Liquid chromatographic monitoring of the depletion of carbadox and its metabolite desoxycarbadox in swine tissues

A liquid chromatographic method was used to monitor a depletion study of carbadox (and its most important metabolite, desoxycarbadox) in young pigs fed carbadox-treated rations for 1 week. Carbadox was found in blood (20 ppb), blood serum (26 ppb), and muscle tissue 24 h after withdrawal from treated ration; residues were reduced to a trace (less than 2 ppb) in 48 h, and eliminated by 72 h. Desoxycarbadox, although not detected in blood, was found in muscle (17 ppb) 24 h after withdrawal; it was reduced to 9 ppb at 48 h and to a trace by 72 h. Although no carbadox was detected in liver 24 h after withdrawal, appreciable desoxycarbadox (125 ppb) was found in liver 24 h after withdrawal; it was reduced to 17 ppb at 48 h and to a trace by 72 h. Whereas only a trace of carbadox was found in kidney 24 h after withdrawal, 186 ppb desoxycarbadox was found in kidney at 24 h, 34 ppb at 48 h, and a trace at 72 h. No metabolite of carbadox other than desoxycarbadox was found in extracts of swine tissues during this medicated feed trial, and no metabolite was found in blood extracts by using the established methodology. The effect of tissue storage (aging) at -20 degrees C on levels of the drug and its metabolite was a modest alteration of residue levels. The inadvertent use of feed adulterated with furazolidone and initially medicated with chlortetracycline, sulfamethazine, and penicillin G, did not affect the uptake of carbadox in this depletion study or interfere with the analytical methodology.     

Liquid chromatographic determination of carbadox, desoxycarbadox, and nitrofurazones in pork tissues

A liquid chromatographic (LC) method has been developed for the determination of carbadox, desoxycarbadox, and nitrofurazones in the 10-40 ppb range in pork muscle, liver, and kidney tissues. Tissues were homogenized in absolute ethanol, and the homogenates were treated with metaphosphoric acid and reduced in volume by rotovaporization. Hexane was added to the concentrates, which were then centrifuged to remove fat. After addition of KH2PO4 to the aqueous phase and extraction with ethyl acetate, the extracts were passed through alumina columns before analysis by reverse phase LC. Overall average recoveries (10-40 ppb range) for carbadox and desoxycarbadox from spiked tissues were 53% +/- 13.6 and 61% +/- 7.2, respectively; overall average recoveries for nitrofurazone and furazolidone were 43% +/- 7.3 and 77% +/- 10.9, respectively. Before these optimum determinations, degradation by even minimal incandescent light was found to reduce recovery especially of desoxycarbadox. The results of this photochemical degradation are reported and briefly discussed.     

Liquid chromatographic determination of carbadox in complete feeds, premixes, and concentrates: interlaboratory study

A liquid chromatographic (LC) method previously published for the determination of carbadox in finished feeds and premixes was slightly modified and tested in an interlaboratory study. The feed samples are extracted with methanol-acetonitrile (50 + 50) after wetting with water. The extracts are purified over a short alumina column. An aliquot of the eluate is analyzed with reverse phase liquid chromatography with ultraviolet detection. Before the actual interlaboratory study, a prestudy with 2 familiarization feed samples was performed. For the interlaboratory study, 2 series of meal and pelleted samples were prepared with carbadox from different suppliers. Eight collaborating laboratories received 6 feed samples previously milled and ground and 4 pelleted samples which had to be ground by the collaborator's in-house method. Collaborators also received 3 carbadox concentrates (about 10% w/w) and 4 premix samples derived from the concentrates (about 1% w/w). Coefficients of variation under reproducibility conditions were 8.3% for meal samples and 4.9% for pellets. A minor but significant effect was noted for the influence of pelleting temperature on the carbadox content. A minor and insignificant effect was observed for the influence of the milling and grinding procedure on the carbadox content. Alumina cleanup of 1% premixes was not essential, although the resulting chromatograms were cleaner. A slight difference in reproducibility was observed with concentrates (10%) when 0.2 or 0.5 g sample size was used, although the average carbadox concentration found was the same. For premixes and concentrates, coefficients of variation under reproducibility conditions were low, ranging from 2.9 to 7.5%.     

