Liquid chromatographic determination of multiple sulfonamide residues in bovine milk

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.     

Rapid procedure for determination of nicarbazin residues in chicken tissues

A procedure is described for the quantitation of nicarbazin residues in chicken tissues. The method includes extraction of tissue with chloroform-ethyl acetate-dimethyl sulfoxide (50 + 50 + 0.8), adsorption on neutral alumina, and subsequent elution of the residues with methanol-pH 6.0 phosphate buffer (1 + 1). Extracts are separated on a 15 cm, 5 micron C18 column with methanol-pH 6.0 phosphate buffer (6.5 + 3.5) as the mobile phase. The dinitrocarbanilide portion of the complex is detected and quantitated with an electrochemical detector in the reductive mode. Recoveries, based on dinitrocarbanilide, were greater than 95% in liver, breast, and thigh muscle tissues fortified with 0.25-8.0 ppm nicarbazin.     

Gas chromatographic-mass spectrometric determination of six sulfonamide residues in egg and animal tissues

A gas chromatographic-mass spectrometric method using selected ion monitoring mode for simultaneous determination of 6 sulfonamides in egg and edible animal tissues has been developed. Sulfonamides are extracted from a sample with acetonitrile. The extract is passed through a silica cartridge column and concentrated. Diazomethane in ether is added to methylate sulfonamides. After evaporation, the residue is dissolved in methylene chloride and cleaned up by silica gel column chromatography. The methylene chloride eluate containing sulfonamide-methyl derivatives is evaporated to dryness, redissolved in ether and partitioned between 6N hydrochloric acid. The acid phase is made alkaline, extracted with ether, and the ether solution, after concentration, is analyzed by gas chromatography-mass spectrometry in selected ion monitoring mode. Average recoveries from egg and silver salmon fortified at 1 and 0.2 ppm levels with 6 sulfonamides are 99.2 and 84.3%, respectively; coefficients of variation are 7.03 and 11.20%, respectively. Detection limits are 0.01-0.05 ppm.     

Liquid chromatographic determination of cephapirin residues in milk

A liquid chromatographic (LC) method was developed for quantitative determination of cephapirin residues in milk that also resolved cephapirin from ampicillin, cloxacillin, and penicillin G. Diluted milk was passed through a C18 cartridge on which the cephapirin was adsorbed; then, interfering material was removed by washing with water and methylene chloride and cephapirin residues were eluted with methanol-acetonitrile (25 + 75). After drying, residues were dissolved in the mobile phase for injection. The LC system had an ultrasphere-ODS column with RP-18 Spheri-10 guard column and a UV detector with a 254 nm filter. The mobile phase was 85% sodium acetate (0.01M) and 15% methanol-acetonitrile (25 + 75) with a flow rate of 1 mL/min. Sensitivity was 20 ppb or less with a recovery of 61-80% in the range studied. Other beta-lactam antibiotics tested did not interfere with detection of cephapirin. Analysis of 30 samples of commercial homogenized milk obtained for a survey of antibiotics in consumer milk in Canada revealed no detectable cephapirin residues.     

Monitoring of five sulfonamide antibacterial residues in milk by in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues in milk was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography with an ultraviolet detector. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium for this on-line technique. To obtain optimum extraction efficiency, several parameters relating to in-tube SPME were investigated. By simple extraction with ethanol, dilution with phosphate buffer solution, and centrifugation, the sample solution then could be directly injected into the device for extraction. The calculated detection limits for sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium were 2.0, 2.8, 1.7, 2.5, and 22 ng/mL, respectively. The method was linear over the range of 20-5000 ng/mL (100-5000 ng/mL for sulfacetamide sodium) with a correlation coefficient R (2) value >0.9980. Excellent method reproducibility was found by intra- and interbatch precisions, yielding the relative standard deviations of <10.0 and <9.94%, respectively. The proposed method was proved to be robust in monitoring sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium residues in milk.     

