Simultaneous analysis of four sulfonamides in chicken muscle tissue by HPLC

The aim of this study was to develop a simple high-performance liquid chromatography (HPLC) with UV detection method for the determination of four sulfonamides in chicken muscle tissue. The sulfonamides were extracted with acetonitrile, acetone and dichloromethane. Separation was carried out on a C18 column using as the mobile phase a mixture of 6‰ disodium hydrogen phosphate and methanol. The analytes were detected by UV in one run. Calibration curves were linear with very good correlation coefficients for concentration ranging from 30 to 150?µg?kg^?1. The limits of detection (LOD) for sulfonamides ranged from 6.5 to 0.14?µg?kg^?1. The recovery for spiked chicken muscle with 50–150?µg?kg^?1 was more than 70%. The relative standard deviations (RSDs) of the sulfonamides for six measurements at 50, 100 and 150?µg?kg^?1 were less than 15%. These parameters met the European Union criteria for method validation. The results were confirmed by LC-MS/MS using multiple reaction monitoring as the operating mode. Confirmation required the retention times of the analytes to be within ±2.5% of the retention times of the standards, the presence of the parent ion and two characteristic fragment ions (product ions) per analyte, as well as the relative ion abundance ratios of the fragment ions corresponding to ratios obtained for the standards, within permitted limits. The transition of two common product ions at m/z 155.7 and 107.5 were monitored for all sulfonamides. Each of the analytes, in all tested samples, met the confirmation criteria. Thus, the applicability of the HPLC-UV method for routine analysis of chicken muscle tissue was demonstrated.     

Mass spectrometric analysis of muscle samples to detect potential antibiotic growth promoter misuse in broiler chickens

Mass spectrometric methods were developed and validated for the analysis in chicken muscle of a range of antibiotic growth promoters: spiramycin, tylosin, virginiamycin and bacitracin, and separately for two marker metabolites of carbadox (quinoxaline-2-carboxylic acid and 1,4-bisdesoxycarbadox), and a marker metabolite of olaquindox (3-methyl-quinoxaline-2-carboxylic acid). The use of these compounds as antibiotic growth promoters has been banned by the European Commission. This study aimed to develop methods to detect their residues in muscle samples as a means of checking for the use of these drugs during the rearing of broiler chickens. When fed growth-promoting doses for 6 days, spiramycin (31.4?μg?kg(-1)), tylosin (1.0?μg?kg(-1)), QCA (6.5?μg?kg(-1)), DCBX (71.2?μg?kg(-1)) and MQCA (0.2?μg?kg(-1)) could be detected in the muscle 0 days after the withdrawal of fortified feed. Only spiramycin could consistently be detected beyond a withdrawal period of 1?day. All analytes showed stability to a commercial cooking process, therefore raw or cooked muscle could be used for monitoring purposes.     

Determination of Sulfonamides in Chicken Meat by Magnetic Molecularly Imprinted Polymer Coupled to HPLC-UV

In this work, Fe₃O₄ magnetic nanoparticles were synthesized and modified by a molecularly imprinted polymer for easy and selective extraction and determination of sulfonamides in chicken meat samples. Imprinted polymer magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, X-ray diffraction pattern, thermal analysis, and scanning electron microscope micrograph. The template was removed by methanol elution. The effective parameters on extraction and determination of sulfonamides on the sorbent such as eluent type, extraction solvent, and adsorption and desorption times were optimized. Sulfonamide separation and determination were performed by high-performance liquid chromatography–UV. The linear ranges for sulfonamides were 0.5–150 μg/L and the limits of detection were 0.1–0.5 μg/L. Relative standard deviations were within 4.3–5.4 %. The method showed good selectivity for extraction of sulfonamides in real samples.     

