Detection method for mycotoxin in medicine and application of detection method

Abstract

The invention provides a detection method of mycotoxin in a medicine and application of the detection method. The mycotoxin comprises aflatoxin B1, aflatoxin B2, ochratoxin A, ochratoxin B, zearalenone and fumonisins B1. The method comprises the following steps: preparing a standard working solution: respectively and accurately weighing a proper amount of mycotoxin standard substances, and preparing the standard working solution with the concentration of 100mu g / mL by using acetonitrile; preparing a mixed standard working solution: sucking the standard working solution, and preparing the mixed standard working solution with the concentration of 100ng / mL; preparing a standard working solution: preparing the mixed standard working solution into the standard working solution with concentration gradient by using acetonitrile; pretreatment of a sample: fully dissolving the sample in an acetonitrile solution, after complete extraction and purification, dissolving the sample with acetonitrile, fixing the volume and uniformly shaking; loading the sample on a machine; and result analysis: determining the nature of the fungaltoxin according to the retention time, and quantifying the fungaltoxin according to the standard curve. The detection method disclosed by the invention is simple, convenient and rapid, the result is accurate, and the content of various fungaltoxins in different medicines can be detected at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and specifically relates to a method for detecting mycotoxins in drugs and application thereof. Background Art

[0002] Pharmaceutical excipients play a vital role in drug production, and they have a direct impact on the quality, safety and effectiveness of drugs. Pharmaceutical excipients include excipients, stabilizers, solubilizers and cosolvents, controlled release agents, flavoring agents and colorants, and absorption enhancers. Excipients can change the physical form of drugs to make them easier to use and store, such as fillers and lubricants in tablets, capsule materials in capsules, solvents and emulsifiers in injections, etc. Stabilizers can improve the stability of drugs and prevent the decomposition, degradation or inactivation of the main drug. For example, antioxidants can prevent drug oxidation, and pH regulators can maintain a stable pH value of drugs; solubilizers and cosolvents can help the main drug dissolve better in solvents and improve its bioavailability; controlled release agents can achieve the timing, positioning and quantitative release of drugs, thereby changing the drug efficacy curve; flavoring agents and colorants can improve the taste and appearance of drugs and improve patient compliance; absorption enhancers can enhance the absorption of drugs through the skin or mucous membranes, which is particularly important for topical preparations. For example, transdermal absorption enhancers can improve the skin permeability of drugs. The quality of pharmaceutical excipients is directly related to the quality of the final drug.

[0003] Most pharmaceutical excipients are made from grain crops. For grain crops, mycotoxin contamination is a common phenomenon. Grain crops are susceptible to mycotoxin contamination throughout the entire process from growth to storage. Once contaminated crops are used as raw materials in the pharmaceutical field, they will inevitably have a negative impact on the drug effect and human health. Among them, aflatoxins, ochratoxins, zearalenone, fumonisins and Fusarium toxins are common mycotoxins in grain crops. There are relevant standards for the detection of mycotoxins in food, but there are very few reports on the detection of mycotoxins in drugs. At present, the common detection methods for mycotoxins include thin layer chromatography, enzyme-linked immunosorbent assay and immunoaffinity chromatography, but the above methods have the problem of cumbersome operation and inability to accurately determine the content of mycotoxins. Therefore, it is urgent to develop a detection method that can achieve rapid, simple and accurate quantification of mycotoxins in drugs. Summary of the invention

[0004] The present invention provides a method for detecting mycotoxins in medicines and application thereof. The detection method of the present invention is simple, rapid and accurate in results and can realize simultaneous detection of the contents of multiple mycotoxins in different medicines.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: a method for detecting mycotoxins in drugs, characterized in that the mycotoxins include aflatoxin B1, aflatoxin B2, ochratoxin A, ochratoxin B, zearalenone and fumonisin B1; the detection method comprises the following steps:

