Validation of HPLC method of analysis of tetracycline residues in eggs and broiler meat and its application to a feeding trial

HPLC with ion-pairing chromatography and diodearray detection at 355nm was used to determine tetracycline antibiotics in eggs and broiler meat. The analytical methods were optimized and validated. The mean recovery values for oxytetracycline for eggs and for tetracycline for breast meat were 76% . The withinday precision ranged from 8.0 to 11.8% for oxytetracycline in eggs and from 6.1 to 15.5% for tetracycline in breast meat. The between-day precision was 4.8% and 5.0% respectively for oxytetracycline in eggs and tetracycline in breast meat. The limit of detection and the limit of quantitation for oxytetracycline in eggs were 2.2 and 13.0ng/g respectively. These limits for tetracycline in breast meat were 10.5 and 20.9ng/g respectively. Residue values of tetracycline antibiotics in eggs and broiler meat were determined after oral administration of medicated feed. Medicated feed with 840mg/kg oxytetracycline was provided to laying hens for seven successive days. Two days after the administration was stopped, the mean oxytetracycline residue value in the eggs was already lower than the Maximum Residue Limit (MRL)-level and reached 118ng/g. Broilers were supplied with medicated feed containing 480mg/kg tetracycline for seven successive days. Four days after the administration was stopped, the mean tetracycline residue value in breast meat decreased below the MRL and was 86ng/g.     

Nicarbazin contamination in feeds as a cause of residues in eggs

A survey was carried out to investigate the prevalence of nicarbazin residues in eggs in Northern Ireland. Nicarbazin, in the form of 4,4-dinitrocarbanilide (DNC), was detected in 39 of the 190 eggs analysed. An experiment was designed to establish the relationship between nicarbazin-contaminated feed and nicarbazin residues in eggs. The concentrations of both the DNC and 4,6-dimethyl-2-hydroxypyrimidine (DHP) components of the drug in eggs were proportional to feed levels. The maximum feed nicarbazin concentration of 12.1mg/kg (8.6 mg/kg DNC and 3.5mg/kg DHP) gave rise to mean maximum whole egg concentrations of 631 mu g/kg DNC and 51.8 mu g/kg DHP. After withdrawal of the experimental diet, DNC was undetectable in eggs after 12 days and DHP after 3 days. Feed contaminated with nicarbazin at concentrations greater than about 2mg/kg gave rise to egg DNC residues at concentrations greater than the Differential Action L imit (DAL) set by the UK (100 mu g/kg). DNC was contained almost entirely in the yolk of the egg, whereas DHP was distributed between albumen and yolk in a ratio of approximately 3:1.     

Evaluation of the residues of furazolidone and its metabolite, 3-amino-2-oxazolidinone (AOZ), in eggs

The use of furazolidone in food-producing animals is banned within the EU. Detection of the protein-bound side-chain metabolite, 3-amino-2-oxazolidinone (AOZ), in animal tissues is the most effective method of enforcing the ban. The study was undertaken to find out if the same applies to eggs. The concentrations of furazolidone and AOZ in eggs reached a plateau of ~360-380 μgkg -1 by the fourth day of treating birds with 400mgkg -1 furazolidone. After a 4-day withdrawal from treatment, intact furazolidone could not be detected. AOZ residues could still be detected up to 21 days following withdrawal from treatment. During treatment, most intact furazolidone residues occur in the albumen. For AOZ, there is a more even distribution of residues between albumen and the yolk. The concentration of furazolidone in egg homogenates stored at -20°C decreases by 44% after 55 days. AOZ residues are stable during this period. From these results, it is clear that AOZ is a more suitable marker residue than the parent compound for monitoring concentrations of the drug in eggs.     

Incidence of residues of nine anticoccidials in eggs

A survey of the presence of residues of anticoccidials was performed. Three hundred and twenty egg samples, purchased in eight different European countries, were analysed for the presence of nine different compounds: dimetridazole, diclazuril, halofuginone, robenidine, nicarbazin, narasin, salinomycin, lasalocid and monensin. Analyses were performed by LC-MS/MS. Of the samples analysed, 114 (35.6%) contained one or more of the nine anticoccidials in concentrations ranging from 0.1 to 63 µg kg^-1. Salinomycin and lasalocid account for more than 60% of all positive samples. Almost 90% of all positive samples contained less than 2 µg kg^-1. Results were put into perspective of the farming method and country of origin.     

