Determination of eight sulfonamides in bovine kidney by liquid chromatography/tandem mass spectrometry with on-line extraction and sample clean-up

A sensitive, high performance liquid chromatography/tandem mass spectrometric (i.e. mass spectrometry/mass spectrometry; LC/MS/MS) method with on-line extraction and sample clean-up for the screening and confirmation of residues of sulfonamides in kidney is described. The sulfonamides are extracted from homogenized kidney with methanol. After centrifugation of the extract, an aliquot of the extract is directly injected on the LC/MS/MS system with further extraction and clean-up of the sample on-line. Detection of the analytes was achieved by positive electrospray ionization (ESI) followed by multiple reaction monitoring. For each sulfonamide the collisional decomposition of the protonated molecule to a common, abundant fragment ion was monitored. The method has been validated for sulfadimethoxine, sulfaquinoxaline, sulfamethazine, sulfamerazine, sulfathiazole, sulfamethoxazole, sulfadiazine and sulfapyridine. Calibration curves resulting from spiked blank kidney samples at the 10-200 microg/kg level showed good linear correlation. At the level of 50, 100 and 200 microg/kg both within- and between-day precision, as measured by relative standard deviation (RSD), were less than 16%. The limits of detection (LODs) ranged from 5 to 13.5 microg/kg. The recoveries ranged from 78 to 82%. The procedure provides a rapid, reliable and sensitive method for the determination of residues of sulfonamides in bovine kidney. The advantage of this method over existing methods is its decreased sample preparation and analysis time, which makes the method more suitable for routine analysis.     

Quantitative determination of sulfonamide residues in foods of animal origin by high-performance liquid chromatography with fluorescence detection

An HPLC method with fluorescence detection is proposed for the quantitative determination of residues of ten of the most used sulfonamides as their derivatives. Sulfonamides were isolated from meat, mix meat and kidney with ethyl acetate (first extraction) and acetone (second extraction) and further purified by partitioning three times with water-methylene chloride. The recovery for mix meat spiked with 1, 5 and 10 microg/kg of sulfonamides averaged 64%, 68% and 75%, respectively. Limits of quantitation were 1 microg/kg for sulfaquinoxaline and 0.5 microg/kg for the remaining sulfonamides.     

Determination of tilmicosin residues in chicken, cattle, swine, and sheep tissues by liquid chromatography

A method was developed and validated for determination and quantitation of tilmicosin residues in swine, cattle, and sheep edible tissues, as well as chicken fat, skin, and muscle over a concentration range of 0.025 microg/g-20 microg/g. For chicken kidney and liver, the method was validated over a range of 0.060 microg/g-20 microg/g. The tissue sample was extracted with methanol and a C18 cartridge was used for solid-phase extraction cleanup. A reversed-phase gradient liquid chromatographic method with detection at 280 nm was used to separate the tilmicosin from matrix components in 30 min run time. The limit of quantitation (LOQ) of the method was 0.025 microg/g for all tested tissues except chicken kidney and liver, for which the LOQ was 0.06 microg/g. Average recoveries for tissue samples ranged from 73 to 98%. Relative standard deviation values ranged from 0.6 to 14.7%.     

Multiresidue determination of sulfonamides in a variety of biological matrices by supported liquid membrane with high pressure liquid chromatography-electrospray mass spectrometry detection

