Determination of beta-lactams in milk using a surface plasmon resonance-based biosensor

Two surface plasmon resonance (SPR)-based biosensor assays for detection of beta-lactam antibiotics in milk are reported. The assays are based on the enzymatic activity of a carboxypeptidase converting a 3-peptide into a 2-peptide, a reaction that is inhibited in the presence of beta-lactams. Antibodies were used to measure either the amount of formed enzymatic product or the amount of remaining enzymatic substrate. Both assays detected different beta-lactams at or below European maximum residue limits (MRLs), and the detection limit for penicillin G was 1.2 microg/kg and 1.5 microg/kg for the 2- and 3-peptide assays, respectively. The precision (CV) was < 5%, both within and between assays at the penicillin G MRL (4 microg/kg). The biosensor results obtained upon analysis of incurred milk samples were compared with results obtained by liquid chromatography (HPLC), and the method agreements were, in general, good.     

Development of an immunoaffinity chromatography purification and ultra performance liquid chromatography tandem mass spectrometry method for determination of 12 sulfonamides in beef and milk

A highly selective and sensitive method was developed for the simultaneous determination of 12 sulfonamides in beef and milk by immunoaffinity chromatography purification coupled to ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The MS/MS conditions, UPLC mobile phase, injection solution, sample purification process, and matrix effect were studied to optimize the operating conditions. The limits of detection (LODs) of the instrument for the studied sulfonamides ranged from 0.4 to 2.0 μg L(-1), being 1.6-8.0 μg kg(-1) for beef and 1.8-6.4 μg kg(-1) for milk. The standard solution was diluted with blank beef or milk matrix for the construction of calibration curves, which had a linear range from 10 to 200 μg kg(-1) and regression coefficients higher than 0.990 (n=10) for all the studied sulfonamides. Samples spiked at 10, 20, and 100 μg kg(-1) showed recoveries above 70% and relative standard deviations below 10%.     

Simultaneous determination of oxytetracycline, tetracycline, and chlortetracycline in milk by liquid chromatography

A simple, rapid procedure is described for the simultaneous quantification of 3 tetracycline drugs in bovine milk. Samples are prepared by dilution with an EDTA/phosphate buffer solution and filtration through a molecular weight cutoff filter. Analytes are concentrated on-column using a reverse-phase gradient elution system of oxalic acid, acetonitrile, and methanol. The limits of quantitation are approximately 15-50 ng/mL and the limits of detection are 10-20 ng/mL, depending on the compound. For oxytetracycline, over the range 50-1200 ng/mL, the average recovery and intralaboratory coefficient of variation were 97% and 4.1%, respectively. Over the same range, these parameters were, respectively, 97% and 5.0% for tetracycline, and 89% and 6.4% for chlortetracycline. The applicability of this procedure is demonstrated by separation and detection of incurred tetracycline residues in milk from treated animals.     

Development of an indirect competitive ELISA for ciprofloxacin residues in food animal edible tissues

An indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect ciprofloxacin (CPFX) in food animal edible tissues. CPFX was converted by an active ester method into conjugates CPFX-bovine serum albumin (CPFX-BSA) and CPFX-human serum albumin (CPFX-HSA), which both allowed production of CPFX-specific rabbit antisera. In the ELISA, CPFX-HSA was coated onto the microtiter plate, followed by incubation with standard CPFX and anti-CPFX antibody. The indirect competitive ELISA revealed that the antisera have no cross-reactivity with penicillin, gentamicin, neomycin, sulfadiazine, and chlortetracycline. The antisera cross-reacted with enrofloxacin and norfloxacin about 69.8 and 44.6% as much as they did with CPFX. This ELISA was highly sensitive (0.32 ng/mL) to CPFX determination. Recovery of CPFX at 40 microg/kg was 75.58% in pork, 81.29% in chicken, and 84.30% in milk. The coefficients of variation varied from 3.7 to 9.2% over the range of CPFX concentrations studied. The linear detection range was between 1.6 and 1000 ng/mL. The results suggest that this ELISA is a specific, accurate, and convenient method for the detection of CPFX residues in food animal edible tissues.     