Determination of ivermectin and moxidecin residues in bovine milk and examination of the effects of these residues on acid fermentation of milk

Ivermectin (IVM) and moxidectin (MXD) are broad-spectrum antiparasitic drugs not approved for use in dairy animals, although their use in dairy sheep, goats and cattle nevertheless occurs in many parts of the world. The work reported here describes (1) the application of an HPLC method (including milk samples clean-up and chemical extraction) to quantify IVM and MXD residues in bovine milk, and (2) an assessment of the effect of different IVM and MXD concentrations on bovine milk acid fermentation. The latter was carried out using the 'yoghurt test' to determine the minimum IVM and MXD concentrations affecting milk acid fermentation. The sample clean-up, chemical extraction and the validated HPLC method allowed the quantification of IVM and MXD up to 0.1 ng ml^-1 in milk with acceptable validation coefficients. Drug recoveries from fortified milk samples ranged between 72% (CV = 9.1%) and 75% (CV = 13.3%) for MXD and IVM, respectively. Neither IVM nor MXD affected the acid fermentation of bovine milk. In fact, there was no drug-induced changes on milk acidity even at IVM and MXD concentrations as high as 1000 ng ml^-1. These results indicate that the yoghurt biological test is not suitable to evaluate the presence of milk residues for these antiparasitic compounds. Thus, a highly sensitive HPLC technique is the only reliable method for determining the presence of residual concentrations of IVM and MXD in milk and dairy products to assure consumer safety.     

Sulphonamide residues in milk of dairy cows following intravenous injection

An investigation was conducted into the elimination into milk of four sulphonamides (sulphadimidine, sulphamethoxypyridazine, sulphadimethoxine and sulphadoxine) after intravenous injections of drugs available in France. Sulphonamide determination in milk was performed using a high-performance liquid chromatography (HPLC) method with detection limit of 0.01 micrograms/ml. These results were used to determine the withdrawal times (two to six milkings) required for these drugs with a tolerance of 0.1 micrograms/ml as proposed by the European countries.     

Determination of (fluoro)quinolones in eggs by liquid chromatography with fluorescence detection and confirmation by liquid chromatography-tandem mass spectrometry

A multiresidue analytical procedure for determination of seven fluoroquinolones (marbofloxacin, norfloxacin as internal standard, ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin), and three quinolones (oxolinic acid, nalidixic acid and flumequine) in eggs is presented. The procedure is based on dispersive solid-phase extraction technique with acetonitrile as extractant. Norfloxacin and ciprofloxacin - d8 were used as internal standards to quantify the (fluoro)quinolones. Analyses were realised by LC-FLD for screening and LC-MS/MS for confirmatory purposes. The whole procedure was evaluated according to the Commission Decision 2002/657/EC. Specificity, decision limit (CCα), detection capacity (CCβ), recovery (absolute and relative), precision (repeatability and reproducibility) were determined during validation process. Recoveries (relative) for the LC-FLD screening determination ranged from 85% to 93%, repeatability and reproducibility were in the range of 5-9% to 9-16%, respectively. CCα and CCβ were 13-37 and 17-43 μg/kg pending on analite. For the LC-MS/MS confirmatory method, the relative recoveries were satisfactory (92-99%) with repeatability and reproducibility in the range of 4-7% to 6-12%, respectively. CCα and CCβ were 3-7 and 7-11 μg/kg depending on the analite. The results of both prepared methods showed these analytical procedures simple, rapid, sensitive and suitable for routine control of eggs.     

Comparison of extraction techniques for the recovery of veterinary drug residues from animal tissues

Four different techniques for the extraction of veterinary drugs were compared. The use of a high speed mixer/emulsifier, an ultrasonic bath, a Stomacher and an end-over-end mixer were used to extract both incurred and fortified residues of chlortetracycline, sulphadiazine and flumequine from chicken muscle. For each drug, similar analytical recoveries from fortified muscle samples were obtained using each of the extraction techniques. However, for each analyte the highest drug concentration detected in incurred samples was obtained following preparation using a mixer/ emulsifier extraction. Residue concentrations obtained using sonication, Stomacher or end-over-end mixer procedures were as low as 20% of those obtained using the mixer/emulsifier. This highlights the need to measure both incurred and spiked samples during method validation. A survey of published methods indicated that 75% of laboratories use some form of high-speed homogenization for the extraction of drug residues from tissue. However only 52% reported detection of incurred residues. More than two-thirds of methods that used other forms of extraction did not measure incurred residues. The use of such methods has implications for the statutory detection of veterinary drug residues.     

