills (concentrated pills), licorice is used both as a raw powder and as a decocted extract. Given that ochratoxin A is readily soluble in water, stable in polar organic solvents, heat-resistant, and not detrimental to its toxicity during cooking, the presence of these two forms in the preparation process increases the risk of residual ochratoxin A. Regarding the current status of mycotoxin testing, licorice is not included in the Chinese Pharmacopoeia, and Xiaoyao Pills (concentrated pills), as a traditional Chinese medicine, are not included. No other literature has identified an effective method for determining ochratoxin A in Xiaoyao Pills (concentrated pills). Summary of the Invention

[0006] The present invention aims to address the deficiencies of the prior art by providing a method for detecting ochratoxin A in Xiaoyao Pills. This method utilizes liquid chromatography-tandem mass spectrometry for detection, effectively saving sample pretreatment and detection time. Furthermore, quantitative determination using a matrix-mixed ochratoxin A standard solution can reduce the impact of the matrix on the accuracy of the detection results.

[0007] To solve the above technical problems, the technical solution adopted by the present invention is: a method for detecting ochratoxin A in Xiaoyao Pills, characterized in that it comprises the following steps:

[0008] Step 1, preparing a matrix-mixed ochratoxin A standard solution: preparing an ochratoxin A standard solution; treating Xiaoyao pills that do not contain ochratoxin A as a blank matrix, adding the treated Xiaoyao pills to the prepared ochratoxin A standard solution to obtain a matrix-mixed ochratoxin A standard solution;

[0009] Step 2: Constructing a standard curve: performing high performance liquid chromatography tandem mass spectrometry on the ochratoxin A standard solution mixed with the matrix described in step 1, and plotting a standard curve with the peak area as the ordinate and the injection concentration as the abscissa;

[0010] Step 3. Prepare the test solution: grind the Xiaoyao pills to be tested into powder, dissolve them in 70% methanol solution, ultrasonicate for 30 minutes, centrifuge, accurately measure 3 mL of the supernatant and pass it through the HLB column to collect the eluate; then elute with 3 mL of methanol, collect the eluate, combine the two eluates, slowly blow nitrogen at 40°C until almost dry, add 50% acetonitrile solution to make the volume to 1 mL, filter with a microporous filter membrane, and take the filtrate to obtain the test solution;

[0011] Step 4: Detect the test solution: Perform high performance liquid chromatography tandem mass spectrometry on the test solution of step 3, and then use the standard curve drawn in step 2 to quantitatively calculate the quantitative ion to obtain the concentration of ochratoxin A in the test solution.

[0012] The above-mentioned method for detecting ochratoxin A in Xiaoyao Pills is characterized in that detection in steps 2 and 4 is performed using a triple quadrupole mass spectrometer, and the triple quadrupole mass spectrometer is an Agilent 1290INfinityⅡ-6470 liquid chromatography-mass spectrometry instrument.

[0013] The above-mentioned method for detecting ochratoxin A in Xiaoyao Pills is characterized in that the concentrations of the ochratoxin A standard solution in step 1 are 0.25 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL and 10.0 ng / mL, respectively.

[0014] The above-mentioned method for detecting ochratoxin A in Xiaoyao Pills is characterized in that the preparation method of the ochratoxin A standard solution in step 1 includes: dissolving the ochratoxin A standard in methanol to prepare a methanol solution containing 1000 ng of ochratoxin A per 1 mL, and then diluting it with 50% acetonitrile solution to obtain a standard solution of the required concentration.

[0015] The above-mentioned method for detecting ochratoxin A in Xiaoyao pills is characterized in that the method for treating the blank matrix in step 1 includes: grinding the Xiaoyao pills that do not contain ochratoxin A, dissolving them in 70% methanol solution, ultrasonically treating them for 30 minutes, centrifuging them, accurately measuring 3 mL of the supernatant, passing it through an HLB column, and collecting the eluate; then eluting with 3 mL of methanol, collecting the eluate, combining the two eluates, and slowly blowing them with nitrogen at 40°C until they are almost dry.

