Residue study of doxycycline and 4-epidoxycycline in pigs medicated via-drinking water.

A study was performed to determine the residues in edible tissues of healthy pigs after continuous administration of doxycycline with drinking water for five consecutive days at a dose rate of 10.5 mg doxycycline kg-1 body weight (BW) per day. Quantitation was performed using a validated HPLC method with fluorescence detection. The method was able to separate doxycycline and its 4-epimer, 4-epidoxycycline. This epimer was found in kidney, liver, skin, fat and muscle tissue. The method was validated at the maximum residue limit (MRL), at half the MRL and at double the MRL for both doxycycline and 4-epidoxycycline. Linear calibration curves were obtained in spiked tissues (r > 0.99). The accuracy of the calibrators of the calibration curves was within -20% to +10%. The accuracy and precision (expressed as the within-run repeatability) were found to be within the required ranges for the specific concentration. The limits of detection and limits of quantification were below one-half of the MRL. The quantification limits were 50 micrograms kg-1 for doxycycline and 100 micrograms kg-1 for 4-epidoxycycline in kidney and liver, 20 micrograms kg-1 for doxycycline and 50 micrograms kg-1 for 4-epidoxycycline in skin and fat and 10 micrograms kg-1 for doxycycline and 50 micrograms kg-1 for 4-epidoxycycline in muscle tissue. The withdrawal time was calculated according to the recommendations of the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/036/95) and was set at 3 days. The plasma concentration of doxycycline and the stability of doxycycline in drinking water were also determined during the treatment period. The mean plasma concentration of doxycycline during the treatment period ranged from 0.83 to 0.96 microgram ml-1. Thirty-six hours after the withdrawal from medicated drinking water, no plasma levels could be detected. Samples of medicated water were taken at time zero and at 24 h after addition of doxycycline to the drinking water. No statistically significant difference in the mean drinking water concentration was seen at time zero and at time 24 h (Student's t-test, alpha = 0.05).     

Production of CTC-containing porcine reference materials.

The European Commission (EC) established the Standards, Measurements and Testing programme for the preparation of Reference Materials (RMs) as an aid to harmonise testing for veterinary drug residues throughout the European Union (EU). The production of chlortetracycline (CTC)-free and CTC-incurred pig tissues as candidate RMs is described. High performance liquid chromatography (HPLC) with fluorescence detection of CTC and 4-epi-CTC was used for all tissue analyses. A pilot study revealed that incurred CTC residues were stable in pig kidney, liver and muscle lyophilised powders during storage for 10 weeks at -70, -20 and +37 degrees C, obviating the need for addition of a stabiliser (thimerosal). In the main study, 500 vials each of CTC-free and CTC-incurred kidney, liver and muscle were produced. Target concentrations in the CTC-incurred lyophilised tissue powders were 750-1500, 500-1000 and 300-600 micrograms kg-1 for kidney, liver and muscle, respectively. Following lyophilisation, the mean +/- s concentrations of CTC in the incurred positive RMs were 1,315 +/- 56.9, 765 +/- 35.3 and 378 +/- 16.8 micrograms kg-1 for kidney, liver and muscle respectively. Residual moisture in the RMs ranged from 1.6 +/- 0.53% for muscle to 3.0 +/- 0.50% for liver. Between-vial homogeneity for incurred powders was determined for 20 vials of each material, which had been removed at regular intervals during the filling process. Relative standard deviations (RSDs) for kidney, liver and muscle were 4.3, 4.6 and 4.4% respectively, being within the interassay RSD of the method and indicating that mixing was effective. Stability of powders stored at -18, 4, 20 and 37 degrees C was assessed over a period of 79 weeks. No measurable degradation occurred over this time period at any of the storage temperatures. It is concluded that these candidate RMs are homogenous, stable and are suitable for certification.     

Determination of fenbendazole and its metabolites in trout by a high-performance liquid chromatographic method.