Modified method for carbadox in feeds: collaborative study

The official first action method for carbadox in swine feed, 42.C01-42.C04, was modified in 2 respects. First, the samples were leached overnight at room temperature instead of boiled for 1 hr. This change avoided problems with overheating and excessive evaporation. Second, the dilution scheme for samples spiked with carbadox standard solution was changed to give absorbance values that were within the optimum working range of all types of spectrophotometers. The modified procedure was collaboratively studied by 21 laboratories. The repeatability standard deviation (sigma0) and reproducibility standard deviation (sigmax) were sigma0 = 0.00029% and sigmax = 0.00056% (8.9% of grand mean) for feeds containing 0.00617% carbadox; and sigma = 0.0012% and sigmax = 0.0019% (9.3% of grand mean) for feeds containing 0.0198% carbadox. The between-laboratory variance ratio was significant for feeds containing 0.0198% carbadox. The mean per cent of intent values for feeds containing 0.00617% carbadox and 0.0198% carbadox were 102% and 104%, respectively. In general, the statistical results were comparable to those previously obtained for the official first action method. Consequently, the modified procedure is not recommended as a replacement for the official first action method.     

Detection of low-level carbadox residues in animal feeds by high pressure liquid chromatography

The high pressure liquid chromatographic (HPLC) method is capable of detecting from 1 to 0.024 ppm methyl 3-(2-quinoxalinyl-methylene)carbazate-N1,N4-dioxide (carbadox). Carbadox is extracted from the feed with 2% NH4OH in acetone, passed through a liquid-liquid partition, subjected to HPLC, and detected by using a 365 nm detector. No feed materials or other active drug ingredients produced false positive results.     

Liquid chromatographic determination of carbadox residues in animal feed

A liquid chromatographic (LC) method for determining residues of carbadox in the 0.01-10 ppm range in swine feed is described. Carbadox is extracted from ground feed with 25% acidified methanol-CHCl3, removed from emulsion-forming coextractables via an alumina column, separated from highly colored pigments by acid-base liquid-liquid partitioning, and finally isolated from interferences on a second alumina column. Isocratic reverse phase LC at 305 nm is used for quantitation. The average overall recovery at the 0.1, 0.5, and 1.0 ppm spike levels was 83.0% with a standard deviation of 2.04% and a coefficient of variation of 2.46%.     

Liquid chromatographic determination of strychnine in stomach contents

A chloroform extract of stomach contents at basic pH is concentrated and then extracted with 0.1 M phosphoric acid. The acid extract is chromatographed on a 10 cm reverse phase column, using 0.005 M phosphate buffer (pH 3.0)-acetonitrile-tetrahydrofuran (750 + 135 + 115) containing 0.01 M octanesulfonic acid at a flow rate of 1.0 mL/min for elution. Strychnine eluted in 7.3 min. Recoveries from spiked stomach contents averaged 92%. The method can be used without modification for other alkaloids.     

Determination of polybrominated biphenyl residues in dry animal feeds.

A new procedure is described for the determination of polybrominated biphenyls (PBBs) in dry animal feeds and developmental results are discussed. Finely ground feed is packed into a chromatographic column containing Celite and then eluted with methylene chloride. The concentrated extract is cleaned up by elution with petroleum ether through Florisil before gas-liquid chromatographic quantitation. Chromatograms thus obtained were essentially free of the interfering peaks encountered when using AOAC methods for pesticide residues in dry products. Results of feed analyses by the proposed procedure averaged 30% higher than those obtained by AOAC procedures. Recoveries of PBBs from samples fortified at levels of 0.04 to 0.4 ppm ranged from 90 to 104%, with an average of 98%.     

Microbiological plate and turbidimetric assays of chlortetracycline in feeds: collaborative study.

The manual and automated turbidimetric assays and a modified official plate assay for chlortetracycline (CTC-HCl) in feed were collaboratively studied. Three feed samples (swine feed, 100 g CTC-HCl/ton; premix I, 20 g each of CTC-HCl and sulfamethazine/lb, and 10 g penicillin/lb; and premix II, 50 g CTC-HCl/lb) were analyzed at 2 dilutions. Twelve laboratories conducted the plate assay; 8 laboratories the manual turbidimetric method; and 7 laboratories, the Autoturb analysis. Within a method, there was no significant difference between dilutions. Between methods, there was a significant difference between the manual turbidimetric plate assays only for swine feed. However, the same sample dilutions or the average values of the 2 dilutions for both methods showed no statistical difference. Among the collaborators, the slope of CTC-HCl standard curve varied between about 2.0 and 3.0 for the plate method. The turbidimetric assay has been adopted as official first action for feeds containing larger than or equal to 20 g CTC-HCl/lb.     