液相色谱法快速测定鸡肉中氧氟沙星手性对映体残留量

建立了鸡肉样品中氧氟沙星对映体液相色谱荧光检测(liquid chromatography fluorescence detector,LC-FLD)残留量快速分析方法。鸡肉样品经磷酸盐缓冲液提取后,用C18固相萃取小柱净化,洗脱液吹干后用流动相复溶即可进行液相色谱分析。分析时采用LeapsilTM C18色谱柱,以水相(含2 mmol/L硫酸铜和2.5 mmol/L异亮氨酸,pH3.5)和甲醇作为流动相进行梯度分析,荧光检测器检测,其中激发波长为297 nm,发射波长为487 nm,外标法定量。本方法在5.0、50、100μg/kg和150μg/kg 4个添加水平下,左氧氟沙星对映体平均添加回收率在75.5%～86.1%之间,批内变异系数在1.97%～4.42%之间,批间变异系数在3.02%～6.02%之间;右氧氟沙星对映体平均添加回收率在77.3%～84.9%之间,批内变异系数在2.15%～4.21%之间,批间变异系数在3.87%～5.84%之间。方法对两种对映体的检测限均为1.5μg/kg,定量限均为5.0μg/kg。     

Development of an HPLC–UV method for the simultaneous determination of tetracyclines in muscle and liver of porcine,chicken and bovine with accelerated solvent extraction

A simple and especially rapid method-using accelerated solvent extraction (ASE) and HPLC has been developed for the quantitative determination of oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, demeclocycline and doxycycline in muscle and liver of porcine, chicken and bovine. Samples of muscle and liver were extracted with trichloracetic acid/acetonitrile using ASE instrument, parameters such as extraction temperature (40–80 °C) and pressure (45–85 bar) were investigated and the selected extraction (60 °C, 65 bar) was most effective. The limits of detection were lower than 10 μg/kg and limits of quantification no more than 15 μg/kg for all compounds in muscle and liver. The recoveries of tetracyclines spiked at levels of muscle 50–150 μg/kg, liver 150–450 μg/kg, averaged from 75.0% to 104.9% with the relative standard deviation values less than 10%. The method was applied to determine 30 real porcine livers. It is demonstrated that the new method is robust for detection and quantification of seven tetracycline residues in muscle and liver of porcine, chicken and bovine.     

Quantification of lead and cadmium in poultry and bird game meat by square-wave anodic-stripping voltammetry

A square-wave anodic-stripping voltammetric method for the analysis of lead and cadmium in chicken muscle and liver was developed and validated, and the results of a monitoring study relative to chicken and pigeon meat are reported. The voltammetric method allows the analysis of lead and cadmium at the same time in samples after acid digestion. The use of perchloric acid for digestion and of acetate buffer in the supporting electrolyte are suitable to reduce matrix interferences and obtain limits of quantification which were below 10 ng g?1 for meat and liver samples. The regression between the analytical signal and the concentration of the target analytes in spiked samples and Certified Reference Materials proved to be linear within the 10-100 ng g?1 range for meat and within the 50-500 ng g?1 range for liver. The analytical method was verified using available Certified Reference Materials BCR-184 (cattle meat) and BCR-185R (cattle liver) as well as with spiked chicken samples. Precision (i.e. repeatability and intermediate precision) and accuracy (percentage recovery and bias) were of the order of 0.3-4.5% for both lead and cadmium The level of lead in muscle was in the range between 6.4 and 59.8 ng g?1 in chickens and between 7.9 and 63.6 ng g?1 in farmed pigeons, whereas it was between 8.0 and 84.4 ng g?1 in chicken liver. The cadmium concentration was 0.4-10.4 ng g?1 in chicken muscle, 10.4-90.6 ng g?1 in chicken liver and 2.2-8.0 ng g?1 in farmed pigeons.     

多壁碳纳米管小柱-液质联用法测定鸡肉中兽药残留方法的研究

本实验建立了鸡肉中喹诺酮类、磺胺类、氯霉素类、四环素类兽药残留的液相色谱-串联质谱快速测定方法.研究了鸡肉中4类兽药残留的提取、净化前处理条件,试样用2.5％甲酸乙腈溶液提取,滤过型多壁碳纳米管小柱净化,Accucore VDX色谱柱梯度分离,三重四极杆质谱测定,基质标准曲线法定量.同时以电喷雾离子源正模式和负模式扫描...     

Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle,chicken muscle,egg, fish,milk and kidney

A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1 μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5 μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10 μg/kg in kidney and 20 μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015 μg/kg, and the detection limits were 1.5 μg/kg in pig muscle and 6 μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r²) which was greater than 0.9.     

QuEChERS-高效液相色谱法检测不同畜禽产品中5种氟喹诺酮类的药物残留

目的 建立QuEChERS法检测多种畜禽产品中5种氟喹诺酮类药物残留.方法 采用酸化的乙腈溶液提取猪、牛、羊、鸡和鸭等畜禽的肌肉、皮脂、脂肪、肝和肾组织中氟喹诺酮类残留,用微型高速分散机在离心管内同时完成均质与提取,采用增强型脂质去除过滤柱对提取物进行2次洗脱,用高效液相色谱-可变波长荧光检测器法检测.结果 达氟沙星和...     

Development and application of a method for the analysis of two trichothecenes: deoxynivalenol and T-2 toxin in meat in China by HPLC-MS/MS

A reliable and sensitive method was developed and successfully applied for the determination of deoxynivalenol and T-2 toxin simultaneously in pig dorsal muscle, pig back fat and chicken muscle by high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) analysis. Limit of detection of deoxynivalenol and T-2 was 0.02 μg/kg and 0.007 μg/kg, and limit of quantification of deoxynivalenol and T-2 was 0.07 μg/kg and 0.02 μg/kg, respectively. Sixty-six meat samples were analyzed and deoxynivalenol was detected in the samples of pig back fat, with concentrations lower than 0.5 μg/kg, and T-2 toxin was detected in the samples of pig dorsal muscle, pig back fat and chicken muscle, with concentrations lower than 0.5 μg/kg. The results of sample analysis show that only trace residues of deoxynivalenol and T-2 toxin were detected in the samples analyzed.     

高效液相色谱-串联质谱法测定动物肌肉组织中氨基甲酸酯类杀虫剂及其代谢物残留

建立动物组织中氨基甲酸酯类杀虫剂及其代谢物(共16种)残留的高效液相色谱-串联质谱分析方法。样品经乙腈提取、浓缩、净化,液相色谱串联质谱测定,内标法定量。16种杀虫剂在1.0～100μg/L范围内线性关系良好(r＞0.9959);方法定量限为0.5～2.5μg/kg;样品添加5.0、10.0、20μg/kg时,加标回收率为71.4%～105.5%;相对标准偏差为3.2%～13.7%。该方法具有简便快捷、灵敏度高的特点,适用于动物肌肉中氨基甲酸酯类杀虫剂及其代谢物残留量的检测。     

Preconcentration and Simultaneous Analysis of Benzimidazole Anthelmintics in Milk Samples by Ultrasound-Assisted Surfactant-Enhanced Emulsification Microextraction and High-Performance Liquid Chromatography

The proposed ultrasound-assisted surfactant-enhanced emulsification microextraction (UASEME) coupled with high-performance liquid chromatography-photodiode array detection (HPLC-PDA) has been developed for the preconcentration and simultaneous analysis of five benzimidazole anthelmintics. Dichloromethane (extraction solvent) and Triton X-114 (emulsifier) was used for extraction of the target analytes. The parameters affecting the extraction efficiency were investigated and optimized. Under the optimum conditions, linearity was in the range from 10 to 150 μg?L^?1 with good coefficients of determination (R ²) higher than 0.994. Preconcentration factors were obtained up to 60, corresponding to limits of detection range of 1.8???3.6 μg?L^?1. Intra-day (n?=?5) and inter-day (n?=?4?×?3) precisions were obtained with relative standard deviation of retention time and peak area below 0.8 and 9.2 %, respectively. Good recoveries for the spiked target anthelmintics at different concentrations (e.g., 20, 50, and 100 μg?L^?1) of milk samples were obtained in 72.5–113.5 %. The results demonstrated that the proposed UASEME-HPLC-PDA can be used as an alternative powerful method for the simultaneous determination of the target analytes in milk samples.     

