Rapid detection method for illegal antibiotic additives in plant extract

Abstract

The invention relates to the technical field of medicine safety, and discloses a method for rapidly detecting illegal antibiotic additives in plant extracts, which comprises the following steps: S1, sample treatment: respectively pretreating a solid sample and a liquid sample, then extracting target antibiotics by adopting an optimized solvent, s2, purification treatment: removing interfering substances by adopting a PRIMEHLB solid-phase extraction or QUNCHERS purification method, S3, instrument analysis: preparing a standard curve, and performing qualitative and quantitative analysis on target antibiotics by adopting a high performance liquid chromatography-mass spectrometry technology after preparation, and S4, result judgment: determining the content of the target antibiotics according to the detection result in combination with regulatory standards. The method is used for judging whether illegally added antibiotics exist in a sample or not. By adopting an optimized solvent and purification technology, complex matrix interfering substances such as astragalus polysaccharide, vitamins and amino acids in the plant extract can be effectively removed, the extraction efficiency of target antibiotics is remarkably improved, background interference is reduced, and the detection sensitivity is improved.

Description

Technical Field

[0001] The invention relates to the technical field of medical safety, and in particular to a rapid detection method for illegal antibiotic additives in plant extracts. Background Art

[0002] Plant extracts refer to substances with biologically active ingredients extracted from plants by physical or chemical methods. They are usually rich in natural chemical components in plants, such as alkaloids, phenolic compounds, flavonoids, volatile oils, sugars, proteins, etc. These components have multiple uses such as medicine, health care, cosmetics or food additives.

[0003] Plant extracts are widely used in many fields such as medicine, food, and feed, and their quality and safety are of vital importance. In recent years, some unscrupulous companies have illegally added antibiotics to plant extracts in pursuit of profits, such as adding various antibiotics to extracts made from astragalus. These illegal additions not only violate relevant laws and regulations, but also pose a serious threat to animal and human health. Long-term consumption of plant extracts containing illegally added antibiotics can cause drug resistance in animals and even lead to drug accumulation in animal-derived products, posing potential threats to humans such as teratogenicity, carcinogenicity, and mutagenicity, and is also not conducive to the healthy development of the breeding industry.

[0004] At present, plant extracts themselves have complex compositions. For example, astragalus extracts contain astragalus polysaccharides, vitamins, amino acids, etc. These matrix interfering substances will affect the accuracy of antibiotic detection. Existing detection technologies are difficult to effectively remove interfering substances when processing complex matrix samples, resulting in low extraction efficiency of target antibiotics and large background interference, which in turn affects detection sensitivity and accuracy and cannot meet the needs of rapid and accurate detection. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a rapid detection method for illegal antibiotic additives in plant extracts to solve the problem that it is difficult to effectively remove interfering substances, resulting in low extraction efficiency of target antibiotics and large background interference, which in turn affects the detection sensitivity and accuracy and cannot meet the needs of rapid and accurate detection.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: A rapid detection method for illegal antibiotic additives in plant extracts comprises the following steps:

[0007] S1. Sample processing: Pre-treat solid samples and liquid samples separately, and then use optimized solvents to extract target antibiotics;

[0008] S2. Purification: Use PRIMEHLB solid phase extraction or QUNCHERS purification method to remove interfering substances;

[0009] S3, instrumental analysis: prepare the standard curve, and then use high performance liquid chromatography-mass spectrometry to perform qualitative and quantitative analysis of the target antibiotics;

[0010] S4. Result determination: Based on the test results and combined with regulatory standards, it is used to determine whether there are illegally added antibiotics in the sample.

[0011] Preferably, in S1, the optimized solvent includes acetonitrile, n-hexane, and formic acid acetonitrile aqueous solution, the solid sample is astragalus granules, whose main components include astragalus polysaccharides, vitamin A, vitamin B, vitamin C and amino acids, and the pretreatment step includes: weighing 2.00 g of sample, adding 10 mL of acetonitrile, vortex mixing at 3000 r / min for 2 minutes, ultrasonicating at 25°C for 30 minutes, centrifuging at 10000 r / min for 10 minutes, taking the supernatant, repeating the extraction twice and combining the supernatants, adding 10 mL of n-hexane for oscillation, and discarding the upper solution after centrifugation.