鸡蛋烘烤工艺技术条件研究

对鸡蛋烘烤工艺条件进行了一系列研究。确定采用配方Ⅳ浸泡、煮制时间7min、烘烤温度80℃、烘烤时间22h，成品呈琥珀色、光亮，有浓郁的烤、卤蛋混合香味。开发出了一种新型的鸡蛋制品。     

Residues of doxycycline and oxytetracycline in eggs after medication via drinking water to laying hens

Doxycycline (DOTC) and oxytetracycline (OTC) were dissolved in drinking water (0.5 g/l) and supplied to laying hens for 7 consecutive days. Eggs laid were collected daily during and after medication, and the antibiotic concentrations in the yolk and albumin were determined by the cup-plate method with Bacillus cereus var. mycoides ATCC 11778. The concentrations of both antibiotics were increased in yolk day by day with the advance in medication, reached peaks 2 days after withdrawal and then declined gradually. Mean peak concentrations in the yolk were 6.70 micrograms/g for DOTC and 1.42 micrograms/g for OTC. Peak concentrations in the albumin occurred in the middle stage of medication, where the mean values were 12.24 micrograms/g for DOTC and 1.03 micrograms/g for OTC. DOTC was detected in albumin until 24 days after withdrawal and for 2 days more in yolk than in albumin. OTC was detected in yolk until 9 days after withdrawal. The depletion period of OTC was shorter for the albumin, where the residue disappeared in all eggs 6 days after withdrawal. In spite of similarities between DOTC and OTC in structure, DOTC was deposited in higher concentrations and lasted for a longer period in eggs. This characteristic was considered due to its greater lipophilicity, closely correlated with oral absorption and tissue penetration.     

鸡蛋中药物残留的危害与现状

鸡蛋是居民餐桌上重要的食材,鸡蛋的质量和安全是人们关注的焦点。长期食用药物残留超标的鸡蛋,当体内药物蓄积到一定浓度会对人体产生多种急、慢性中毒,严重时会致癌、致畸、致突变,甚至死亡。因此需要健康养殖、合理规范用药,监控和减少药物残留的发生,提高食品安全生物水平工作十分迫切。本文介绍了鸡蛋中药物残留的危害和现状,以期为防范药物滥用或误用造成安全隐患和引导养殖户规范用药提供一定的参考作用。     

Clenbuterol residues in pig muscle after repeat administration in a growth-promoting dose

The aim of this study was to determine the level of clenbuterol residues in muscle tissue of pigs after repeat administration in a growth-promoting dose. An anabolic dose of clenbuterol (20 μg/kg body mass per day) was administered orally to experimental group (n = 12) for 28 days, whereas control animals (n = 3) were left untreated. Clenbuterol treated pigs were randomly sacrificed (n = 3) on days 0, 3, 7 and 14 of treatment discontinuation and clenbuterol residues determined in muscle tissue. Determination of residual clenbuterol was by enzyme-linked immunosorbent assay (ELISA) as a screening method and liquid chromatography tandem mass spectrometry (LC-MS/MS) as a confirmation method. The highest clenbuterol content in the muscle of treated animals was recorded on day 0 of treatment cessation (4.40 ± 0.37 ng/g) and significantly (p < 0.05) exceeded the maximum residue limit (MRL) of 0.1 ng/g. On day 3 of withdrawal, it was 0.49 ± 0.22 ng/g and on day 7 0.10 ± 0.02 ng/g (at MRL); on day 14 of treatment discontinuation, clenbuterol content was below the limit of detection (< 0.1 ng/g) in all samples. Administration of clenbuterol as a growth promoter in pig production could lead to residues in meat for human consumption up to 7 days after treatment discontinuation.     

Effect of cooking on the stability of veterinary drug residues in chicken eggs

ABSTRACT

The available information on drug residue stability in chicken egg is scarce. The objective of this study was to evaluate the stability of drug residues in egg under different traditional cooking procedures. Fresh eggs were spiked with different drug concentrations of albendazole (ABZ) and its albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO₂) metabolites; flubendazole (FLBZ) and its reduced flubendazole (R-FLBZ) and hydrolyzed flubendazole (H-FLBZ) metabolites; amoxicillin (AMX); and enrofloxacin (EFX) and its ciprofloxacin (CFX) metabolite. The egg samples were cooked in different ways, namely, boiling, microwaving, and omelette making. Drug residue concentrations in egg were quantified by HPLC with UV or fluorescence detectors. ABZ and ABZSO concentrations in egg were not affected by boiling and microwaving, while the omelette processing significantly reduced these molecules. Residues of ABZSO₂ in egg were stable or increased after all cooking procedures. In contrast, FLBZ and its metabolites FLBZ-H and FLBZ-R residues in egg decreased after all treatments. The residue concentration quantified for EFX and CFX did not show significant changes after any cooking method. AMX residues were unstable, with extremely significant drug reduction after all cooking processes. Conventional methods of egg cooking cannot be considered a tool to eliminate all veterinary drug residues.     