A high-performance liquid chromatographic (HPLC) method based on solid phase extraction was developed for the simultaneous determination of fenbendazole (FBZ), fenbendazole sulfoxide (FBZ-SO) and fenbendazole sulfone (FBZ-SO2) in trout muscle and skin tissues. The compounds were extracted with acetonitrile and the extract was concentrated and cleaned up by solid phase extraction on C18 and CN cartridges. The extract was analysed by reversed-phase gradient HPLC on a C18 column followed by ultraviolet detection at 290 nm. The method's detection limits were 4.0 micrograms kg-1 for FBZ, 4.5 micrograms kg-1 for FBZ-SO and 3.8 micrograms kg-1 for FBZ-SO2. The mean recovery in muscle was 88% for FBZ, 94% for FBZ-SO and 92% for FBZ-SO2 at a level of 5-150 micrograms kg-1. The corresponding mean recoveries in skin tissue were 88, 81 and 86% at a level of 10-100 micrograms kg-1. The mean relative repeatability standard deviation was 9.2% at a level of 5 micrograms kg-1, 5.9% at a level of 10-100 micrograms kg-1 and 2.3% at a level of 150 micrograms kg-1. The method was applied to trout given feed containing FBZ in an aquaculture pilot plant. The three compounds FBZ, FBZ-SO and FBZ-SO2 were all detected in muscle and skin tissues shortly after administration. The concentrations were generally highest in skin tissue.     

Detection of incurred dihydrostreptomycin residues in milk by liquid chromatography and preliminary confirmation methods.

An LC method for the determination of the aminoglycosides streptomycin (STR) and dihydrostreptomycin (DHS) in milk was developed/modified on the basis of published papers. Mean recoveries were 87 and 95% for STR and DHS, respectively. Recoveries are dependent on the concentration level and batch of solid-phase extraction columns used, and independent of fat content and homogenization. The relative standard deviations are 15.6 and 9.6% for STR and DHS, respectively, at a level of 100 micrograms kg-1. Limits of detection (8 and 12 micrograms kg-1, respectively) and quantification (12 and 18 micrograms kg-1, respectively) are far below the EU maximum residue limit of 200 micrograms kg-1. Lyophilized DHS samples can be used for internal control of the analysis as the DHS concentration is not influenced by the lyophilization process and subsequent storage at 6 degrees C. In incurred milk samples no false negative results of preliminary confirmation tests (Charm II Aminoglycoside test, Ridascreen Streptomycin ELISA) with respect to DHS concentrations > or = 20 micrograms kg-1 as determined by the LC method are observed. DHS concentrations of incurred samples determined by ELISA are higher than those obtained by the LC method. These differences were more pronounced with incurred than with spiked milk samples, thus leading to the conclusion that in incurred samples substances are present which co-react in the ELISA and which are not detected by the LC method.     

Residues of oxytetracycline and its 4'-epimer in edible tissues from turkeys

Residues of oxytetracycline (OTC) in edible tissues (muscle, liver, and kidney) of 18 turkeys were determined after continuous administration of the drug for 3 days in drinking water at the maximum recommended concentration of 400 mg/L. The European Union (EU) maximum residue limits (MRLs) set for OTC are 100 microg/kg in muscle tissues, 300 microg/kg in liver, and 600 microg/kg in kidney, as the sum of the parent compound and its derivative 4'-epi-oxytetracycline (4-epi-OTC). Cleanup of tissue samples was performed by metal chelate affinity chromatography (MCAC), but the original technique was miniaturized by the adoption of a mini solid-phase extraction column, allowing reduction of solvents, time, and hazardous waste. OTC and its 4'-epimer were quantitated by an isocratic liquid chromatography elution with UV detection. After 1 day of withdrawal, OTC plus 4-epi-OTC residues were greater than MRL values in muscle and liver; 3 days after the end of treatment, all tissue residues were far lower than the MRL values. At the first day after the end of treatment, 4-epi-OTC was detected at very low concentrations only in muscle, in liver after 1 and 3 days of withdrawal, and in kidney at all sampling times. The withdrawal time was calculated according to EU recommendations and was set at 5 days.     

