Matrix solid-phase dispersion (MSPD) isolation and liquid chromatographic determination of oxytetracycline, tetracycline, and chlortetracycline in milk

A multiresidue method for the isolation and liquid chromatographic determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) antibiotics in milk is presented. Blank and tetracycline (OTC, TC, and CTC) fortified milk samples (0.5 mL) were blended with octadecylsilyl (C18, 40 microns, 18% load, endcapped, 2 g) derivatized silica packing material containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetic. A column made from the C18/milk matrix was first washed with hexane (8 mL), following which the tetracyclines were eluted with ethyl acetate-acetonitrile (1 + 3; v/v). The eluate contained tetracycline analytes that were free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 365 nm). Correlation coefficients of standards curves for individual tetracycline isolated from fortified samples were linear (from 0.982 +/- 0.009 to 0.996 +/- 0.004) with average percentage recoveries from 63.5 to 93.3 for the concentration range (100, 200, 400, 800, 1600, and 3200 ng/mL) examined. The inter-assay variability ranged from 8.5 +/- 2.4% to 20.7 +/- 13.0% with an intra-assay variability of 1.0-9.3%.     

Validation of a method for determination of tetracycline antibiotics in salmon muscle tissue

A method was validated for analysis of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in fortified salmon muscle tissue. Recoveries of OTC were 100 +/- 6, 86 +/- 6, and 82 +/- 5% (n = 6) at fortification levels of 1.0, 0.5, and 0.2 ppm, respectively. Recoveries of TC were 68 +/- 4, 65 +/- 6, and 66 +/- 7%; recoveries of CTC were 45 +/- 9, 48 +/- 8, and 0%, respectively. Detection limits for OTC and TC were 0.08 and 0.09 ppm, respectively.     

Screening of tetracycline residues in fish muscles by CCD camera-based solid-surface fluorescence

A methodology for the screening of tetracyclines (TCs), including tetracycline (TC), oxytetracycline (OTC), and chlorotetracycline (CTC), in different fish muscle matrices has been proposed. This method was based on in situ fluorescent derivation of TCs, transferring weakly fluorescing TCs to highly fluorescent species, on alkaline-activated solid silica gel G plates (SGGPs). By coupling solid-surface fluorescence (SSF) with charge-coupled device (CCD) camera imaging, a CCD camera-based SSF (CCD-SSF) methodology has been developed. Calibration curve, repeatability, selectivity, limit of detection (LOD), and limit of quantification (LOQ) have been explored for evaluating the performance of the method itself. Linear calibration curves were obtained over a range of 0.20-1.0 ng/spot for all three TCs. The LODs, defined as 3sigma, for TC, OTC, and CTC were 0.14, 0.15, and 0.16 ng/spot, respectively. The trueness of method was validated by HPLC, and no significant difference between CCD-SSF and HPLC was found, on a basis of 95% confidence level. By spiked recovery studies, a linear calibration curve ranging from 20 to 300 microg/kg of TC in fish muscle samples with a correlation coefficient (R 2) equal to 0.994 was obtained. The total average recovery for TC in fish muscle samples from six different fish matrices, fortified with TC at 50, 100, and 200 microg/kg levels, was 75.7% with average relative standard deviations (RSDs) ranging from 2.0 to 7.7%. RSDs ranged from 2.5 to 5.8% and from 5.2 to 7.6% for in-day and interday repeatability, respectively. The detection and quantification limits in fish muscle matrices were 16 and 53 microg/kg of TCs, respectively. The newly developed CCD-SSF method has been applied to the screening of the TC residues in fish muscle samples. The method has been demonstrated to bear some advantages, such as its simplicity, high throughput, low cost, use of fewer pollutants, and reasonable sensitivity.     

Liquid chromatographic determination of epimerization of chlortetracycline residue to 4-epi-chlortetracycline residue in animal feed, using McIlvain's buffer as extractant

A method for studying McIlvain's solution as a factor in epimerization of chlortetracycline in animal feed has been developed. McIlvain's solution (McBuffer) was used previously to extract oxytetracycline (OTC), tetracycline-HCl (TC-HCl), and chlortetracycline-HCl (CTC-HCl) from animal feed because it gave the best recoveries for all 3 tetracyclines; however, the McBuffer solution caused epimerization of CTC-HCl to 4-epi-chlortetracycline (4-epi-CTC). Recovery results were 30-40% lower for CTC than for OTC and TC from the same sample extract. The levels of concentration tested for CTC and 4-epi-CTC were 10 ppm, the detection limits for both were 3 ppb, and the calibration curves were linear between 5 and 80 ppm for both CTC-HCl and 4-epi-CTC. The present study shows that CTC recoveries averaged 35% lower because of epimerization initiated by the use of McBuffer in the extraction procedure.     

