CN105548392A - Method for simultaneously detecting various antibiotics in livestock and poultry manure by high performance liquid chromatography - Google Patents
Method for simultaneously detecting various antibiotics in livestock and poultry manure by high performance liquid chromatography Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
本发明涉及一种抗生素检测方法，具体为利用高效液相色谱同时检测畜禽粪便中多种抗生素的方法，对畜禽粪便样品进行预处理，得到待测液，将待测液通过高效液相色谱仪进行分析物的分离，计算分离出的残留抗生素对应的峰面积，根据标准曲线进行定量计算，得出残留的含量。该方法应用于畜禽粪便中11种残留抗生素的检测，与高效液相色谱仪—质谱联用技术相比，检测成本较低；检测效率高，可以同时分离11种抗生素；分析物无主要干扰物干扰，基本不影响分析物定性和定量。The invention relates to an antibiotic detection method, specifically a method for simultaneously detecting multiple antibiotics in livestock and poultry feces by using high-performance liquid chromatography, pretreating the livestock and poultry feces samples to obtain a test solution, and passing the test solution through a high-performance liquid phase The chromatograph separates the analytes, calculates the peak area corresponding to the separated residual antibiotics, performs quantitative calculations according to the standard curve, and obtains the residual content. This method is applied to the detection of 11 kinds of residual antibiotics in livestock and poultry feces. Compared with high-performance liquid chromatography-mass spectrometry, the detection cost is lower; the detection efficiency is high, and 11 kinds of antibiotics can be separated at the same time; the analyte has no major interference The interference of the analyte basically does not affect the qualitative and quantitative analysis of the analyte.
Description
技术领域technical field
本发明涉及一种抗生素检测方法，具体为利用高效液相色谱同时检测畜禽粪便中多种抗生素的方法。The invention relates to an antibiotic detection method, in particular to a method for simultaneously detecting multiple antibiotics in livestock and poultry feces by using high performance liquid chromatography.
背景技术Background technique
抗生素是指由细菌、放线菌、真菌等微生物经培养而获得的或是用化学合成法合成的结构相同或类似的具有杀灭或抑制病原微生物的物质。近年来大量的抗生素作为兽药和饲料添加剂广泛应用于畜禽养殖业中，在动物疾病防治、提高饲料转化效率、促进动物生长发育方面发挥着重要作用。但是，通过口服或肌肉注射进入动物体内的抗生素并不能被完全吸收利用，以原形或初级代谢产物的形式随尿或粪便排出体外的量约占用药量的40％—90％。因此，兽用抗生素极易随着粪尿等进入农田土壤等环境中，造成环境抗生素残留以及耐药细菌的传播扩散，严重影响整个农业生态系统的安全，最终危及人类健康。建立同时检测环境介质中多种抗生素的分析方法是研究其环境行为和生态环境风险的基础。Antibiotics refer to substances with the same or similar structure that are obtained from bacteria, actinomycetes, fungi and other microorganisms through cultivation or synthesized by chemical synthesis, and have the ability to kill or inhibit pathogenic microorganisms. In recent years, a large number of antibiotics have been widely used in livestock and poultry breeding as veterinary drugs and feed additives, and play an important role in the prevention and control of animal diseases, improving feed conversion efficiency, and promoting animal growth and development. However, antibiotics introduced into animals through oral or intramuscular injection cannot be fully absorbed and utilized, and the amount excreted in urine or feces in the form of prototype or primary metabolites accounts for about 40%-90% of the drug dose. Therefore, veterinary antibiotics are very easy to enter the environment such as farmland soil with feces and urine, causing environmental antibiotic residues and the spread of drug-resistant bacteria, seriously affecting the safety of the entire agricultural ecosystem, and ultimately endangering human health. Establishing an analytical method for simultaneous detection of multiple antibiotics in environmental media is the basis for studying their environmental behavior and ecological environment risk.
