Application of ion-exchange cartridge clean-up in food analysis IV. Confirmatory assay of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin, in bovine tissues by liquid chromatography-electrospray ionization tandem mass spectrometry.

A multiresidue analytical method was developed for the confirmation of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in bovine tissues using electrospray ionization liquid chromatography-tandem mass spectrometry (LC-ESI-MS-MS) with a product ion scan mode. All penicillins gave [M-H]-, [M-H-CO2]- and [M-H-141]- as the product ion, when [M-H]- was selected as the precursor ion. Combination of an ion-exchange cartridge clean-up and the LC-ESI-MS-MS method can reliably identify all of these penicillins fortified at a concentration of 0.05 mg/kg in bovine tissues, including liver, kidney and muscle. The limits of confirmation satisfy the maximum residue limits for each of the penicillins established by the World Health Organization, US Food and Drug Administration, European Union and Japan.

Application of ion-exchange cartridge clean-up in food analysis III. Determination of benzylpenicillin, phenoxymethyl-penicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in bovine liver and kidney by liquid chromatography with ultraviolet detection.

A multiresidue analytical method was developed for the simultaneous determination of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in bovine liver and kidney. The method involves the use of an ion-exchange cartridge for sample clean-up followed by ion-pair high-performance liquid chromatography with ultraviolet detection. The recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from bovine liver spiked at levels of 0.5 mg/kg and 0.1 mg/kg were in the range of 73-91% and 83-96% with coefficients of variation of 1.4-4.2% and 3.4-8.7%, respectively. For bovine kidney spiked at levels of 0.5 mg/kg and 0.1 mg/kg, the recoveries of these compounds were 79-92% and 82-92% with RSDs of 1.8-5.9% and 2.7-7.8%, respectively. The detection limits for the six penicillins were 0.02-0.05 mg/kg in bovine liver and kidney.

Simultaneous determination of triclabendazole and its sulphoxide and sulphone metabolites in bovine milk by high-performance liquid chromatography.

A simple method has been developed for the simultaneous determination of triclabendazole and its metabolites (sulphoxide and sulphone) in bovine milk by reversed-phase high-performance liquid chromatography (HPLC). A milk sample was homogenized with sodium sulfate anhydrous and acetonitrile, and centrifuged. The supernatant was isolated, rinsed with n-hexane saturated with acetonitrile, and evaporated. The residue was dissolved with 0.1 M potassium dihydrogenphosphate, and 0.1 M sodium hydrogencarbonate, and then cleaned up on a Bond Elut C18 cartridge. The three compounds were separated on a Capcell Pak C18 UG 120 (5 microm, 150x4.6 mm I.D.) column and determined by UV detection at 295 nm. The mobile phase was acetonitrile-0.05 M ammonium acetate (50:50), and the flow-rate was 0.8 ml/min at 40 degrees C. The mean recoveries (n=4) were 89.1-95.0% with a relative standard deviation of 1.1-2.6%. The detection limits were 0.004-0.006 microg/g in milk. The proposed method was used to monitor raw milk samples for the market, and applied to the analysis of milk samples from 10 cows which had been administered with triclabendazole to control the liver fluke. The confirmation of the triclabendazole and its metabolites in the above milk sample has been achieved by electrospray LC-MS for the first time.

Application of ion-exchange cartridge clean-up in food analysis. II. Determination of benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in meat using liquid chromatography with ultraviolet detection.

A multiresidue analytical method was developed for the simultaneous determination of benzylpenicillin (PCG), phenoxymethylpenicillin (PCV), oxacillin (MPIPC), cloxacillin (MCIPC), nafcillin (NFPC) and dicloxacillin (MDIPC) in meat. The method involves the use of an ion-exchange cartridge for sample clean-up followed by ion-pair high-performance liquid chromatography with ultraviolet detection. The recoveries of PCG, PCV, MPIPC, MCIPC, NFPC and MDIPC from pork muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg were in the range of 77-90, 73-95 and 80-93% with coefficients of variation of 0.5-1.7, 1.6-4.4 and 3.2-6.6%, respectively. For beef muscle spiked at levels of 0.5, 0.1 and 0.05 mg/kg, the recoveries of these compounds were 83-92, 71-86 and 77-90% with coefficients of variation of 1.7-4.4, 2.6-7.0 and 3.9-6.4%, respectively. The detection limits for each penicillin were 0.02 mg/kg in meat.

Simultaneous determination of beta-lactam antibiotics in milk by ion-pair liquid chromatography.

A simple and rapid ion-pairing liquid chromatographic method was developed for the simultaneous determination of five penicillins (PCs), ampicillin (AB-PC), benzylpenicillin (PC-G), Dicloxacillin (MDI-PC) and nafcillin (NF-PC) in milk. These PCs are most frequently used for the treatment of mastitis of cows. These antibiotics were extracted with acetonitrile from milk and cleaned up by solid-phase extraction with a C18 cartridge. PCs were separated on a Kaseisorb LC ODS-300-5 column with a mobile phase (1 ml/min) of acetonitrile-methanol-0.05 M potassium dihydrogenphosphate (20:10:80, v/v/v) mixture containing 5 mM of sodium 1-decanesulfonate adjusted to pH 3.5 and UV detection at 210 nm. The average recoveries of five PCs from milk fortified at 0.5 and 1.0 micrograms/ml (n = 5) were 79.8-89.4% with relative standard deviations ranging from 2.7 to 7.2% The detection limit of PCs in milk were 0.03-0.05 microgram/ml.