壤和堆肥中四环素类抗生素的检测方法优化及其在土壤中的降解研究

在对土霉素（OTC）、四环素（TC）和金霉素（CTC）3 种四环素类抗生素的高效液相色谱（HPLC）检测分析方法以及在土壤和堆肥中的提取方法进行改进和优化的基础上,采用该方法进行了 3 种抗生素在土壤中的降解试验。结果表明,选用 Agilent Eclipse XDB-C8（4.6150 mm,5 m）色谱柱,以 0.01 mol/L草酸/乙腈/甲醇（79/10.5/10.5,v/v/v）为流动相,紫外检测波长 268 nm,流速 1.0 mL/min,进样量 5 L,采用外标法定量,可使 3 种四环素类抗生素在 20 min 内全部洗脱并达到基线分离; 在 0~10 mg/L 范围内,抗生素浓度与峰面积呈显著的线性关系,相关系数（r）均 0.999。土壤和堆肥样品中的 OTC、TC 和 CTC可用1 mol/L NaCl/0.5 mol/L 草酸/乙醇（25/25/50,v/v/v）混合溶液提取,其回收率在 76.0%~92.5% 之间。加入到土壤中的抗生素在 25℃下避光培养 49 d 后,在壤土和红土中的降解率分别是 67%~72% 和 36%~46%,对应的半衰期分别为 2630 d 和 4675 d,说明抗生素在壤土中比红土中容易降解。此外,3种抗生素在壤土中的半衰期没有显著性差异,而在红土中 CTC 和 TC 的降解速率显著高于 OTC。     

我国有机肥原料及商品有机肥中四环素类抗生素的检出率及含量

[目的]畜禽粪便等有机肥是抗生素污染农田土壤的重要源头之一.本研究通过调查分析我国有机肥原料及商品有机肥中四环素类抗生素的检出率及含量,为有机肥的安全施用提供依据.[方法]在全国采集有机肥生产的主要原料和辅料样品180个及商品有机肥样品244个,进行土霉素(OTC)、四环素(TTC)、金霉素(CTC)和强力霉素(DXC...     

土壤及畜禽粪肥中四环素类抗生素固相萃取-高效液相色谱法的优化与初步应用

本研究优化了土壤和有机肥中3 种四环素类抗生素的提取和测定方法。方法经优化后,土壤和有机肥中的抗生素的提取效率达到52%~95%,且分析时间大大缩短。本研究还利用优化方法在天津进行了有机肥和菜地中3 种抗生素残留的初步调查。集约化养殖场的猪、鸡粪便中四环素类抗生素残留状况为: 金霉素（CTC）检出率达到78%,最高残留值达到563.8 mg/kg（干基）; 四环素（TC）和土霉素（OTC）检出率也高达56%,最高值分别达到34.8 mg/kg 和 22.7 mg/kg。在天津销售的几种商品有机肥中同样检测出四环素类抗生素的残留,残留水平与猪粪和鸡粪相当。菜田土壤样品中TCs的总检出率为64%,3.种抗生素中土霉素检出率最低为18%,最高值达到105.6 g/kg（风干基）; 四环素检出率为36%,最高值达到196.7 g/kg; 金霉素检出率为32%,最高值达到477.8 g/kg。在所调查土壤中,温室和大棚土壤TCs的残留水平高于露地土壤。占调查样品 27.3% 的菜田土壤中TCs总量超过欧盟规定的生态安全触发线（100 g/kg）,存在一定的潜在生态风险。     

江苏省畜禽养殖场水环境中四环类抗生素污染研究

2009年在江苏省范围内采集27个规模化养殖场排水口和周围环境水体样品53个,用高效液相色谱-三重四极杆质谱对其中的土霉素（OTC）、金霉素（CTC）、四环素（TC）和强力霉素（DOX）等四环素类抗生素污染进行了检测。结果显示,土霉素、金霉素、四环素和强力霉素在水体样品中的检出率分别为60.4%、60.4%、34.0%和17.0%,污染量分别在0.07～72.91、0.10～10.34、0.08～3.67μg·L-1和0.11～39.54μg·L-1之间,检出率以土霉素和四环素最高;猪养殖场中土霉素、四环素和强力霉素的污染量比牛和鸡场的要高,牛场中金霉素和强力霉素的污染量明显低于鸡场;从区域上看,苏南地区受到的污染明显高于苏北。     