A high performance liquid chromatography (HPLC) coupled to a mass spectrometer (MS) was used for a simultaneous determination of 16 sulfonamide compounds spiked in water, urine, milk, and bovine liver and kidney tissues. Supported liquid membrane (SLM) made up of 5% tri-n-octylphosphine oxide (TOPO) dissolved in hexyl amine was used as a sample clean-up and/or enrichment technique. The sulfonamides mixture was made up of 5-sulfaminouracil, sulfaguanidine, sulfamethoxazole, sulfamerazine, sulfamethizole, sulfamethazine (sulfadimidine), sulfacetamide, sulfapyridine, sulfabenzamide, sulfamethoxypyridazine, sulfamonomethoxine, sulfadimethoxine sulfasalazine, sulfaquinoxaline, sulfadiazine, and sulfathiazole. Some of these compounds, such as, sulfaquinoxaline, sulfadiazine, sulfabenzamide, sulfathiazole and sulfapyridine failed to be trapped efficiently by the same liquid membrane (5% TOPO in hexylamine). The detection limits (DL) obtained were 1.8 ppb for sulfaguanidine and sulfamerazine and between 3.3 and 10 ppb in bovine liver and kidney tissues for the other sulfonamides that were successfully enriched with SLM; 2.1 ppb for sulfaguanidine and sulfamerazine and between 7.5 and 15 ppb in cow’s urine, whereas the DL values in milk were 12.4 ppb for sulfaguanidine and sulfamerazine and between 16.8 and 24.3 for the other compounds that were successfully enriched by the membrane. Several factors affecting the extraction efficiency during SLM enrichment, such as donor pH, acceptor pH, enrichment time and the membrane solvent were studied.     

Application of ion-exchange cartridge clean-up in food analysis. V. Simultaneous determination of sulphonamide antibacterials in animal liver and kidney using high-performance liquid chromatography with ultraviolet and mass spectrometric detection

A simple, rapid, and reliable method for the determination of residual sulphonamide antibacterials (SAs) (sulfadiazine, sulfamerazine, sulfadimidine, sulfamethoxypiridazine, sulfisozole, sulfamonomethoxine, sulfamethoxazole, sulfisoxazole, sulfadimethoxine, and sulfaquinoxaline) in animal liver and kidney was developed using a combination of clean-up on a Bond Elut PSA cartridge and HPLC with UV detection. The SAs were extracted with ethyl acetate and then dissolved in 5 ml of 50 v/v% ethyl acetate-n-hexane after being evaporated to dryness. For clean-up of the crude sample, the resuspended extract was applied to a Bond Elut PAS (primary/secondary amine cartridge), and then SAs were eluted from the cartridge using 5 ml of 20 v/v% acetonitrile-0.05 M ammonium formate before being analysed by HPLC. Recoveries of the SAs at the levels of 0.5 and 0.1 microg/g were 70.8-98.2%, the rerative standard deviation were less than 7.0%, and the detection limits were 0.03 microg/g. The present analysis method of SAs in animal kidney and liver using HPLC with a clean-up procedure was demonstrated to be highly applicable to the direct LC-MS-MS analysis without any modification.     

Determination of 10 sulfonamide residues in meat samples by liquid chromatography with ultraviolet detection

A liquid chromatography (LC) method is described for the simultaneous determination of 10 commonly used sulfonamide drug residues in meat. The 10 sulfonamide drugs of interest were sulfadiazine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethizole, sulfamonomethoxine, sulfachloropyridazine, sulfadoxine, sulfadimethoxine, and sulfaquinoxaline. The residues were extracted with acetone-chloroform (1 + 1). Sulfonamides were quantitatively retained in the extracting solution and afterwards eluted from a cation-exchanger solid-phase extraction cartridge with a solution of methanol-aqueous ammonia. The solution was dried, reconstituted with 5 mL methanol and filtered before analysis by LC-ultraviolet using a C18 column with a mobile phase gradient of potassium dihydrogen phosphate buffer, pH 2.5, and methanol-acetonitrile (30 + 70, v/v). The method was applied to cattle, swine, chicken, and sheep muscle tissues. The validation was performed with a fortified cattle meat sample at level of 100 ppb, which is the administrative maximum residue limit for sulfonamides in the European Union. The limit of quantitation for all sulfonamides was between 3 and 14 ppb. Recovery was evaluated for different meat matrixes. The mean recovery values were between 66.3% for pork meat samples and 71.5% for cattle meat samples.     

