A combined extract, extraction method and detection method for sulfamethoxazole and its degradation products in pig manure

Abstract

The present invention provides a combined extracting solution, an extraction method and a synchronous detection method for sulfamethoxazole and its degradation products in pig manure. The combined extracting solution for sulfamethoxazole and its degradation products in pig manure provided by the present invention comprises EDTA-MCIlvaine buffer, methanol, acetonitrile and acetone in a volume ratio of (4-6):(1-3):(1-3):1; the pH of the combined extracting solution is 5-7. The combined extracting solution provided by the present invention can be used to extract sulfamethoxazole and its degradation products and other residues from pig manure at the same time. The extraction method provided by the present invention adopts the combined extracting solution, and optimizes the ultrasonic extraction and solid phase extraction methods to achieve the synchronous extraction and high recovery rate of sulfamethoxazole and its degradation products in pig manure. The detection method of sulfamethoxazole and its degradation products in pig manure provided by the present invention adopts the extraction method and high performance liquid chromatography-mass spectrometry combined method for detection, optimizes the liquid chromatography conditions and mass spectrometry conditions, and achieves the synchronous and accurate detection of sulfamethoxazole and its degradation products in pig manure.

Description

Technical Field

[0001] The invention relates to the field of harmless treatment of organic fertilizers, and in particular to a combined extracting solution, an extraction method and a detection method for sulfamethoxazole and its degradation products in pig manure. Background Art

[0002] With the rapid development of livestock and poultry farming, antibiotics are widely used in the farming industry. According to statistics, the output of antibiotics in 2020 reached 223,000 tons, of which 32,700 tons were used in livestock and poultry farming. Sulfonamides (SAs) are one of the most common veterinary antibiotics. Antibiotics ingested by animals cannot be completely metabolized by the body. About 20% to 97% of antibiotics are excreted as active substances and released into the environment, or enter the ecosystem through fertilization, resulting in a large amount of residues in the ecosystem, which will pose a potential threat to the ecosystem and human health. Among them, sulfamethoxazole (SMX), as a kind of sulfonamide antibiotics, has better stability in the environment and is more difficult to degrade. Its degradation will produce byproducts 4-Hydroxyl-N-(5-methyl-1,2-oxazole-3-yl)benzene-1-sulfonamide (4-OH-SMX) and 4-NitroSulfamethoxazole (4-NO2-SMX), which are much more toxic than the parent compound. Their toxicity is 5.7 and 21.4 times that of the parent compound, respectively. In addition, SMX can produce other byproducts through biodegradation, such as sulfonamide, sulfanilic acid, 4-aminophenol, 4-aminothiophenol, 3-amino-5-methylisoxazole, etc.

[0003] At present, the detection of antibiotic residues mainly includes microbiological method, immunoassay, capillary electrophoresis, high performance liquid chromatography and liquid-mass spectrometry, most of which have poor resolution and sensitivity. However, high performance liquid chromatography tandem mass spectrometry combines the high separation ability of liquid chromatography for complex matrices with the unique sensitivity, selectivity, relative molecular mass and structure of mass spectrometry. It can accurately perform qualitative and quantitative analysis on complex matrices and accurately detect multiple antibiotics in livestock and poultry manure or organic fertilizer at the same time.

[0004] The Chinese patent application with publication number CN118275584A discloses a method for rapid and efficient simultaneous extraction of multiple antibiotics in soil or manure, which first uses EDTA-Mcllvaine buffer to perform two ultrasonic extractions on the pretreated soil or pretreated manure, and then uses an organic extractant to perform one ultrasonic extraction on the residue obtained from the first two extractions; then the supernatants obtained from the three ultrasonic extractions are mixed, concentrated and extracted using a solid phase extraction column, and after completion, the solid phase extraction column is eluted with an eluent to obtain an eluent, and the content of multiple antibiotics in the eluent is determined by ultra-high performance liquid chromatography. Among them, in the ultrasonic extraction, the organic extractant is methanol, acetonitrile and acetone in a volume ratio of 1.8~2.2:1. In the solid phase extraction, methanol and water are used to activate the solid phase extraction column in sequence; the eluent is dichloromethane; before eluting the solid phase extraction column with the eluent, the solid phase extraction column is eluted with a methanol solution. When liquid chromatography was used for detection, mobile phase A was acetonitrile and mobile phase B was 0.1% formic acid aqueous solution.