Liquid chromatographic determination of spiramycin residues in chicken tissues

A liquid chromatographic (LC) method is described for determination of spiramycin residues in chicken muscles. The drug is extracted from muscles with acetonitrile, the extract is concentrated to 3-4 mL and rinsed with n-hexane followed by ethyl ether, and the drug is extracted with chloroform. LC analysis is carried out on a Zorbax BP-C8 column, and spiramycin is detected spectrophotometrically at 231 nm. Recoveries of spiramycin added to chicken muscles at 0.2 and 0.1 ppm were 93.9 and 89.0%, respectively. The detection limit was 5 ng for spiramycin standard, and 0.05 ppm in chicken muscles.     

Rapid determination of total cholesterol in homogenized milk

A rapid method that is amenable to automation has been developed for the determination of total cholesterol in homogenized milk. The milk sample is saponified in ethanolic KOH in the presence of an internal standard, cholestane. Cholesterol and the internal standard are then isolated by solid-phase extraction on a nonpolar adsorbent and eluted with organic solvent. The evaporated extract is derivatized and analyzed by capillary gas chromatography. Average recovery of cholesterol acetate added to milk prior to saponification was 95%. The average relative standard deviation for repeated analyses was 2%. The limit of detection for this method is 2 mg/100 g. Twenty samples can be analyzed by one analyst in a normal work day if the gas chromatograph is equipped with an autosampler. This method has been compared with a modified AOAC method for the determination of total cholesterol. At a confidence level of 95%, no difference was observed between the 2 methods.     

N-linked glycan profiling of mature human milk by high-performance microfluidic chip liquid chromatography time-of-flight tandem mass spectrometry

N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.     

Rapid determination of fumigant and industrial chemical residues in food

A gas chromatographic (GC) method is described for the determination of 22 fumigant and industrial chemical residues in a variety of foods. The fumigants and industrial chemicals determined are methyl bromide, methylene chloride, carbon disulfide, chloroform, 1,1-dichloroethane, ethylene dichloride, methyl chloroform, carbon tetrachloride, methylene bromide, propylene dichloride, 2,3-dichloropropene, trichloroethylene, 1,3-dichloropropylene, 1,1,2-trichloroethane, chloropicrin, ethylene dibromide, tetrachloroethylene, propylene dibromide, 1,1,2,2-tetrachloroethane, p-dichlorobenzene, o-dichlorobenzene, and 1,2-dibromo-3-chloropropane. Except for the latter three, the fumigants are determined at 90 degrees C on 3.6 m 20% loaded OV-101 columns with electron-capture and Hall-electroconductivity detectors. The other 3 compounds (o-dichlorobenzene, p-dichlorobenzene, and 1,2-dibromo-3-chloropropane), which elute beyond 30 min on the above columns, are determined at 90 degrees C on 1.8 m 5% loaded OV-101 columns with the same detectors. The ng/g-level fortifications have an overall mean analyte recovery of 70% and a coefficient of variation of 40%. The variety of foods examined includes both fatty and nonfatty food types (e.g., off-the-shelf cooked and uncooked grain-based items, dairy products, fresh and canned fruits and vegetables, and meats). Samples are extracted and cleaned up according to fat content and food type. Samples containing less than 71% fat are extracted by using an aqueous: nonaqueous shakeout (20% acetone solution under isooctane). Most extracts (isooctanes) are analyzed directly. Extracts from samples containing from 21 to 70% fat (e.g., ground beef, pecans, and corn chips) are cleaned up further on micro-Florisil columns to remove excess fat. A few other samples containing more than 71% fat or oil (e.g., butter, salad dressing, and vegetable oil) are diluted directly in isooctane and, depending on the degree of dilution, can be cleaned up further on micro-Florisil columns. Also, clear beverages (e.g., soda and tea) are extracted directly with isooctane. These extraction and cleanup techniques were tested on 231 different table-ready foods. Three-hundred incurred residues of 10 different fumigants were found in 138 items examined; 93 items had no detectable residues. The main advantage of the method is rapid semiquantitative determination of multiple fumigants from all food types.     