Development and optimisation of an improved derivatisation procedure for the determination of avermectins and milbemycins in bovine liver

A robust procedure has been developed to overcome the instability problems experienced with the fluorescent derivative of eprinomectin. The procedure involves addition of acetic acid, together with the typical reagents methylimidazole and trifluoroacetic anhydride, to produce a fluorescent molecule that can be determined by high performance liquid chromatography (HPLC) with fluorescence detection. Derivatisation is completed in 30 min at 65 degrees C. This derivatisation procedure was shown to be suitable, also, for the related compounds, moxidectin, abamectin, doramectin and ivermectin. A multi-residue method for these compounds in bovine liver has been developed using the derivatisation procedure. Samples are extracted with acetonitrile; followed by clean-up on deactivated alumina and C18 solid phase extraction (SPE) cartridges. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery ranged between 73 and 97%. The intra- and inter-assay variations showed relative standard deviations typically of < 6% and < 14%, respectively. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).     

Determination of oxytetracycline, tetracycline, and chlortetracycline in milk by liquid chromatography with postcolumn derivatization and fluorescence detection

A multiresidue method for isolation and liquid chromatographic determination of oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) in milk is presented. The sensitivity of the method is adequate to meet the needs of regulatory agencies. The European Community established 100 micrograms/kg as the maximum residue limit (MRL) in milk for TC, CTC, and OTC. Recoveries exceeded 80% for all tetracyclines at all levels, with good precision. Correlation coefficients of standards curves for individual tetracyclines isolated from fortified samples ranged from 0.991 for CTC to 0.998 for OTC. Other antibiotics that might interfere with analysis did not interfere with elution times of OTC, TC, and CTC. The procedure is rapid, precise, and quantitative and requires minimal preparation and minimal use of organic solvents. It can be applied to routine surveillance programs. We can prepare 10 samples for analysis in about 1.45 h.     

Synthesis of penicillin analogs, (--)-methyl 6-epi-6-bromobisnorpenicillanate and (+/-)-methyl 6-epi-bromopenicillanate.

The [(4-methoxycarbonylthiazolidino)carbonyl-dihalomethyl]phenylmercury compounds 2a,b and 5a,b have been synthesized. When heated in refluxing bromobenzene they form halogenated penicillin analogs. (--)-Methyl 6-epi-6-bromobisnorpenicillinate (3a) and methyl 6-epi-6-bromopenicillinates (6a,b) have been prepared and isolated.     

New lanostane-type triterpene acids from wolfiporia extensa

ABSTRACT: BACKGROUD: Dried sclerotia of Wolfiporia extensa (Polyporaceae) is used to invigorate the spleen and to tranquilize the mind in Chinese herbal medicine. Lanostane-type triterpene acids were regard as major secondary metabolites from dried sclerotia of W. extensa. RESULTS: Three new lanostane-type triterpene acids, 3-epi-benzoyloxyl-dehydrotumulosic acid (1), 3-epi-(3'-O-methyl malonyloxy)-dehydrotumulosic acid (2) and 3-epi-(3'-hydroxy-3'-methylglutaryloxyl)-dehydrotumulosic acid (3), were isolated from the sclerotia of W. extensa, together with 3 known lanostane derivatives (4-6). Their structures were elucidated on the basis of spectroscopic analysis, including 1D and 2D-NMR techniques. CONCLUSION: Six lanostane derivatives including three new triterpene acids and three known compounds were reported from the sclerotia of W. extensa in this paper.     

Flubendazole residues in eggs after oral administration to laying hens: determination with reversed phase liquid chromatography.