[0006] S1: Prepare standard working solution: accurately weigh appropriate amounts of aflatoxin B1, aflatoxin B2, ochratoxin A, ochratoxin B, zearalenone and fumonisin B1 standards, and prepare standard working solution with a concentration of 100 μg / mL using acetonitrile;

[0007] S2: Prepare mixed standard working solution: pipette 100 μL of each standard working solution into a 100 mL volumetric flask, dilute to the mark with acetonitrile, and prepare a mixed standard working solution with a concentration of 100 ng / mL;

[0008] S3: Prepare standard working solution: Use acetonitrile to prepare the mixed standard working solution into a standard working solution with a concentration gradient of 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL;

[0009] S4: Sample pretreatment: fully dissolve the sample in acetonitrile solution and place it in the purification material. Use the extraction solvent to circulate and purify at a temperature of 75-105°C. After the extraction and purification is complete, transfer the solution to a volumetric flask, dilute it with acetonitrile, and shake well to obtain the sample solution.

[0010] S5: Sample loading: The instrument used is a high performance liquid chromatograph, and the detector is a UV absorption detector;

[0011] S6: Result analysis: Inject the standard working solution prepared in S1, the standard working solution prepared in S3 and the sample solution in S4 into a high performance liquid chromatograph, determine the retention time of each fungal toxin based on the peak time of the standard working solution, draw a standard curve based on the mass concentration corresponding to the peak area of ​​each fungal toxin in the standard working solution, and obtain a linear regression equation to determine the content of different fungal toxins in the sample.

[0012] Furthermore, the medicine includes tablets, capsules, creams, gels, injections, oral solutions and suppositories.

[0013] Furthermore, the mass ratio of the sample to acetonitrile in S4 is 1:(10-15).

[0014] Furthermore, the purification material in S4 is an immunoaffinity column.

[0015] Furthermore, the extraction solvent in S4 is acetonitrile-formic acid-water, and the volume ratio of acetonitrile-formic acid-water is (2-6):(1-3):(4-6).

[0016] Furthermore, the model of the high performance liquid chromatograph in S5 is Waters 2695.

[0017] Furthermore, the conditions of the high performance liquid chromatograph are:

[0018] The chromatographic column is a reverse phase C18 chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column temperature is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

[0019] Furthermore, the specification of the reverse phase C18 chromatographic column is 3.0 mm×200 mm, and the particle size of the filling particles is 3 μm.

[0020] The invention also discloses the application of the detection method in detecting mycotoxins in drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] First, the present invention provides a method for simultaneously detecting six kinds of mycotoxins in drugs. The sample is subjected to extraction and purification treatment to reduce the interference of impurities, and to improve the sample concentration and detection sensitivity. The method has the advantages of simple operation, low detection limit, high sensitivity, high accuracy and good precision, and can effectively detect drugs with mycotoxin contamination risks.

[0023] Second, the detection method of the present invention has a spiked recovery rate of each fungal toxin between 87% and 102.3%, indicating that the method can accurately determine the content of aflatoxin B1, aflatoxin B1, ochratoxin A, ochratoxin B, zearalenone and fumonisin B1 in drugs, and the relative standard deviation RSD is between 1.7% and 4.7%, indicating that the method has good precision. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is a liquid chromatographic separation diagram of a mixed standard solution of six different fungal toxins, where 1 is aflatoxin B1, 2 is aflatoxin B2, 3 is ochratoxin A, 4 is ochratoxin B, 5 is zearalenone, and 6 is fumonisin B1. DETAILED DESCRIPTION

[0025] The embodiments of the present application are described in detail below with reference to the accompanying drawings.

[0026] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0027] Example 1

[0028] 1. Experimental Reagents and Materials

[0029] 1.1 Reagents

[0030] Acetonitrile, formic acid, and methanol were all chromatographically pure and ultrapure water.

