Application of a tandem mass spectrometer and core-shell particle column for the determination of 151 pesticides in grains

A comparison of ultrahigh performance liquid chromatography (UHPLC) with a 2.6 μm core-shell particle column (Kinetex C(18)) and conventional liquid chromatography (LC) with a 3 μm porous particle column (Atlantis dC(18)), coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS), for the determination of 151 pesticides in grains is presented in this study. Pesticides were extracted from grain samples using a procedure known as QuEChERS (quick, easy, cheap, effective, rugged, and safe). Quantification, with an analytical range from 5 to 500 μg/kg, was achieved using matrix-matched standard calibration curves with isotopically labeled standards or a chemical analogue as internal standards. The method performance parameters that included overall recovery, intermediate precision, and measurement uncertainty were evaluated using a designed experiment, that is, the nested design. The UHPLC (Kinetex C(18)) was superior to conventional LC (Atlantis dC(18)) as it yielded a shorter analytical run time, increased method sensitivity, and improved method performance. For UHPLC/ESI-MS/MS (Kinetex C(18)), 90% of the pesticides studied had recoveries between 81 and 110%, 88% of the pesticides had intermediate precision ≤20%, and 84% of the pesticides showed measurement uncertainty ≤40%. As compared to UHPLC/ESI-MS/MS (Kinetex dC(18)), the LC/ESI-MS/MS (Atlantis dC(18)) showed a relatively lower sensitivity, less repeatability, and larger measurement uncertainty. UHPLC/ESI-MS/MS with 2.6 μm core-shell particle column and scheduled MRM proved to be a good choice for quantification or determination of pesticides in grains.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue

A method for isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and oxytetracycline-fortified fish muscle tissue samples (0.5 g) were blended with octadecylsllyl (C18, 40 microns, 18% load, endcapped) derivatized silica packing material (2 g) containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetate. A column made from the C18/fish tissue matrix was first washed with hexane (8 mL), following which the oxytetracycline was eluted with acetonitrile-methanol (1 + 1, v/v) containing 0.06% w/v each of butylated hydroxyanisole and butylated hydroxytoluene. The eluate contained oxytetracycline analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array set at 365 nm). Standard curves for oxytetracycline isolated from fortified samples were linear (0.998 +/- 0.002) with an average absolute percentage recovery of 80.9 +/- 6.6% for the concentration range (50, 100, 200, 400, 800, 1600, and 3200 ng/g) examined. The interassay variability was 11.3 +/- 5.2% with an intra-assay variability of 1.1%.     

Determination of vitamin B(6) in cooked sausages

A reverse-phase high-performance liquid chromatography (HPLC) method has been described for the determination of various active forms of vitamin B(6) in meat products. Different extracting agents were tested to solubilize fully the analyte for quantification. The best data were obtained by extracting the samples with 5% (w/v) metaphosphoric acid. Separation by HPLC was performed with fluorescence detection (excitation, 290 nm; emission, 395 nm), on a 10 cm x 0.46 cm i.d. Hypersil BDS C(18) 5 microm column using a mixture of 50 mM phosphate buffer (pH 3.2) and acetonitrile (99:1, v/v) as mobile phase. Precision of the method was 0.5% (within a day) and 4.3% (between days). The detection limits were 0.020 mg/100 g for pyridoxal and pyridoxamine, 0.017 mg/100 g for pyridoxamine phosphate, 0.500 mg/100 g for pyridoxal phosphate, and 0.033 mg/100 g for pyridoxol, with a signal-to-noise ratio of 3. The recovery ranged from 92.0 to 100.0%.     

Simultaneous determination of chloramphenicol and aflatoxin M1 residues in milk by triple quadrupole liquid chromatography-tandem mass spectrometry

A reliable, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of chloramphenicol and aflatoxin M(1) in milk has been developed. This method includes simple extraction of sample with acetonitrile, separation on a MGIII-C(18) column using 5 mM ammonium acetate aqueous solution/methanol (60:40, v/v) as mobile phase, and MS/MS detection using multiple reaction monitoring mode. The method was validated according to Commission Decision 2002/657/EC. The limits of detection (LODs) were 0.05 μg/kg for chloramphenicol and 0.005 μg/kg for aflatoxin M(1.) The limits of quantification (LOQs) were 0.2 μg/kg for chloramphenicol and 0.02 μg/kg for aflatoxin M(1). The recovery values ranged from 88.8% to 100.6%, with relative standard deviation lower than 15% in all cases, when samples were fortified at three different concentrations. The decision limits (CCα) and detection capability (CCβ) of the method were also reported. This method has been successfully applied for simultaneous analysis of chloramphenicol and aflatoxin M(1) residues in milk from local supermarkets in China.     