[0016] The above-mentioned method for detecting ochratoxin A in Xiaoyao Pills is characterized in that the high performance liquid chromatography detection conditions in step 2 and step 4 are:

[0017] Octadecylsilane bonded silica gel was used as the packing material (Agilent Eclipse Plus C18, 2.1×100 mm, 1.8 μm). Mobile phase A consisted of 0.1% formic acid containing 2 mmol / L ammonium acetate, and mobile phase B consisted of methanol. The column temperature was 30°C, the flow rate was 0.3 ml / min, and the injection volume was 5 μL. Gradient elution was performed.

[0018] Mass spectrometry detection conditions are:

[0019] The ion source is electrospray (ESI + ) ion source, drying gas temperature: 250℃, drying gas flow rate: 8L / min, nebulizer pressure: 35psi, sheath gas temperature: 375℃, sheath gas flow rate: 11L / min, capillary voltage: 3000V; detection mode was multiple reaction monitoring (MRM), and the monitored ion pairs and collision voltage (CE) included: quantitative ion (404.0→239.0, CE: 25V) and qualitative ion (404.0→358.0, CE: 15V; 404.0→102.0, CE: 55V).

[0020] The above-mentioned method for detecting ochratoxin A in Xiaoyao Pills is characterized in that the gradient elution procedure includes:

[0021] From 0 min to 2 min, the volume percentage of mobile phase A decreased from 95% to 50%;

[0022] From 2 to 8 minutes, the volume percentage of mobile phase A decreased from 50% to 5%;

[0023] From 8 to 10 minutes, the volume percentage of mobile phase A was maintained at 5%.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The present invention uses an HLB universal cartridge to purify the sample to be tested and adopts liquid chromatography-tandem mass spectrometry for detection, which can effectively save sample pretreatment time and detection time; and uses a matrix-mixed ochratoxin A standard solution for quantitative determination, which can reduce the influence of the matrix on the accuracy of the detection result.

[0026] 2. The present invention adopts positive ion mode (ESI + ), a quantitative ion (404.0→239.0) and two qualitative ions (404.0→358.0; 404.0→102.0) were selected for determination, and the negative ion mode (ESI - ) in positive ion mode (ESI +) The ion pairs under the condition of 404.0→239.0; 404.0→102.1 can more accurately determine the ochratoxin A in the test solution.

[0027] 3. The present invention accurately identifies the characteristic fragment ions of ochratoxin A and constructs a standard curve using a matrix-mixed ochratoxin A standard solution, which can accurately quantify ochratoxin A in the sample to be tested, and the test results are accurate and reliable.

[0028] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The negative ion acquisition mode (ESI - ), and the mass spectrum with monitoring ion pairs of m / z 402.1→358.1 and m / z 402.0→211.0.

[0030] Figure 2 In Example 1, positive ion acquisition mode (ESI + ), the mass spectrum with ion pairs of m / z 404.0→239.0 and m / z 404.0→102.1.

[0031] Figure 3 In Example 1, positive ion acquisition mode (ESI + ), the mass spectrum with ion pairs of m / z 404.0→239.0, m / z 404.0→358.0 and m / z 404.0→102.0.

[0032] Figure 4 This is the mass spectrum of Example 2 using methanol-0.1% formic acid (2 mM ammonium acetate) as the mobile phase.

[0033] Figure 5 This is the mass spectrum of methanol-0.1% formic acid as the mobile phase in Example 2.

[0034] Figure 6 It is the mass spectrum of the solvent standard curve in Example 3.

[0035] Figure 7 This is the solvent standard curve drawn in Example 3.

[0036] Figure 8 This is the mass spectrum of the standard curve of the matrix mixed solution in Example 3.

[0037] Figure 9 This is the standard curve of the matrix mixed solution drawn in Example 3.

[0038] Figure 10This is the mass spectrum of the Xiaoyao Pill sample numbered X156 in Example 3. DETAILED DESCRIPTION

[0039] The present invention is described in detail below with reference to the following examples. These examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of the present invention. Experimental procedures where specific conditions are not specified in the examples are generally based on conventional conditions, those described in the manual, or those recommended by the manufacturer. The equipment, materials, and reagents used are all commercially available unless otherwise specified.

[0040] The ochratoxin A reference substance used in the following examples has a batch number of H3003268, a source of Beijing Manhag Biotechnology Co., Ltd., and a concentration of 100 μg / mL.