Effects of cooking and storage on residues of cyadox in chicken muscle

The aim of this study was to investigate the depletion of residues of cyadox in chicken muscle over time. The heat stabilities of cyadox (CYX) and its two metabolites, 1,4-bisdesoxycyadox (BDCYX) and quinoxaline-2-carboxylic acid (QCA), in water, cooking oil, and as incurred residues in chicken muscle were investigated. CYX was shown to be unstable with a half-life of about 37.7 min in 100 degrees C water. In hot cooking oil at 180 degrees C, all three compounds were unstable. CYX decreased quickly and was not able to be detected after heating for 2 min. Diode-array analysis of CYX standard solution in cooking oil indicated that a portion of BDCYX was formed. The residues of CYX and BDCYX deteriorated rapidly in frozen storage, while that of QCA changed slowly. Muscles containing CYX residues were boiled, microwaved, or fried for the specified times. During boiling, CYX and BDCYX were reduced 94% and 81% in 10 min, respectively. During microwave cooking, CYX and BDCYX were reduced 54% and 47% in 2.5 min, respectively. During frying, CYX and BDCYX were reduced 86% and 76%, respectively. No significant reduction of QCA was found for the three cooking methods. The half-lives of CYX residues in cooked chicken muscles were estimated as follows: 2.22 min for CYX and 4.44 min for BDCYX by boiling; 6.66 min for CYX and 9.36 min for BDCYX by microwaving.     

Rapid and sensitive quantification of amino acids in soil extracts by capillary electrophoresis with laser-induced fluorescence

A growing body of research is arguing that amino acids are key components of the soil nitrogen cycle. For example, we now know that many plants can take up intact amino acids, even in competition with soil microbes. Our growing recognition of the importance of amino acids is not matched by knowledge of the amounts and type of amino acids in the soil, certainly not in comparison with our encyclopaedic knowledge of inorganic N. The primary reason that less is known about amino acids than inorganic N is that measuring the amounts of individual amino acids with conventional chromatographic techniques is slow and typically requires extensive sample clean-up if KCl extracts are analysed. The aim of this study was to develop capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) as a more rapid alternative for measuring individual amino acids in KCl extracts of soil. The CE-LIF method separated 17 common amino acids within 12 min, with detection limits between 7 and 250 nM. One molar KCl extracts could be analysed without any sample clean-up or de-salting, and spike and recovery tests indicated that the complex matrix of soil extracts did not affect quantification. Further evidence of the suitability and robustness of the method came from the repeated analysis (n=5) of the same soil KCl extract. The relative standard deviation of migration times for replicate analyses were <0.2% while relative standard deviations for peak areas were <5%. To demonstrate application of the CE-LIF method to real world problems it was used to analyse amino acids in 1 M KCl extracts from a sub-alpine grassland and a Eucalyptus regnans forest. The most abundant amino acids were Ala, Gly and Arg. Other amino acids present at smaller concentrations or in a minority of samples were Asn, Cit, GABA, Glu, His, Phe, Leu, and Lys. The proposed CE-LIF method offers significant advantages over chromatographic methods via its rapidity, reproducibility and, most importantly, its ability to analyse crude KCl extracts.     

Sensitive determination of ethopabate residues in chicken tissues by liquid chromatography with fluorometric detection

A liquid chromatographic (LC) method is described for determination of ethopabate residues in chicken tissues. The drug is extracted from tissues with acetonitrile, and the extract is concentrated to 2-3 mL. This aqueous solution is rinsed with ethyl acetate and cleaned up by Florisil column chromatography. LC analysis is carried out on a Zorbax ODS column, and ethopabate is quantitated by using a fluorometric detector set at 306 nm (excitation) and 350 nm (emission). Recoveries of ethopabate added to chicken tissues at levels of 0.01 and 0.05 ppm were 87.8 and 92.7%, respectively. The detection limit was 100 pg for ethopabate standard, and 0.5 ppb in chicken tissues.     