Analysis of ochratoxin a in coffee by solid-phase cleanup and narrow-bore liquid chromatography-fluorescence detector-mass spectrometry

A method for the analysis of ochratoxin A (OTA) in green and roasted coffee has been developed. OTA was extracted from coffee with 1% NaHCO(3), and the extract was filtered and purified by solid-phase cleanup using a polymeric column that exhibits reversed-phase and anion-exchange functionalities. OTA was analyzed on a narrow-bore reversed-phase C(18) HPLC column with acetonitrile/water (0.1% formic acid) (40:60) as mobile phase and quantified with a fluorescence detector. The presence of OTA in coffee was confirmed by single-quadruple mass spectrometry using an electrospray ionization source. The method has been validated, obtaining a recovery of 82.5% and a detection limit of 0.1 ng/g. It has been applied to 20 coffee samples from various countries and different manufacturers with no detection of OTA.     

Liquid chromatographic determination of nicarbazin in feed

A liquid chromatographic method was developed for the determination of nicarbazin (4,4'-dinitrocarbanilide.2-hydroxy-4,6-dimethylpyrimidine) in chicken feed. Ground feed was extracted with hot dimethylformamide, filtered, and then cleaned up on an alumina column. The nicarbazin was eluted from the column with ethanol and quantitated using a reverse phase C-18 column, with a methanol-water mobile phase and ultraviolet detection at 344 nm. Recoveries at a typical use level of 100 micrograms/g feed averaged 98% with a standard deviation of 3%. Samples fortified at levels as low as 0.1 micrograms/g were analyzed with 92% recovery. The detection limit is 1 ng, and the response is linear between 4 and 1000 ng. Feed additives in combination with nicarbazin do not interfere with recovery.     

Simultaneous determination of ethidimuron, methabenzthiazuron, and their two major degradation products in soil

An analytical method has been developed for the quantification of two herbicides (ethidimuron and methabenzthiazuron) and their two main soil derivatives. This method involves fluidized-bed extraction (FBE) prior to cleanup and analysis by reverse-phase liquid chromatography with UV detection at 282 nm. FBE conditions were established to provide efficient extraction without degradation of the four analytes. (14)C-labeled compounds were used for the optimization of extraction and purification steps and for the determination of related efficiencies. Extraction was optimal using a fexIKA extractor operating at 110 degrees C for three cycles (total time = 95 min) with 75 g of soil and 150 mL of a 60:40 v/v acetone/water mixture. Extracts were further purified on a 500 mg silica SPE cartridge. Separation was performed on a C18 Purosphere column (250 mm x 4 mm i.d.), at 0.8 mL min(-1) and 30 degrees C with an elution gradient made up of phosphoric acid aqueous solution (pH 2.2) and acetonitrile. Calibration curves were found to be linear in the 0.5-50 mg L(-1) concentration range. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. Recovery values, determined from spiked samples, were close to 100%. Limits of detection ranged between 2 and 3 microg kg(-1) of dry soil and limits of quantification between 8 and 10 microg kg(-1) of dry soil. An attempt to improve these performances by using fluorescence detection following postcolumn derivatization by orthophthalaldehyde-mercaptoethanol reagent was unsuccessful.     

