High pressure liquid chromatographic determination of nitrofurazone in milk.

A rapid high pressure liquid chromatographic (HPLC) screening method for the quantitative determination of nitrofurazone in milk has been developed. The drug is extracted with ethyl acetate from a 2.0 ml milk serum sample, the organic layer is evaporated to dryness, and the residue is dissolved in the mobile phase and injected into the liquid chromatogarph. A reverse phase muBondapak C18 column is used with monitoring at 365 nm. The detection limit is 5 ppb and recoveries are 57--67%. Mass spectroscopic confirmation of the HPLC nitrofuran peak is described.     

Liquid chromatographic determination of aflatoxin M1 in milk

The official AOAC method for aflatoxin M1 in milk was modified by replacing cellulose column chromatography with cartridge chromatographic cleanup and replacing thin layer chromatographic (TLC) determination with liquid chromatographic (LC) quantitation to yield a new method for bovine and porcine milk. An acetone extract of milk is treated with lead acetate and defatted with hexane, and M1 is partitioned into chloroform as in the AOAC method. Chloroform is removed by evaporation under a stream of nitrogen at 50 degrees C. The residue is dissolved in chloroform, the vessel is rinsed with hexane, and the 2 solutions are applied in sequence to a hexane-activated silica Sep-Pak cartridge. Less polar impurities are removed with hexane-ethyl ether, and M1 is eluted with chloroform-methanol, and determined by C18 reverse phase LC using fluorescence detection. Recoveries of M1 added to bovine milk at 0.25, 0.50, and 1.0 ng/mL were 90.8, 93.4, and 94.1%, respectively. The limit of detection was less than 0.1 ng M1/mL for both bovine and porcine milk.     

Liquid chromatographic determination and gas chromatographic-mass spectrometric confirmation of lasalocid sodium in bovine liver: interlaboratory study

An analytical method has been developed that can reliably measure the metabolic marker residue of lasalocid. The method monitors this marker residue in food samples to ensure that the total residue of toxicological concern is not being exceeded. Interlaboratory studies of the liquid chromatographic determinative procedure and the gas chromatographic/mass spectrometric confirmatory procedure for lasalocid sodium at the 0.7 ppm level and higher were successful.     

Solid-phase extraction followed by liquid chromatography-mass spectrometry for trace determination of beta-lactam antibiotics in bovine milk.

A confirmatory assay able to unambiguously identify and quantify 10 approved-for-use beta-lactam antibiotics in milk below stipulated U.S. and EU tolerance levels is presented. beta-Lactams are extracted from 10 mL of intact milk by a Carbograph 4 cartridge. After solvent removal, residue reconstitution, and filtration, a completely transparent and uncolored extract is injected into a liquid chromatography -mass spectrometry (LC-MS) instrument equipped with an electrospray (ES) ion source and a single quadrupole. During the chromatographic run, the ES/MS system is operated first in the positive-ion mode (PI) and then in the negative-ion (NI) mode. This is done to circumvent matrix interferences resulting in remarkable signal weakening of the last-eluted analytes, when detecting them as [M+H]+ adduct ions. MS data acquisition is performed by a time-scheduled three-ion selected ion monitoring program. At the 5 ng/mL level, recoveries of the beta-lactams are between 70 (nafcillin) and 108% (cephalin), with relative standard deviations ranging between 5 (oxacillin) and 11% (amoxicillin and ceftiofur). The response of the ES/MS detector is linearly related to injected amounts up to 500 ng, irrespective of the chemical characteristics of the beta-lactams and the acquisition mode selected (PI or NI modes). Limits of quantification, based on a minimal value of the signal-to-noise ratio of 10, were estimated to be within 0.4 (cephalin) and 3 ng/mL (dicloxacillin). Analyses of milk samples taken after intramammary application of amoxicillin showed that 1.2 ng/mL of this penicillin was still present 6 days after treatment. At this concentration level, the identification power of the method is not weakened, as signals of the three product ions of amoxicillin are still well distinguishable from the background noise.     

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.     

Liquid chromatographic determination and identification of morantel-related residues as precursors of 3-(3-methyl-2-thienyl) acrylic acid (CP-20,107) in bovine milk

A simple liquid chromatographic (LC) method was developed to determine and identify incurred morantel-related residues in bovine milk by converting them to 3-(3-methyl-2-thienyl) acrylic acid (CP-20, 107). Key techniques in this method involve short-term digestion of milk in HCl to release residues convertible to CP-20, 107, isolation and alkaline hydrolysis of these precursors to CP-20, 107, and recovery of the product for LC analysis. Photochemical conversion of CP-20, 107 to its cis-isomer and separation by LC identifies the residue. A homolog (pyrantel), which is used as an internal standard, is hydrolyzed to 3-(2-thienyl) acrylic acid. These acrylic acid isomers are readily resolved by LC. The method was evaluated over the 1-4 ppb (ng/mL) range for accuracy and precision to assess its utility for withdrawal studies. Bovine milk supplemented with morantel at 1, 2, and 4 ppb and assayed in replicate (n = 7-8) over 4 trials gave mean values and standard deviations of 1.0 +/- 0.11, 2.0 +/- 0.24, and 4.0 +/- 0.44 ppb, respectively. A milk specimen containing physiologically incurred residues of morantel assayed 2.1 +/- 0.19 ppb in replicate (n = 5).     