Genotoxicity in German Surface Waters - Results of a Collaborative Study

As surface waters are widely used for the preparation of drinking water, appropriate test systems are required for the monitoring of possible genotoxic contaminations. In the course of a BMBF-funded collaborative project several methods (Ames test, umu test, alkaline elution, DNA unwinding assay, Comet assay and unscheduled DNA-synthesis test) have been examined for their ability to measure genotoxicity particularly in natural surface waters. The project was subdivided into two parts: In the first part appropriate test versions were developed (sensitized Ames test, luminometric umu test, alkaline elution using clams, Comet assay with fish cells or aquatic plants), adapted to the test subject and validated regarding their sensitivity towards standard genotoxins. All test results were statistically evaluated. In the course of the second part both natural and concentrated samples of the rivers Rhine, Elbe, Mulde, Wupper and one drinking water resource (Wahnbachbarrage) were tested. In parallel all samples were chemically analyzed. Among the unconcentrated samples several statistically positive test results were obtained both for the rivers Elbe and Rhine especially with the Ames test and the Comet assay. Only one river (Wupper) showed significant genotoxicity in the umu test. In this case chemical analysis revealed concentrations of about 41 and 47 µg/l of fluoroquinolonic acid, a bacterial gyrase inhibitor which may be responsible for this effect. No genotoxicity could be found in the drinking water resource even after concentration. The water extracts clearly showed different background genotoxicity in the umu test corresponding to increased pollution along the Rhine (Karlsruhe < K ln < D sseldorf). The eucaryotic in vitro tests revealed comparable results with nonconcentrated water samples. As a conclusion of the study we propose a graduated testing battery consisting of a bacterial (umu or Ames test) and an eucaryotic test like the Comet assay or the alkaline elution assay followed by an additional eucaryotic test (UDS test or micronucleus test) in a decisive function. There is a need for further evaluation by effect-orientated chemical analysis which should be done in the case of positive genotoxicity results in at least two tests or persistent positive results in only one.     

浙江省商品有机肥中重金属含量变化趋势及风险管控对策

  【目的】  有机肥是农业生产的重要肥源,了解有机肥质量安全现状,可为有机肥合理施用和管控提供参考。  【方法】  于2017年采集浙江省96家有机肥生产企业共99个商品有机肥样品,研究分析其Hg、As、Cd、Pb、Cr、Cu、Zn和Ni 8种重金属含量、分布特征、污染成因及施用风险。并与2013年文献进行比较,分析重金属含量的变化。  【结果】  1) 商品有机肥中Hg、As、Cd、Pb、Cr、Cu、Zn、Ni的平均含量分别为0.17、5.5、0.63、17.3、33.2、213.8、660.5、17.7 mg/kg,变异系数为54.9%～151.3%,含量差异较大。相比2013年,Cd、Pb、Cr、Hg和As含量在2017年均有所降低,降幅为23.0%～72.7%,其中Hg和As含量大体呈逐年下降趋势,2017年较2009年平均含量分别下降74.2%和51.8%,Cr和Pb平均含量较2010年分别下降74.9%和54.4%,Cd平均含量在近4年降幅明显,2017年较2013年下降72.7%。但商品有机肥中As仍有8.1%超标,Cu、Zn含量偏高,Ni含量有增加趋势。Cu、Zn、Ni是浙江省商品有机肥主要风险因子。2) 主成分和相关性分析表明,Cu-Zn、Cr-Ni-Hg、Cd-Pb重金属污染来源具有同源性。Cu、Zn、As以饲料添加来源为主,Cr、Hg、Ni主要来源于饲料原料本身,Cd、Pb则以饲料原料本身、生产粘结剂或生产环境来源为主。3) 商品有机肥成分配方复杂,猪粪原料占比较大,其次为鸡粪和牛粪。根据NY 525—2012有机肥重金属限量标准,以猪粪为主要原料的有机肥As超标率9.6%,以鸡粪、羊粪为主要原料的有机肥未出现重金属超标,根据德国腐熟堆肥重金属限量标准,以猪粪和鸡粪为主要原料的有机肥Cu、Zn超标样品较多,Ni只在以猪粪和鸡粪为主要原料的有机肥中出现超标。4) 长期施用含重金属商品有机肥,存在土壤重金属积累风险。按有机肥年施肥量30 t/hm2推算,Cu、Zn和Cd风险相对较大,安全使用年限分别为14、18、34 年。  【结论】  浙江省商品有机肥总体质量安全程度较高,有机肥中Cr、Pb、Hg、As、Cd含量近年来得到较好的控制,但Cu、Zn、Ni含量偏高,部分As含量超标,长期施用条件下存在土壤重金属累积风险,因此需加强对有机肥中重金属的管控,制定商品有机肥中Cu、Zn、Ni限量标准,以保障土壤长期可持续利用。     