[0041] Example 1: Selection of ion pairs

[0042] Prepare ochratoxin A solution: Accurately weigh an appropriate amount of ochratoxin A reference substance and add methanol to make a solution containing 1000 ng of ochratoxin A per 1 mL to obtain the ochratoxin A reference substance stock solution; accurately measure an appropriate amount of the stock solution and dilute it with 50% acetonitrile solution to a solution containing 5.0 ng of ochratoxin A per 1 mL.

[0043] An Agilent 1290 INfinityⅡ-6470 liquid chromatography-mass spectrometer was used with octadecylsilane bonded silica gel as the filler (Agilent Eclipse Plus C18, 2.1×100 mm, 1.8 μm), methanol-0.1% formic acid (2 mM ammonium acetate) as the mobile phase, column temperature at 30°C; flow rate at 0.3 mL / min, injection volume of 5 μL, and ochratoxin A solution was used for comparative selection of ion pairs.

[0044] 1. Use negative ion acquisition mode (ESI - ), the monitoring ion pairs were m / z402.1→358.1 and m / z402.0→211.0, with response values of 1000.20 and 402.88, respectively. Figure 1 .

[0045] 2. Use positive ion acquisition mode (ESI + ), the ion pairs are m / z404.0→239.0 and m / z404.0→102.1, and the response values are 32113.61 and 3221.71 respectively. Figure 2 .

[0046] 3. Use positive ion acquisition mode (ESI +), the ion pairs are m / z404.0→239.0, m / z404.0→358.0 and m / z404.0→102.0, and the response values are 32113.61, 14227.93 and 3221.71 respectively. Figure 3 .

[0047] Depend on Figures 1 to 3 It can be seen that the positive ion mode (ESI + ), the ion pairs are m / z404.0→239.0, m / z404.0→358.0 and m / z404.0→102.0. The peak shapes of the three detection ion pairs are good, and the response values are stronger than the other two cases. The positive ion mode (ESI + ), three ion pairs m / z 404.0→239.0, m / z 404.0→358.0, and m / z 404.0→102.0 were detected.

[0048] Example 2: Selection of mobile phase

[0049] Using the ochratoxin A solution prepared in Example 1, a comparison was made between the mobile phases of methanol-0.1% formic acid (2 mM ammonium acetate) and the mobile phase of methanol-0.1% formic acid. The results are shown in FIG. Figure 4 and Figure 5 .

[0050] Depend on Figures 4 and 5 It can be seen that the chromatographic peak shape and response value when the mobile phase is methanol-0.1% formic acid (2 mM ammonium acetate) are better than those when the mobile phase is methanol-0.1% formic acid, so methanol-0.1% formic acid (2 mM ammonium acetate) is selected as the mobile phase.

[0051] Example 3: Selection of solvent standard curve and matrix mixed solution standard curve

[0052] 1. Preparation of solvent standard curve:

[0053] Preparation of Ochratoxin A Standard Solution: Accurately measure 0.5 mL of 100 μg / mL Ochratoxin A reference substance into a 50 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain a 1000 ng / mL Ochratoxin A reference substance stock solution. Accurately measure 1 mL of the Ochratoxin A reference substance stock solution into a 10 mL volumetric flask, dilute to the mark with methanol, and shake well to obtain a 100 ng / mL Ochratoxin A reference substance solution. Dilute the Ochratoxin A reference substance solution with 50% acetonitrile to prepare a series of standard solutions, obtaining Ochratoxin A standard solutions with concentrations of 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL, respectively.

[0054] An Agilent 1290 INfinity II-6470 liquid chromatography-mass spectrometer was used with octadecylsilane bonded silica gel as the packing (Agilent Eclipse Plus C18, 2.1×100 mm, 1.8 μm), 0.1% formic acid (2 mmol / L ammonium acetate) as the mobile phase A, methanol as the mobile phase B, the column temperature was 30°C, the flow rate was 0.3 mL / min, and the injection volume was 5 μL. Gradient elution was performed. The gradient elution procedure included:

[0055] From 0 min to 2 min, the volume percentage of mobile phase A decreased from 95% to 50%;