[0012] Preferably, in S1, the liquid sample is a liquid livestock compound premix feed type III, whose main ingredients include hawthorn, licorice and crude extracts of traditional Chinese medicine and feed-grade vitamin B1, vitamin B2, vitamin B6, and sodium selenite. The pretreatment step includes: measuring 2 mL of the sample, adding 30 mL of a mixture of formic acid and acetonitrile water, centrifuging at 10,000 r / min, and taking the supernatant.

[0013] Preferably, in S2, the purification using the QUNCHERS antibiotic column is to directly inject the extract into the purification column for filtration, or to add the QUNCHERS purification material into the extract, vortex mix at 3000 r / min and then centrifuge, and take the supernatant for later use.

[0014] Preferably, in S2, the purification using a PRIMEHLB solid phase extraction column specifically comprises: activating the solid phase extraction column with 3 mL of methanol and 3 mL of water in sequence, passing all the extract through the column, eluting the interfering substances with 3 mL of 75% methanol aqueous solution, and eluting the target antibiotic with 3 mL of methanol.

[0015] Preferably, in S3, the high performance liquid chromatography-mass spectrometry technology includes LC-MS / MS, the optimized mobile phase is a gradient elution system of acetonitrile and 0.1% formic acid water, the flow rate is 1.0 mL / min, the column temperature is 40°C, and the detection wavelength is in the range of 220 to 280 nm.

[0016] Preferably, in S3, the preparation of the standard curve includes: adding a standard antibiotic solution with a concentration of 10, 25, 75, 150 or 200 ng / mL to a blank sample, completing the pretreatment steps together with the sample, and diluting to 1 mL as a working solution.

[0017] Preferably, in S4, in the result determination step, based on the detection limit (S / N=3) and quantification limit (S / N=10) of the antibiotics, combined with regulatory standards, it is determined whether the antibiotics in the sample are illegally added.

[0018] The present invention provides a rapid detection method for illegal antibiotic additives in plant extracts. It has the following beneficial effects:

[0019] 1. The present invention can effectively remove complex matrix interfering substances in plant extracts, such as astragalus polysaccharides, vitamins and amino acids, by adopting optimized solvents and purification technologies, thereby ensuring that the extraction efficiency of the target antibiotics is significantly improved, while reducing background interference and improving detection sensitivity.

[0020] 2. The present invention achieves qualitative and quantitative analysis of target antibiotics through high performance liquid chromatography-mass spectrometry technology, combined with optimized mobile phase and multiple reaction monitoring mode. It has high sensitivity and high specificity, and can accurately identify and quantify illegally added antibiotic ingredients.

[0021] 3. The entire method of the present invention includes optimized extraction, purification and detection steps, which is easy to operate and time-saving. It can quickly detect different types of samples, solid astragalus granules and liquid livestock compound premix feed, and is suitable for routine detection and large-scale screening of illegally added antibiotics in plant extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a flow chart of the method of the present invention; DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Please see attached Figure 1 The embodiment of the present invention provides a rapid detection method for illegal antibiotic additives in plant extracts, comprising the following steps:

[0025] S1. Sample processing: Pre-treat solid samples and liquid samples separately, and then use optimized solvents to extract target antibiotics;

[0026] S2. Purification: Use PRIMEHLB solid phase extraction or QUNCHERS purification method to remove interfering substances;

[0027] S3, instrumental analysis: prepare the standard curve, and then use high performance liquid chromatography-mass spectrometry to perform qualitative and quantitative analysis of the target antibiotics;

[0028] S4. Result determination: Based on the test results and combined with regulatory standards, it is used to determine whether there are illegally added antibiotics in the sample.

[0029] The optimized solvents in S1 include acetonitrile, n-hexane, and formic acid acetonitrile aqueous solution. The solid sample is Astragalus granules, whose main components include Astragalus polysaccharides, vitamin A, vitamin B, vitamin C and amino acids. The pretreatment steps include: weighing 2.00g sample, adding 10mL acetonitrile, vortex mixing at 3000r / min for 2 minutes, ultrasonic at 25℃ for 30 minutes, centrifuging at 10000r / min for 10 minutes, taking the supernatant, repeating the extraction twice and combining the supernatants, adding 10mL n-hexane to oscillate, and discarding the upper solution after centrifugation. The liquid sample in S1 is liquid livestock compound premix feed type III, whose main components include hawthorn, licorice and crude extracts of traditional Chinese medicine and feed-grade vitamin B1, vitamin B2, vitamin B6, and sodium selenite. The pretreatment steps include: measuring 2mL sample, adding 30mL formic acid acetonitrile water mixture, centrifuging at 10000r / min, and taking the supernatant.