环境残留的抗生素属于痕量物质，现有方法多采用高效液相色谱仪—质谱联用技术进行研究，但该技术成本相对较高，目前对兽用抗生素残留量的检测研究多集中在动物性食品领域，且检测的抗生素种类单一，清楚进入农田环境的畜禽粪便中多种抗生素残留量，对确保生态环境安全具有重要意义。Environmental residual antibiotics belong to trace substances. Existing methods mostly use high-performance liquid chromatography-mass spectrometry technology for research, but the cost of this technology is relatively high. At present, the research on the detection of veterinary antibiotic residues mostly focuses on animal-derived antibiotics. In the food field, and the types of antibiotics detected are single, it is of great significance to ensure the safety of the ecological environment to know the residues of various antibiotics in the livestock and poultry manure entering the farmland environment.
发明内容Contents of the invention
为了克服上述现有技术的抗生素检测方法成本高、检测抗生素种类少且停留在动物性食品检测领域的缺点与不足，本发明提供一种利用高效液相色谱快速检测多种兽用抗生素的方法。In order to overcome the shortcomings and deficiencies of the above-mentioned antibiotic detection method in the prior art, such as high cost, few types of antibiotics and staying in the field of animal food detection, the present invention provides a method for rapidly detecting various veterinary antibiotics by using high performance liquid chromatography.
本发明以高效液相色谱仪为平台，建立了畜禽粪便中多种抗生素残留的检测方法，同时检测多种兽用抗生素的方法在检测生态环境残留抗生素中的应用。特别是应用于检测畜禽粪便中残留的11种抗生素。The invention uses a high-performance liquid chromatography as a platform to establish a detection method for multiple antibiotic residues in livestock and poultry feces, and simultaneously detects multiple animal antibiotics. The method is applied to the detection of residual antibiotics in an ecological environment. Especially for the detection of 11 kinds of antibiotics remaining in livestock and poultry manure.
本发明的目的通过下述方案实现：The object of the present invention is achieved through the following solutions:
利用高效液相色谱同时检测畜禽粪便中多种抗生素的方法，包含以下步骤：The method for simultaneously detecting multiple antibiotics in livestock and poultry manure by high performance liquid chromatography comprises the following steps:
(1)对畜禽粪便样品进行预处理，得到待测液；(1) Pretreating the livestock and poultry feces samples to obtain the liquid to be tested;
将采集回的畜禽粪便样品冷冻干燥后，用EDTA-Mcllvaine缓冲液和有机混合提取剂提取，提取液先用正己烷去脂，然后过活化后的HLB小柱进行富集：用25％甲醇溶液淋洗HLB小柱去除部分杂质，并抽干5min，再用65％甲醇溶液洗脱富集的HLB小柱，洗脱液旋蒸吹干，用初始流动相定容至1mL，过0.22μm膜，得到待测液；After the collected livestock and poultry feces samples were freeze-dried, they were extracted with EDTA-Mcllvaine buffer and organic mixed extractant. The extract was first degreased with n-hexane, and then enriched with an activated HLB column: use 25% methanol Rinse the HLB column with the solution to remove some impurities, and drain it for 5 minutes, then elute the enriched HLB column with 65% methanol solution. film, to obtain the liquid to be tested;
所用EDTA-Mcllvaine缓冲液配制方法为：和Na2HPO4分别配制成0.1mol/L柠檬酸溶液和0.2mol/L的溶液按照体积比8:5混合，配制成Mcllvaine溶液；取37.2g的Na2-EDTA与Mcllvaine溶液中，配得0.1mol/LEDTA-Mcllvaine提取液，以HCl或NaOH调至pH＝4.0；The preparation method of the EDTA-Mcllvaine buffer solution used is: and Na 2 HPO 4 respectively prepared into 0.1mol/L citric acid solution and 0.2mol/L solution according to the volume ratio of 8:5 and mixed to prepare Mcllvaine solution; take 37.2g of Na 2 -EDTA and Mcllvaine solution, prepared 0.1mol/LEDTA-Mcllvaine extract, adjusted to pH=4.0 with HCl or NaOH;
所用的有机混合提取剂为甲醇：乙腈：丙酮按照体积比2:2:1混合，用H3PO4调节pH为4。The organic mixed extractant used is methanol: acetonitrile: acetone mixed according to the volume ratio of 2:2:1, and the pH is adjusted to 4 with H 3 PO 4 .