A simple and sensitive liquid chromatography-mass spectrometry confirmatory method for analyzing sulfonamide antibacterials in milk and egg

A simple and specific method able to identify and quantify traces of 14 sulfonamide antibacterials (SAs) in milk and eggs is presented. This method uses a single solid-phase extraction (SPE) cartridge for simultaneous extraction and purification of SAs in the above matrices. Milk and egg samples are passed through a Carbograph 4 sorption cartridge. After analyte desorption, an aliquot of the final extract is injected into a liquid chromatography-mass spectrometry (LC-MS) instrument equipped with an electrospray ion source (ESI) and a single quadrupole. MS data acquisition is performed in the positive-ion mode and by a time-scheduled multiple-ion selected ion monitoring program. Compared to two published methods, the present protocol extracted larger amounts of SAs from both milk and egg and decreased the analysis time by a factor of 3 with milk samples and by a factor of 2 with egg samples. Recovery of SAs in milk at the 5 ppb level ranged between 76 and 112% with relative standard deviations (RSDs) of 相似文献   

Rapid confirmatory assay for determining 12 sulfonamide antimicrobials in milk and eggs by matrix solid-phase dispersion and liquid chromatography-mass spectrometry

A rapid confirmatory method for determining 12 sulfonamide (SAs) antibacterials in whole milk and eggs is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-mass spectrometry (MS). The LC-MS instrument was equipped with an electrospray ion source and a single quadrupole. After 4 mL of a milk sample containing the analytes had been deposited on sand (crystobalite), this material was packed into an extraction cell. SAs were extracted by flowing 4 mL of water through the cell heated at 75 degrees C. With some modifications, this procedure was applied also to eggs. After pH adjustment and filtration, 0.5 mL of the final extracts was then injected into the LC column. MS data acquisition was performed in the positive-ion mode and by monitoring at least three ions for each target compound. The in-source collision-induced dissociation process produced confirmatory ions. At the 50 ng/g level, recovery of the analytes in milk and eggs was 77-92% with relative standard deviations ranging between 1 and 11%. Estimated limits of quantification (S/N = 10) were 1-3 ng/g of SAs in milk and 2-6 ng/g in eggs. With both matrices, attempts to reduce the analysis time by using a short chromatographic run time caused severe ion signal suppression for the early-eluted SAs. This effect was traced to competition effects by polar endogenous coextractives, maybe proteinaceous species, which are eluted in the first part of the chromatographic run. This unwelcome effect was almost completely removed by simply adopting more selective chromatographic conditions.     

Rapid fluorescence screening assay for enrofloxacin and tetracyclines in chicken muscle

A simple, rapid fluorescence assay was developed for screening both enrofloxacin (ENRO) and tetracyclines in chicken muscle at the U.S. tolerance levels (300 ng/g and 2 microg/g, respectively). Screening for both classes of antibiotics is accomplished using one extraction, thus simplifying and expediting the process. The method requires an initial extraction of chicken muscle with 1% acetic acid in acetonitrile, centrifugation, and analysis of the supernatant for ENRO fluorescence. After addition of ammonium hydroxide, magnesium chloride, and methanol, followed by centrifugation and filtration, the supernatant can be measured for tetracycline fluorescence. Chlortetracycline (CTC) was chosen as a representative tetracycline to demonstrate the method, as it displays intermediate sensitivity among the three tetracyclines approved in the U.S. Comparison of the fluorescence of control and tolerance-level-fortified samples of both ENRO and CTC shows no overlap. Setting a threshold as the average fortified fluorescence minus 3sigma allows for successful screening, as illustrated with blind samples as controls or fortified with ENRO and/or CTC over a range of concentrations. This method can provide an alternative or supplemental approach to currently used microbial screening assays.     