[0030] Specifically, an optimized solvent system (including acetonitrile, n-hexane, and formic acid acetonitrile aqueous solution) is used to extract and process the target antibiotics in solid and liquid plant extract samples. For solid samples (such as Astragalus granules), multiple extractions with acetonitrile, ultrasound-assisted extraction, and high-speed centrifugation are combined with n-hexane oscillation to remove non-polar interfering substances, which significantly improves the extraction efficiency of antibiotics and reduces matrix interference; for liquid samples (such as liquid compound premixed feed for livestock), formic acid acetonitrile aqueous solution is used for direct extraction, and precipitation and complex matrices are removed by centrifugation. The optimized solvent system makes full use of polarity differences and physical separation principles to efficiently remove interfering substances such as astragalus polysaccharides, vitamins, amino acids, etc. in the sample, while stabilizing the target antibiotics, providing pure sample extracts for subsequent purification and mass spectrometry analysis, and ensuring the accuracy and sensitivity of the test results;

[0031] Mixed standard solution of reagents: pipette appropriate amount of the above single standard stock solution respectively, and dilute step by step with acetonitrile to a mixed standard solution with a concentration of about 1 μg / mL;

[0032] Preparation of other reagents: 1% formic acid water acetonitrile (1:9): 1mL formic acid is diluted to 100mL volumetric flask with water, 10mL of 1% formic acid solution is added to 90mL acetonitrile; 1% formic acid water acetonitrile (5:95): 1mL formic acid is diluted to 100mL volumetric flask with water, 5mL of 1% formic acid solution is added to 95mL acetonitrile; 5% acetonitrile water: 5mL acetonitrile is added to 95mL water and mixed evenly;

[0033] Mobile phase: Phase A (0.1% formic acid in water) + Phase B (0.1% formic acid in acetonitrile)

[0034] Mobile phase flow rate: 0.30mL / min

[0035] Table 1 Gradient elution program

[0036]

[0037] In S2, the QUNCHERS antibiotic column is used for purification, which is to directly inject the extract into the purification column for filtration, or add the QUNCHERS purification material to the extract, vortex mix at 3000r / min and centrifuge, and take the supernatant for later use. The PRI MEHLB solid phase extraction column is used for purification, and the specific method includes: activating the solid phase extraction column with 3mL methanol and 3mL water in turn, passing all the extract through the column, eluting the interfering substances with 3mL 75% methanol aqueous solution, and eluting the target antibiotic with 3mL methanol.

[0038] Specifically, the method of using the QUNCHERS antibiotic column for purification is based on the principle of efficient solid phase extraction and provides two optional operation modes to meet different sample processing requirements. The first method is to directly inject the extract into the QUNCHERS antibiotic column, and through the selective binding of the adsorption material in the column, the impurities and interferences in the sample are quickly removed. After filtration, the purified extract can be obtained, which is simple and efficient.

[0039] The second method is to add QUNCHERS purification material to the extract, vortex mix at 3000r / min to ensure that the purification material is fully in contact with the extract, separate the supernatant by centrifugation, and retain the target compound. Both methods can effectively remove non-target components in the sample, such as complex matrices such as astragalus polysaccharides, vitamins, and amino acids in plant extracts, while retaining antibiotic components to ensure the accuracy and reliability of downstream testing;

[0040] The method of using PR I MEHLB solid phase extraction column for purification is based on the selective adsorption and separation principle of solid phase extraction, which can efficiently remove interferences in complex matrices and enrich the target antibiotics at the same time. The specific operation includes the following steps: First, 3 mL of methanol and 3 mL of water are used to activate the solid phase extraction column in turn to ensure that the filler in the column reaches the optimal adsorption state; then, the extract is completely passed through the extraction column to allow the target antibiotics in it to combine with the adsorption material in the column, and some impurities are initially separated; then, 3 mL of 75% methanol aqueous solution is used to elute the column to further remove interfering substances such as astragalus polysaccharides, vitamins and other polar impurities; finally, 3 mL of pure methanol is used to elute the target antibiotics to fully release the adsorbed antibiotic components and obtain the purified eluent. This method achieves efficient purification through step-by-step operation to ensure the sensitivity and accuracy of downstream detection results.