[0005] Since the soil and manure matrix is ​​relatively complex and their physical properties are very different, the composition and content of livestock manure of different livestock species vary greatly. Pig manure contains a variety of substances such as lipids and proteins, and its composition is relatively complex. The above existing technologies used in the extraction and detection of antibiotics in pig manure have the problem of low extraction recovery rate, which needs to be further improved. Summary of the invention

[0006] The first purpose of the present invention is to overcome the problem in the prior art that the recovery rate of antibiotic residues (especially sulfamethoxazole and its degradation products) in the extraction and detection of antibiotics in pig manure needs to be further improved, and provide a combined extracting solution for sulfamethoxazole and its degradation products in pig manure, which includes EDTA-MCIlvaine buffer, methanol, acetonitrile and acetone in a volume ratio of (4-6):(1-3):(1-3):1; the pH of the combined extracting solution is 5-7. The combined extracting solution for sulfamethoxazole and its degradation products in pig manure provided by the present invention can simultaneously extract sulfamethoxazole and its degradation products and other residues from pig manure, and the extraction recovery rate of sulfamethoxazole and its degradation products in pig manure is as high as 60.5-99.7%.

[0007] Among them, the degradation products of sulfamethoxazole include 4-nitro-sulfamethoxazole (4-NO2-SMX), 4-amino-N-(5-methyl-1,2-oxazol-3-yl)benzenesulfonamide (4-OH-SMX), sulfonamide, sulfanilic acid, 4-aminophenol, 4-aminothiophenol, and 3-amino-5-methylisoxazole; sulfamethoxazole and its degradation products can be collectively referred to as sulfamethoxazole residues.

[0008] The pH of the combined extract is 5-7, and the extraction recovery rate of the residues of sulfamethoxazole and its degradation products in pig manure is high; especially when the pH is 6, the extraction recovery rate of the residues of sulfamethoxazole and its degradation products in pig manure is higher.

[0009] The second object of the present invention is to provide a method for extracting sulfamethoxazole and its degradation products in pig manure, comprising: adding the aforementioned combined extract to a pig manure sample and performing ultrasonic extraction to obtain an ultrasonic extract; preferably, the ultrasonic extraction is performed three times.

[0010] The method for extracting sulfamethoxazole and its degradation products in pig manure provided by the present invention adopts the aforementioned combined extracting solution, and can simultaneously extract the residual sulfamethoxazole and its degradation products in the degradation process from pig manure, and the recovery rate is high. Compared with the prior art, the extraction method provided by the present invention directly adopts the combined extracting solution for extraction three times, and the ultrasonic time can be 10 to 20 min, such as 10 min, 15 min and 20 min, and the extraction recovery rate of sulfamethoxazole and its degradation products in pig manure is high.

[0011] In some embodiments of the present invention, the extraction method further comprises subjecting the ultrasonically obtained extract to solid phase extraction to obtain a solid phase extraction solution; wherein the eluent in the solid phase extraction comprises component A and component B in a volume ratio of (5-7):(3-5); the component B comprises methanol, and the component A comprises dichloromethane and acetone in a volume ratio of (5-7):(3-5).

[0012] The method for extracting sulfamethoxazole and its degradation products in pig manure provided by the present invention is further combined with solid phase extraction, especially with the above-mentioned eluent, so as to further improve the recovery rate of sulfamethoxazole and its degradation products in pig manure.

[0013] In some embodiments of the present invention, before subjecting the ultrasonic extract to solid phase extraction, the solid phase extraction column is activated with hydrochloric acid.

[0014] In some embodiments of the present invention, when the ultrasonic extract is subjected to solid phase extraction, the solid phase extraction column is eluted with methanol and water respectively.

[0015] In some embodiments of the present invention, when the ultrasonic extract is subjected to solid phase extraction, the solid phase extraction column is first eluted with n-hexane, and then the solid phase extraction column is eluted with the eluent to obtain an extract of sulfamethoxazole and its degradation products.