Improved enzyme-linked immunosorbent assay for aflatoxin B1 in agricultural commodities

An improved enzyme-linked immunosorbent assay (ELISA) for aflatoxin B1 in cornmeal and peanut butter was developed. Aflatoxin B1 in cornmeal and peanut butter samples was extracted with 70% methanol in water containing 1% dimethylformamide diluted with assay buffer to a final concentration of 7.0% methanol, and directly subjected to an ELISA procedure that took less than 1 h for quantitative analysis and less than 30 min for screening tests. Analytical recoveries for 5-100 ppb B1 added to the cornmeal and peanut butter were 91 and 95.4%, respectively. The interwell and interassay coefficient of variation was 10% or less at the 20 ppb level and above. Agreement for B1 levels in more than 30 naturally contaminated corn, mixed feed, and peanut butter samples was excellent between the ELISA data and the data obtained from different independent laboratories using TLC or other analytical methods.     

浙江省重点地区猪粪中重金属含量及安全施用评估

【目的】 浙江的规模养殖集中度高,畜禽排泄物资源化利用率达96%以上,通过堆肥和制作有机肥还田是主要的消纳途径,因此粪便中的重金属含量影响土壤安全及作物的生长。饲料是猪粪重金属最主要的来源,因此,本研究检测了浙江省猪饲料和排泄物中的重金属,为作物施用的安全性评价提供基础数据。 【方法】 采集35家不同规模养殖场的仔猪、生长猪和育肥猪3个阶段饲喂的饲料和粪便样本共566个,采用干灰化法、高压消解法消解,用AAS、AFS、ICP-MS法分析了铁、铜、锰、锌、砷、铅、铬、镉和汞9种重金属含量。 【结果】 1) 粪便中8种重金属平均含量趋势为Zn > Cu > Mn > As > Cr > Pb > Cd > Hg,主要残留元素为Zn、Cu、Mn和As,按干物质计其平均值分别为2405、1288、528和28.2 mg/kg,变异系数 ( CV) 分别为83%、34%、6%和15%。猪粪中Cr、Pb、Cd元素含量较低,在0.29～3.52 mg/kg之间,Hg含量仅3.97 μg/kg。猪粪中重金属含量仔猪阶段趋高,育肥猪阶段趋低,各个阶段变异都较大,变异系数CV值介于21%～112%之间,显示各地的生猪饲养中微量元素添加还比较混乱。2) 饲料有机砷的使用存在明显区域差异,萧山地区占72.7%的饲料样本在各阶段猪饲料中均添加有机砷,衢州地区的占33.3%,两个地区各有1家养殖场未使用有机砷。猪粪中54.9%样本砷含量 > 15 mg/kg,16.9%样本砷含量 > 50 mg/kg,2.8%的粪便砷超过70 mg/kg。3) 按畜禽粪便安全使用准则进行判定,69.9%猪粪铜超标,16.9%猪粪砷超标。按有机肥料标准进行判定,猪粪中54.9%样本砷含量超标。按照饲料中金属元素在粪便中的浓缩率:Fe、Zn、Cu为7～10倍,锰约为3倍,当前饲养水平下,Fe、Zn、Cu元素存在过量添加。4) 以浙江全省旱地单位面积承载量估算,当前能消纳猪粪中Zn、Cu的排放,但当每公顷猪粪施用量超过2.27 t (干计) 时,局部地区重金属就有累积风险。 【结论】 应降低猪饲料中微量元素的添加量,禁止饲用有机砷制剂,才能有效降低猪粪施用所导致的重金属污染;按照目前监测猪粪肥重金属元素含量水平,有机肥的施用量用应控制在2.27 t/hm2 (干基) 以内,以防止对土壤造成污染。      

Determination of metaldehyde in suspected cases of animal poisoning using gas chromatography-ion trap mass spectrometry.