[0041] S3 medium and high performance liquid chromatography-mass spectrometry technology includes LC-MS / MS, the optimized mobile phase is a gradient elution system of acetonitrile and 0.1% formic acid water, the flow rate is 1.0mL / min, the column temperature is 40°C, and the detection wavelength is in the range of 220 to 280nm. The preparation of S3 medium and standard curves includes: adding 10, 25, 75, 150 or 200ng / mL concentration of antibiotic standard solution to the blank sample, completing the pretreatment steps with the sample, and diluting to 1mL as the working solution.

[0042] Specifically, the mass spectrometry conditions were: ion source: ES I source, positive and negative ion mode; IS voltage: 5500 V; curtain gas CUR: 35 psi; nebulizer gas GS1: 55 psi; auxiliary gas GS2: 55 psi; source temperature TEM: 600 °C; collision gas CAD: 8 psi;

[0043] Table 2 Mass spectrometry analysis parameters of plant extracts illegally added with antibiotics

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050] Experimental method sample:

[0051] Solid sample: Astragalus granules. Main ingredients: Astragalus polysaccharide, vitamins A, B, C and amino acids, etc. Manufacturer: Qiqihar Shuangfu Biotechnology Co., Ltd. Mixed samples, crushed;

[0052] Liquid sample: Liquid compound premixed feed type III for livestock. Main ingredients: crude extracts of Chinese medicines such as hawthorn and licorice, feed-grade vitamin B1, vitamin B2, vitamin B6, sodium selenite. Carrier: purified water.

[0053] In S4, in the result determination step, based on the detection limit (S / N=3) and quantification limit (S / N=10) of the antibiotics, combined with regulatory standards, it is determined whether the antibiotics in the sample are illegally added.

[0054] Specifically, the chemical properties of the antibiotics illegally added to plant extracts were determined from the properties of the target test objects. It was found that the chemical structures of the target objects all contained unstable amide groups, -OH, -COOH, -COOR, -NH2, -O- structures, etc., which were highly polar. Solvents with high polarity were suitable as extractants. Since there were many target objects, it was convenient to use the QUNCHERS method or the pre-column method.

[0055] Weigh 2.00 / 2.00mL of sample, add 45 kinds of antibiotic standard solutions to prepare 100ng / g positive sample, perform pretreatment according to 3.4, test the sample values ​​at 0.5h, 1h, 4h, 8h, 12h, 24h, and 48h respectively, measure 6 samples at each time point, and calculate the average value and relative standard deviation (RSD%);

[0056] Table 1 Solid sample 100ng / g positive addition recovery test results at different time points:

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] Table 2 RSD% of the recovery rate of 6 samples measured at different time points for the solid sample 100 ng / g positive addition

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Table 3 Recovery test results of liquid samples 100 ng / mL positive addition at different time points

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Table 4 RSD% of the recovery rate of 6 samples measured at different time points of liquid sample 100 ng / mL positive addition

[0075]

[0076]

[0077]

[0078]

[0079] As can be seen from Tables 1 and 2, the recovery rate of the solid sample and liquid sample positive addition experiments has a downward trend over time, but the decline is small, and all 45 antibiotics are ≥70%. As can be seen from Tables 3 and 4, the RSD% of the 6 samples at different time points is ≤7.0%, and the parallelism of the test results is good;

[0080] Weigh 1.00g, 2.00g, 3.00g, 4.00g, 5.00g, and 10.00g of solid samples, add 45 kinds of standard solutions, and prepare 10, 25, 75, 125, and 150ng / g (ng / mL) positive samples according to different sample weights. Perform pretreatment according to 3.4, and expand the extraction solution and re-dissolution solution to the corresponding volume in proportion. 3.00g, 4.00g, 5.00g, and 10.00g require a water bath rotary evaporator due to the large volume of the extraction solution. Determine the detection limit with S / N>3 times the signal-to-noise ratio;

[0081] The results show that the influence of 10.00g matrix is ​​significantly increased, and the detection limit of 45 antibiotics is ≥150ng / g. Therefore, 10g is not selected. When the sample weight is 2.00g, 3.00g, 4.00g, and 5.00g, when the concentration of 45 antibiotics is 1ng / g, the peak area and baseline noise are greater than 3 times the signal-to-noise ratio, and the results are reliable. However, the sample weight of 3.00g, 4.00g, and 5.00g is larger than that of 2.00g of extraction solution, and the operation of the rotary evaporator is complicated, so the sample weight of 3.00g, 4.00g, 5.00g, and 10.00 is not selected. When the sample weight is 1.00g, the final volume is 0.5mL according to the pretreatment method, which is very small. Therefore, the sample weight of 1.00g is not selected. The sample weight is established as 2.00g.