[0016] The third object of the present invention is to provide a method for detecting sulfamethoxazole and its degradation products in pig manure, comprising: Step (1): extracting sulfamethoxazole and its degradation products from pig manure to obtain an extract of sulfamethoxazole and its degradation products; Step (2): using high performance liquid chromatography-mass spectrometry to detect the extract of sulfamethoxazole and its degradation products; Wherein, in step (1), sulfamethoxazole and its degradation products are extracted from pig manure by the aforementioned extraction method to prepare an extract of sulfamethoxazole and its degradation products.

[0017] The method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention can simultaneously extract sulfamethoxazole and its degradation products from pig manure with a recovery rate of up to 60.5% to 99.7%, and can also realize the synchronous and accurate detection of sulfamethoxazole and its degradation products.

[0018] In some embodiments of the present invention, the HPLC method uses C18 as the stationary phase to fill the chromatographic column; the mobile phase includes mobile phase A and mobile phase B; the mobile phase A includes an aqueous solution of 2 mM ammonium formate, and the mobile phase B is methanol.

[0019] The detection method provided by the present invention adopts the mobile phase to achieve effective separation of sulfamethoxazole and its degradation products from other components in the extract of porcine sulfamethoxazole and its degradation products. At the same time, sulfamethoxazole and its degradation products can also be effectively separated from each other, thereby achieving synchronous and accurate detection of sulfamethoxazole and its degradation products and other residues in the extract of sulfamethoxazole and its degradation products.

[0020] In some embodiments of the present invention, the chromatographic column is ACQUITY UPLC HSS T3, 100 mm×2.1 mm, 1.8 μm.

[0021] In some embodiments of the present invention, the mobile phase is eluted using the following gradient by volume percentage: 0-1.2 min, 3% of the mobile phase B, 97% of the mobile phase A; 1.2-5 min, the mobile phase B gradually increased from 3% to 55%, and the mobile phase A gradually decreased from 97% to 45%; 5-6.5 min, 55% of the mobile phase B, 45% of the mobile phase A; 6.5-7 min, the mobile phase B gradually increased from 55% to 95%, and the mobile phase A gradually decreased from 45% to 5%; 7-8.5 min, 95% of the mobile phase B, 5% of the mobile phase A; 8.5-8.6 min, the mobile phase B gradually decreased from 95% to 3%, and the mobile phase A gradually increased from 5% to 97%; 8.6~10 min, 3% of the mobile phase B, 97% of the mobile phase A.

[0022] In the present invention, the above-mentioned flow is combined with the above-mentioned gradient elution, which can further improve the effective separation of sulfamethoxazole and its various degradation products from other components in the extract of sulfamethoxazole and its degradation products, and can also improve the effective separation of sulfamethoxazole and its various degradation products from each other, and the mass spectrum peak distribution of the sulfamethoxazole and its various degradation products obtained by detection is clearer, more specific, and easier to identify.

[0023] The combined extracting solution and extraction method for sulfamethoxazole and its degradation products in pig manure provided by the present invention achieve separation and purification of target objects by optimizing the type and pH value of the combined extracting solution and optimizing ultrasonic extraction and solid phase extraction in the extraction process, so that sulfamethoxazole and its degradation products and other residues can be extracted from pig manure at the same time, and a high recovery rate of 60.5% to 99.7% can be guaranteed.

[0024] The present invention provides a method for detecting sulfamethoxazole and its degradation products in pig manure. The above-mentioned extraction method can be used to simultaneously extract sulfamethoxazole and its degradation products and other residues from pig manure; at the same time, a solid phase extraction-ultra-high performance liquid chromatography-mass spectrometry (SPE-UPLC / MS / MS) method can be used to simultaneously detect SMX and its seven degradation byproducts (sulfonamide, sulfanilic acid, 4-aminophenol, 4-aminothiophenol, 3-amino-5-methylisoxazole, 4-OH-SMX, 4-NO2-SMX) in pig manure and optimize the method, optimize the chromatographic conditions and mass spectrometry conditions, establish and improve a method for detecting multiple antibiotic residues in complex environmental samples, and provide a technical guarantee for monitoring the environment and antibiotic residues in livestock and poultry manure; in particular, the extraction and detection of sulfamethoxazole and its degradation products and other residues in pig manure improve the harmless treatment level of pig manure and other livestock and poultry manure.