Thin-layer Chromatographic Method for Quantification of Sulfonamides in Chicken Meat

The sulfonamides are a widely used group of antimicrobials in veterinary practice especially in developing countries due to their broad spectrum of activity, availability, and low cost. The objective of this study was to establish a thin-layer chromatographic (TLC) method to detect residues of commonly used sulfonamides in Sri Lanka in chicken. The TLC method separated sulfadiazine (SDZ), sulfadoxine (SD), sulfamethazine (SMZ), sulfathiazole (STZ), and sulfaquinoxaline (SQ) on silica gel plates using chloroform: n_butanol (90:10). Sulfonamides densitometric measurements were conducted by derivatising with fluorescamine and scanning the separated bands at 366 nm. The quantity of the sulfonamide in samples was calculated using a standard calibration curve obtained by plotting values of peak area against sulfonamide concentrations at 200, 150, 100, 50, 30, and 25 ng/g. The mean percentage recoveries were 80–120% while the intra-day and inter-day precision was 3–22% and 6–42% respectively. The SD had the highest limit of detection (LOD) and limit of quantification (LOQ) values of 60 and 100 ng/g respectively. The LOD of all the other analytes were 40 ng/g, and STZ had the lowest LOQ values of 50 ng/g. A reliable, inexpensive, simple, liquid-liquid extraction method for the rapid analysis of five sulfonamides at maximum residue limit (MRL) in chicken using TLC was developed.     

三穗鸭中磺胺类和喹诺酮类药物残留检测及风险评估

建立了15种磺胺类和6种氟喹诺酮类药物残留的超高效液相色谱法-串联质谱法,并采用此法对2018年贵州省三穗县三穗鸭肉进行了15种磺胺类和6中氟喹诺酮类药物残留检测,在此基础上对三穗鸭肉产品中的磺胺类和氟喹诺酮类药物的残留风险进行了分析。结果显示,21种药物在5~200 μg/L质量浓度范围内线性关系良好,决定系数(R²)均大于0.990,药物的检出限为0.5~2.0 μg/kg,定量限2.0~5.0 μg/kg,在添加浓度5~50 μg/kg的范围内,21种药物回收率为70.3%~113.2%,相对标准偏差为2.1%~11.5%(n=6),该方法简单、快捷、精密度和准确度高,能满足鸭肉中两类兽药检测;三穗鸭肉中氟喹诺酮类均未检出,磺胺类仅检出磺胺氯哒嗪,检出率2.17%,检出值为2.37~3.68 μg/kg,均值3.33 μg/kg,未超过国家规定限量(100 μg/kg),其余磺胺类指标均未检出;参照国际上磺胺类ADI值规定,三穗鸭肉中磺胺氯哒嗪的食品安全指数值平均值为0.00014,最小值为0.00010,最大值为0.00016,均远远低于1;结果均表明,三穗鸭肉中氟喹诺酮类和磺胺类药物残留对人体的潜在危害水平是可以接受的。     

Development of an accelerated solvent extraction, ultrasonic derivatisation LC-MS/MS method for the determination of the marker residues of nitrofurans in freshwater fish

A rapid method using accelerated solvent extraction (ASE) and ultrasound enhanced derivatisation has been developed for the quantitative determination of metabolites of nitrofurans, namely 3-amino-2-oxalidinone (AOZ), 5-morpholinomethyl-3-amino-2-oxalidinone (AMOZ), 1-amino-hydantoin (AHD) and semicarbazide (SEM), in muscle and skin of carp and finless eel. The target analytes were extracted using ASE, ultrasonic derivatisation for 1?h and then purified by solid phase extraction. Averaged decision limits (CCα) and detection capability (CCβ) of the method were in the range of 0.07-0.13 and 0.31-0.49?μg?kg?1 in carp and finless eel, respectively. The accuracy in terms of recovery was in the range 77.2-97.4%. The simplified and traditional methods were compared with incurred residue samples. The simplified method reduced the derivatisation time and has been applied to the determination of nitrofurans residues in fish.     