[0031] 1.2 Preparation of standard working solution

[0032] Accurately weigh appropriate amounts of aflatoxin B1, aflatoxin B2, ochratoxin A, ochratoxin B, zearalenone and fumonisin B1 standards, and prepare standard working solutions with acetonitrile at a concentration of 100 μg / mL.

[0033] 1.3 Preparation of mixed standard solution

[0034] 100 μL of each standard working solution was pipetted into a 100 mL volumetric flask, and the volume was made up to the mark with acetonitrile to prepare a mixed standard working solution with a concentration of 100 ng / mL.

[0035] 1.4 Preparation of standard working solution

[0036] The mixed standard working solution was prepared with acetonitrile into standard working solutions with concentration gradients of 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL.

[0037] 2. Instruments and Equipment

[0038] Waters 2695 high performance liquid chromatograph, with ultraviolet absorption detector and analytical balance as detector.

[0039] 3. Liquid chromatography conditions

[0040] The chromatographic column is a reverse phase C18 chromatographic column (specification: 3.0 mm×200 mm, filling particle size: 3 μm), mobile phase A is acetonitrile, mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column oven is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

[0041] 4. Detection of standard solution

[0042] Under the above chromatographic conditions, the prepared 6 kinds of mycotoxin standard working solutions were subjected to gradient elution and detection, and the peak time of each mycotoxin was determined, and the retention time was used for qualitative analysis. Under the same conditions, the mixed standard working solution was subjected to gradient elution and detection to obtain the liquid chromatographic separation diagram of the mixed standard working solution of 6 different mycotoxins. The results are as follows: Figure 1 The standard curve was drawn with the mass concentration (X, mg / L) corresponding to the peak area (Y) of each fungal toxin in the standard working solution, and the linear regression equation was obtained to determine the content of different fungal toxins in the sample, and the detection limit (S / N=3) and quantification limit (S / N=10) were calculated. The results are shown in Table 1.

[0043] Table 1. Linear equations, correlation coefficients, detection limits and quantification limits of various mycotoxins

[0044] Mycotoxins Linear equations <![CDATA[Coefficient of correlation R 2 > Detection limit Limit of Quantitation Aflatoxin B1 Y=3.477X-0.667 0.9989 0.34 1.12 Aflatoxin B2 Y=1.721X+0.124 0.9997 0.41 1.31 Ochratoxin A Y=2.322X+0.346 0.999 0.53 1.62 Ochratoxin B Y=1.225X+0.296 0.9997 0.45 1.43 Zearalenone Y=1.272X+0.241 0.9998 0.14 0.45 Fumonisin B1 Y=3.1762X+0.6341 0.9991 0.39 1.31

[0045] 5. Precision and recovery experiments

[0046] The blank matrix was subjected to 3 different concentrations and 6 parallel levels of spike recovery test to determine the recovery rate and calculate the relative standard deviation RSD, and the results are shown in Table 2. The spike recovery rate of each mycotoxin in the blank matrix was between 87% and 102.3%, indicating that the method can accurately determine the content of aflatoxin B1, aflatoxin B2, ochratoxin A, ochratoxin B, zearalenone and fumonisin B1 in the drug, and the relative standard deviation RSD was between 1.7% and 4.7%, indicating that the method has good precision.

[0047] Table 2. Results of spike recovery tests for various mycotoxins

[0048]

[0049] Example 2 Detection of Mycotoxins in Tablet Drug Aspirin

[0050] 1. Sample Processing

[0051] Dissolve 1 g of sample in 10 g of acetonitrile solution and place it in an immunoaffinity column. Use the extraction solvent acetonitrile-formic acid-water (the volume ratio of acetonitrile-formic acid-water is 2:1:4) for cyclic extraction and purification at 75°C. After the extraction and purification is complete, transfer the solution to a volumetric flask, make up to volume with acetonitrile, and shake well to obtain the sample solution.