[0025] The present invention improves the recovery rate of SMX and its degradation products in pig manure by providing an extraction method and a detection method for SMX and its degradation products in pig manure, especially by optimizing the extract (composition and pH, etc.) and ultrasonic extraction conditions (such as ultrasonic time, ultrasonic extraction times, etc.) in ultrasonic extraction, and controlling and optimizing the eluent and various links in solid phase extraction, and optimizing the chromatographic column, mobile phase composition and elution conditions in the detection process, and finally improving the recovery rate of SMX and its degradation products in pig manure. At the same time, the detection method provided by the present invention has better sensitivity and higher recovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 It is one of the mass spectra of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0028] Figure 2 This is the second mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0029] Figure 3 This is the third mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0030] Figure 4 This is the fourth mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0031] Figure 5 This is the fifth mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0032] Figure 6 This is the sixth mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0033] Figure 7 This is the seventh mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0034] Figure 8 This is the eighth mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention.

[0035] Fig. 9 This is the ninth mass spectrum diagram of the method for detecting sulfamethoxazole and its degradation products in pig manure provided by the present invention. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are 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.

[0037] The present invention relates to raw material information as follows: Sulfamethoxazole, 3-amino-5-methylisoxazole, 4-aminophenol, sulfanilic acid, and sulfonamide were purchased from Shandong Xiya Chemical Co., Ltd. with purities of 98%, 97%, 98%, 98%, and 99%, respectively; 4-aminothiophenol was purchased from Sinopharm Chemical Reagent Co., Ltd. with a purity of 97%; 4-OH-SMX and 4-NO2-SMX were synthesized by Beijing Fusion Venture Technology Co., Ltd. with a purity of 98%.

[0038] Solution preparation: Preparation of Na2EDTA-McIlvaine buffer solution: weigh 11 g C6H8O7·H2O (citric acid), 17.74 g Na2HPO4 (disodium hydrogen phosphate), 10 H 14 Dissolve 60.5 g of N2Na2O8·2H2O (ethylenediaminetetraacetic acid disodium salt dihydrate) in 1.624 L of deionized water, adjust the pH to 4.00±0.05 with NaOH and HCl, and stir with a magnetic stirrer until all dissolved. Transfer the dissolved solution to a high pressure sterilizer and sterilize at 121°C for 20 min. Standard stock solutions of SMX and its seven degradation products (3-amino-5-methylisoxazole, 4-aminophenol, 4-aminothiophenol, sulfanilic acid, sulfonamide, 4-NO2-SMX, and 4-OH-SMX): Weigh 10 mg of each of SMX and its seven degradation products into a 10 mL brown volumetric flask. Dissolve sulfamethoxazole with 0.03 mol / L NaOH and make up to 10 mL with methanol. Dissolve the remaining seven degradation products with methanol and make up to 1 g / L.

[0039] Example 1 This embodiment provides a method for extracting sulfamethoxazole and its degradation products from pig manure, and the specific operation steps are as follows.

[0040] Processing of pig manure samples: One portion of fresh pig manure was taken from a pig farm in the suburbs of Beijing (no antibiotics were used in the breeding process and the target substance was not contained after testing), freeze-dried in a freeze dryer for 48 h, ground in a ball mill, and passed through a 100-mesh sieve to obtain pig manure pellets; The target solution mixture (8 target substances) was added at 10 mg / kg (dry weight). Specifically, 1 g of pig manure pellets was weighed, and 0.01 mL of each target substance stock solution was added to the pig manure pellets to obtain a pig manure sample.

[0041] Extraction of sulfamethoxazole and its degradation products from pig manure: Ultrasonic extraction: weigh 1 g of the pig manure sample and put it into a 15 mL glass centrifuge tube. After adding 10 mL of EDTA-MCIlvaine buffer: methanol: acetonitrile: acetone = 5:2:2:1 (volume ratio, pH = 6.0) extract to the glass centrifuge tube, vortex it for 45 s; put the vortexed glass centrifuge tube into an ultrasonic cleaner and perform ultrasonic extraction at 25 °C, 40 KHz, and 150 W for 10 min; wipe it dry, take it out and put it into a centrifuge for centrifugation at 4000 rpm for 15 min, take the supernatant and pour it into a 500 mL brown bottle; repeat the above operation 3 times, and combine the supernatants of the three extractions into the brown bottle; add 275 mL of deionized water to the brown bottle to ensure that the methanol content is less than 5%; use a Jinteng filtration device to filter out impurities in the extract with a 0.45 μm fiber filter membrane to obtain an ultrasonic extract.