[0082] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A rapid detection method for illegal antibiotic additives in plant extracts, characterized in that: The following steps are involved: S1. Sample processing: Pre-treat solid samples and liquid samples separately, and then use optimized solvents to extract target antibiotics; S2. Purification: Use PRIMEHLB solid phase extraction or QUNCHERS purification method to remove interfering substances; S3, instrumental analysis: prepare the standard curve, and then use high performance liquid chromatography-mass spectrometry to perform qualitative and quantitative analysis of the target antibiotics; S4. Result determination: Based on the test results and combined with regulatory standards, it is used to determine whether there are illegally added antibiotics in the sample.

2. The rapid detection method for illegal antibiotic additives in plant extracts according to claim 1, characterized in that: In S1, the optimized solvent includes acetonitrile, n-hexane, and formic acid acetonitrile aqueous solution. The solid sample is astragalus granules, whose main components include astragalus polysaccharides, vitamin A, vitamin B, vitamin C and amino acids. The pretreatment step includes: weighing 2.00 g of sample, adding 10 mL of acetonitrile, vortex mixing at 3000 r / min for 2 minutes, ultrasonicating at 25°C for 30 minutes, centrifuging at 10000 r / min for 10 minutes, taking the supernatant, repeating the extraction twice and combining the supernatants, adding 10 mL of n-hexane for oscillation, and discarding the upper solution after centrifugation.

3. The rapid detection method for illegal antibiotic additives in plant extracts according to claim 1, characterized in that: In S1, the liquid sample is liquid livestock compound premix feed type III, whose main ingredients include hawthorn, licorice and crude extracts of traditional Chinese medicine and feed-grade vitamin B1, vitamin B2, vitamin B6, and sodium selenite. The pretreatment step includes: measuring 2 mL of the sample, adding 30 mL of a mixture of formic acid and acetonitrile water, centrifuging at 10,000 r / min, and taking the supernatant.

4. The rapid detection method for illegal antibiotic additives in plant extracts according to claim 1, characterized in that: In S2, the purification using the QUNCHERS antibiotic column is to directly inject the extract into the purification column for filtration, or to add the QUNCHERS purification material into the extract, vortex mix at 3000r / min and then centrifuge, and take the supernatant for later use.

5. The rapid detection method for illegal antibiotic additives in plant extracts according to claim 1, characterized in that: In S2, the PRIMEHLB solid phase extraction column is used for purification, and the specific method includes: activating the solid phase extraction column with 3 mL of methanol and 3 mL of water in turn, passing all the extract through the column, eluting the interfering substances with 3 mL of 75% methanol aqueous solution, and eluting the target antibiotic with 3 mL of methanol.

6. The rapid detection method for illegal antibiotic additives in plant extracts according to claim 1, characterized in that: In S3, the high performance liquid chromatography-mass spectrometry technology includes LC-MS / MS, the optimized mobile phase is a gradient elution system of acetonitrile and 0.1% formic acid water, the flow rate is 1.0 mL / min, the column temperature is 40°C, and the detection wavelength is in the range of 220 to 280 nm.

7. The rapid detection method for illegal antibiotic additives in plant extracts according to claim 1, characterized in that: In S3, the preparation of the standard curve includes: adding 10, 25, 75, 150 or 200 ng / mL of antibiotic standard solution to the blank sample, completing the pretreatment steps together with the sample, and diluting to 1 mL as the working solution.

8. The rapid detection method for illegal antibiotic additives in plant extracts according to claim 1, characterized in that: In S4, in the result determination step, based on the detection limit (S / N=3) and quantification limit (S / N=10) of the antibiotics, combined with regulatory standards, it is determined whether the antibiotics in the sample are illegally added.

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