Doxycycline depletion and residues in eggs after oral administration to laying hens

The depletion of doxycycline (DC) residues in eggs was determined after oral drug administration by drinking water to laying hens. The antibiotic was supplied to birds for 5 consecutive days and the eggs were collected during medication and 18 days after withdrawal. DC residues were determined by LC-MS/MS. DC was isolated from eggs with a solution of 0.02 M of oxalic acid (pH 4), 0.1 M Na₂EDTA and acetonitrile. The limit of detection (LOD) and limit of quantification (LOQ) of the method were 2 and 5 µg kg^–1, respectively. Analyses were performed on whole egg, egg white and yolk separately. DC was detectable 24 h after the beginning of administration. The concentration of antibiotic increased daily, resulting in the highest DC concentration in whole eggs at the first day of the withdrawal period. Thirteen days after withdrawal, the content of DC in whole eggs was below the LOQ of the method. However, some differences were found in the depletion curve of DC between egg white and yolk. Residues of DC in egg white were much higher during treatment and 1 day after withdrawal, but later the concentration in egg white decreased fairly rapidly and a higher DC content in egg yolk was observed. The depletion period was shorter for egg white than for yolk, and DC was detected in the egg white until 12 days after withdrawal and 2 days more in egg yolk than in white. DC reached a peak faster in egg white, but the residues were detectable for longer period in the yolk.     

大米中敌稗残留量测定方法的研究

为贯彻实施敌稗农药最大残留限量国家标准 ,建立大米中敌稗残留量测定方法 ,试样用丙酮提取 ,石油醚萃取和弗罗里硅土净化 ,乙醚 -正己烷 (1∶1)淋洗。净化液用配有电子捕获检测器的气相色谱仪测定 ,外标法定量。方法最低检测浓度 4× 10 -4mg kg ,敌稗浓度在 0 2 0～ 2 0 0mg kg范围内 ,平均回收率为 89 7%～ 90 7% ,相对标准偏差为 4 4 6 %～ 5 2 1% ,符合农药残留分析的要求。     

高效液相色谱法测定鸡蛋中角黄素的不确定度评定

依据JJF1135-2005化学分析测量不确定度评定和 JJF1059.1-2012测量不确定度评定与表示,分析了高效液相色谱(HPLC)法测量鸡蛋中黄角素的整个测量过程不确定度来源;并对各个因素引入的不确定度进行计算和合成;最后给出扩展不确定度。当鸡蛋中黄角素测定结果为0.39 mg/kg 时,扩展不确定度为0.017 mg/kg,k=2。评定结果表明, 实验过程中的不确定度主要来源于标准曲线配制、标准曲线拟和样品测量。     

固相萃取-高效液相色谱-串联质谱法同时测定鸡蛋中42种农药残留

目的 建立固相萃取-高效液相色谱-串联质谱法同时测定鸡蛋中42种农药残留的检测方法。方法 样品经加水分散后,用乙腈提取,PRiME HLB固相萃取柱进行净化处理,采用Shim-pack XR-ODSⅡ色谱柱(75 mm×2.0 mm, 2.2 μm)分离,5 mmol乙酸铵水溶液(A)-甲醇溶液(B)为流动相,进行梯度洗脱;质谱法选择电喷雾离子源ESI,正负离子分段扫描,多反应监测(MRM)模式检测,基质匹配标准溶液外标法定量。结果 42种农药的峰面积与质量浓度在0.1~2 ng/mL范围内呈现良好的线性关系,相关系数r均大于0.994;方法检出限(LOD, S/N=3)为0.04~0.3 μg/kg,定量限(LOQ, S/N=10)为0.1~1.0 μg/kg。在1.0、2.0、5.0、10 μg/kg四个添加水平下,42种农药平均加标回收率为61.0%~118.5%,相对标准偏差为0.8%~9.9%。结论 该方法具有操作简单、分析时间短、试剂用量少、灵敏度高且重复性好等特点,适用于鸡蛋中农药残留的高通量快速检测分析。     