[0042] Solid Phase Extraction: 3 mL of methanol, 3 mL of 0.5 mol / L hydrochloric acid, and 3 mL of deionized water were added to the HLB solid phase extraction column in order for activation. During the activation, the vacuum pump was not turned on to allow the liquid to flow down naturally. Turn on the vacuum pump and pass about 300 mL of the pre-treated solution through the HLB solid phase extraction column at a flow rate of 5 mL / min; Turn off the vacuum pump and sequentially add 5 mL of 5% methanol and 5 mL of deionized water to flow through the HLB solid phase extraction column at a natural flow rate for cleaning; Turn on the vacuum pump to drain the liquid in the solid phase extraction column; Turn off the vacuum pump and add 3 mL of n-hexane to flow through the HLB solid phase extraction column at a natural flow rate; Add 10 mL (dichloromethane:acetone = 3:2):methanol = 3:2 (volume ratio) to the solid phase extraction column for elution, and use a 15 ml glass centrifuge tube at the bottom to receive the eluted solution; The eluted eluate was placed in a nitrogen blower and blown to near dryness at 35 °C; 600 μL of methanol and 400 μL of water were added to the eluate blown to near dryness, and vortexed for 45 s using a vortexer; after filtering through a 0.22 μm glass fiber filter membrane, the solution was injected into a sample injection bottle to obtain a solid phase extraction solution, which was then tested on the machine.

[0043] The extraction method provided in this embodiment achieves the simultaneous extraction of sulfamethoxazole and its seven degradation products, and the recovery rates of the eight target detection substances are between 60.5% and 99.7%.

[0044] Example 2 The present embodiment provides a method for detecting sulfamethoxazole and its degradation products in pig manure, including extraction and detection of sulfamethoxazole and its degradation products in pig manure, wherein the extraction method of sulfamethoxazole and its degradation products in pig manure is the same as that in Example 1; and detection is performed by high performance liquid chromatography-mass spectrometry combined method.

[0045] Liquid phase conditions: Chromatograph: Sciex ExionLC AE; Chromatographic column: The specifications of the chromatographic column are ACQUITY UPLC HSS T3 (100 mm×2.1 mm, 1.8 μm); column temperature: 40°C; Mobile phase: Mobile phase A is 2 mM ammonium formate; Mobile phase B is methanol; Elution gradient: 0-1.2 min, 3% B; 1.2-5 min, 3% B-55% B; 5-6.5 min, 55% B; 6.5-7 min, 55% B-95% B; 7-8.5 min, 95% B; 8.5-8.6 min, 95% B-3% B; 8.6-10 min, 3% B; Flow rate: 0.2 mL / min; Injection volume: 10 μL.

[0046] The elution gradient table is shown in Table 1.

[0047] Table 1 Gradient elution program

[0048] Mass spectrometry conditions: Mass spectrometer: Triple Quad TM 7500; ion source: electrospray ionization source (ESI); spray voltage: ±2000V; detection method: multiple reaction monitoring (MRM); ion source temperature: 450 ℃; curtain gas: 40 psi; nebulizer gas: 30 psi; auxiliary heater: 50 psi.

[0049] According to the above mass spectrometry conditions, the MRM mode detection parameters of the target are shown in Table 2.