Gas chromatographic determination of N-nitrosamines, aromatic amines, and melamine in milk and dairy products using an automatic solid-phase extraction system

A reliable analytical method was presented for the simultaneous determination of six N-nitrosamines, nine aromatic amines, and melamine in milk and dairy products using gas chromatography coupled with mass spectrometry. The sample treatment includes the precipitation of proteins with acetonitrile, centrifugation, solvent changeover by evaporation, and continuous solid-phase extraction for cleanup and preconcentration purposes. Samples (5 g) containing 0.15-500 ng of each amine were analyzed, and low detection limits (15-130 ng/kg) were achieved. Recoveries for milk and dairy products samples spiked with 1, 10, and 50 μg/kg ranged from 92% to 101%, with intraday and interday relative standard deviation values below 7.5%. The method was successfully applied to determine amine residues in several milk types (human breast, cow, and goat) and dairy products.     

Determination of nitrate in dried foods by gas chromatography-thermal energy analyzer

The method described for determining NO3- in dried foods is based on extraction of NO3- from the sample with subsequent nitration of benzene. The nitrobenzene is extracted with ethyl acetate, analyzed by using a gas chromatograph-thermal energy analyzer (GC-TEA), and quantitated against a nitrobenzene standard. Sensitivity is 100-200 micrograms/kg. Coefficients of variation for analyses of dried foods were 3-13%. Recovery of NO3- from nonfat dry milk spiked at 10 mg/kg averaged 100%.     

Determination of polysorbates in foods by colorimetry with confirmation by infrared spectrophotometry, thin-layer chromatography, and gas chromatography

A method is presented for the detection of polysorbates (PSs) in 8 kinds of processed foods by colorimetric and thin-layer chromatographic (TLC) techniques. The PSs are extracted from processed foods with a mixture of methylene chloride and ethanol by using an Extrelut column. The extract is further purified by using a silica gel column. The PS extract is complexed with cobalt-thiocyanate (Cothiocyanate) reagent and is determined spectrophotometrically at 620 nm. The recoveries and coefficients of variation for 8 kinds of processed foods fortified with 0.1% PS 80 were 67.9-94.6% and 4.0-11.3%, respectively. The detection limit of TLC corresponded to 50 mg PS 80/kg. PS identity was confirmed by infrared spectrophotometry of PS extract, and gas chromatography of fatty acids and thin layer chromatography of POE-sorbitan residues after saponification.     

Capillary column gas chromatographic determination of ethyl carbamate in alcoholic beverages with confirmation by gas chromatography/mass spectrometry

A method is described for determining ethyl carbamate at low microgram/kg levels in several types of alcoholic beverages by capillary column gas chromatography with Hall electrolytic conductivity detection and confirmation by mass spectrometry. Samples are diluted to obtain a uniform concentration of ethanol (ca 10%) then saturated with NaCl and extracted with methylene chloride. Extracts are evaporated to a small volume and injected in ethyl acetate solution for chromatographic analysis. The method was evaluated by 5 laboratories, 4 employing the Hall detector and one using mass spectrometric detection. Overall between-laboratory mean percent recoveries were: wine, 85.3 +/- 21.0% coefficient of variation (CV) (spiking level 20-45 micrograms/kg); sherry, 83.8 +/- 16.1% CV (spiking level, 81-142 micrograms/kg); whiskey, 79.5 +/- 13.9% CV (spiking level 127-190 micrograms/kg); and brandy, 85.0 +/- 12.5% CV (spiking level 297-446 micrograms/kg). Mass spectrometric results agreed well with the Hall results for all commodities. Detection limits were about 5 micrograms/kg for the Hall detector and about 0.5 microgram/kg for mass spectrometric detection.     