超高效液相色谱法检测化妆品中的12种磺胺抗生素

建立了采用超高效液相色谱(UPLC)-二极管阵列检测器（PDA）测定化妆品中12种常见的磺胺抗生素(磺胺、磺胺间甲氧嘧啶、磺胺醋酰、磺胺甲基异唑、磺胺嘧啶、磺胺二甲异唑、磺胺噻唑、磺胺二甲氧嘧啶、磺胺甲基嘧啶、磺胺喹啉、磺胺二甲嘧啶、磺胺硝苯)的方法。采用Acquity UPLCTM BEHC C18 色谱柱(50 mm×2.1 mm, 1.7 μm),流动相为乙腈/0.1%的甲酸水溶液,梯度洗脱。样品经提取、反萃取后,用UPLC-PDA进行分析检测,结合保留时间和紫外光谱进行定性分析,定量检测波长268 nm。12种磺胺的检出限(S/N=3)均为1 μg/g,定量下限(S/N=10)为2～3 μg/g,在1～25 mg/L(磺胺硝苯为0.5～12.5 mg/L)范围内,峰面积和质量浓度的线性关系良好(r>0.9997)。添加水平为40, 8 μg(磺胺硝苯为20, 4 μg)时,12种磺胺的平均回收率分别为86.8%～98.1%和80.1%～96.9%,相对标准偏差小于10%(n=6)。结果表明该方法简单,分离效果好,速度快,能够满足检测化妆品中12种常见的磺胺抗生素的需要。     

High-performance liquid chromatographic procedure for routine residue monitoring of seven sulfonamides in milk

A simplified method for routine monitoring of 7 residual sulfonamides (SAs) (sulfadiazine (SDZ), sulfamerazine (SMR), sulfadimidine (SDD), sulfamonomethoxine (SMM), sulfamethoxazole (SMX), sulfadimethoxine (SDM), and sulfaquinoxaline (SQ)) in milk using high-performance liquid chromatography (HPLC) with a photodiode array detector is described. The spiked and blank samples were cleaned up by using an Ultrafree-MC/PL centrifugal ultrafiltration unit. For determination/identification, a Mightysil RP-4 GP column and a mobile phase of 25% (v/v) ethanol in water with a photodiode array detector were used. Average recoveries from milk samples spiked with 0.05, 0.1, 0.2, and 0.5 microg mL(-1) of each drug were >82%. The inter- and intra-assay variabilities were 2.0-3.1%. The practical detection limits for 7 SAs were 0.005-0.02 microg mL(-1). The total time and amount of solvent required for the analysis of one sample were <40 min and <6 mL of ethanol, respectively. No toxic solvents were used.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

Simplified high-performance liquid chromatographic determination of residual amprolium in edible chicken tissues

A simplified determining method for the routine monitoring of residual amprolium in edible chicken tissues (muscle and liver) is developed using a high-performance liquid chromatographic (HPLC) method with a photodiode-array detector after sample cleanup by an Ultrafree-MC/PL centrifugal ultrafiltration unit. For the HPLC determination and identification, a Mightysil RP-4 GP column and a mobile phase of an ethanol-5 mM 1-heptanesulfonic acid sodium salt solution (35:65, v/v) using an ion-pairing system with a photodiode-array detector are used. Average recoveries (spiked at 0.3-3.0 microg/g) are > 90%. The inter- and intravariabilities are 1.9-2.4%. The limits of quantitation are 0.22 microg/g for muscle and 0.25 microg/g for liver. The total time and solvent required for the analysis of one sample are < 20 min and < 2 mL of ethanol, respectively. No toxic solvents and regents are used.     