Metabolites of oxytetracycline, tetracycline, and chlortetracycline and their distribution in egg white, egg yolk, and hen plasma

4-epioxytetracycline and N-demethyloxytetracycline, as metabolites of oxytetracycline (OTC), 4-epitetracycline and N-demethyltetracycline, as metabolites of tetracycline (TC), and 4-epichlortetracycline, isochlortetracycline (ICTC), 4-epi-ICTC, and N-demethyl-ICTC, as metabolites of chlortetracycline (CTC), were detected in egg yolk and plasma obtained from feeding studies with either OTC, TC, or CTC. In egg white, only OTC, TC with its 4-epimer, and ICTC with its 4-epimer were detected in substantial concentrations. The ratios of epimerization and N-demethylation in the eggs did not change during the medication period. The samples were analyzed by an automated HPLC system (ASTED) with UV, fluorescence, or MS-MS detection.     

致谢审稿专家

Two agricultural soils were collected from Dahu and Pinchen counties and swine manure compost (SMC) from Pingtung County in Taiwan, China to investigate the sorption and dissipation of three tetracyclines (TCs), i.e., oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC), in compost, soils and soil/compost mixtures with different organic carbon (OC) contents. There were seven treatments in total. TCs were most strongly adsorbed to SMC in all treatments due to the high OC content. When SMC was present in the soils, the sorption of TCs was significantly enhanced, which might be attributed to the increased OC content and CEC. The adsorption of TCs showed non-linear adsorption isotherms and fitted well to the Freundlich model. After 49 d of incubation at 25 oC in soils and soil/compost mixtures in the dark, TCs elapsed in all substrates, with the time required for 50% degradation (DT50) between 20 and 41 d, and the time for 90% degradation (DT90) between 68 and 137 d. Soil amended with compost enhanced the stability of TCs and reduced their mobility. The dissipation of TCs in a soil environment was slow, indicating that these compounds might be persistent in soil.     

Limited survey of residual tetracyclines in tissues collected from diseased animals in Aichi Prefecture, Japan.

Tissues were collected to survey the actual conditions of tetracycline antibiotics (TCs) residues in slaughtered animals that did not pass inspection at slaughterhouses in Aichi Prefecture, Japan, because of the presence of disease symptoms. Tissues were analyzed by liquid chromatography. Among 271 samples, 49 (18.1%) were positive for oxytetracycline (OTC), 5 (1.8%) for chlortetracycline (CTC), and 5 (1.8%) for doxycycline (DC), respectively. One sample (cattle kidney) was positive for both OTC and DC. However, tetracycline was not detected in any samples. Percentage frequencies of TCs residues were 29.1% (37/127) and 15.2% (22/144) for cattle and hogs, respectively. Kidney samples showed higher incidence of TCs residues and 1.5-7 times higher residual concentrations than liver and miscellaneous samples.     

固相萃取-高效液相色谱法测定有机肥中四环素类抗生素药物残留

建立了固相萃取-高效液相色谱(SPE-HPLC)法测定有机肥中土霉素(OTC)、四环素(TC)和金霉素(CTC)3种四环素类抗生素残留的方法。肥料样品采用甲醇、EDTA和McIlvaine缓冲液的混合液(pH=7.2)为提取液,用强阴离子交换柱(SAX)-亲水亲脂萃取柱(HLB)串联进行纯化和富集。采用甲醇-乙腈-0.01mol/L草酸溶液为流动相,进行HPLC分析。3种抗生素的线性范围为0.10～20 mg/L,OTC、TC和CTC的检测限分别为0.03、0.03和0.05 mg/L。不同添加水平的样品加标回收率为64%～86%,RSD在4.14%～8.16%之间。该方法测定了上海市场上40种肥料,发现部分样品有四环素类抗生素的残留物。     