Determination of diagnostic levels of arsenic in animal tissue: collaborative study

The method chosen for this collaborative study is a modification of the AOAC method for As residues, 41.009-41.012. The tissue is dry-ashed overnight at 500 degrees C, and then dissolved in dilute HCl. The solution is diluted and an aliquot is reacted with zinc metal to evolve arsine gas. The gas is trapped in AgDDC solution and As is quantitated at 540 nm. Nine collaborating laboratories performed single analyses on 4 blind duplicate pairs of bovine liver samples which were spiked at 0, 4.3, 10.8, or 21.6 mg As/kg liver. A National Bureau of Standards control (SRM 1566 Oyster Tissue, 13.4 +/- 1.9 mg As/kg) and a 1000 mg As/L standard were also submitted to the collaborators. Intralaboratory coefficients of variation ranged from 7.7 to 17.8%; interlaboratory coefficients of variation ranged from 10.9 to 19.0%. The method has been adopted official first action.     

Residues of azoxystrobin from grapes to raisins

Azoxystrobin, a fungicide of the strobilurin group, has an European Union maximum residue level (MRL) of 2 mg/kg for grapes. This work aimed to assess residues on fresh and washed grapes and on raisins following processing with (i) alkali treatment and sun drying and (ii) sun drying only. QUADRIS 25% SC was applied according to good agricultural practice for two consecutive years on a typical cv. Thomson seedless and a seed-producing clone. Samples were collected 0, 15, and 21 days postapplication and analyzed using gas chromatography/electron capture detection; recoveries were 86 +/- 12% for grapes and 99 +/- 15% for raisins. Residues on grapes were 0.49-1.84 mg/kg, and washing removed 75% of the residue. Residues in raisins produced from seedless grapes were 0.51-1.49 (treatment 1) and 1.42-2.08 mg/kg (treatment 2), with residue transfer factors sometimes >1, even following alkali treatment, which reduced residues considerably. To avoid trade problems, a higher MRL for raisins is necessary.     

Determination of 49 organophosphorus pesticide residues and their metabolites in fish, egg, and milk by dual gas chromatography-dual pulse flame photometric detection with gel permeation chromatography cleanup

A new method for the quantitative determination of 49 kinds of organophosphorus pesticide residues and their metabolites in fish, egg, and milk by dual gas chromatography-dual pulse flame photometric detection was developed. Homogenized samples were extracted with acetone and methylene chloride (1 + 1, v/v), and then the extracts were cleaned up by gel permeation chromatography (GPC). The response of each organophosphorus pesticide showed a good linearity with its concentration; the linearity correlation was not less than 0.99. The detection limits (S/N = 3) of pesticides were in the range of 0.001-0.025 mg kg?1. The recovery experiments were performed by blank sample spiked at low, medium, and high fortification levels. The recoveries for fish, egg, and milk were 50.9-142.2, 53.3-137.2, and 50.3-139.4% with relative standard deviations (RSD, n = 6) of 2.3-24.9, 4.3-26.7, and 2.8-32.2%, respectively. The method was applied to detect organophosphorus pesticides in samples collected from the market, and satisfactory results were obtained. This quantitative method was highly sensitive and exact and could be applied to the accurate determination of organophosphorus contaminants in fish, egg, and milk.     

Multiresidue analysis of 58 pesticides in bean products by disposable pipet extraction (DPX) cleanup and gas chromatography-mass spectrometry determination