A clean and rapid liquid chromatographic technique for sulfamethazine monitoring in pork tissues without using organic solvents

A rapid method for the isolation and high-performance liquid chromatographic (HPLC) determination of sulfamethazine (SMZ) in pork tissues (kidney, liver, and muscle) without using organic solvents is developed. The isolation is performed by homogenization with an acid solution using an ultrasonic-homogenizer, followed by centrifugation. The HPLC analyses are performed using a reversed-phase C(4) column (150- x 4.6-mm i.d.), a mobile phase of 0.02 mol/L citric acid solution, and a photodiode array detector. The resulting HPLC chromatograms are free from interferences for determination and identification. The proposed technique is shown to be linear (r > 0.99) over the concentration range 0.1-2.0 microg/g for all pork tissues. Average recoveries of SMZ (spiked 0.1-2.0 microg/g) range from 87.6% to 90.2%, with inter- and intra-assay variabilities of less than 4%. The total time required for the analysis of one sample and limit of quantitation is less than 20 min and 0.09 microg/g, respectively.     

固相萃取-高效液相色谱法同时测定牛奶中残留的9种磺胺类药物

建立了同时测定牛奶中残留的9种磺胺类药物的固相萃取-高效液相色谱分析方法。牛奶样品经磷酸盐缓冲液稀释后高速离心去除脂肪,过C18小柱,用水淋洗,甲醇洗脱,洗脱液经氮气吹干后用乙酸乙酯溶解,并过氨基固相萃取小柱净化,用正己烷及水淋洗,以甲醇-乙腈-水（含1%乙酸）(体积比为1∶1∶8)洗脱,洗脱液用于高效液相色谱分析。采用Inertsil ODS-3 C18柱分离,以水-乙酸和甲醇-乙腈为流动相进行梯度洗脱,二极管阵列检测器检测,外标法定量。9种磺胺类药物标准曲线的线性回归系数均在 0.9999 以上,线性范围为25～5000 μg/L,检出限为1.7～2.8 μg/L,定量限为5.7～9.2 μg/L。在10,20,40 μg/L 添加水平下的添加回收率为72.1%~88.3%,相对标准偏差为2.3%～5.0%。该方法具有快速、灵敏的特点,符合现行兽药残留分析的要求。     

Pre-column derivatization of sulfa drugs with fluorescamine and high-performance liquid chromatographic determination at their residual levels in meat and meat products.

A rapid, sensitive and selective high-performance liquid chromatographic method is described for simultaneous determination of eight sulfa drugs in meat and meat products using pre-column derivatization with fluorescamine. The drugs are sulfisomidine, sulfadiazine, sulfamerazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline. The method includes blender extraction of 3-g samples with chloroform, partition with 3 M hydrochloric acid, derivatization with fluorescamine at pH 3.0 and subsequent high-performance liquid chromatographic analysis on a C18 column with fluorescence detection at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. The drugs were separated with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55 degrees C. The average recovery from samples fortified at 0.1 ng/g was 92.6% with a coefficient of variation of 6.2%. The detection limit was 0.01 ng/g for sulfaquinoxaline and 0.005 ng/g for the other seven drugs. The method was field-tested in a survey of 37 samples including beef (five), pork (seven), chicken (seven), ham (five), sausage (eight), bacon (two) and roast beef (three). Sulfadimidine was detected in one pork sample at the level of 0.295 ng/g and in ham at 0.178 ng/g.     

固相萃取–高效液相色谱法同时测定饲料中4种磺胺类药物残留

建立固相萃取–高效液相色谱法同时测定饲料中的磺胺嘧啶、磺胺二甲嘧啶、磺胺甲恶唑、磺胺喹恶啉4种磺胺类药物残留的方法。样品用乙腈提取,然后用碱性氧化铝固相萃取柱净化,色谱柱为C_(18)柱(250 mm×4.6mm,5μm),以水–乙腈(体积比为75∶25,含0.3%乙酸)为流动相,流量为1.0 mL/min,检测波长为270 nm。4种磺胺类药物的质量浓度在1~10μg/mL范围内与其色谱峰面积呈良好的线性,相关系数均大于0.999,检出限为0.025~0.133μg/g。磺胺类药物测定结果的相对标准偏差为0.22%~0.30%(n=6),样品加标回收率为93.6%~106.7%。实际饲料样品中均未检出这4种磺胺组分。该方法具有干扰少,灵敏度高,重复性好的优点,可以作为饲料中的磺胺类药物残留的一种检测方法。     

High-performance liquid chromatographic determination of penicillin G, penicillin V and cloxacillin in beef and pork tissues.