[0056] From 2 to 8 minutes, the volume percentage of mobile phase A decreased from 50% to 5%;

[0057] 8min-10min, maintain the volume percentage of mobile phase A at 5%;

[0058] The mass spectrometry detection conditions were as follows: the ion source was electrospray (ESI + ) ion source, drying gas temperature: 250℃, drying gas flow rate: 8L / min, nebulizer pressure: 35psi, sheath gas temperature: 375℃, sheath gas flow rate: 11L / min, capillary voltage: 3000V; detection mode was multiple reaction monitoring (MRM), and the monitored ion pairs and collision voltage (CE) included: quantitative ion (404.0→239.0, CE: 25V) and qualitative ion (404.0→358.0, CE: 15V; 404.0→102.0, CE: 55V).

[0059] The ochratoxin A standard solution was tested according to the above chromatographic and mass spectrometric conditions. The results are shown in Figure 6 , with the peak area as the ordinate and the injection concentration as the abscissa, the solvent standard curve is drawn, see Figure 7 ; Solvent standard curve is y = 3250.5x + 426.79, R 2 =0.9947.

[0060] 2. Preparation of standard curve of matrix mixed solution:

[0061] Take 5 g of Xiaoyao Pills (concentrated pills) without ochratoxin A as the blank matrix test sample powder, accurately weigh it, accurately add 50 mL of 70% methanol solution, ultrasonicate for 30 minutes, centrifuge (3000 rpm, 10 minutes), accurately measure 3 mL of supernatant, slowly pass it through a treated HLB column [specification: 3 mL (60 mg), eluted with 3 mL of methanol and water respectively] until an appropriate amount of air passes through, and collect the eluate; then elute with 3 mL of methanol, collect the eluate, combine the two eluates, slowly blow nitrogen at 40°C until nearly dry, accurately add 1.0 mL of ochratoxin A standard solution of 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL, respectively, vortex mix, and filter with a microporous filter membrane (0.22 μm). The filtrate is the matrix-mixed ochratoxin A standard solution.

[0062] The matrix-mixed ochratoxin A standard solution was tested under the above chromatographic and mass spectrometric conditions. The results are shown in Figure 8 , with the peak area as the ordinate and the injection concentration as the abscissa, the standard curve of the matrix mixed solution was drawn, see Figure 9 The standard curve of the matrix mixed solution is y = 3249.4x - 46.943, R 2 =0.9999.

[0063] 3. Take 5 g of the test sample powder of Xiaoyao Pills (concentrated pills) No. X156, accurately weigh it, accurately add 50 mL of 70% methanol solution, ultrasonically treat it for 30 minutes, centrifuge it (3000 rpm, 10 minutes), accurately measure 3 mL of the supernatant, slowly pass it through a treated HLB column [specification: 3 mL (60 mg), eluted with 3 mL of methanol and water respectively] until an appropriate amount of air passes through, and collect the eluate; then elute with 3 mL of methanol, collect the eluate, combine the two eluates, slowly blow it with nitrogen at 40°C until it is almost dry, add 50% acetonitrile solution to make the volume 1 mL, filter it with a microporous filter membrane (0.22 μm), and the filtrate is the test solution.

[0064] The solution to be tested was tested according to the above chromatographic conditions and mass spectrometry conditions. The results are shown in Figure 10 The test results of the test solution were analyzed according to the solvent standard curve and the matrix mixed solution standard curve respectively.

[0065] Depend on Figure 6 、 Figure 8 and Figure 10As can be seen, the mass spectrum of the solvent standard curve has a smooth baseline, while the mass spectrum of the matrix mixed solution standard curve is affected by the blank matrix, resulting in a fluctuating baseline that is similar to the baseline of the test solution, more consistent with the actual sample testing situation. The recovery calculated using the solvent standard curve was 67.2% with an RSD of 4.0%, while the recovery calculated using the matrix mixed solution standard curve was 81.8% with an RSD of 3.3%. The recovery results indicate that the values determined using the matrix mixed solution standard curve are higher than those determined using the solvent standard curve. Therefore, the matrix mixed solution was used as the standard curve to calculate the ochratoxin A levels in the samples.