A method was developed to detect the molluscicide metaldehyde in samples of stomach contents for forensic toxicology investigations. Gas chromatography-ion trap mass spectrometry in full-scan mode was used to identify and quantify metaldehyde. The limit of detection based on mass chromatograms for the m/z 89 ion was 3 microg/g. Mean recoveries from six different spiked samples were 74% at 25 microg/g and 94% at 500 microg/g. The relative standard deviation of six replicate determinations of a sample containing 632 microg/g metaldehyde was 7.3%.     

Determination of oxytetracycline in premixes and veterinary products by liquid chromatography

A liquid chromatographic method for the assay of oxytetracycline in premixes and veterinary products is described. Premix samples are extracted with acidified methanol, diluted with mobile phase, and filtered before chromatography on a C-8, reverse phase column. The assay method separates oxytetracycline from epioxytetracycline, tetracycline, and chlortetracycline. Total elution time for oxytetracycline is less than 5 min at 1.5 mL/min. Five spiked premix samples each of 2 and 50 g/lb had a coefficient of variation of 3.5 and 4.5% and a mean recovery of 99 and 104%, respectively. The results of premixes and veterinary products assayed by this method compared closely with those of the same assayed by the official AOAC microbiological method.     

Near-infrared reflectance spectroscopy (NIRS) enables the fast and accurate prediction of essential amino acid contents. 2. Results for wheat,barley, corn,triticale, wheat bran/middlings,rice bran,and sorghum

Further NIRS calibrations were developed for the accurate and fast prediction of the total contents of methionine, cystine, lysine, threonine, tryptophan, and other essential amino acids, protein, and moisture in the most important cereals and brans or middlings for animal feed production. More than 1100 samples of global origin collected over five years were analyzed for amino acids following the Official Methods of the United States and European Union. Detailed data and graphics are given to characterize the obtained calibration equations. NIRS was validated with 98 independent samples for wheat and 78 samples for corn and compared to amino acid predictions using linear crude protein regression equations. With a few exceptions, validation showed that 70-98% of the amino acid variance in the samples could be explained using NIRS. Especially for lysine and methionine, the most limiting amino acids for farm animals, NIRS can predict contents in cereals much better than crude protein regressions. Through low cost and high speed of analysis NIRS enables the amino acid analysis of many samples in order to improve the accuracy of feed formulation and obtain better quality and lower production costs.     

Enzyme-linked immunosorbent assay for screening aflatoxin B1 in cottonseed products and mixed feed: collaborative study

A joint AOAC/IUPAC (International Union of Pure and Applied Chemistry) interlaboratory study of an enzyme-linked immunosorbent screening assay (ELISA) for aflatoxins was conducted in laboratories in Canada, France, Japan, South Africa, Switzerland, The Netherlands, Tunisia, and the United States. Twenty-eight samples of raw and roasted peanuts, corn, whole cottonseed, cottonseed meal, ammoniated cottonseed meal, and poultry feed containing various quantities of natural aflatoxins and supplemented when appropriate with aflatoxin B1 were distributed to participating laboratories for testing. The assay is based on conjugation of pure aflatoxin B1 to an enzyme and the competition between this conjugate and (free) aflatoxins in the product for aflatoxin-specific antibodies coated onto microtiter well walls. After a wash step to remove all unbound aflatoxins, a substrate, added to each well, is catalyzed from a colorless to a green solution by any bound enzyme-conjugated aflatoxin B1 present. The intensity of the color decreases as the amount of free aflatoxin B1 in the product increases. Overall correlation was good between ELISA and thin-layer chromatographic (TLC) results for cottonseed products and mixed feed. Variable results were reported for corn and peanut product samples. Although some positive samples (greater than 15 ng/g) of cottonseed products and mixed feed were reported to contain less than 15 ng/g by visual determination, a review of data for absorbance measurements showed that the contamination level was close to the greater than or equal to 15 ng/g standard and would not have been reported as negative under routine screening.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the test tube and the dialysis tubing in vitro methods for estimating the bioavailability of phosphorus in feed ingredients for swine