Modified liquid chromatographic method for determination of gentian violet in animal feed

A modified liquid chromatographic method is described for the determination of Gentian Violet (GV) in animal feed. The reliable detection limit is 0.5 ng (reference standards), and 1 ppm GV was reliably determined in feed. The calibration curve was linear between 1 and 40 micrograms/mL. The method, developed in a study by the National Center for Toxicological Research, was modified to use methanol-water (9 + 1) instead of benzene-methanol as the eluting solution in the column cleanup. GV is extracted from feed with methanol-1N HCl (99 + 1), cleaned up on a Sephadex LH-20 column to remove any remaining interferences, separated on a Nova-Pak C18 column fitted with a precolumn filter, and determined at 588 nm. The identity of GV is confirmed by thin-layer chromatography (Rf = 0.47) by comparison with a reference standard. Average recoveries from 3 sets of 5 feed samples containing 2.5, 5.0, and 10.0 ppm GV were 115, 95, and 102%, respectively.     

Liquid chromatographic determination of tetracycline residues in animal feeds

A liquid chromatographic method for the multiresidue determination of tetracyclines (TCs) in feeds is described. The levels of quantitation were 10 ppm each for tetracycline-HCl (TC), oxytetracycline (OTC), and chlortetracycline-HCl (CTC); the detection limit was 40 ppb for each. The calibration curves were linear between 2.5 and 100 ppm. The procedure involved double extraction with pH 2.0 and pH 4.5 McIlvain buffers, cleanup on a Sephadex LH-20 column, separation on a Nova-Pak C18 column, and detection at 370 nm. Recoveries of 10 micrograms/g of each TC in multiresidue feed samples ranged from 55.8 to 75.5% for OTC, 71.6 to 100% for TC, and 22.4 to 60.6% for CTC. The identities of the TCs were confirmed by thin layer chromatography.     

基于离子排斥色谱的饲料酸化剂中多种有机酸同步检测

为了规范饲用酸化剂的使用和管理,建立一种快速、准确的多种有机酸同步检测方法应需而生。该文以实现饲料酸化剂中多种有机酸的同步检测为目标,基于离子排斥色谱技术,对饲料酸化剂中多种有机酸(柠檬酸、酒石酸、苹果酸、乳酸、甲酸、乙酸、富马酸、丙酸和丁酸)的检测条件进行了优化研究。研究采用Bio-Rad Aminex HPX-87H(300mm×7.8 mmΦ,9μm)离子排斥色谱柱及二极管阵列检测器,以硫酸溶液作为流动相,比较了不同流动相浓度、不同柱温、不同流动相流速、不同检测波长等色谱条件对同步检测饲料酸化剂中9中有机酸的影响;以上述优选的条件建立分析方法,考察此方法的线性范围和检出限、回收率和精密度,并用实际样品进行了验证。研究结果表明:以0.025 mol/L硫酸溶液为流动相,流速0.70 m L/min,柱温30℃,DAD检测波长为210 nm,以保留时间结合待测物质的紫外特征吸收光谱进行定性分析,外标法进行定量分析。9种有机酸组分的质量浓度与其峰面积在一定范围内呈良好线性关系,决定系数0.999,9种有机酸组分在20 min内可以得到良好分离;方法检出限为0.01~2.4 mg/L,加标回收率为90.7%~101.1%,相对标准偏差为0.91%~3.33%。基于上述条件建立的检测方法能够满足饲料酸化剂中多种有机酸成分的同步定量分析。     

Purification of fusaproliferin from cultures of Fusarium subglutinans by preparative high-performance liquid chromatography

Thirty-six Fusarium strains were grown on cracked yellow corn and evaluated for optimum fusaproliferin production, with Fusarium subglutinans E-1583 producing the highest levels (1600 microg/g). Three solvent systems were tested for extracting fusaproliferin from the cultures of F. subglutinans E-1583. Methanol gave the highest fusaproliferin recovery, followed by methanol/1% aqueous NaCl (55:45, v/v) and acetonitrile/methanol/H(2)O (16:3:1, v/v/v). Hexane partitioning was effective in removing many impurities from the crude fusaproliferin extracts prior to the liquid chromatography step. Fusaproliferin samples were further purified by high-performance liquid chromatography (HPLC) with a C18 preparatory column using a mobile phase of acetonitrile/H(2)O (80:20, v/v). The purity of the fusaproliferin was verified by analytical HPLC, GC/MS, (1)H NMR spectroscopy, and electrospray ionization (ESI) MS. The isolated fusaproliferin was shown to be free of impurities and can be used as a standard for routine analysis. Fusaproliferin was shown to be temperature-sensitive when samples were stored at room temperature (20-24 degrees C) for more than several days. After 30 days at 4 degrees C, approximately 8% of the fusaproliferin had been transformed to deacetyl-fusaproliferin; however, samples stored at -20 degrees C for 1 year contained only trace amounts of the deacetylated form.     

