Analysis of trace residues of tetracyclines in dark-colored honeys by high-performance liquid chromatography using polymeric cartridge and metal chelate affinity chromatography

An efficient clean-up procedure was developed for the trace residue determination of tetracyclines (TCs) in dark-colored honeys. TCs were extracted from samples with McIlvaine buffer (pH 4.0) containing 0.01 mol/L Na(2)EDTA. The extracts were treated with both a polymeric cartridge (GL-Pak PLS-2) and a metal chelate affinity column (MCAC) preloaded with copper(II). TCs were eluted and analyzed by high-performance liquid chromatography (HPLC) using fluorescence detection. The method was evaluated for the determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in buckwheat honey, because its color is the darkest. The mean recoveries of OTC, TC and CTC from spiked samples, at three fortification levels, were >70%, and the relative standard deviations (RSDs) were <10%. Limits of quantitation (LOQs) of OTC, TC, and CTC were estimated to be 0.015 mg/kg, 0.019 mg/kg, and 0.024 mg/kg, respectively.     

Development of an ELISA and Immunochromatographic Assay for Tetracycline,Oxytetracycline, and Chlortetracycline Residues in Milk and Honey Based on the Class-Specific Monoclonal Antibody

A sensitive class-specific monoclonal antibody against tetracyclines (TCs) was generated and used to develop an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic assay for TC, oxytetracycline (OTC), and chlortetracycline (CTC) detection in milk and honey samples. The dynamic range of detection for TC in ELISA was 0.26–2.00 μg L^?1 with an IC₅₀ of 0.72 μg L^?1. The IC₅₀ value of OTC and CTC was 3.2 and 6.4 μg L^?1, respectively. The recovery of TC, OTC, and CTC in milk samples was 82–102, 91–105, and 90–101 %, respectively, and 88–101, 89–101, and 89–95 % in honey samples, respectively. In the immunochromatographic assay, the cutoff values for TC, OTC, and CTC were 15, 15, and 50 μg L^?1 in milk, respectively, and 40, 40, and 40 μg L^?1 in honey, respectively. The results revealed that ELISA and the immunochromatographic assay can be applied for the rapid and sensitive detection of TC, OTC, and CTC in milk and honey samples.     

SPE and MSPD as pre-separation techniques for HPLC of tetracyclines in meat, milk and cheese

 Solid phase extraction (SPE) and Matrix solid phase dispersion (MSPD) have been tested as pre-separation procedures for high-performance liquid chromatography (HPLC) determination of tetracycline antibiotics in milk, meat and cheese. The extraction recoveries ranged from 48% to 86% for SPE and from 89% to 93% for MSPD at concentration levels of the maximal residual limits (MRL) recommended by the European Union to be 100 ng/g for the tetracyclines oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC). The detection limits were 15 – 22 ng/g for SPE and 30 ng/g for MSPD. Following the relatively simple SPE procedure we used a Lichrosorb RP-18 column and a diode array detector for HPLC. Results dealing with analysis of tetracyclines HPLC in cheese are published for the first time. Received: 7 March 1997     

Rapid determination of residues of 4 tetracyclines in meat by a microbiological screening, HPLC and LC/MS/MS

A rapid and precise determination residues of 4 tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DOXY)) in meat was developed by employing three analyses; a microbiological screening, HPLC and LC/MS/MS. TCs were extracted with pH 4.0 McIlvaine buffer containing 0.01 mol/L EDTA from a meat sample, and then purified using a mixed mode, reversed-phase and cation-exchange cartridge. The mean recoveries (n=5) of 0.2 microg/g OTC, TC and CTC, 0.05 microg/g DOXY spiked in meat samples were 76.6-99.0% (C.V. 1.6-5.4%). In 13 meat samples in which the microbiological screening indicated the presence of TCs, CTC (9 samples) and DOXY (4 samples) were identified with HPLC and LC/MS/MS.     