The validity of a simplified in vitro test tube (TT) method was compared with a more complicated dialysis tubing (DT) method to estimate the percentage of available phosphorus (P) in 41 plant origin and five animal origin feed ingredients for swine. The TT method using 1.0 or 0.25 g samples was compared with the DT method using 1.0 g samples at two pancreatic incubation times (2 vs 4 h) in a 3 x 2 factorial arrangement of treatments. Each DT and TT method treatment was replicated three and six times, respectively. Both methods utilize three enzymatic digestions: (i) predigestion with endoxylanase and beta-glucanase for 1 h, (ii) pepsin digestion for 2 h, and (iii) pancreatin digestion for 2 or 4 h. For the TT method, the entire procedure was conducted in a 50 mL conical centrifuge tube and replicated six times. For the DT method, the first two digestions were conducted in a 10 mL plastic syringe before the contents were quantitatively transferred into a segment of DT for the pancreatic digestion. The percentages of hydrolyzed P for plant origin ingredients measured by the DT method using 1.0 g samples and the TT method using 0.25 g samples were highly correlated (r = 0.94-0.97, P < 0.001) with each other and with published in vivo available P values for swine. Repeatabilities for these two methods ranged from 99.64 to 99.86%. The TT method using 1.0 g samples, however, did not provide valid estimates of P availability for all ingredients. For animal origin ingredients, neither method was significantly correlated (r = 0.1-0.6, P >or = 0.4) with published in vivo available P values. In conclusion, the accuracy and validity of the TT method using 0.25 g samples with a 2 h pancreatic digestion time was equal to or superior to the DT method using 1.0 g samples with a 4 h pancreatic digestion time for estimating P availability in plant origin feed ingredients.     

Determination and depletion of residues of carbadox, tylosin, and virginiamycin in kidney, liver, and muscle of pigs in feeding experiments

The results of residue determinations of the growth promotors carbadox, tylosin, and virginiamycin in kidney, liver, and muscle from pigs in feeding experiments are described as well as the analytical methods used. Residues of the carbadox metabolite quinoxaline-2-carboxylic acid were found in liver from pigs fed 20 mg/kg in the diet with a withdrawal time of 30 days. No residues were detected in muscle with zero withdrawal time. The limit of determination was 0.01 mg/kg for both tissues. No residues of virginiamycin and tylosin were found in pigs fed 50 and 40 mg/kg, respectively, in the diet, even with zero withdrawal time. Residues of tylosin of 0.06 mg/kg and below were detected in liver and kidney from pigs fed 200 or 400 mg/kg and slaughtered within 3 h after the last feeding.     

The stomach as a "bioreactor": when red meat meets red wine

To determine the stomach bioreactor capability for food oxidation or antioxidation, rats were fed red turkey meat cutlets (meal A) or red turkey meat cutlets and red wine concentrate (meal B). The hydroperoxides (LOOH) and malondialdehyde (MDA) levels of the stomach contents were evaluated during and after digestion; the postprandial plasma MDA level was also evaluated. In independently fed rats, the stomach LOOH concentration fell substantially 90 min following the meal, and the addition of red wine polyphenols enhanced LOOH reduction 3-fold. A similar trend was obtained for MDA. After pyloric ligation, the stomach contents of rats fed red meat homogenate showed >2-fold increases in LOOH and MDA accumulation. The postprandial plasma MDA level increased significantly by 50% following meal A and was maintained or even fell by 34% below basal level following meal B. The findings show that consumption of partially oxidized food could increase lipid peroxidation in the stomach and the absorption of cytotoxic lipid peroxidation products into the body. The addition of antioxidants such as red wine polyphenols to the meal may alter these outcomes. These findings explain the potentially harmful effects of oxidized fats in foods and the important benefit of consuming dietary polyphenols during the meal.