Determination of thiamin in cooked sausages

A reliable and rapid high-performance liquid chromatography (HPLC) method has been set up for the determination of total thiamin in difficult sample matrices such as cooked sausages. Different hydrolysis conditions and enzymes were tested to release the vitamin from its phosphate ester. The best data in the enzymatic digestion were obtained by incubating the samples with 6% clara-diastase at 50 degrees C for 3 h. After oxidation of thiamin to thiochrome, the sample extracts were purified by using a C(18) Sep-Pak cartridge. Final determination was performed by reversed-phase HPLC with fluorescence detector (excitation 360 nm, emission 430 nm), on a low-cost 25 cm x 4 mm i.d. Spherisorb C(8) cartridge using a mixture of 5 mM phosphate buffer pH 7.0 and acetonitrile (70:30, v/v) as mobile phase. Precision of the method was 1.5% (within a day) and 5. 2% (between days). The detection limit was 0.015 mg/100 g. All the recoveries from the different cooked sausages were better than 90% of thiamin hydrochloride added to samples of meats. In the samples analyzed, the mean value for thiamin was between 0.039 and 0.508 mg/100 g fresh weight.     

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%.     

Matrix solid phase dispersion (MSPD) isolation and liquid chromatographic determination of furazolidone in pork muscle tissue

A method for the isolation and liquid chromatographic (LC) determination of furazolidone in pork muscle tissue is presented. Blank or furazolidone-fortified pork muscle tissue samples (0.5 g) were blended with octadecylsilyl (C18, 18% load, endcapped, 2 g) derivatized silica. A column made from C18/pork matrix was first washed with hexane (8 mL), followed by elution of furazolidone with ethyl acetate. The ethyl acetate extract was then passed through an activated alumina column. The eluate contained furazolidone that was free from interfering compounds when analyzed by LC with UV detection (photodiode array, 365 nm). Detector response with increasing concentrations of furazolidone isolated from fortified samples was linear (r = 0.998 +/- 0.002) with an average percentage recovery of 89.5 +/- 8.1% for the concentration range (7.8-250 ng/g) examined and resulted in a minimum detectable limit of 390 pg on column, and a detector response of more than 5 times baseline noise. The inter-assay variability was 9.9 +/- 5.4% with an intra-assay variability of 1.5%.     

Simultaneous liquid chromatographic screening of five coccidiostats in chicken liver

A reverse-phase liquid chromatographic (LC) method is described for simultaneously determining 5 coccidiostats--aklomide, dinsed, ethopabate, nitromide, and zoalene in chicken liver. The method entails blender extraction of 10 g liver with ethyl acetate, column chromatography through Sephadex LH-20 and neutral alumina, and LC analysis on a C18 column with UV detection at 260 nm. The drugs were eluted from Sephadex with methanol-benzene (10 + 90), from alumina with methanol-dichloromethane (10 + 90), and from C18 with acetonitrile-water (linear gradient: 25% acetonitrile for 10 min, increasing to 55% over 15 min; flow rate 1 mL/min). Liquid chromatography was completed in 40 min and calculations were based on peak height measurements. Average recoveries of the coccidiostats from fortified liver ranged from 72 to 97%, except for dinsed, which showed a relatively constant average recovery of 57%. The detection limit for the standards was 2.5 ng on column. Levels as low as 50 ng/g were detected in fortified liver samples.     