Analysis of tetracyclines in honey and royal jelly by LC/MS/MS

A simple and accurate method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) was developed for the determination of tetracyclines (TCs), i.e., oxytetracycline (OTC), chlortetracycline (CTC) and tetracycline (TC), in honey and royal jelly. Mass spectral acquisition was performed in the positive mode. In LC separation, L-column ODS and 0.01% formic acid-acetonitrile were used as the column and mobile phase, respectively. TCs in a honey sample were diluted with water, while TCs in royal jelly were extracted with 2% metaphosphoric acid-methanol (6:4). They were cleaned up with Oasis HLB and Sep Pak C18 cartridges, respectively. The quantification limits of TC, OTC, and CTC were 5, 5, and 10 ng/g, respectively, while those in royal jelly were 25, 25, and 50 ng/g, respectively. The recoveries of TCs from both honey and royal jelly were 75-120%.     

Analysis of enrofloxacin and its metabolite ciprofloxacin in bovine and porcine muscle by high-performance liquid chromatography following cation exchange clean-up.

A simple and rapid method of analysis for the trace residue determination of enrofloxacin and its metabolite ciprofloxacin has been developed. Clean-up of the samples is by cation exchange solid phase extraction (SPE) and determination made by high-performance liquid chromatography using a base-deactivated column and fluorescence detection. The method has been validated for the determination of residues in bovine and porcine muscle tissue and bacon. Recoveries at the 0.010 mg kg-1 level for enrofloaxacin and ciprofloxacin respectively were 90%, 75% in bovine muscle, 75%, 54% in porcine muscle and 81%, 63% in bacon. Determination to the 0.001 mg kg-1 level in bovine muscle and to the 0.002 mg kg-1 level in porcine muscle and bacon was also carried out. The method has been used as a quantitative screening procedure.     

Automated determination of oxytetracycline residues in muscle, liver, milk and egg by on-line dialysis and post-column reaction detection HPLC.

An automated method for control of oxytetracycline (OTC) residues in chicken and bovine muscle, salmon liver, bovine milk and hen egg has been developed. Tissue homogenate, decreamed milk or whole egg solution was dialysed and the dialysate enriched on a small polystyrene column on-line to HPLC. OTC and the internal standard (tetracycline) were separated on a polystyrene column by ion-pair chromatography. The column effluent was mixed with sodium hydroxide and irradiated at 366 nm. Monitoring the resulting derivatives with a fluorescence detector (excitation: 358 nm, emission: 460 nm), OTC could be detected at 1 ng/ml in milk, 1 ng/g in egg, 3-4 ng/g in muscle and 8 ng/g in liver. Relative standard deviations at 50 and 200 ng/g (milk: 20 and 100 ng/ml) ranged from 1.6 to 3.1%.     

Tetracycline antibiotics transfer from contaminated milk to dairy products and the effect of the skimming step and pasteurisation process on residue concentrations

The presence of antibiotics in raw milk and milk derivatives poses a threat to human health and can negatively affect the dairy industry. Therefore, the main object of this study was to investigate the transfer of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC) from raw, experimental milk contaminated with tetracyclines (TCs) to different dairy products: cream, butter, buttermilk, sour milk, whey, curd and cheese. Additionally the effect of the skimming process on TCs concentrations was tested, as well as the influence of low-temperature long-time pasteurisation. The analyses of TCs in milk and dairy products were performed by an LC-MS/MS method. In order to determine TCs residues in dairy products, an analytical method was developed with the same extraction step for all matrices. TCs molecules were inhomogenously distributed between the milk derivative fractions. The highest concentrations were determined in curd and cheese in the ranges 320–482 µg/kg and 280–561 µg/kg, respectively. Low levels of TCs in butter and whey were observed (11.8–41.2 µg/kg). TCs were found in sour milk (66.0–111 µg/kg), cream (85.0–115 µg/kg) and buttermilk (196–221 µg/kg) at much higher levels than in butter and whey, but lower than in curd and cheese. During the skimming process, the highest yield of cream was obtained after the raw milk was held at 2–8°C for 24 h. The differences in concentrations of TCs between whole milk and skimmed milk, expressed as percentages of recovery, were below 19% (recoveries in excess of 81%). The highest content was observed in milk and cream skimmed at 2–8°C. The degradation percentages for TCs during the pasteurisation process (63°C for 30 min) were below 19%.     