Liquid chromatographic determination of depletion of bithionol sulfoxide and its two major metabolites in bovine milk

A liquid chromatographic method is described for determining bithionol sulfoxide and its metabolites, bithionol and bithionol sulfone, in milk. Samples are treated with HCl to precipitate proteins and to permit extraction of bithionol sulfoxide in nonionized form. Tetrahydrofuran is added to the organic phase to facilitate extraction in diethyl ether; the dried residue is dissolved in chloroform, hexane, and sodium hydroxide and subjected to LC analysis. Residues of bithionol sulfoxide and its 2 metabolites were determined in milk of lactating cows. Holstein-Friesian dairy cows were administered a single oral dose of bithionol sulfoxide (50 mg/kg). Milk samples were analyzed with a reliable detection level of 0.025 microgram/mL for each compound. Residues of bithionol sulfoxide and bithionol were detected during 30 and 16 milkings, respectively; bithionol sulfone was never present at detectable levels.     

Liquid chromatographic determination of sulfamethazine in milk

A simple, relatively rapid liquid chromatographic method has been developed for the determination of sulfamethazine (SMZ) in milk at levels in the low ppb range. The method is based on extracting SMZ from milk with chloroform, evaporating the chloroform, dissolving the residues in hexane, extracting into buffers, and chromatographing the buffer solution. The method has been shown to determine levels as low as 5 ppb reliably. Levels greater than or equal to 7 ppb have been confirmed by gas chromatography/mass spectrometry after derivatization of extracts from fortified, incurred, and shelf milk. Intralaboratory recoveries and percent coefficients of variation are satisfactory. Sulfadimethoxine and sulfaquinoxaline can also be determined by the method. Application of the method to other dairy products is being investigated.     

Determination of aflatoxin M1 in milk by liquid chromatography with fluorescence detection

A liquid chromatographic (LC) method is proposed for the determination of aflatoxin M1 in milk. The method was successfully applied to both liquid whole and skim milk and also whole and skim milk powder. The samples are initially extracted with acetonitrile-water followed by purification using a silica gel cartridge and a C18 cartridge. Final analysis by LC was achieved using a radial compression module equipped with a 5 micron C18 column and a fluorescence detector. The method was successfully applied to samples at levels of 10 to 0.08 ppb added aflatoxin M1 with recoveries in the range of 70-98%.     

Determination of coumaphos and its oxygen analog in eggs and milk by using a multiresidue method with liquid chromatographic quantitation and capillary gas chromatographic/mass spectrometric confirmation

A multiresidue method for carbamate insecticides was adapted for the determination of coumaphos and its oxygen analog in eggs and milk. Eggs were extracted with acetonitrile and milk was extracted with acetone. Co-extractives were removed using liquid partitioning and charcoal column procedures described in the carbamate method. Coumaphos and its oxygen analog were determined by using a high performance liquid chromatograph equipped with a fluorescence detector. Recovery studies were performed for the 2 compounds at levels of 0.01 and 0.10 ppm in eggs and 0.01 and 0.02 ppm in milk. Overall average recovery was 100% (range 95-109%). In a trial of the method by another laboratory, the recovery of coumaphos and its oxygen analog from milk averaged 87 and 96%, respectively. Data are presented on the capillary gas chromatographic/mass spectrometric confirmation of coumaphos residues.     

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.     

Optimum conditions for formation of aflatoxin M1-trifluoroacetic acid derivative

Because thin-layer chromatographic (TLC) confirmation of identity and reverse-phase liquid chromatographic (LC) determination with fluorescence detection of aflatoxin M1 both require the derivative formed in the reaction of M1 and trifluoroacetic acid (TFA), various reaction conditions were studied to obtain complete derivative formation. Of the various organic solvents tested, the reaction between M1 and TFA proceeded best in the nonpolar solvents hexane and isooctane. Other parameters investigated were reaction temperature and time, aflatoxin M1 concentration, and solvent volume. The following procedure is considered optimum: 200 microL each of hexane and trifluoroacetic acid are mixed with M1 standard in a silylated glass vial or with milk residue in a regular glass vial with a Teflon-lined screw cap and heated 10 min at 40 degrees C. The mixture is evaporated to dryness under N2, and the derivative is saved for TLC or LC. No unreacted aflatoxin M1 was detected by reverse-phase LC after this procedure was incorporated for analysis of milk samples.     