(2)将待测液通过高效液相色谱仪进行分析物的分离；(2) Carry out the separation of analyte by high-performance liquid chromatograph by liquid to be tested;
将待测液通过高效液相色谱仪进行分析物的分离，测定的色谱条件为：The liquid to be tested is separated by a high-performance liquid chromatograph, and the chromatographic conditions for determination are:
采用AgilentEclipsePlusC18色谱柱：150mm×4.6mm，3.5μm，Agilent公司；二元梯度泵；可变波长扫描紫外检测器；流动相A为0.1％甲酸溶液，流动相B为甲醇与乙腈按照体积比1:1；流速为0.6mL/min；进样体积20μL；柱温35℃；检测波长为274nm；梯度洗脱顺序：0～18min，80％A；18～20min，80％～40％A；20～30min，40％A；30～32min，40％～80％A；32～35min，80％A。Adopt AgilentEclipsePlusC18 chromatographic column: 150mm * 4.6mm, 3.5 μ m, Agilent Company; Binary gradient pump; Variable wavelength scanning ultraviolet detector; Mobile phase A is 0.1% formic acid solution, and mobile phase B is methanol and acetonitrile according to volume ratio 1: 1; flow rate is 0.6mL/min; injection volume is 20μL; column temperature is 35°C; detection wavelength is 274nm; gradient elution sequence: 0～18min, 80%A; 30min, 40%A; 30-32min, 40%-80%A; 32-35min, 80%A.
(3)计算分离出的残留抗生素对应的峰面积，根据标准曲线进行定量计算，得出残留的含量；(3) Calculate the peak area corresponding to the residual antibiotics separated, carry out quantitative calculation according to the standard curve, and obtain the residual content;
所述标准曲线依照步骤(1)、步骤(2)，采用相同的试剂以及参数，在相同实验条件下得出。The standard curve is obtained under the same experimental conditions using the same reagents and parameters according to step (1) and step (2).
该方法用于同时检测11种抗生素，包括土霉素(OTC)、金霉素(CTC)、磺胺二甲嘧啶(SDMe)、磺胺甲恶唑(SMZ)、磺胺噻唑(ST)、磺胺间甲氧嘧啶(SMN)、环丙沙星(CIP)、诺氟沙星(NOR)、恩诺沙星(ENR)、泰乐菌素(TYL)、氯霉素(CAP)。The method was used for the simultaneous detection of 11 antibiotics, including oxytetracycline (OTC), chlortetracycline (CTC), sulfamethazine (SDMe), sulfamethoxazole (SMZ), sulfathiazole (ST), sulfamethazine Oxypyrimidine (SMN), ciprofloxacin (CIP), norfloxacin (NOR), enrofloxacin (ENR), tylosin (TYL), chloramphenicol (CAP).
本发明提供的利用高效液相色谱同时检测多种抗生素的方法，应用于畜禽粪便中11种残留抗生素的检测，11种目标抗生素在高效液相色谱仪紫外检测器274nm波长下均具有良好吸收，检测效果最佳，通过对流动相及各组分配比、流速、温度等条件的优化组合，确定11种目标抗生素同时检测的最佳条件。与高效液相色谱仪—质谱联用技术相比，检测成本较低；检测效率高，可以同时分离11种抗生素；分析物无主要干扰物干扰，基本不影响分析物定性和定量。The method for simultaneously detecting multiple antibiotics by using high performance liquid chromatography provided by the present invention is applied to the detection of 11 kinds of residual antibiotics in livestock and poultry feces, and all 11 kinds of target antibiotics have good absorption under the 274nm wavelength of the ultraviolet detector of the high performance liquid chromatography , the detection effect is the best, through the optimized combination of mobile phase and the distribution ratio of each component, flow rate, temperature and other conditions, the best conditions for the simultaneous detection of 11 target antibiotics are determined. Compared with high-performance liquid chromatography-mass spectrometry, the detection cost is lower; the detection efficiency is high, and 11 kinds of antibiotics can be separated at the same time; the analyte has no interference from major interfering substances, and basically does not affect the qualitative and quantitative analysis of the analyte.
具体实施方式detailed description
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.