[0066] Example 4:

[0067] 1. Selection of extraction solvent

[0068] In the preparation of the test solution according to the method of Example 3, 50% methanol and 30% methanol were used instead of 70% methanol as the extraction solvent, respectively. The test results of the test solution were analyzed using the standard curve of the matrix mixed solution. The results showed that the average recovery rate of 50% methanol as the extraction solvent was 27.6%, and the RSD% was 1.1%; the average recovery rate of 30% methanol as the extraction solvent was 20.65%, and the RSD% was 3.6%, which was significantly lower than the recovery rate of 70% methanol as the extraction solvent.

[0069] 2. Selection of methanol elution volume:

[0070] Take 5 g of Xiaoyao Pills (concentrated pills) test sample powder, accurately weigh it, accurately add 50 mL of 70% methanol solution, ultrasonically treat for 30 minutes, centrifuge (3000 rpm, 10 minutes), accurately measure 3 mL of supernatant, slowly pass it through a treated HLB column [specification: 3 mL (60 mg), eluted with 3 mL of methanol and water in turn] until an appropriate amount of air passes through, and collect the eluate; then elute with 6 mL of methanol, collect the eluate, combine the two eluates, slowly blow nitrogen at 40°C until almost dry, add 50% acetonitrile solution to make the volume 1 mL, filter with a microporous filter membrane (0.22 μm), and the filtrate is the solution to be tested.

[0071] The test solution was tested according to the chromatographic conditions and mass spectrometry conditions of Example 3, and the test results of the test solution were analyzed using the matrix mixed solution standard curve. The average recovery rate was 69.25% and the RSD% was 0.9%, which was significantly lower than the recovery rate using 3 mL of methanol elution.

[0072] Example 5: Detection of ochratoxin A in Xiaoyao Pills

[0073] Ochratoxin A was detected in 41 Xiaoyao Pill samples using the following methods:

[0074] Step 1: Prepare matrix-mixed ochratoxin A standard solution:

[0075] Prepare ochratoxin A standard solution: accurately weigh an appropriate amount of ochratoxin A reference substance and add methanol to make a solution containing 1000 ng of ochratoxin A per 1 mL to obtain the ochratoxin A reference substance stock solution; accurately measure an appropriate amount of the stock solution and dilute it with 50% acetonitrile solution to a series of concentrations containing 0.25 ng, 0.5 ng, 1.0 ng, 5.0 ng, and 10.0 ng per 1 mL to obtain the ochratoxin A standard solution;

[0076] Preparation of matrix-mixed ochratoxin A standard solution: Take 5 g of the blank matrix test sample powder of Xiaoyao Pills (concentrated pills) without ochratoxin A, accurately weigh it, accurately add 50 mL of 70% methanol solution, ultrasonicate for 30 minutes, centrifuge (3000 rpm, 10 minutes), accurately measure 3 mL of the supernatant, and slowly pass it through a pre-treated HLB column [specification: 3 mL (60 mg), eluted with 3 mL of methanol and 3 mL of water in sequence] until an appropriate amount of Air was passed through and the eluate was collected; then eluted with 3 mL of methanol, the eluate was collected, the two eluates were combined, and slowly blown to dryness with nitrogen at 40°C. 1.0 mL of ochratoxin A reference solution of 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL was accurately added, respectively, vortexed to mix, and filtered through a microporous filter membrane (0.22 μm). The filtrate was the matrix-mixed ochratoxin A standard solution;

[0077] Step 2, constructing a standard curve: The ochratoxin A standard solution mixed with the matrix described in step 1 was subjected to high performance liquid chromatography tandem mass spectrometry detection using an Agilent 1290INfinityⅡ-6470 liquid chromatography-mass spectrometry instrument, with octadecylsilane bonded silica as the filler (Agilent Eclipse Plus C18, 2.1×100 mm, 1.8 μm), 0.1% formic acid (2 mmol / L ammonium acetate) as the mobile phase A, methanol as the mobile phase B, the column temperature was 30°C; the flow rate was 0.3 mL / min, the injection volume was 5 μL, and gradient elution was performed; the gradient elution procedure included:

[0078] From 0 min to 2 min, the volume percentage of mobile phase A decreased from 95% to 50%;

[0079] From 2 to 8 minutes, the volume percentage of mobile phase A decreased from 50% to 5%;