[0052] 2. Test conditions

[0053] Waters 2695 high performance liquid chromatograph, the detector is an ultraviolet absorption detector, the chromatographic column is a reverse phase C18 chromatographic column (specifications are 3.0 mm×200 mm, and the filling particle size is 3 μm), the mobile phase A is acetonitrile, the mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column oven is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

[0054] Example 3 Detection of Mycotoxins in Capsule Drug Vitamin E Capsules

[0055] 1. Sample Processing

[0056] Dissolve 1 g of sample in 12 g of acetonitrile solution and place it in an immunoaffinity column. Use the extraction solvent acetonitrile-formic acid-water (the volume ratio of acetonitrile-formic acid-water is 2:3:4) for cyclic extraction and purification at 80°C. After the extraction and purification is complete, transfer the solution to a volumetric flask, make up to volume with acetonitrile, and shake well to obtain the sample solution.

[0057] 2. Test conditions

[0058] Waters 2695 high performance liquid chromatograph, the detector is an ultraviolet absorption detector, the chromatographic column is a reverse phase C18 chromatographic column (specifications are 3.0 mm×200 mm, and the filling particle size is 3 μm), the mobile phase A is acetonitrile, the mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column oven is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

[0059] Example 4 Detection of Mycotoxins in Gel Drug Chlortetracycline Ointment

[0060] 1. Sample Processing

[0061] Dissolve 1 g of sample in 13 g of acetonitrile solution and place it in an immunoaffinity column. Use the extraction solvent acetonitrile-formic acid-water (the volume ratio of acetonitrile-formic acid-water is 4:3:5) for cyclic extraction and purification at 85°C. After the extraction and purification is complete, transfer the solution to a volumetric flask, make up to volume with acetonitrile, and shake well to obtain the sample solution.

[0062] 2. Test conditions

[0063] Waters 2695 high performance liquid chromatograph, the detector is an ultraviolet absorption detector, the chromatographic column is a reverse phase C18 chromatographic column (specifications are 3.0 mm×200 mm, and the filling particle size is 3 μm), the mobile phase A is acetonitrile, the mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column oven is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

[0064] Example 5 Detection of Mycotoxins in Posaconazole Injection

[0065] 1. Sample Processing

[0066] Dissolve 1 g of sample in 14 g of acetonitrile solution and place it in an immunoaffinity column. Use the extraction solvent acetonitrile-formic acid-water (the volume ratio of acetonitrile-formic acid-water is 5:3:5) for cyclic extraction and purification at 90°C. After the extraction and purification is complete, transfer the solution to a volumetric flask, make up to volume with acetonitrile, and shake well to obtain the sample solution.

[0067] 2. Test conditions

[0068] Waters 2695 high performance liquid chromatograph, the detector is an ultraviolet absorption detector, the chromatographic column is a reverse phase C18 chromatographic column (specifications are 3.0 mm×200 mm, and the filling particle size is 3 μm), the mobile phase A is acetonitrile, the mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column oven is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

[0069] Example 6 Detection of Mycotoxins in Prednisone Oral Solution

[0070] 1. Sample Processing

[0071] Dissolve 1 g of sample in 14 g of acetonitrile solution and place it in an immunoaffinity column. Use the extraction solvent acetonitrile-formic acid-water (the volume ratio of acetonitrile-formic acid-water is 1:2:1) for cyclic extraction and purification at 95°C. After the extraction and purification is complete, transfer the solution to a volumetric flask, make up to volume with acetonitrile, and shake well to obtain the sample solution.

[0072] 2. Test conditions

[0073] Waters 2695 high performance liquid chromatograph, the detector is an ultraviolet absorption detector, the chromatographic column is a reverse phase C18 chromatographic column (specifications are 3.0 mm×200 mm, and the filling particle size is 3 μm), the mobile phase A is acetonitrile, the mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column oven is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

[0074] Results Analysis

[0075] The method of the present invention was used to detect mycotoxins in different drugs. The detection results of Examples 2-6 are shown in Table 1.