利用高效液相色谱同时检测畜禽粪便中多种抗生素的方法的仪器条件如下：The instrument conditions of the method for simultaneously detecting multiple antibiotics in livestock and poultry manure by high performance liquid chromatography are as follows:
检测器为可变波长扫描紫外检测器即VWD，检测波长为274nm，柱温35℃，流速为0.6mL/min，进样体积20μL，流动相A为0.1％甲酸溶液，流动相B为甲醇/乙腈按照v/v为1:1混合，梯度洗脱顺序：0～18min，80％A；18～20min，80％～40％A；20～30min，40％A；30～32min，40％～80％A；32～35min，80％A。The detector is a variable wavelength scanning ultraviolet detector (VWD), the detection wavelength is 274nm, the column temperature is 35°C, the flow rate is 0.6mL/min, the injection volume is 20μL, the mobile phase A is 0.1% formic acid solution, and the mobile phase B is methanol/ Acetonitrile was mixed according to v/v ratio of 1:1, gradient elution sequence: 0～18min, 80%A; 18～20min, 80%～40%A; 20～30min, 40%A; 30～32min, 40%～ 80%A; 32～35min, 80%A.
猪粪中11种抗生素残留的分离监测：Separation and monitoring of 11 kinds of antibiotic residues in pig manure:
(1)所用仪器为美国安捷伦1200高效液相色谱仪，色谱柱规格为AgilentEclipsePlusC18(150mm×4.6mm，3.5μm)，可变波长扫描紫外检测器(VWD)，检测波长274nm，柱温35℃，流速为0.6mL/min，进样体积20μL，流动相A为0.1％甲酸溶液，流动相B为甲醇/乙腈按照v/v为1:1混合，梯度洗脱顺序：0～18min，80％A；18～20min，80％～40％A；20～30min，40％A；30～32min，40％～80％A；32～35min，80％A。Agilent1200LC工作站。(1) The instrument used is Agilent 1200 high performance liquid chromatography in the United States, the chromatographic column specification is Agilent Eclipse Plus C18 (150mm×4.6mm, 3.5μm), variable wavelength scanning ultraviolet detector (VWD), the detection wavelength is 274nm, and the column temperature is 35°C. The flow rate is 0.6mL/min, the injection volume is 20μL, the mobile phase A is 0.1% formic acid solution, the mobile phase B is methanol/acetonitrile mixed according to v/v ratio of 1:1, gradient elution sequence: 0～18min, 80%A ; 18-20min, 80%-40%A; 20-30min, 40%A; 30-32min, 40%-80%A; 32-35min, 80%A. Agilent1200LC workstation.
(2)标准曲线的测定：准确称取11种目标抗生素标准品各10mg于10mL棕色容量瓶中，诺氟沙星用0.03mol/LNaOH溶解并定容至10mL，其余10种抗生素目标物用甲醇溶解并定容，配制成1mg/mL单标储备液，于4℃避光保存。准确移取各种单标储备液，用棕色容量瓶配制11种抗生素浓度均为0.05、0.1、0.5、1.0、5.0、10.0、50.0、100mg/L的混合标准溶液，采用步骤(1)的条件进行测试，得到工作曲线。11种抗生素的线性关系、相关系数及检出限见表1。(2) Determination of the standard curve: Accurately weigh 10 mg of each of the 11 target antibiotic standards in a 10 mL brown volumetric flask, dissolve norfloxacin with 0.03 mol/L NaOH and dilute to 10 mL, and use methanol for the remaining 10 target antibiotics Dissolve and dilute to volume, prepare a 1 mg/mL single-label stock solution, and store at 4°C in the dark. Accurately pipette various single-standard stock solutions, prepare 11 kinds of antibiotic concentrations in brown volumetric flasks, and mix standard solutions with concentrations of 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100 mg/L, using the conditions of step (1) Carry out the test and get the working curve. The linear relationship, correlation coefficient and detection limit of the 11 antibiotics are shown in Table 1.
(3)HLB小柱的活化：HLB小柱在使用前，先后用5mL甲醇、10mL纯水进行活化。(3) Activation of the HLB cartridge: Before use, the HLB cartridge was activated with 5 mL of methanol and 10 mL of pure water.