A method based on disposable pipet extraction (DPX) sample cleanup and gas chromatography with mass spectrometric detection by selected ion monitoring (GC/MS-SIM) was established for 58 targeted pesticide residues in soybean, mung bean, adzuki bean and black bean. Samples were extracted with acetonitrile and concentrated (nitrogen gas flow) prior to being aspirated into DPX tubes. Cleanup procedure was achieved in a simple DPX-Qg tube. Matrix-matched calibrations were analyzed, and the limits of quantification (LOQ) of this method ranged from 0.01 mg kg(-1) to 0.1 mg kg(-1) for all target compounds. Coefficients of determination of the linear ranges were between 0.9919 and 0.9998. Recoveries of fortified level 0.02 mg kg(-1) on soybean, mung bean, adzuki bean and black bean were 70.2-109.6%, 69.1-119.0%, 69.1-119.8%, and 69.0-120.8%, respectively, for all studied pesticides. Moreover, pesticide risk assessment for all the detected residues in 178 market samples at Beijing market area was conducted. A maximum 0.958% of ADI (acceptable daily intake) for NESDI (national estimated daily intake) and 55.1% of ARfD (acute reference dose) for NESTI (national estimated short-term intake) indicated low diet risk of these products.     

Ion-pair liquid chromatographic determination of some penicillins in canine and equine sera

A sensitive liquid chromatographic method was developed for determining oxacillin, cloxacillin, and dicloxacillin (simultaneously), and penicillin G, amoxicillin, carbenicillin, and ticarcillin in canine and/or equine serum. The method involves filtering diluted serum through a 30,000 molecular weight cut-off filter and separating penicillins from other serum components by ion-pair liquid chromatography using a reverse-phase column eluted with acetonitrile-water solutions. The ultraviolet absorbance of the column effluent was monitored at 230 nm. Recoveries of oxacillin, cloxacillin, dicloxacillin, and penicillin G (spiked at 2.5 micrograms/mL), amoxicillin (spiked at 5 micrograms/mL), and carbenicillin and ticarcillin (spiked at 10 micrograms/mL) from canine and equine serums ranged from 78.3 to 104.4% with coefficients of variation ranging from 3.35 to 5.95%. The limit of detection for these penicillins was 0.02-0.05 microgram/mL.     

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.     

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.     

Determination of nitrofuran residues in milk of dairy cows using liquid chromatography-tandem mass spectrometry

An analytical method has been developed for the determination of total bound and extractable residues of the nitrofuran drugs furazolidone, nitrofurazone, furaltadone, and nitrofurantoin in milk of dairy cows. The method involves overnight acid hydrolysis and simultaneous derivatization of the released side chains with 2-nitrobenzaldehyde. During hydrolysis, the bound metabolites are hydrolyzed to the side chains. After pH adjustment and solid-phase extraction cleanup, the derivatives are detected and quantitated using a liquid chromatography-tandem mass spectrometry system with an atmospheric pressure chemical ionization interface. Validation of the method is accomplished by fortifying control milk with a mixture of side chains at 1, 2, and 4 ng/g. Internal standards are added at the beginning of the procedure to compensate for matrix effects and recovery losses. Method accuracies range from 83 to 104% with coefficients of variation less than 13% for all four analytes. The limits of detection are相似文献   

New methodology for the determination of phthalate esters, bisphenol A, bisphenol A diglycidyl ether, and nonylphenol in commercial whole milk samples

This paper reports a new methodology aimed at determining dimethyl phthalate, diethyl phthalate, di-n-butyl phthalate, butylbenzyl phthalate, bis(2-ethylhexyl) phthalate, nonylphenol, bisphenol A, and bisphenol A diglycidyl ether in commercial whole milk. These compounds are used as plastic additives, lacquers, resins, or surfactants and can be found in milk due to contact with plastic materials during food processing and storage. They are all suspected endocrine disrupters or mutagens. A multiresidue method based in solid-phase extraction with C-18 cartridges followed by a cleanup step using disposable cartridges was developed. Detection and quantification were performed by gas chromatography coupled to mass spectrometric (GC-MS) detection using an appropriate surrogate (4-n-nonylphenol) and internal standard [deuterated bis(2-ethylhexyl) phthalate]. Limits of detection were from 0.06 to 0.36 microg/kg and intraday variation from 3 and 27%, with recoveries between 73 and 119%. Five brands of commercial whole milk processed and packed in different ways were analyzed. All samples contained target compounds at concentrations between 0.28 and 85.3 microg/kg, and the total concentration ranged between 79.3 and 187.4 microg/kg, the levels being higher in sterilized milks. Nonylphenol, diethyl phthalate, dibutyl phthalate, and bis(2-ethylhexyl) phthalate were the major contributors.     