Development of Multiresidue Analysis for 21 Synthetic Colorants in Meat by Microwave-Assisted Extraction–Solid-Phase Extraction–Reversed-Phase Ultrahigh Performance Liquid Chromatography

A novel ultrahigh performance liquid chromatographic method is developed for analysis of 21 synthetic colorants with different acid–base property, solubility, and polarity. The meat samples were extracted by microwave-assisted extraction followed by cleanup with solid-phase extraction. The effective separation of the colorants in meat matrixes was achieved, and no interfering peaks could be detected at the retention time of the analytes. The calibration curves showed good linearity with correlation coefficients of 0.9940–0.9999. The limits of quantification were 0.48–7.19 μg/kg. The average recovery of the 21 analytes from meat samples spiked with 25 and 75 μg?kg^?1 was 61.29–116.1 % with relative standard deviation (RSD) of <11 %. For blank beef sausage spiked with 50 μg?kg^?1 for each analyte, the intraday precision (as RSD) for 21 analytes was 1.45–9.21 % for six determinations within a day. This method has the advantages of being rapid, sensitive, accurate, and with high-throughput and can be applied for multiresidue analysis of meat samples, including six allowable azo food colorants, ten banned azo food colorants, four banned triphenylmethanes, and rhodamin B food colorant.     

In-house validation of PremiTest, a microbiological screening test with solvent extraction, for the detection of antimicrobial residues in poultry muscles

PremiTest, a microbial inhibition test for the screening of antimicrobial residues, was validated according to the criteria established by Decision 2002/657/EC. Sensitivity, detection capability (CCβ), specificity, selectivity, robustness and applicability were evaluated. The methodology involves the technique of solvent extraction, which increases the detection capability of the test for a wider range of antibiotics. The following CCβ values in poultry muscle were found: penicillin G ≤ 12.5 μg kg(-1), total sulfonamides ≤ 75 μg kg(-1), erythromycin 75 μg kg(-1) and lincomycin 50 μg kg(-1). The detection capability of chlortetracycline was equal to its maximum residue limit (100 μg kg(-1)) and the method did not detect gentamicin (1000 μg kg(-1)), for which no MRL is established in poultry muscle. Specificity evaluated in relation to different analytes and matrices did not detect any interferences in the tests results; whilst the robustness showed that the pH neutralisation point of the extract affects the analytical results and the kits' performance. Only the screening of tetracyclines requires the analysis of extracts without pH neutralisation. The results of the validation process showed that this method is acceptable for screening β-lactam, sulfonamide and macrolide antimicrobial groups in the National Residues and Contaminants Control Programme (PNCRC), and that for this it is fit for purpose.     