[0050] Table 2 MRM mode detection parameters of target compounds Determination of standard curve: Accurately pipette the single standard stock solutions of SMX and its 7 degradation products, dilute to volume with a brown volumetric flask, prepare 8 mixed standard solutions with target concentrations of 1, 10, 100, 125, 200, 500, and 1000 μg / L, and test them under optimized HPLC-MS conditions to obtain the working curve, such as Figure 1The mass spectra obtained by detecting 8 kinds of targets are shown in Figure 2~Figure 9 As shown, from Figure 2 It can be seen that the retention time of 4-aminophenol is 3.29 min. Figure 3 It can be seen that the retention time of 3-amino-5-methylisoxazole is 4.53 min. Figure 4 It can be seen that the retention time of sulfamethoxazole is 6.11 min. Figure 5 It can be seen that the retention time of 4-aminothiophenol is 8.85 min. Figure 6 It can be seen that the retention time of sulfanilic acid is 1.29 min. Figure 7 It can be seen that the retention time of sulfonamide is 1.18 min. Figure 8 It can be seen that the retention time of 4-NO2-SMX is 6.82 min. Figure 8 It can be seen that the retention time of 4-OH-SMX is 6.38 min. By calculating the peak area of ​​the standard solution in the mass spectrometer, a standard curve is drawn and the regression equation and correlation coefficient are calculated (see Table 3).

[0051] Table 3 Standard curves, correlation coefficients, detection limits and quantification limits of 8 target compounds

[0052] Sample detection: The standard solution and solid phase extraction solution were detected with an injection volume of 10 μL, wherein the liquid chromatography conditions and mass spectrometry conditions were as described above.

[0053] According to the regression equation of each target in Table 3 and the peak area data in each target mass spectrum, the concentration of each target in the solid phase extraction solution was calculated, and then the detection recovery rate of the target in the pig manure sample was obtained by comparing and calculating with the original concentration.

[0054] The detection recoveries of eight substances in pig manure samples of SMX and its seven degradation products (sulfamethoxazole and its degradation products) determined by the above method were 60.5%~99.7%, as shown in Table 4 for details.

[0055] Table 4 Detection recovery of sulfamethoxazole and its degradation products

[0056] The detection method provided in this embodiment can achieve the synchronous separation and detection of 8 target detection objects and obtain a mass spectrum within 10 minutes from sample injection to completion. The detection method has the characteristics of high detection sensitivity, high accuracy, simple operation, and short detection cycle.

[0057] In particular: It can be seen from Table 3 that in this embodiment, the detection limits of SMX and its degradation products are no higher than 0.069 μg / kg, and the quantification limits are no higher than 0.2 μg / kg. In particular, the detection limit of SMX is 0.00009 μg / kg, the quantification limit is 0.00025 μg / kg, and the recovery rate is 94.3%.

[0058] In the prior art, sulfamethoxazole and its degradation products have not been found to be extracted from pig manure. Instead, the extraction and detection of antibiotics in soil, sludge, etc. are more focused on. However, the recovery rate of sulfonamide antibiotics is generally only about 70%, especially the quantitative limit of sulfonamide antibiotics is generally 10 μg / kg (such as CN 117630215 A).

[0059] It can be seen from this that compared with the detection method in the prior art, the detection method provided by the present invention has better sensitivity (the detection limit is significantly lower than the detection method in the prior art) and higher recovery rate.

[0060] Example 3 This embodiment provides a method for detecting sulfamethoxazole and its degradation products in pig manure, which is basically the same as that of Example 2, except that the ultrasonic time is 20 min.

[0061] In this embodiment, the detection recovery rate of each target object is shown in Table 5.

[0062] Table 5 Detection recovery of sulfamethoxazole and its degradation products

[0063] Comparative Example 1 This comparative example provides a method for detecting sulfamethoxazole and its degradation products in pig manure, which is basically the same as Example 2, except that the pH of the extract in the ultrasonic extraction is 4.

[0064] In this comparative example, the recovery rates of the various target substances are shown in Table 6.

[0065] Table 6 Detection recovery of sulfamethoxazole and its degradation products

[0066] It can be seen from Table 5 that in the prior art, the pH of the composition extracted in ultrasonic extraction is different from that in the present application, and the final detection recovery rates of each target object are significantly worse than those of each target object in Example 2 of the present application. For example, the recovery rate of SMX is only 57.13% in this comparative example, while the recovery rate of SMX in Example 2 of the present application can reach 94.3%.

[0067] Comparative Example 2 This comparative example provides a method for detecting sulfamethoxazole and its degradation products in pig manure, which is basically the same as Example 2, except that the extracting solution in the ultrasonic extraction is different. The ultrasonic extraction disclosed in CN118275584A is adopted: first, two ultrasonic extractions are performed with EDTA-MCIlvaine buffer having a pH of 4.0±0.1, and then one ultrasonic extraction is performed with methanol: acetonitrile: acetone = 2:2:1.