Flubendazole residues in eggs were experimentally induced by providing groups of 8 laying hens feed with approximately 3, 10 and 30 mg kg-1 flubendazole for 21 days. Eggs were sampled during this period and one week after the administration. Samples of both whole egg and egg white/yolk were analysed separately. Flubendazole analysis was performed by reversed phase HPLC and UV detection at 250 nm (eggs) or 320 nm (feed). The limit of detection (LOD) for flubendazole in feed was 0.3 mg kg-1 and in whole egg 0.012 mg kg-1. Both the hydrolysed and reduced metabolites of flubendazole were also determined quantitatively. The eggs of control hens housed in the same room during the study period did not contain any detectable flubendazole or metabolite residue. The eggs from the lowest dosed group (3 mg kg-1 feed) did contain residues, but most of them were only slightly higher than the LOD. Residues in eggs collected from the laying hens that obtained feed with 10 and 30 mg kg-1 flubendazole reached a plateau level after some 10 days and there was a good dose response relation between levels in feed and those in eggs. The residues of parent compound and metabolites almost exclusively occurred in yolk, the metabolites accounting for some 60-65% of the total residue. The residues of the parent compound and its metabolites declined below 100 micrograms kg-1 5 days after the administration of dosed feed had ended.     

Multi-residue analysis of tranquillizers in meat: confirmatory assays using mass spectrometry.

A rapid and sensitive multi-residue method was developed to attempt to confirm the presence of the beta-blocker carazolol and the tranquillizers acepromazine, azaperone, chlorpromazine, propionylpromazine and xylazine in pig muscle tissues. The procedure involves determination by liquid chromatography coupled with tandem mass spectrometry. The liquid chromatographic separation was performed on a Symmetry C18 column with gradient elution. A mixture of aqueous buffer, containing 0.01% m/v trifluoroacetic acid (pH 3.5), and acetonitrile at a flow rate of 0.4 ml min-1 was used as the mobile phase. The abundant parent ions [M+ H+] produced by positive electrospray ionisation were selected for collisional dissociation with argon. Fragment ions were recorded with daughter ion scan and multiple reaction monitoring. The analytes were identified unambiguously by assessing retention times and diagnostic ions in meat samples spiked from 50 micrograms kg-1 [maximum residue limit (MRL) for azaperone and azaperol] to 5 micrograms kg-1 (MRL for carazolol).     

LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver.

European legislation forbids the use of beta-agonists as growth-promoting substances in cattle raised for human consumption. However, the use of beta-agonists is allowed as a therapeutic treatment of tocolysis for female cattle during calving and of respiratory diseases and tocolysis for horses not raised for human consumption. A maximum residue limit (MRL) of 0.5 microgram kg-1 for clenbuterol in the liver of cattle and horses is proposed by law. Residues of beta-agonists in liver are identified with LC-MS-MS. Using ion trap technology, it was possible to identify each analyte without the need to resolve completely the chromatographic peaks. For each analyte, specific fragment ion spectra were obtained. The coeluting or incompletely resolved peaks were separated mass spectrometrically. For tulobuterol, bromobuterol and mabuterol, qualitative information was obtained. All beta-agonists could be detected up to a concentration of 0.1 microgram kg-1. For clenbuterol, a limited quantitative validation was performed. A working range was defined for which the method was applicable. Quantification was based on the integration of the response of the analytes in spiked blank liver samples. The mean recovery was 15%. The relative standard deviation (RSD) values at different concentrations were below the maximum allowed RSD. The limit of detection of clenbuterol was 0.11 microgram kg-1. The limit of quantification was 0.21 microgram kg-1. It was possible to quantify clenbuterol below one-half of the MRL. The advantage of this method is the ease of use of the mass spectrometric separation to qualify and quantify the presence of four beta-agonists in liver.     

毛细管电泳法检测蜂蜜中残留的抗生素

 用毛细管电泳法对 5种常用的抗生素四环素 (TC) ,土霉素 (OTC) ,多西环素 (DOC) ,金霉素 (CTC)和氯霉素 (CP)进行了分离。研究了电泳缓冲液的 pH、不同有机试剂添加剂、温度等因素对抗生素分离的影响。实验结果表明 ,体积分数为 4%的N 甲基吗啡啉的加入可大大改善分离效果。在各自相应的浓度范围内 ,峰面积和样品浓度之间呈现良好的线性关系 ,重现性好。氯霉素的检测极限为 10 μg/L ,四环素、土霉素、多西环素为 2 0 μg/L ,金霉素为 40 μg/L(信噪比 >5 )。该方法成功地应用于蜂蜜中残留抗生素的测定 ,具有操作简单、快速方便、灵敏度及自动化程度高。     

Oxacillin residues in milk after drying off with Stapenor Retard TS.