Possible causes of nicarbazin residues in chicken tissues

Two experiments were carried out to investigate possible causes of nicarbazin residues in broiler chicken tissues. The first experiment was designed to establish whether feeding nicarbazin as stipulated in the product license can result in 4,4'-dinitrocarbanilide (DNC) tissue residues exceeding the JECFA MRL (200 micrograms/kg). It was shown that the MRL was exceeded in the livers of broilers housed on deep litter, but not in those of broilers housed on wire flooring. Muscle DNC concentrations were well below the MRL. The higher residual tissue concentrations in birds housed on deep litter were attributed to faecal recycling. The second experiment was to establish the relationship between nicarbazin-contaminated withdrawal ration up to the point of slaughter and DNC residues in the tissues of broilers that had not been previously exposed to nicarbazin. Tissue DNC concentrations were found to be proportional to feed concentrations. The housing method caused no significant difference in tissue residues. Meal containing nicarbazin at a concentration of 2.4 mg/kg or greater caused liver DNC residues above the JECFA MRL. Violative residues may, therefore, occur in chickens not exposed to nicarbazin during rearing, but fed withdrawal ration contaminated at 2.4 mg/kg or greater, or in chickens housed on deep litter and fed nicarbazin-medicated meal according to the product license even when the withdrawal ration is nicarbazin-free.     

The residue levels of narasin in eggs of laying hens fed with unmedicated and medicated feed

Laying hens were fed contaminated feed containing narasin 2.5 mg/kg for 21 days followed by a 7 day withdrawal period, hens in the control group were fed unmedicated feed. Eggs were collected during trial days 0, 3, 7, 14, 21 and after the withdrawal period of 7 days. The concentration of narasin in yolks and egg whites was analyzed by a liquid chromatography-mass spectrometry method. Narasin was found to accumulate in yolks, where the narasin concentration increased during the treatment. The concentration of narasin varied from 5.9 to 13.8 microg/kg (mean 10.6 microg/kg) in yolks after 21 day feeding periods. The concentrations of narasin ranged from < 0.9 to 1.4 microg/kg after the withdrawal period. Narasin residues were not found in egg whites of the laying hens fed contaminated feed nor in either yolks or egg whites of the laying hens fed unmedicated feed. The effect of cooking was also tested on the amount of narasin residues in eggs. Cooking for 10 min did not significantly influence the narasin residues in eggs. Traces of lasalocid were also found in the yolks. The traces of lasalocid are attributable to an accidental contamination of the feed during its manufacture.     

Carbendazim and metalaxyl residues in post-harvest treated apples

Bramley apples were treated with Ridomil mbc 60 WP, containing carbendazim and metalaxyl, at half and full rate application at 10 and 20°C, and with diphenylamine to control disease during storage. Carbendazim and metalaxyl residues were determined by HPLC and GC/MS, respectively, in apple core, flesh and peel at the initial time, 3 and 6 months after storage under controlled atmosphere conditions of 4.5°C, 5% CO₂ and 1% O₂. The concentration of carbendazim residues in apple flesh was ≤ 0.43 mg kg^-1 in all treatments; the mean concentrations in apple core and peel were 2.2 ±1.1 and 5.2 ±2.2 mg kg^-1, respectively. The MRL for carbendazim in apple was 2.0 mg kg^-1. The concentration of metalaxyl residues in apple flesh was ≤ 0.22 mg kg^-1; the mean concentrations in apple core and peel were 0.41 ±0.18 and 0.79 ±0.94 mg kg^-1, respectively. The MRL for metalaxyl in apple was 1.0 mg kg^-1. The temperature of the fungicide solution had little effect on the carbendazim residues but did have some effect on metalaxyl residues. Carbendazim residue content per apple was decreased when diphenylamine was included. A change in the ratio of carbendazim to metalaxyl was noted in the flesh, core and peel of the stored apple. The ratio of carbendazim to metalaxyl was 5:1 in the fungicide mix as applied. The ratio remained fairly constant in the core during storage. However, the ratio changed significantly in the peel and, to a lesser extent, and in a different direction, in the flesh. In the peel, the ratio was around 15:1 at 3 months of storage and 12:1 after 6 months of storage, whereas for flesh the ratios were 2:1 and 4:1, respectively.