[0068] In this comparative example, the detection recovery rates of each target substance are shown in Table 7.

[0069] Table 7 Detection recovery of sulfamethoxazole and its degradation products

[0070] It can be seen from Table 7 that, in the extraction method of the prior art, although the composition of the extract is the same as that of the present application, and the proportion of the organic extractant therein is also the same, the actual use and dosage of the buffer solution are different from those of the present application, and the final detection recovery rate of each target object is significantly worse than the detection recovery rate in Example 2 of the present application. For example, the recovery rate of SMX in this comparative example is 81%, while the recovery rate of SMX in Example 2 of the present application can reach 94.3%.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A combined extract of sulfamethoxazole and its degradation products in pig manure, characterized in that: The method comprises EDTA-MCIlvaine buffer, methanol, acetonitrile and acetone in a volume ratio of (4-6):(1-3):(1-3):1; the pH value of the combined extract is 5-7.

2. A method for extracting sulfamethoxazole and its degradation products from pig manure, characterized in that: include: The combined extract according to claim 1 is added to a pig manure sample and subjected to ultrasonic extraction to obtain an ultrasonic extract.

3. The extraction method according to claim 2, characterized in that The ultrasonic extraction was performed three times.

4. The extraction method according to claim 2 or 3, characterized in that The method also includes subjecting the ultrasonic extract to solid phase extraction; the eluent in the solid phase extraction includes component A and component B in a volume ratio of (5-7):(3-5); the component B includes methanol, and the component A includes dichloromethane and acetone in a volume ratio of (5-7):(3-5).

5. The extraction method according to any one of claims 2 to 4, characterized in that: Before subjecting the ultrasonic extract to solid phase extraction, the solid phase extraction column is activated with hydrochloric acid.

6. The extraction method according to claim 5, characterized in that After subjecting the ultrasonic extract to solid phase extraction, the solid phase extraction column is rinsed with methanol and water respectively; And / or, after subjecting the ultrasonic extract to solid phase extraction, the solid phase extraction column is firstly eluted with n-hexane, and then the solid phase extraction column is eluted with the eluent to obtain an extract of sulfamethoxazole and its degradation products.

7. A method for detecting sulfamethoxazole and its degradation products in pig manure, characterized by: Step (1): extracting sulfamethoxazole and its degradation products from pig manure to obtain an extract of sulfamethoxazole and its degradation products; Step (2): using high performance liquid chromatography-mass spectrometry to detect the extract of sulfamethoxazole and its degradation products; Wherein, in step (1), sulfamethoxazole and its degradation products are extracted from pig manure by the extraction method described in any one of claims 3 to 6 to prepare an extract of sulfamethoxazole and its degradation products.

8. The detection method according to claim 7, characterized in that: The high performance liquid chromatography method uses C18 as the stationary phase to fill the chromatographic column; And / or, the mobile phase comprises mobile phase A and mobile phase B; the mobile phase A comprises an aqueous solution of 2 mM ammonium formate, and the mobile phase B comprises methanol.

9. The detection method according to claim 8, characterized in that: The chromatographic column is ACQUITY UPLC HSS T3, 100 mm×2.1 mm, 1.8 μm.

10. The detection method according to claim 8 or 9, characterized in that: By volume percentage, the mobile phase was eluted using the following gradient: 0-1.2 min, 3% of the mobile phase B, 97% of the mobile phase A; 1.2-5 min, the mobile phase B gradually increased from 3% to 55%, and the mobile phase A gradually decreased from 97% to 45%; 5-6.5 min, 55% of the mobile phase B, 45% of the mobile phase A; 6.5-7 min, the mobile phase B gradually increased from 55% to 95%, and the mobile phase A gradually decreased from 45% to 5%; 7-8.5 min, 95% of the mobile phase B, 5% of the mobile phase A; 8.5-8.6 min, the mobile phase B gradually decreased from 95% to 3%, and the mobile phase A gradually increased from 5% to 97%; 8.6~10 min, 3% of the mobile phase B, 97% of the mobile phase A.

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