Sorption of tetracycline and chlortetracycline on K- and Ca-saturated soil clays, humic substances, and clay-humic complexes

Tetracycline (TC) and chlortetracycline (CTC) are used extensively for growth promotion and therapeutic purposes in livestock production. The sorption of TC and CTC on clays, humic substances (HS), and clay-humic complexes (clay-HC) derived from two agricultural soils was quantified using dilute CaCl2 (Ca) and KCI (K) as background solutions. In all systems, the soil components sorbed > 96% of added tetracyclines. Strongest sorption was observed for clays, followed by HS, and then clay-HC. Greater sorption by the Ca systems than the K systems and decreased sorption with increasing pH suggests that cation bridging and cation exchange contribute to sorption. X-ray diffraction analysis showed that TC and CTC were sorbed in the interlayers of smectites and that the presence of HS reduced interlayer sorption of tetracyclines by smectites in clay-HC. The results indicate that tetracyclines are dominantly sorbed on soil clays and that HS in clay-HC either mask sorption sites on clay surfaces or inhibit interlayer diffusion of tetracyclines.     

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.     

RP-HPLC快速检测牛奶中7中四环素类药物残留量的研究

目的：建立快速检测牛奶中7 种四环素类药物残留的反相高效液相色谱法。方法：用0.1mol/L McIlvaine-EDTA 溶液和50% 三氯乙酸(TCA)溶液共同处理牛奶样品,通过Waters Oasis HLB 固相萃取柱进行净化处理。仪器条件中所用色谱柱为SunfireTM C18(250mm × 4.6mm,5μm)柱,以甲醇- 乙腈-0.01mol/L 草酸(pH2)为流动相,采用梯度洗脱模式。结果：7 种四环素类药物线性范围宽,峰面积和样品浓度在0.05～1μg/ml 范围内呈很好的线性关系,加标平均回收率为78.66%～106%,最低检测限为0.02μg/ml。结论：该方法操作简单、重现性好、检测限低、灵敏可靠,可在14min 内同时将四环素等7 种四环素类药物达到基线分离。     

Liquid Chromatography-Tandem Mass Spectrometry Determination and Depletion Profile of Chlortetracycline,Doxycycline, and Oxytetracycline in Broiler Chicken Muscle After Oral Administration

A simple and fast method based on liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) was developed and validated for determination of tetracyclines in broiler chicken muscle. Sample preparation was performed using extraction with acetonitrile, followed by low-temperature purification (at ??20 °C) and further concentration. The chromatographic analysis was carried out using a Zorbax SB-C18 column with gradient elution using water and methanol both acidified with 0.025 M of formic acid. Mass spectrometry with electrospray ionization was operated in positive polarity using selected reaction monitoring (SRM) analysis mode, achieving the requirements of four identification points for each compound. Demeclocycline was used as internal standard. The method validation was done according to the criteria of Commission Decision 2002/657/EC. Parameters such as recovery, matrix effects, selectivity/specificity, linearity, precision (intra- and inter-day precision), accuracy, limits of detection (LOD) and quantification (LOQ), decision limit (CCα), detection capability (CCβ), and robustness were determined. Intra-day precision values were within the range 2.2–5.8% and inter-day precision was less than 10% for all analytes. Accuracy ranged from 98.2 to 103.2%. The method was successfully applied for depletion studies of chlortetracycline, doxycycline, and oxytetracycline in broiler chicken tissues after multiple oral administrations. After the depletion studies, the present study support more prudent use of CTC, DOX, and OTC for treatment of chickens and suggest a dose of 60 mg kg^?1 body weight for CTC and OTC and 20 mg kg^?1 body weight for DOX, orally administrated for five consecutive days.     

Validation of HPLC method for determination of tetracycline residues in chicken meat and liver