(4)畜禽粪便样品的预处理及测试：将采集回的猪粪样品，试验样品选用山西省寿阳县一生态农场，在养殖过程中未使用抗生素，经检测不含抗生素目标物，冷冻干燥，研磨过2mm筛，准确称取1.0g研磨样品，将11种抗生素混合于猪粪中得到混合猪粪样品，首先加入10mLEDTA-Mcllvaine缓冲液涡旋混匀30s，常温超声15min，在4℃下8000r/min离心15min，上清液转移至另外棕色容器中，重复提取一次，再用5mL有机混合提取液重复提取两次，步骤同上。(4) Pretreatment and testing of livestock and poultry manure samples: The collected pig manure samples were selected from an ecological farm in Shouyang County, Shanxi Province. No antibiotics were used in the breeding process, and no antibiotic targets were detected, and frozen. Dry, grind through a 2mm sieve, accurately weigh 1.0g of the ground sample, mix 11 kinds of antibiotics in pig manure to obtain a mixed pig manure sample, first add 10mL LEDTA-Mcllvaine buffer solution, vortex and mix for 30s, ultrasonic at room temperature for 15min, at 4°C Centrifuge at 8000r/min for 15min, transfer the supernatant to another brown container, repeat the extraction once, and repeat the extraction twice with 5mL organic mixed extract, the steps are the same as above.
合并提取液，加入10mL正己烷去脂后，过0.45μm滤膜，将过膜后提取液使用旋转蒸发仪浓缩至3-5mL，浓缩后的提取液以1mL/min流速过活化后的HLB柱，用5mL的甲醇和水v/v为25:75混合液淋洗HLB小柱，并抽干5min，用10mL的甲醇和水v/v为65:35混合液洗脱目标抗生素，收集洗脱液，于旋转蒸发仪上浓缩至干，用初始流动相定容至1mL，过0.22μm膜，上机待测，按照步骤(1)的条件进行高效液相色谱分离测试。Combine the extracts, add 10 mL of n-hexane to degrease, pass through a 0.45 μm filter membrane, concentrate the extracts to 3-5 mL using a rotary evaporator, and pass the concentrated extracts through an activated HLB column at a flow rate of 1 mL/min , rinse the HLB cartridge with 5 mL of methanol and water v/v 25:75 mixture, and drain for 5 min, elute the target antibiotic with 10 mL of methanol and water v/v 65:35 mixture, collect the eluted The solution was concentrated to dryness on a rotary evaporator, adjusted to 1 mL with the initial mobile phase, passed through a 0.22 μm membrane, and tested on the machine, and carried out high performance liquid chromatography separation test according to the conditions of step (1).
(5)对上述测试得到的曲线分析，对分离出的残留目标抗生素面积进行积分，依据标准品制作的标准曲线进行定量计算，得出残留的含量，计算回收率，添加回收率见表2。(5) To the curve analysis that above-mentioned test obtains, the residual target antibiotic area that isolates is integrated, carry out quantitative calculation according to the standard curve that standard substance is made, draw residual content, calculate recovery rate, add recovery rate and see Table 2.
表1紫外检测器下11种抗生素线性关系、相关系数及检出限Table 1 The linear relationship, correlation coefficient and detection limit of 11 kinds of antibiotics under the ultraviolet detector
表211种目标抗生素的回收率、标准偏差、检出限及定量限The recovery rate, standard deviation, detection limit and limit of quantification of table 211 kinds of target antibiotics
注：ST-磺胺噻唑；CIP-环丙沙星；OTC-土霉素；NOR-诺氟沙星；SDMe-磺胺二甲嘧啶；ENR-恩诺沙星；SMN-磺胺间甲氧嘧啶；SMZ-磺胺甲恶唑；CTC-金霉素；CAP-氯霉素；TYL-泰乐菌素。Note: ST-sulfathiazole; CIP-ciprofloxacin; OTC-oxytetracycline; NOR-norfloxacin; SDMe-sulfamethazine; ENR-enrofloxacin; SMN-sulfamethoxine; SMZ - sulfamethoxazole; CTC - aureomycin; CAP - chloramphenicol; TYL - tylosin.
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