超高效液相色谱串联质谱法同时检测复杂基质中四环素类抗生素及其代谢产物

为同时检测复杂基质中（由猪粪和蘑菇渣混合而成的堆肥原料）3种四环素类抗生素（四环素TC、土霉素OTC和金霉素CTC）及其代谢产物,建立了超高效液相色谱串联质谱检测方法（UPLC—MS／MS）。该方法同时采用pH值=4的Na2EDTA-Mellvaine缓冲溶液和乙腈为提取溶液,经过固相萃取净化后,以乙腈和0．1％的甲酸水溶液为流动相,采用超高效液相色谱柱进行分离,在电喷雾正离子模式下,用四极杆串联质谱仪进行定性和定量分析。3种四环素类抗生素及其代谢产物均在7min内完成分离,总共分析时间为12min。在0-6mg·kg^-1DW（Dryweight）浓度范围内,3种四环素类抗生素及其代谢产物的标准曲线线性良好,线性相关系数R2均大于0．9960,重现l生也较好（n=11,相对标准偏差均小于15％）。在3个加标水平0．2mg·kg^-1DW、1mg·kg^-1DW和4mg·kg^-1DW下,TC、OTC、CTC的回收率分别为71％-89％、66％～94％和66％～84％;去甲基金霉素（DMCTC）的回收率为52％-64％;差向异构产物的回收率在32％～51％之间;脱水产物以及差向脱水产物的回收率均低于30％。3种四环素类抗生素及其代谢产物的检出限和定量限分别在1．668～17．270μg·kg^-1和5．561—45．918μg·kg^-1范围内,表明该方法具有较高的灵敏度。对北京市某露天堆肥场中的样品进行测定发现,TC、OTC、CTC的浓度分别为0．4、1．6、2．9mg·kg^-1,检测到的代谢产物主要为相应的差向异构体,其中差向金霉素（ECTC）的浓度最高,达到2．7mg·kg^-1,和母体的含量水平比较接近,其他代谢产物也有不同程度的检出。     

Gas chromatographic determination of captan, folpet, and captafol residues in tomatoes, cucumbers, and apples using a wide-bore capillary column: interlaboratory study.

An interlaboratory study of the determination of captan, folpet, and captafol in tomatoes, cucumbers, and apples was conducted by 4 laboratories using wide-bore capillary column gas chromatography with electron capture detection. The 3 fungicides were determined using the Luke et al. multiresidue method modified to include additional solvent elution in the optional Florisil column cleanup step used with this method. The crops were fortified with each fungicide at 3 levels per crop. Mean recoveries ranged from 86.2% for a 25.1 ppm level of captan in apples to 115.4% for a 0.288 ppm level of captafol in apples. Interlaboratory coefficients of variation ranged from 3.4% (24.7 ppm folpet) to 9.7% (0.243 ppm captafol) for tomatoes; from 2.8% (2.0 ppm captafol) to 8.2% (24.8 ppm captan) for cucumbers; and from 1.5% (0.234 ppm folpet) to 22.1% (0.266 ppm captafol) for apples.     

猪粪中3种四环素类抗生素在土壤中的动态变化及降解途径

采用室内模拟培养试验,研究了180 d内不同用量猪粪中3种四环素类抗生素（TCs）（包括四环素TTC,土霉素OTC,金霉素CTC）在土壤中降解的动态变化规律及降解途径。结果表明,猪粪中3种四环素类抗生素进入土壤后含量均呈现前期迅速下降,中后期减缓的规律,但不同种类和用量处理的变化速率和减少幅度有显著差异（P0.05）。180d时,猪粪处理土壤中的降解率为金霉素土霉素四环素,平均半衰期为17.43d、31.32d、49.48d。降解率与猪粪用量呈负相关,同培养时间呈正相关。猪粪中抗生素在土壤中的降解率要高于纯品抗生素,降解率的增加以外源微生物降解为主,外源微生物降解对四环素的作用最好。综上所述,随着培养时间的延长,猪粪中四环素类抗生素在土壤中能随微生物降解等作用而逐渐减少,但短期内仍可能产生环境危害。     

Turbidimetric assay for tetracyclines in feeds using a microtiter plate system

The microtiter plate system for turbidimetric assay of chlortetracycline (CTC) and oxytetracycline (OTC) levels in feeds uses a 96 well microtiter plate, a multichannel pipette, and an ELISA reader to measure turbidity. Feeds are extracted for both tetracyclines using AOAC extraction systems. For CTC, the range of the standard curve is 0.001-0.005 microgram CTC/mL; for OTC, the range is 0.004-0.016 microgram OTC/mL. Repeatability of CTC assays, as shown by the coefficient of variation (CV), ranged from 0.54 to 5.65% for same-day assays and from 2.01 to 9.39% for assays on different days. For OTC, CVs ranged from 2.69 to 10.01% for same-day assays and 3.24 to 9.08% for different-day assays. Average recoveries for CTC were 108.7% for same-day assays and 106.8% for different-day assays; for OTC, average recoveries were 112.4% and 106.5% for same-day and different-day assays, respectively.     

Liquid chromatographic determination of chlortetracycline hydrochloride in ruminant and poultry/swine feeds.