The objective was to develop confirmatory high-performance liquid chromatographic methods for penicillin residues in animal tissues with detection limits of less than or equal to 10 ng/g. A previously described procedure was modified by using a larger sample size and isocratic analysis. Tissues (15 g) were blended with 45 ml of water and 20 ml of homogenate were mixed with 40 ml acetonitrile and filtered. The filtrate (30 ml) was mixed with 10 ml of 0.2 M H3PO4 and extracted with methylene chloride. The combined methylene chloride layers were mixed with acetonitrile and hexane, washed with two 4-ml portions of water and then extracted with four 1-ml portions of 0.01 M phosphate buffer (pH 7). The combined buffer extracts were concentrated to 1 ml under reduced pressure. Analysis was isocratic during 0.01 M phosphate buffer (pH 7)-acetonitrile with proportions 85:15 (penicillin G), 82:18 (penicillin V) or 78:22 (cloxacillin). A polystyrene-divinylbenzene copolymer column, 150 x 4.6 mm I.D. (Polymer Labs. PLRP-S), was used with a flow-rate of 1 ml/min and detection at 210 nm. The presence of penicillins was confirmed by treating a duplicate sample with penicillinase. Recoveries were greater than 90% in most instances. Detection limits were 5 ng/g in muscle and higher in liver and kidney. The procedure is a simple and sensitive method for confirming the presence of penicillins in animal tissues.     

流动注射在线预富集-高效液相色谱法测定水产品中4种磺胺类药物

建立了以活性碳纤维作吸附剂,流动注射在线预富集与高效液相色谱联用法测定水产品中4种磺胺类药物(磺胺噻唑、磺胺二甲嘧啶、磺胺间甲氧嘧啶、磺胺喹啉)残留含量的方法。样品经磷酸盐缓冲溶液提取后,采用活性碳纤维作吸附剂流动注射在线预富集,在pH 3.0的甲酸溶液-甲酸甲醇溶液(60∶40,V/V)的流动相体系中,用Eclipse XDB-C18色谱柱(250 mm×4.6 mm,5μm)分离,流速1.0 mL/min;检测波长270nm。结果表明,4种磺胺类药物在0.001～0.064 mg/L浓度范围内呈良好的线性关系,相关系数r为0.9997～0.9999;检出限为0.48～1.15μg/L(S/N=3);富集倍数在137～406之间;加标回收率为93.5%～104.0%。本方法操作简便、结果准确、灵敏度高。     

Determination of sulfonamides in swine meat by immunoaffinity chromatography

A procedure was developed for the preparation of anti-sulfonamide (SA) group-specific antibodies and immunosorbents. Sulfonamide haptens and conjugates were synthesized by building spacer arms on an N1 group of 4-aminobenzensulfonamide. The anti-SA group-specific antibodies and immunosorbents were prepared successfully. After extraction with methanol-water (8 + 2), sulfamonomethoxine, sulfadimethoxine, and sulfaquinoxaline were cleaned up on immunoaffinity columns and determined by reversed-phase liquid chromatography with UV detection at 270 nm. The recoveries from fortified swine meat (10-100 microg/kg) ranged from 70.8 to 94.1%, with coefficients of variation of 3.4-12.9%. Limits of detection were 1-2 microg/kg.     