Screening and mass spectral confirmation of beta-lactam antibiotic residues in milk using LC-MS/MS.

Milk is typically screened for beta-lactam antibiotics by nonspecific methods. Although these methods are rapid and sensitive, they are not quantitative and can yield false positive findings. A sensitive and specific method for the quantitation and mass spectral confirmation of five beta-lactam and two cephalosporin antibiotics commonly or potentially used in the dairy industry is described using high-performance liquid chromatography with tandem mass spectrometry. The antibiotics studied were ampicillin, amoxicillin, penicillin G, penicillin V, cloxacillin, cephapirin, and ceftiofur. The antibiotics were extracted from milk with acetonitrile, followed by reversed-phase column cleanup. The extract was analyzed by liquid chromatography coupled with a mass spectrometer, using a water/methanol gradient containing 1% acetic acid on a C-18 reversed-phase column. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Quantitation was based on the most abundant product ions from fragmentation of the protonated ion for amoxicillin, cephapirin, ampicillin, and ceftiofur and on the fragmentation of the sodium adduct for penicillin G, penicillin V, and cloxacillin. The method was validated at the U.S. FDA tolerance or safe level and at 5 or 2.5 ng/mL for these compounds in bovine milk. Theoretical method detection limits in milk based on a 10:1 signal to noise ratio were 0.2 ng/mL (ampicillin), 0.4 ng/mL (ceftiofur), 0.8 ng/mL (cephapirin), 1 ng/mL (amoxicillin and penicillin G), and 2 ng/mL (cloxacillin and penicillin V) using a nominal sample size of 5 mL.     

Simultaneous determination of 20 pharmacologically active substances in cow's milk, goat's milk, and human breast milk by gas chromatography-mass spectrometry

This paper reports a systematic approach to the development of a method that combines continuous solid-phase extraction and gas chromatography-mass spectrometry for the simultaneous determination of 20 pharmacologically active substances including antibacterials (chloramphenicol, florfenicol, pyrimethamine, thiamphenicol), nonsteroideal anti-inflammatories (diclofenac, flunixin, ibuprofen, ketoprofen, naproxen, mefenamic acid, niflumic acid, phenylbutazone), antiseptic (triclosan), antiepileptic (carbamazepine), lipid regulator (clofibric acid), β-blockers (metoprolol, propranolol), and hormones (17α-ethinylestradiol, estrone, 17β-estradiol) in milk samples. The sample preparation procedure involves deproteination of the milk, followed by sample enrichment and cleanup by continuous solid-phase extraction. The proposed method provides a linear response over the range of 0.6-5000 ng/kg and features limits of detection from 0.2 to 1.2 ng/kg depending on the particular analyte. The method was successfully applied to the determination of pharmacologically active substance residues in food samples including whole, raw, half-skim, skim, and powdered milk from different sources (cow, goat, and human breast).     

Effect of a pilot washing system on dicofol levels in orange matrix

An efficient analytical method is described for the analysis of dicofol residues in pulp and orange peel. Samples are mixed with Celite and transferred to chromatographic columns prepacked with silica gel. Dicofol is eluted with ethyl acetate, and the extracts are analyzed by gas chromatography with electron capture detection. Mean recoveries for dicofol at levels of 0.5, 2.0, 5.0, and 10 mg/kg ranged from 87 to 95% with relative standard deviation values between 2.6 and 9.0%. To investigate the effect of a pilot washing system on dicofol residues in oranges, the analytical procedure was applied to samples submitted to different treatments with commercial formulations under field and laboratory conditions. The orange samples with and without washing were analyzed in duplicate, and the results indicated that washing under the described conditions did not allow a complete removal of dicofol residues from orange peel.