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   

Optimization and validation of the reversed-phase high-performance liquid chromatography with fluorescence detection method for the separation of tocopherol and tocotrienol isomers in cereals, employing a novel sorbent material

The separation and determination of tocopherols (Ts) and tocotrienols (T3s) by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed and validated after optimization of various chromatographic conditions and other experimental parameters. Analytes were separated on a PerfectSil Target ODS-3 (250 × 4.6 mm, 3 μm) column filled with a novel sorbent material of ultrapure silica gel. The separation of Ts and T3s was optimized in terms of mobile-phase composition and column temperature on the basis of the best compromise among efficiency, resolution, and analysis time. Using a gradient elution of mobile phase composed of isopropanol/water and 7 °C column temperature, a satisfactory resolution was achieved within 62 min. For the quantitative determination, α-T acetate (50 μg/mL) was used as the internal standard. Detection limits ranged from 0.27 μg/mL (γ-T) to 0.76 μg/mL (γ-T3). The validation of the method was examined performing intraday (n = 5) and interday (n = 3) assays and was found to be satisfactory, with high accuracy and precision results. Solid-phase extraction provided high relative extraction recoveries from cereal samples: 87.0% for γ-T3 and 115.5% for δ-T. The method was successfully applied to cereals, such as durum wheat, bread wheat, rice, barley, oat, rye, and corn.     

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.     

Conversion of cephapirin to deacetylcephapirin in milk and tissues of treated animals

Cephapirin is one of six beta-lactam antibiotics approved for use in the treatment of food-producing animals in the United States. When used for treatment of mastitis by intramammary infusion, it is partially converted to a microbiologically active metabolite identified as deacetylcephapirin (DACEP). The degradation was followed in four cows with naturally acquired mastitis which were treated with cephapirin. DACEP persisted longer than the parent compound in the milk. When a calf was treated with cephapirin by intramuscular injection, the compound was almost completely converted to DACEP in tissues. The deacetyl form must be considered in the determination of residues in treated animals.     

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.     

Determination of aflatoxicol and aflatoxins B1 and M1 in eggs

Procedures from 2 methods, one for aflatoxins B1 and M1 in eggs and one for aflatoxicol in milk, blood, and liver, have been combined to determine the 3 toxins in eggs. The sample is blended with sodium chloride-saturated water and this mixture is then blended with acetone. After separation from the solid residue, the aqueous acetone extract is defatted with petroleum ether. The toxins are next partitioned into chloroform and separated from interferences on a silica gel column. Aflatoxicol is determined by fluorescence measurement after separation on a C18 reverse phase liquid chromatographic column, and aflatoxins B1 and M1 are determined by fluorescence densitometry after separation on a silica gel thin layer chromatographic plate. In a recovery study with eggs, mean recoveries of aflatoxicol added at levels of 0.1, 0.05, and 0.025 ng/g were 87, 77, and 78%, respectively. Mean recoveries of aflatoxins B1 and M1 added at a level of 0.1 ng/g were 75 and 87%, respectively, and at an added level of 0.05 ng/g were 86 and 75%. The within-laboratory precision (repeatability) ranged from 2 to 13%.     

Improved liquid chromatographic determination of riboflavin in milk and dairy products

Reported here is a simple liquid chromatographic (LC) method for the determination of riboflavin in milk (liquid, evaporated, and dry), yogurt, and cheese. The method involves passing liquid samples or filtrates of semisolid and solid samples through a C18 cartridge. Retained riboflavin is then eluted with an aliquot of 50% methanol in 0.02M acetate buffer of pH 4. A volume of the eluate is injected into the LC system consisting of a C18 column, a solvent of water-methanol-acetic acid (65 + 35 + 0.1, v/v) with a flow rate of 1 mL/min, and a UV detector set at 270 nm. The method is precise and accurate and compares favorably with the present AOAC method. Moreover, it involves fewer sample preparation steps and has a total analysis time of less than 1 h.     

Analysis of nitrate ion in nettle (Urtica dioica L.) by ion-pair chromatographic method on a C30 stationary phase

Nitrate ion is a frequent pollutant not only in soil and natural water resources but in vegetables and foods as well. In our study we focused on nettle due to its increased ability to accumulate nitrate ions. A new, simple method for the separation and determination of nitrate ion based on reversed-phase ion-pair chromatography has been elaborated. A new four-step sample pretreatment method enables the precipitation of proteins and oxidative degradation of compounds that may disturb the identification of the nitrate ion: (1) extraction of the total nitrate content, (2) precipitation of proteins with acetonitrile, (3) oxidative degradation of the organic contaminants with H2O2, (4) evaporation of the solvent and taking up of the residue in water. The chromatographic separations were carried out on a high-density C30 stationary phase under isocratic conditions. The optimal mobile-phase composition was 10% (v/v) acetonitrile and 90% (v/v) 20 mmol L(-1) phosphate buffer, containing 2 mmol of tetrabutylammonium hydroxide at pH 6.0. The method could also be used for the separation of IO3(-), SeO3(2-), BrO3(-), NO2(-), Br-, SeO4(2-), and I- ions. The validated method is sensitive (the detection limit is 0.18 ng of nitrate ion). The method is linear in a high concentration range (0.031-30.66 microg mL(-1)). Recoveries varied between 98% and 103%. Reproducibility of the elaborated sample pretreatment method showed 1.54%. The method can be used for the determination of nitrate ion from different plants.