High-performance liquid chromatographic method with diode-array detection (HPLC–DAD) was optimised and validated for determination of tetracyclines (TCs) residue in chicken meat and liver through evaluating each step of various methods. The principle steps involved ultrasonic-assisted extraction of TCs from chicken samples by 2 ml of 20% trichloroacetic acid and citrate buffer (pH 4) which gave a clearer supernatant and high recovery, followed by centrifugation and purification on SPE (Strata C18-E cartridge) using 10 ml of 0.01 M methanolic oxalic acid for TCs elution. Separation was on reversed-phase column (Nuclosil 100 C18, 25 cm × 4.6 mm ID, 5 μ) by multisteps gradient elution which provided a better chromatographic peak resolution and the late eluting peaks were as sharp as those eluting earlier. Monitoring was at 351 nm which gave a higher detector response factor. Validity study of the method revealed that all obtained calibration curves showed good linearity (r² > 0.999) over the range of 50–5000 ng. Sensitivity was found to be 1.44, 1.90, 0.95 and 1.23 ng for OTC, TC, CTC and DC, respectively. Accuracy was in the range of 71.88–92.44.3% and 68.88–84.84% for meat and liver, respectively. Precision was lower than 10% in all cases indicating that the method can be used as a validated method. Limit of detection (LOD) was found to be 4.4, 5, 13 and 10 ng for OTC, TC, CTC and DC, respectively. The corresponding values of limit of quantitation (LOQ) were 10, 13, 27 and 22 ng.     

毛细管电泳-电化学检测法测定鸡蛋中残留四环素类抗生素

本研究运用毛细管电泳-电化学检测法(CE-ED)对鸡蛋中残留的四环素(TC)、土霉素(OTC)进行快速测定分析。考察了缓冲液浓度和pH值、TW-20、盐浓度、分离电压、进样时间、检测电位等因素对分离检测的影响。结果表明,TW-20体积分数为2.5%时,在检测电位为1.2V,20mmol/L缓冲溶液(pH2.6),25kV分离电压,进样8s的条件下,TC和OTC有很好的分离效果。TC和OTC的线性范围分别为:2.0×10-6～5.0×10-4g/ml和8.0×10-6～3.0×10-4g/ml,检测限分别为1.9×10-8g/ml和4.1×10-8g/ml。     

Incidence of tetracycline residues in chicken meat and liver retailed to consumers

The incidence of tetracyclines’ (TCs) residue (oxytetracycline, tetracycline, chlorotetracycline and doxycycline) in fresh chicken samples (meat and liver) collected during one year was recorded. TC residues were analysed using an HPLC-DAD method. The limit of detection for meat was 4.4, 5, 10 and 7?ng?g^?1 for OTC, TTC, CTC and DOC, respectively, compared with 5.5, 6, 12 and 9?ng?g^?1 stated for liver. The recovery of the method ranged from 91% to 70% depending upon both matrix type and tetracycline kind. The results revealed that 66 (44%) samples contained TC residues including 21 (42%) breast, 19 (38%) thigh and 26 (52%) liver samples. The corresponding contaminated ranges were 124–5812, 107–6010 and 103–8148?µg?kg^?1. A total of 12 (8%), 13 (7.33%) and 20 (13.33%) samples of breast, thigh and liver, respectively, had TC residues above the Codex maximum residue limit (MRL). Liver samples had a higher incidence and level than those found in breast or thigh samples.     

Simultaneous determination of tetracycline,oxytetracycline, and 4-epitetracycline in milk by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography with photodiode-array detection (HPLC–PAD) was optimized and validated for the simultaneous determination of tetracycline (TC), 4-epitetracycline (4-epiTC) and oxytetracycline (OTC) in milk. Milk samples were extracted and cleaned-up using solid-phase extraction Discovery SPE DSC-18 tubes. The separation were accomplished in less than 8 min in a Waters Symmetry C18 column at ambient temperature with a mobile phase consisted of 0.010 M aqueous oxalic acid:acetonitrile:methanol (150:20:20 by volume). Quantitation was carried out by the peak area method, with detection limits of 2.0 μg/l of each tetracycline. Average recoveries of TC, 4-epiTC and OTC from spiked samples at the four concentrations (0.25, 0.5, 1.0 and 1.5 μg/ml) were 91.5, 71.5 and 83.1, respectively, with their standard deviations less than 4% within a day and 7% between days. This method was applied for the simultaneous determination of TC, 4-epiTC and OTC in market milk samples purchased from local supermarkets. Oxytetracycline was found being present in all samples in a concentration range of 13–106 μg/l, 4-epiTC in most samples at 18–65 μg/l, TC in one sample at 44 μg/l.     