石斑鱼中阿维菌素类药物多残留测定及食用安全风险评估

建立高效液相色谱-串联质谱同时定量检测石斑鱼血浆、肌肉组织、肝脏组织中阿维菌素、伊维菌素、甲氨基阿维菌素苯甲酸盐方法。样品经乙腈提取,碱性氧化铝固相萃取柱和LC-C18固相萃取柱串联净化,Thermo Hypersil Gold C18色谱柱分离,10 mmol/L乙酸铵-0.1%甲酸溶液和乙腈梯度洗脱,电喷雾正离子模式下以多反应监测方式检测,基质匹配法外标定量。分别以环境水体中阿维菌素上下限质量浓度(4、8 ng/mL)、伊维菌素上下限质量浓度(6、12 ng/mL)作为受试质量浓度开展生物富集、消除实验,并对石斑鱼的食用安全进行了风险评估。结果表明,阿维菌素和伊维菌素在2.5~200 ng/mL范围内,甲氨基阿维菌素苯甲酸盐在0.25~20 ng/mL范围内,线性回归系数均大于0.99。方法检出限分别为2.5、2.5、0.25 ng/mL(血浆),1、1、0.1μg/kg(肌肉组织),2.5、2.5、0.25μg/kg(肝脏组织),方法定量限分别为5、5、0.5 ng/mL(血浆),2、2、0.2μg/kg(肌肉组织),5、5、0.5μg/kg(肝脏组织)。3个添加量的平均回收率为74.6%~93.6%,日内相对标准偏差为2.3%~10.9%,日间相对标准偏差为9.2%~12.6%。阿维菌素、伊维菌素均属于非生物累积性物质,在石斑鱼体内代谢规律相同,均按一级动力学过程从体内消除。本研究条件下,环境水体中药物质量浓度是石斑鱼肌肉组织中药物残留质量浓度及消除时间的重要因素。为保证食用安全,环境水体中阿维菌素质量浓度达到4~8 ng/mL时,石斑鱼浸浴72 h后安全食用时间为22 d;环境水体中伊维菌素质量浓度达到6~12 ng/mL时,石斑鱼浸浴72 h后安全食用时间为39 d。     

Determination of Sulfonamide Residues in Chicken Liver Using High-Performance Liquid Chromatography

A novel high-performance liquid chromatography method was adopted for determination of sulfacetamide, sulfathiazole, sulfamethazine, and sulfamethoxypyridazine residues in chicken liver based on the coupling of dispersive solid-phase extraction (DSPE) and dispersive liquid-liquid microextraction (DLLME). In the DSPE-DLLME method, chicken livers were firstly extracted with 50 mM phosphoric acid:MeCN (30:70), then cleaned up with a solid adsorbent. One milliliter of the resulting liquid supernatant extract and 650 μL of 1,2-dichloroethane were then added to a centrifuge tube containing 10 mL water. The analytes were extracted under optimized pretreatment conditions. Signal linearity over the concentration range of 0.006–4.00 mg/kg was observed (regression coefficients, 0.9988–0.9992), and limits of detection were in the range of 0.0004–0.0084 mg/kg. The present method was applied to the determination of the sulfonamides in chicken liver samples. The spiked recovery values ranged from 70.2 to 91.4 % and the relative standard deviations were less than 5.35 % (n?=?6).     

磺胺甲噁唑和磺胺嘧啶在青石斑鱼组织中的分布及代谢规律

通过一次性投喂各含有200 mg/kg磺胺甲噁唑（sulfamethoxazole,SMZ）和磺胺嘧啶（sulfadiazine,SDZ）的饲料,研究两种药物在青石斑鱼中各组织分布与消除规律。采用超高效液相色谱-串联质谱检测青石斑鱼各组织中SMZ和SDZ的含量,并用内标法定量。结果表明,SMZ在青石斑鱼各组织中的最大含量：肝脏、背肌、血浆、肾脏和鳃依次为827.97μg/kg、776.70μg/kg、610.29μg/L、432.14μg/kg和345.18μg/kg。SDZ在青石斑鱼各组织中的最大含量：肝脏、背肌、鳃、血浆和肾脏依次为895.30μg/kg、660.55μg/kg、431.88μg/kg、419.56μg/L和310.67μg/kg。SMZ在青石斑鱼各组织中的半衰期：肾脏、鳃、背肌、血浆和肝脏半衰期依次为26.65、21.00、20.38、18.73h和16.90h。SDZ在青石斑鱼各组织中的半衰期：肾脏、血浆、鳃、背肌和肝脏依次为31.50、27.72、24.75、21.66h和18.24h。SMZ和SDZ在青石斑鱼肝脏中半衰期最短,代谢速度最快;在肾脏中半衰期最长,代谢速度最慢。在水温（25±2）℃条件下,SMZ和SDZ各200mg/kg的剂量同时单次投喂青石斑鱼,建议休药期不低于3d。SMZ和SDZ代谢规律研究为磺胺类药物在水产品中的合理使用提供了参考。