A liquid chromatographic (LC) method is described for the determination of chlortetracycline hydrochloride (CTC) in poultry/swine and ruminant feeds in the 10-100 ppm range and in premix. CTC is extracted from ground feed/premix with acidified acetone, and the extract is filtered through a Millex-HV filter or disposable C18 column. The filtrate is partitioned with methylene chloride when additional cleanup is necessary. A Nova-Pak C18 column is used for LC separation with determination at 370 nm. The average recovery of CTC from premix was 95% with a standard deviation (SD) of 1.70 and a coefficient of variation (CV) of 1.79%. The overall average recovery from feeds was 77% with an SD of 3.18 and a CV of 4.10%.     

Liquid chromatographic determination of monensin in chicken tissues with fluorometric detection and confirmation by gas chromatography-mass spectrometry

An accurate, sensitive method is described for the determination of monensin residue in chicken tissues by liquid chromatography (LC), in which monensin is derivatized with a fluorescent labeling reagent, 9-anthryldiazomethane (ADAM), to enable fluorometric detection. Samples are extracted with methanol-water (8 + 2), the extract is partitioned between CHCl3 and water, and the CHCl3 layer is cleaned up by silica gel column chromatography. Free monensin, obtained by treatment with phosphate buffer solution (pH 3) at 0 degrees C, is derivatized with ADAM and passed through a disposable silica cartridge. Monensin-ADAM is identified and quantitated by normal phase LC using fluorometric detection. The detection limit is 1 ppb in chicken tissues. Recoveries were 77.6 +/- 1.8% at 1 ppm, 56.7 +/- 7.1% at 100 ppb, and 46.5 +/- 3.7% at 10 ppb fortification levels in chicken. Gas chromatography-mass spectrometry is capable of confirming monensin methyl ester tris trimethylsilyl ether in samples containing residues greater than 5 ppm.     

HPLC-UV method for nicotine, strychnine, and aconitine in dairy products

The toxic nitrogen alkaloids nicotine, strychnine, and aconitine were quantitated in whole milk, skim milk, and cream using solid-phase extraction cleanup and HPLC-UV with dual wavelength detection. Samples were extracted in McIlvaine's buffer with EDTA and then partitioned with aqueous acetonitrile and hexane. The aqueous phase was concentrated and passed through an OASIS HLB column. The column was eluted with methylene chloride/ammonium hydroxide, 1 mL/1 microL, v/v. The eluent was acidified with hydrochloric acid and evaporated. The sample was diluted for HPLC with acetonitrile/phosphate buffer pH 7.4. Chromatography was performed on an Xterra RP-18 column using a gradient of acetonitrile and ammonium bicarbonate buffer at pH 9.8. Nicotine and strychnine were monitored at 260 nm; aconitine was monitored at 232 nm. Calibration curves were generated from external standards in the range 0.2-10 microg/mL using 1/x weighting. Mean recoveries in whole milk spiked between 0.1 and 10 ppm were the following: nicotine 89.2%, strychnine 75.7%, and aconitine 85.1%. Mean recoveries in skim milk spiked between 0.1 and 10 ppm were the following: nicotine 72.1%, strychnine 78.2%, and aconitine 82.9%. Mean recoveries in cream spiked between 0.2 and 20 ppm were the following: nicotine 87.9%, strychnine 76.9%, and aconitine 82.0%. Relative standard deviations of recovery were less than 20% in each case.     

土壤中土霉素残留的高效液相色谱检测方法

为有效测定土壤中土霉素残留量,建立了固相萃取－高效液相色谱法提取以及测定潮土、红壤、紫色土中土霉素残留量的方法。土壤中土霉素残留经提取缓冲溶液进行有效提取,经过DVB固相萃取小柱纯化、无水甲醇洗脱和氮气流浓缩后,经HPLC测定。对提取缓冲液、流动相以及流动相pH值、有机相与无机相的比例以及流速等测定条件进行优化研究。结果表明：提取液为Na2EDTA-Mcllvaine,流动相为乙腈∶0.01mol/L磷酸二氢钠（pH值2.5,V∶V=10∶90）,温度25℃,流速1.2ml/min,检测波长350nm对3种不同性质的土壤中土霉素残留量的测定最为合适。应用本方法进行土壤中土霉素残留量的测定,土霉素含量与峰面积具有良好的线性关系,相关系数（n=9）分别为红壤0.997,紫色土0.995,潮土0.987;检出限分别为红壤0.11mg/kg,紫色土0.17mg/kg,潮土0.09mg/kg;回收率(n=18)分别为红壤80.7%~128.8%,紫色土70.5%~100.0%,潮土61.5%~103.9%;相对标准偏差(RSD, n=18)分别为红壤7.1%~28.2%,紫色土11.9%~38.1%,潮土4.1%~17.0％。本方法简便、准确,适合于测定不同土壤中土霉素残留量,结果可靠。