生鲜乳中兽药残留的快速筛查与风险预警体系构建

该研究探索并构建一种基于历史数据分析的安全预警方法,对生鲜乳中兽药残留情况进行识别、控制与评价,以便在风险发生之前做出准确判断。首先利用高效液相色谱-高分辨飞行时间串联质谱(High Performance Liquid Chromatography-high Resolution Time-of-flight Tandem Mass Spectrometry,HPLC-TOF-MS/MS)方法对上海市各牧场的生鲜乳进行兽药残留筛查,然后以生鲜乳中泼尼松(Prednisone,Pre)残留检出数为例,利用休哈特控制图(Shewhart control charts)构建风险预警体系。结果表明:在300个生鲜乳样品中共筛查出42种兽药残留,涉及类固醇类(12种)、糖皮质类(6种)和镇静剂(5种)等12大类;在筛查的6个星期中,泼尼松检出数(Number of Prednisone Detected,Pn)控制图呈现稳定的状态;当假设第7周泼尼松检出数为6时,Pn控制图呈现稳态,未触发预警;当假设第7周泼尼松检出数为10时,Pn控制图出现异常,稳态遭到破坏,此时该批样品触发风险预警。综上,利用HPLC-TOF-MS/MS能对生鲜乳样品进行高通量的兽药残留筛查,再利用休哈特控制图结合历史数据可以对生鲜乳样品的兽药残留进行有效的风险监测和预警。     

Rapid and sensitive determination of microbial activity in soils and in soil aggregates by dimethylsulfoxide reduction

Summary Based on the reduction of dimethylusulfoxide (DMSO) to dimethylsulfide (DMS) by microorganisms, a simple, rapid, sensitive and inexpensive method for the determination of microbial activity in soil samples was developed. When DMSO was added to samples, DMS appeared immediately in the gas phase, which was quantitatively analyzed by gas chromatography. The DMS liberation rate was constant for several hours. The reaction immediately starts and its linearity indicate that neither the physiological state nor the number of organisms were changed by the assay. DMSO reduction is widespread among microorganisms; out of 144 strains tested (both fungi and bacteria) only 5 were unable to carry out this reaction. The reaction in soil samples was strongly inhibited by toluene, cyanide, azide, or by fumigation, but was considerably stimulated by glucose. These findings demonstrate that the reaction was due to the activity of microorganisms. The DMSO reduction in different soil samples was significantly correlated with arginine ammonification and heat output (r>0.9). A good correlation was observed with the organic-matter content (r = 0.74), but not with microbial numbers, clay content, or the pH of the soil. Standard deviations of less than 10% were routinely found. Furthermore, the method is sufficiently sensitive to allow measurements of activity in very small samples (< 0.1 g). For example, a microbial activity profile can be established for a single soil aggregate, revealing marked differences in activity on the outside and in the interior.     

Rapid and sensitive determination of phenol in honey by high-performance liquid chromatography with fluorescence detection

The objective of this research was to develop a novel high-performance liquid chromatographic (HPLC) method involving a simple sample preparation procedure for the rapid, low-cost, and sensitive quantitation of phenol in honey at levels of regulatory and practical importance. After proper dilution of honey with water, the samples were analyzed by a gradient HPLC system, using a reversed-phase column with fluorescence detection at excitation and emission wavelengths of 270 and 300 nm, respectively. The eluents applied were water-acetonitrile-85% orthophosphoric acid (10:10:0.01, v/v/v) and water-85% orthophosphoric acid (20:0.01, v/v). The retention time of phenol was found to be 14.1 min, and the limit of quantitation for phenol in honey was set at 5 microg/kg. Overall recovery was 98%. The proposed method has been successfully applied to real sample analysis.     

Microbiological determination of penicillin G, ampicillin, and cloxacillin residues in milk

A fast cylinder plate microbiological method was developed for the quantitative determination of penicillin G, ampicillin, and cloxacillin in milk. Agar plates seeded with stable spores of Bacillus stearothermophilus var. calidolactis were used and incubated at 64 degrees C for 4 1/2 hr. Standard curves were obtained for the following ranges of concentration of antibiotics: 0.004-0.064 IU penicillin G/mL, 0.0025-0.04 microgram ampicillin/mL, and 0.03-0.48 microgram cloxacillin/mL. The method is suitable for detecting penicillin residues in milk and for quantitative milk-out studies of the above antibiotics used in treatment of bovine mastitis.