Development of liquid chromatographic methods for determination of quinocetone and its main metabolites in edible tissues of swine and chicken

Quinocetone (QCT), a new antimicrobial growth promotant of quinoxalines, can effectively improve the growth and feed efficiency of food animals with more safety than is provided by olaquindox and carbadox. To clarify its metabolism and residue levels in animals, a liquid chromatographic method with UV-Vis detection was developed for the determination of QCT and its main metabolites, desoxyquinocetone (DQCT) and 3-methylquinoxaline-2-carboxylic acid (MQCA), in muscle, liver, kidney, and fat of swine and chicken. For sample pretreatment, QCT and DQCT were extracted with ethyl acetate and purified with iso-octane; after alkaline hydrolysis of the tissue, MQCA was extracted with ethyl acetate and citric acid buffer (pH 6.0), and the extract was purified over a cation-exchange column (AG MP-50 resin). Detection was at 312 and 320 nm for QCT and DQCT, respectively, and at 320 nm for MQCA. The observed limit of detection for the 3 compounds was 0.025 microg/g in various tissues. The methods were linear over the concentrations range of 0.01-0.64 microg/mL with mean recoveries of approximately 71-86% and relative standard deviations of about 4-12% at the levels of 0.05, 0.10, and 0.20 microg/g. The method is highly selective and can be applied to the determination of QCT and its main metabolites in animal tissues, which would accelerate the pharmacokinetic and residue study of QCT in food animals.     

固相萃取-高效液相色谱法测定动物肉组织中磺胺类药物的残留

建立了一种快速、灵敏、环保的固相萃取-反相高效液相色谱同时分析动物肉组织中5种磺胺类药物残留的方法。将样品加入到盛有无水硫酸钠的离心管中,再用乙酸乙酯提取;提取液经氨基固相萃取柱净化后,用1.5%（体积分数）乙酸乙醇溶液洗脱。洗脱液用高效液相色谱分离,二极管阵列检测器检测,外标法定量。5种磺胺类药物的线性关系良好,磺胺二甲基嘧啶(SM2)、磺胺间甲氧嘧啶(SMM)、磺胺甲唑(SMZ)的线性范围均为30～5000 μg/L,磺胺二甲氧嘧啶(SDM)、磺胺喹啉(SQ)的线性范围均为60～5000 μg/L。2种动物肉组织(鸡肉、猪肉)中5种磺胺类药物的加标回收率在73.2%至97.3%范围内,当添加水平为50 μg/kg时,加标回收率的相对标准偏差在2.5%至11.6%范围内;SM2,SMM和SMZ的检测限（S/N=3）和定量限（S/N=10）分别为3 μg/kg和10 μg/kg,SDM和SQ的检测限和定量限分别为7 μg/kg和25 μg/kg。     

Confirmatory method for macrolide residues in bovine tissues by micro-liquid chromatography-tandem mass spectrometry

A new confirmatory method for three macrolides (tylosin, tilmicosin and erythromycin) in bovine muscle, liver and kidney by micro-LC-MS-MS using an atmospheric pressure ionisation source and an ionspray interface has been developed. Roxithromycin was used as internal standard. The molecular related ions, [M+2H]2+, at m/z 435 for tilmicosin, and [M+H]+, at m/z 734 and 916 for erythromycin and tylosin, respectively, were the precursor ions for collision-induced-dissociation and two diagnostic product ions for each macrolide were identified for the unambiguous confirmation by selected reaction monitoring LC-MS-MS. Precision values (relative standard deviations) were all below 14.9%, whereas the overall accuracy (relative error) ranged from -17.7 to -9.8% for tylosin, from -17.5 to -10.7% for tilmicosin and from -19.6 to -13.7% for erythromycin, in all the investigated bovine tissues. The limits of quantification were 30 (muscle) or 40 (liver, kidney) microg kg(-1), 20 (muscle) or 150 (liver, kidney) microg kg(-1), 50 (muscle, liver) or 80 (kidney) microg kg(-1), 20 (muscle, liver) or 50 (kidney) microg kg(-1) for tylosin, tilmicosin, erytromycin and roxithromycin, respectively.