[0080] 8min-10min, maintain the volume percentage of mobile phase A at 5%;

[0081] The mass spectrometry detection conditions were as follows: the ion source was electrospray (ESI +) ion source, drying gas temperature: 250°C, drying gas flow rate: 8 L / min, nebulizer pressure: 35 psi, sheath gas temperature: 375°C, sheath gas flow rate: 11 L / min, capillary voltage: 3000 V; detection mode was multiple reaction monitoring (MRM), and the monitored ion pairs and collision voltage (CE) included: quantification ion (404.0→239.0, CE: 25 V) and qualification ion (404.0→358.0, CE: 15 V; 404.0→102.0, CE: 55 V);

[0082] With the peak area as the ordinate and the injection concentration as the abscissa, draw the standard curve (see Figure 9 ), the linear regression equation of ochratoxin A was obtained as y = 3249.4x - 46.943, R 2 It is 0.999, and the linear relationship is good;

[0083] Step 3. Prepare the test solution: grind the Xiaoyao pills to be tested, accurately weigh 5 g, accurately add 50 mL of 70% methanol solution, ultrasonicate for 30 minutes, centrifuge (3000 rpm, 10 minutes), accurately measure 3 mL of the supernatant, slowly pass it through the treated HLB column [specification: 3 mL (60 mg), eluted with 3 mL of methanol and water in turn] until an appropriate amount of air passes through, and collect the eluate; then elute with 3 mL of methanol, collect the eluate, combine the two eluates, slowly blow nitrogen at 40 ° C to near dryness, add 50% acetonitrile solution to 1 mL, filter with a microporous filter membrane (0.22 μm), and the filtrate is the test solution;

[0084] Step 4. Detection of the test solution: The test solution of step 3 was subjected to high performance liquid chromatography tandem mass spectrometry detection using an Agilent 1290 INfinity II-6470 liquid chromatography-mass spectrometry instrument, with octadecylsilane bonded silica as the filler (Agilent Eclipse Plus C18, 2.1×100 mm, 1.8 μm), 0.1% formic acid (2 mmol / L ammonium acetate) as the mobile phase A, methanol as the mobile phase B, column temperature 30°C; flow rate 0.3 mL / min, injection volume 5 μL, gradient elution; the gradient elution program includes:

[0085] From 0 min to 2 min, the volume percentage of mobile phase A decreased from 95% to 50%;

[0086] From 2 to 8 minutes, the volume percentage of mobile phase A decreased from 50% to 5%;

[0087] 8min-10min, maintain the volume percentage of mobile phase A at 5%;

[0088] The mass spectrometry detection conditions were as follows: the ion source was electrospray (ESI+ ) ion source, drying gas temperature: 250°C, drying gas flow rate: 8 L / min, nebulizer pressure: 35 psi, sheath gas temperature: 375°C, sheath gas flow rate: 11 L / min, capillary voltage: 3000 V; detection mode was multiple reaction monitoring (MRM), and the monitored ion pairs and collision voltage (CE) included: quantification ion (404.0→239.0, CE: 25 V) and qualification ion (404.0→358.0, CE: 15 V; 404.0→102.0, CE: 55 V);

[0089] Then, the standard curve drawn in step 2 was used to quantitatively calculate the quantitative ion to obtain the concentration of ochratoxin A in the test solution, and the content of ochratoxin A in the tested Xiaoyao Pills was further converted. The specific results are shown in the table below.

[0090] Table 1 Content of ochratoxin A in Xiaoyao Pills

[0091]

[0092]

[0093] The present invention adopts HLB universal small column to purify the sample to be tested and adopts liquid chromatography-tandem mass spectrometry for detection, which can effectively save sample pretreatment time and detection time; adopts matrix-mixed ochratoxin A standard solution for quantitative determination, which can reduce the influence of the matrix on the accuracy of the detection result.