Milk samples from 28 cows were analysed for residues of oxacillin after drying off with Stapenor Retard TS (oxacillin). Analysis was performed with an automated HPLC system consisting of an on-line solid-phase extraction and photochemical post-column derivatization with UV-detection at 300 nm. Although the time interval between treatment and parturition was less than the demanded 55 days, the maximum residue limit of 30 micrograms kg-1 was only exceeded in one case, in which the withdrawal time was 28 days.     

Immunochemical screening for antimicrobial drug residues in commercial honey.

Honey samples (n = 100; origin: various countries from Eurasia, Oceania, and the Americas) were analysed by enzyme immunoassays (EIA) for tetracyclines, streptomycin, and sulfathiazole. Considering antibody specificity, these EIAs are either quantitative (streptomycin) or qualitative (tetracyclines, sulfathiazole) tests. Honey extract purification was achieved by liquid-liquid partition (tetracyclines), and by solid phase extraction-immunoaffinity chromatography (streptomycin, sulfathiazole). Detection limits were 20 micrograms kg-1 (tetracycline equivalents), 10 micrograms kg-1 (streptomycin), and 50 micrograms kg-1 (sulfathiazole equivalents), with mean recoveries of 100-117%. A total of 42% of the samples was found positive by EIA; 25% were positive in one assay, 13% in two, and 3% were positive in all three tests. In the EIA for tetracyclines, 26% were positive, with 12 samples exceeding a level of 50 micrograms kg-1 (tetracycline equivalents). In the EIA for streptomycin, 19% were positive, with a mean concentration of 19 +/- 12 micrograms kg-1. In the sulfathiazole EIA, 16% of the samples were positive, with 13 samples exceeding a level of 100 micrograms kg-1 (sulfathiazole equivalents). However, when samples which were positive in the sulfathiazole EIA were reanalysed for sulfonamides by HPLC, no sulfa drugs could be detected. Experimental heating (40 degrees C) of honey spiked with sulfathiazole indicated that the sulfa drug(s) responsible for positive EIA results could be present a sugar derivatives.     

New labdane diterpenes from Solidago canadensis

The ethanol extract of roots of Solidago canadensis yielded eight labdane-type diterpenes. Five of those were new natural compounds (9,13,15,16-bisepoxy-labdane-7-ene-6,15-dione (3a), 13-epi-9,13,15,16-bisepoxy-labdane-7-ene-6,15-dione (3b), 15,16-epoxy-labdane-7,13-diene-6,16-dione (5), 15-ethoxy-9,13,15,16-bisepoxy-labdane-7-ene-6-one (6a) and 13-epi-15-ethoxy-9,13,15,16-bisepoxy-labdane-7-ene-6-one (6b). The known labdane diterpenes deoxysolidagenone (1), solidagenone (2) and 15,16-epoxy-labdane-7,13-diene-6,15-dione (4) were also isolated. Chemical structures were determined using 1D and 2D NMR techniques and MS analysis.     

Characterization and determination of organic compounds in the mutagenic XAD-2 extracts of drinking water.

Amberlite XAD-2 extracts, which exhibit mutagenicity in the Ames assays, of drinking water sampled each month during the period from April 1988 to March 1989 were studied in order to characterize and determine the organic pollutants. The major organic pollutants were phthalate ester plasticizers such as dibutyl and di(2-ethylhexyl) phthalate. Several polynuclear aromatic hydrocarbons (PAHs) and the organocholorine pesticide oxadiazon were also identified to be present at low concentrations. The XAD-extractable and chromatographable organic pollutants were found to be composed of PAHs with a mean concentration of 0.136 micrograms/l(ca. 10% of the total amount of organic compounds detected), phthalates with a mean value of 0.405 micrograms/l(ca. 30%) and other compounds with a mean value of 0.845 micrograms/l(ca. 60%). The concentrations and compositions of these organic pollutants were correlated with the effective rainfall content of the river and with the water temperature.     