Matrix solid phase dispersion isolation and liquid chromatographic determination of five benzimidazole anthelmintics in fortified beef liver

A multiresidue method for isolation and liquid chromatographic determination of 5 benzimidazole anthelmintics (thiabendazole, oxfendazole, mebendazole, albendazole, and fenbendazole) in beef liver tissue is presented. Blank or benzimidazole-fortified liver samples (0.5 g) were blended with octadecylsilyl derivatized silica packing material (C18, 18% load, endcapped, 2 g). A column made from the C18/liver matrix was first washed with hexane (8 mL), following which the benzimidazoles were eluted with acetonitrile. The acetonitrile extract was then passed through an activated alumina column. The eluate contained benzimidazole analytes that were free from interfering compounds as determined by UV detection (photodiode array, 290 nm). Correlation coefficients of standard curves for individual benzimidazoles isolated from fortified samples, using internal standardization, were linear (0.996 +/- 0.002 to 0.999 +/- 0.001) with average relative percentage recoveries from 62.0 +/- 6.7 to 86.8 +/- 8.6% for the concentration range (100-3200 ng/g) examined. The interassay variability was 7.0 +/- 4.1 to 12.9 +/- 10.2% with an intra-assay variability from 2.2 to 4.0%.     

Monitoring of artemisinin, dihydroartemisinin, and artemether in environmental matrices using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The area cultivated with Artemisia annua for the extraction of the antimalarial compound artemisinin is increasing, but the environmental impact of this cultivation has not yet been studied. A sensitive and robust method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of artemisinin in soil. Dihydroartemisinin and artemether were included in the method, and performance on analytical columns of both traditional C(18) phenyl-hexyl and porous shell particles-based Kinetex types was characterized. The versatility of the method was demonstrated on surface water and groundwater samples and plant extracts. The limit of detection was 55, 30 (25 ng/g soil), and 4 ng/mL for dihydroartemisinin, artemisinin, and artemether, respectively. Method performance was demonstrated using naturally contaminated soil samples from A. annua fields in Kenya. The highest observed concentrations were above EC(10) for lettuce growth. Monitoring of artemisinin in soil with A. annua crop production seems necessary to further understand the impact in the environment.     

Quantitative analysis of tylosin in eggs by high performance liquid chromatography with electrospray ionization tandem mass spectrometry: residue depletion kinetics after administration via feed and drinking water in laying hens

Maximum residue limits (MRLs) have been established by the European Union when tylosin is used therapeutically. They are fixed at 200 microg/kg for eggs. A highly sensitive and selective quantitative liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method suitable for monitoring tylosin residues in eggs to determine its depletion kinetics was developed and validated. For sample pretreatment all samples were liquid-liquid extracted with citrate buffer (pH 5.0) and acetonitrile. Liquid chromatographic separation was carried out on a reversed phase C18 column employing a 0.5% formic acid/acetonitrile gradient system. The tylosin recovery in eggs at a concentration range from 1.0-400 microg/kg was >82% with relative standard deviations between 1.5 and 11.0%. In two experimental studies administrating tylosin via feed (final dosage: 1.5 g/kg) or drinking water (final dosage: 0.5 g/L), no residues above the MRL were found during and after treatment. Moreover, all samples were well below the actual MRL of 200 microg/kg. Therefore, our residue data suggest that a withholding period for eggs is not required when laying hens are treated with tylosin in recommended dosages via feed or drinking water. Keywords: Tylosin; residue; depletion; laying hen; withholding period; mass spectrometry.     

Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products and animal feed

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples.     

Organic solvent-free reversed-phase ion-pairing liquid chromatography coupled to atomic fluorescence spectrometry for organoarsenic species determination in several matrices

A novel method has been developed to determine As-containing animal feed additives including roxarsone (ROX), p-arsanilic acid (p-ASA) and nitarsone (NIT), as well as other organic As species (dimethylarsonic acid (DMAA) and monomethylarsonic acid (MMAA)) by ion-pairing high-performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry (IP-HPLC-HG-AFS). A simple isocratic reversed-phase (RP) HPLC method with a mobile phase containing citric acid and sodium hexanesulfonate (pH 2.0) was developed using a C(18) column. The use of an organic solvent free mobile phase turns this methodology into an environmentally friendly alternative. Several ion pair forming agents, such as sodium hexanesulfonate, tetrabutylammonium bisulfate and perfluoroheptanoic acid, were studied. The limits of detection for As species were calculated in standard solution and resulted to be 0.2, 0.5, 0.6, 1.6, and 1.6 μg As L(-1) for MMAA, DMAA, p-ASA, ROX and NIT, respectively. This method exhibited convenient operation, high sensitivity and good repeatability. It was applied to As speciation in different samples including arugula, dog food, dog urine and chicken liver.