[0094] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment based on the technical essence of the invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for detecting ochratoxin A in Xiaoyao pills, characterized in that: The following steps are involved: Step 1, preparing a matrix-mixed ochratoxin A standard solution: preparing an ochratoxin A standard solution; treating Xiaoyao pills that do not contain ochratoxin A as a blank matrix, adding the treated Xiaoyao pills to the prepared ochratoxin A standard solution to obtain a matrix-mixed ochratoxin A standard solution; Step 2: Constructing a standard curve: performing high performance liquid chromatography tandem mass spectrometry on the ochratoxin A standard solution mixed with the matrix described in step 1, and plotting a standard curve with the peak area as the ordinate and the injection concentration as the abscissa; Step 3. Prepare the test solution: grind the Xiaoyao pills to be tested into powder, dissolve them in 70% methanol solution, ultrasonicate for 30 minutes, centrifuge, accurately measure 3 mL of the supernatant and pass it through the HLB column to collect the eluate; then elute with 3 mL of methanol, collect the eluate, combine the two eluates, slowly blow nitrogen at 40°C until almost dry, add 50% acetonitrile solution to make the volume to 1 mL, filter with a microporous filter membrane, and take the filtrate to obtain the test solution; Step 4: Detect the test solution: Perform high performance liquid chromatography tandem mass spectrometry on the test solution of step 3, and then use the standard curve drawn in step 2 to quantitatively calculate the quantitative ion to obtain the concentration of ochratoxin A in the test solution.

2. The method for detecting ochratoxin A in Xiaoyao Pills according to claim 1, wherein: In step 2 and step 4, a triple quadrupole mass spectrometer is used for detection, and the triple quadrupole mass spectrometer is an Agilent 1290 INfinity II-6470 liquid chromatography-mass spectrometer.

3. The method for detecting ochratoxin A in Xiaoyao Pills according to claim 1, wherein: The concentrations of the ochratoxin A standard solution in step 1 are 0.25 ng / mL, 0.5 ng / mL, 1.0 ng / mL, 5.0 ng / mL and 10.0 ng / mL, respectively.

4. The method for detecting ochratoxin A in Xiaoyao Pills according to claim 1, wherein: The preparation method of the ochratoxin A standard solution in step 1 includes: dissolving the ochratoxin A standard in methanol to prepare a methanol solution containing 1000 ng of ochratoxin A per 1 mL, and then diluting it with 50% acetonitrile solution to obtain a standard solution of the required concentration.

5. The method for detecting ochratoxin A in Xiaoyao Pills according to claim 1, characterized in that: The method for treating the blank matrix in step 1 includes: grinding Xiaoyao pills that do not contain ochratoxin A, dissolving them in 70% methanol solution, ultrasonically treating them for 30 minutes, centrifuging them, accurately measuring 3 mL of the supernatant, passing it through an HLB column, and collecting the eluate; then eluting with 3 mL of methanol, collecting the eluate, combining the two eluates, and slowly blowing them with nitrogen at 40°C until they are almost dry.

6. The method for detecting ochratoxin A in Xiaoyao Pills according to claim 1, characterized in that: The HPLC detection conditions in step 2 and step 4 are: Octadecylsilane bonded silica gel was used as the packing material (Agilent Eclipse Plus C18, 2.1×100 mm, 1.8 μm). Mobile phase A consisted of 0.1% formic acid containing 2 mmol / L ammonium acetate, and mobile phase B consisted of methanol. The column temperature was 30°C, the flow rate was 0.3 ml / min, and the injection volume was 5 μL. Gradient elution was performed. Mass spectrometry detection conditions are: The ion source is electrospray (ESI + ) ion source, drying gas temperature: 250℃, drying gas flow rate: 8L / min, nebulizer pressure: 35psi, sheath gas temperature: 375℃, sheath gas flow rate: 11L / min, capillary voltage: 3000V; detection mode was multiple reaction monitoring (MRM), and the monitored ion pairs and collision voltage (CE) included: quantitative ion (404.0→239.0, CE: 25V) and qualitative ion (404.0→358.0, CE: 15V; 404.0→102.0, CE: 55V).

7. The method for detecting ochratoxin A in Xiaoyao Pills according to claim 6, characterized in that: The gradient elution procedure includes: From 0 min to 2 min, the volume percentage of mobile phase A decreased from 95% to 50%; From 2 to 8 minutes, the volume percentage of mobile phase A decreased from 50% to 5%; From 8 to 10 minutes, the volume percentage of mobile phase A was maintained at 5%.

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