[0076] Table 1. Test results of Examples 2-6

[0077]

[0078] Note: “-” means not detected

[0079] Third, the present invention adopts a pretreatment method in which the sample is circulated through a purification column for extraction and purification, and combines it with high performance liquid chromatography to determine the content of different fungal toxins in the drug. It has the advantages of simple operation, low detection limit, high sensitivity, high accuracy and good precision, and can effectively detect drugs with risk of fungal toxin contamination.

[0080] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable ordinary technicians in the field to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made based on the essence of the content of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting mycotoxins in drugs, characterized in that: The mycotoxins include aflatoxin B1, aflatoxin B2, ochratoxin A, ochratoxin B, zearalenone and fumonisin B1; the detection method comprises the following steps: S1: Prepare standard working solution: accurately weigh appropriate amounts of aflatoxin B1, aflatoxin B2, ochratoxin A, ochratoxin B, zearalenone and fumonisin B1 standards, and prepare standard working solution with a concentration of 100 μg / mL using acetonitrile; S2: Prepare mixed standard working solution: pipette 100 μL of each standard working solution into a 100 mL volumetric flask, dilute to the mark with acetonitrile, and prepare a mixed standard working solution with a concentration of 100 ng / mL; S3: Prepare standard working solution: Use acetonitrile to prepare the mixed standard working solution into a standard working solution with a concentration gradient of 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, and 20 ng / mL; S4: Sample pretreatment: fully dissolve the sample in acetonitrile solution and place it in the purification material. Use the extraction solvent to circulate and purify at a temperature of 75-105°C. After the extraction and purification is complete, transfer the solution to a volumetric flask, dilute it with acetonitrile, and shake well to obtain the sample solution. S5: Sample loading: The instrument used is a high performance liquid chromatograph, and the detector is a UV absorption detector; S6: Result analysis: Inject the standard working solution prepared in S1, the standard working solution prepared in S3 and the sample solution in S4 into a high performance liquid chromatograph, determine the retention time of each fungal toxin based on the peak time of the standard working solution, draw a standard curve based on the mass concentration corresponding to the peak area of ​​each fungal toxin in the standard working solution, and obtain a linear regression equation to determine the content of different fungal toxins in the sample.

2. The detection method according to claim 1, characterized in that The medicines include tablets, capsules, creams, gels, injections, oral solutions and suppositories.

3. The detection method according to claim 1, characterized in that The mass ratio of the sample to acetonitrile in S4 is 1:(10-15).

4. The detection method according to claim 1, characterized in that The purification material in S4 is an immunoaffinity column.

5. The detection method according to claim 1, characterized in that The extraction solvent in S4 is acetonitrile-formic acid-water, and the volume ratio of acetonitrile-formic acid-water is (2-6):(1-3):(4-6).

6. The detection method according to claim 1, characterized in that The model of the high performance liquid chromatograph in S5 is Waters 2695.

7. The detection method according to claim 6, characterized in that The conditions of the high performance liquid chromatograph are: The chromatographic column is a reverse phase C18 chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is 1% (v / v) formic acid-water, the flow rate is 1.2 mL / min, the injection volume is 10 μL, the column temperature is 45° C., the detection wavelength is 240 nm, and the elution gradient program is: 0-5 min, 10%-15% A and 90%-85% B; 5-10 min, 20%-25% A and 80%-75% B; 10-15 min; 25%-40% A and 75%-60% B; 15-20 min, 40%-60% A and 60%-40% B; 20-30 min, 60%-85% A and 40%-15% B.

8. The detection method according to claim 7, characterized in that The specification of the reverse phase C18 chromatographic column is 3.0 mm×200 mm, and the particle size of the filling particles is 3 μm.

9. Use of the detection method according to any one of claims 1 to 8 in detecting mycotoxins in drugs.

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