Gas chromatographic method for the analysis of residues of seven penicillins in food of animal origin]

A capillary gas chromatographic method is described for the determination of residues of benzylpenicillin, phenoxymethylpanicillin, methicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin in bovine muscle, liver, kidney, adipose tissue and milk. The samples are extracted with acetonitrile under slightly acidic conditions, the co-extracted water is separated with the addition of sodium chloride and dichloromethane and discarded. Clean-up is performed by liquid/liquid partitioning steps and anion exchange chromatography. The penicillin residues are methylated with diazomethane. After derivatization, only the extracts from liver and kidney needed further clean-up using cartridges with a polar diol sorbent. The gas chromatographic procedure is based on split/splitless injection, programmed temperature vaporization, separation on a methyl silicone fused silica column and nitrogen-specific thermionic detection. Internal standardization is used for quantification. The limits of detection for all penicillins are well below 3 microgram/kg in milk and all tissues. Recoveries of spiked samples at 3 and 10 micrograms/kg are in the range of 65-80% for milk and 50-70% for bovine tissues.     

基于分子印迹整体柱的在线固相萃取-液相色谱联用测定奶粉中四环素类兽药残留

目的建立高交联结构的分子印迹整体柱(molecularly imprinted polymer monolithic column,MIP-MC)制备方法,利用在线固相萃取与液相色谱联用技术检测奶粉样品中四环素类兽药残留。方法在不锈钢色谱柱中,以土霉素为模板,甲基丙烯酸和甲基丙烯酸羟乙酯为功能单体,乙二醇二甲基丙烯酸酯和双季戊四醇六丙烯酸酯为交联剂,偶氮二异丁腈为引发剂,在丙酮-甲醇-十二醇混合溶剂中,制备了分子印迹整体柱。将整体柱与液相色谱联用,在线固相萃取奶粉中的四环素类兽药残留。结果在最佳在线固相萃取条件下,获得了较高的富集因子(19.3)和净化效果。四环素类在0.05、0.25和0.5 mg/kg 3个加标水平下,回收率为84.2%~103.4%,相对标准偏差为1.37%~4.87%,方法检出限(S/N=3)和定量限(S/N=10)分别为8.48~11.74?g/kg和28.24~39.09?g/kg。结论该在线固相萃取方法简单快速、灵敏性高、选择性好,适用于奶粉中四环素类抗生素残留的测定。     

食品中四环素类残留的酶联免疫检测试剂盒的研制

目的：基于定量检测动物源性食品中四环素类抗生素的残留量,开发研制四环素类抗生素竞争酶联免疫(ELISA)检测试剂盒。方法：采用杂交瘤技术,筛选并克隆稳定分泌抗四环素(CEL)的杂交瘤细胞株,利用免疫BALB/c 小白鼠制备抗四环素单克隆抗体。结果：logit/log 拟合标准曲线为y= － 2.3x ＋ 2.79,相关系数r=0.9971,线性检测范围为0.05～4.05ng/mL,半数抑制剂量(IC50)为0.293ng/mL,灵敏度为0.05ng/mL,检测限为3～5ng/mL;组织、牛奶、蜂蜜的添加回收率分别为(90 ± 10)%、(85 ± 10)%、(90 ± 10)%;平均批内和批间变异系数均小于15%;四环素与金霉素等同类抗生素的交叉反应率大于60%,与其他类抗生素无交叉反应;此试剂盒在4℃可保存180d 以上。结论：建立的试剂盒是符合技术指标的要求,可用于动物源性食品中四环素类抗生素残留的定量检测。     

Residue analysis of metoclopramide in bovine and swine tissues by HPLC and administration experiment

A reversed-phase ion-pair HPLC method with ultraviolet detection has been developed for determination of metoclopramide (MCP) in bovine and swine muscle, liver, kidney, fat and intestine. MCP was extracted from samples with acetonitrile, and the extracts were cleaned up on an Oasis HLB cartridge (60 mg) after liquid-liquid extraction. The limit of detection of MCP was 0.002 microg/g and the limit of quantitation was 0.007 microg/g. Recoveries of MCP spiked at 0.03 ppm ranged from 74.1 to 93.3% for bovine tissues and from 86.1 to 92.7% for swine tissues. The present method was used for the analysis of bovine and swine tissues 1 day after withdrawal following drug administration. The MCP concentrations in all tissues were lower than the Japanese provisional MRLs.