Total synthesis of the originally proposed and revised structures of palmerolide A and isomers thereof

Palmerolide A is a recently disclosed marine natural product possessing striking biological properties, including potent and selective activity against the melanoma cancer cell line UACC-62. The total syntheses of five palmerolide A stereoisomers, including the originally proposed (1) and the revised [ent-(19-epi-20-epi-1)] structures, have been accomplished. The highly convergent and flexible strategy developed for these syntheses involved the construction of key building blocks 2, 19-epi-2, 20-epi-2, ent-2, 3, ent-3, 4, and ent-4, and their assembly and elaboration to the target compounds. For the union of the building blocks, the Stille coupling reaction, Yamaguchi esterification, Horner-Wadsworth-Emmons olefination, and ring-closing metathesis reaction were employed, the latter being crucial for the stereoselective formation of the macrocycle of the palmerolide structure. The Horner-Wadsworth-Emmons olefination and the Yamaguchi lactonization were also investigated and found successful as a means to construct the palmerolide macrocycle. The syntheses were completed by attachment of the enamide moiety through a copper-catalyzed coupling process.     

液相色谱-四极杆/ 飞行时间质谱快速筛查与确证苹果、番茄和甘蓝中的281种农药残留

建立了苹果、番茄和甘蓝中281种农药残留的QuEChERS结合液相色谱-四极杆/飞行时间质谱(LC-Q-TOF/MS)快速筛查方法。方法采用1%醋酸乙腈提取样品,经丙基乙二胺(PSA)净化,LC-Q-TOF/MS测定。281种农药在苹果、番茄和甘蓝中3个添加水平下的回收率在70%～120%范围的比例分别为98.6%、99.3%和98.2%;回收率的相对标准偏差(RSD)均≤20%(n=5);在0.25～10倍最大残留限量(MRL)的含量范围内,线性相关系数r²≥0.99的农药比例分别为95.7%、96.1%和98.2%;检出限分别为0.03～4.47、0.01～4.49和0.02～3.61 μg/kg。本方法采用精确质量数据库和谱图库检索的方式,实现了不用农药标准品对照而完成对果蔬中农药残留的快速筛查,提高了定性鉴别的准确性。应用所建立的方法对30个市售果蔬样品进行筛查,共检出13种农药残留。其中,甘蓝中甲胺磷的含量超过了GB 2763-2012《食品安全国家标准\5食品中农药最大残留限量》和欧盟的MRL;番茄中鱼藤酮的含量超出欧盟的MRL。     

液相色谱-四极杆/ 飞行时间质谱快速筛查与确证苹果、番茄和甘蓝中的281种农药残留

建立了苹果、番茄和甘蓝中281种农药残留的QuEChERS结合液相色谱-四极杆/飞行时间质谱(LC-Q-TOF/MS)快速筛查方法。方法采用1%醋酸乙腈提取样品,经丙基乙二胺(PSA)净化,LC-Q-TOF/MS测定。281种农药在苹果、番茄和甘蓝中3个添加水平下的回收率在70%～120%范围的比例分别为98.6%、99.3%和98.2%;回收率的相对标准偏差(RSD)均≤20%(n=5);在0.25～10倍最大残留限量(MRL)的含量范围内,线性相关系数r²≥0.99的农药比例分别为95.7%、96.1%和98.2%;检出限分别为0.03～4.47、0.01～4.49和0.02～3.61 μg/kg。本方法采用精确质量数据库和谱图库检索的方式,实现了不用农药标准品对照而完成对果蔬中农药残留的快速筛查,提高了定性鉴别的准确性。应用所建立的方法对30个市售果蔬样品进行筛查,共检出13种农药残留。其中,甘蓝中甲胺磷的含量超过了GB 2763-2012《食品安全国家标准\5食品中农药最大残留限量》和欧盟的MRL;番茄中鱼藤酮的含量超出欧盟的MRL。