Simultaneous determination of five fasciolicides in milk by liquid chromatography with electrochemical detection.

A sensitive and specific method is described for determination of 5 fasciolicides in milk. The drugs are used to control liver flukes in cattle. The milk sample was homogenized with acetone and acetonitrile, sonicated, and centrifuged. The supernatant was extracted with dichloromethane. The extract was evaporated to dryness, dissolved in 1% sodium hydrogen carbonate, and purified on a C18 cartridge. The 5 drugs were separated from the matrix by reversed-phase liquid chromatography (LC) and determined by dual-electrode coulometric detection on a Kaseisorb LC ODS-300-5 column. The mobile phase was acetonitrile-0.05M potassium dihydrogen phosphate (55 + 45) at pH 3.0. The flow rate was 1 mL/min at 40 degrees C. The applied potentials of detectors 1 and 2 were set at 0.20 and 0.55 V, respectively. The average recovery of the drugs added to milk at 0.01 and 0.1 micrograms/mL was 89.6%, and the coefficient of variation was 4.7%. The detection limits of the drugs in milk were 4-20 ng/mL. The method is used to monitor commercial milk samples and to determine the residual levels of these drugs in milk from cows treated with a fasciolicide.

Determination of dephosphate bromofenofos in milk by liquid chromatography with electrochemical detection.

A sensitive, specific method for the determination of dephosphate bromofenofos (DBFF) in milk by liquid chromatography (LC) with electrochemical detection is described. DBFF, the only metabolite of bromofenofos (BFF, a fasciolicide), was extracted from milk by liquid-liquid partition with acetone, acetonitrile, and dichloromethane and purified by using a C18 cartridge. The compound was separated from the matrix peaks by reversed-phase LC and detected by dual-electrode coulometric detection on a Kaseisorb LC ODS-300-5 (250 x 4.6 mm id, 5 microns) column. The mobile phase was acetonitrile-0.05M potassium dihydrogen phosphate (55 + 45, v/v) at pH 3.0. The flow rate was 1 mL/min at 40 degrees C. The applied potentials of detectors 1 and 2 were maintained at 0.30 and 0.45 V, respectively. Average recoveries (n = 5) of DBFF from milk spiked at 1 and 10 ng/mL were 73.1 and 82.7%, respectively; and coefficients of variation were 8.4 and 2.8%, respectively. The detection limit of DBFF in milk was 0.2 ng/mL. Fifty-nine raw and 181 commercial milks were analyzed. DBFF was detected in 4 raw milks (0.2-1.5 ng/mL; average, 0.6 ng/mL) and in 3 normal liquid commercial milks (0.3-0.7 ng/mL; average, 0.5 ng/mL). The identity of DBFF from milk was confirmed by gas chromatography/mass spectrometry.

Determination of nitroxynil in cow milk by reversed-phase high-performance liquid chromatography with dual-electrode coulometric detection.

A sensitive and specific method is described for the determination of nitroxynil (fasciolicide) residues in cow milk. The milk samples were extracted with acetone and acetonitrile, following clean-up using a simple liquid-liquid extraction step. Nitroxynil was separated from the matrix peaks by reversed-phase high-performance liquid chromatography and detected using dual-electrode coulometric detection. The mobile phase was a mixture (20:80, v/v) of acetonitrile and 0.05 M potassium dihydrogenphosphate with the pH adjusted to 4.0. The flow-rate was 1 ml/min at 40 degrees C. The applied potentials of detectors 1 and 2 were maintained at -0.7 and +0.2 V, respectively. Average recoveries (n = 5) of nitroxynil from milk samples spiked at concentrations of 0.01 and 0.1 micrograms/ml were found to be 92.0% and 97.0% with coefficients of variation of 6.2% and 2.2%, respectively. The detection limit of nitroxynil in milk was 0.7 ng/ml. During the analysis of 30 raw cow milk and 140 market milk samples, nitroxynil was detected at a level of 4 ng/ml in one raw cow milk sample.

[Investigation of the residue of the fasciolicide nitroxynil in milk and dairy products].

A sensitive gas chromatographic method with electron capture detection of the fasciolicide, nitroxynil, in milk and dairy products was developed and was applied to assess nitroxynil concentration in cow's milk after subcutaneous injection of three lactating cows. The level of nitroxynil in cow's milk reached a maximum (0.25-0.26 micrograms/ml) in 6-30 hours, and was undetectable within 8 weeks. Analysis of nitroxynil concentrations in cream, skimmed milk, curd and whey prepared from nitroxynil excreting cow's milk showed that the chemical became concentrated in cream and curd. Nitroxynil appeared to be stable at temperature used in LTLT and HTST pasteurization with the rate of degradation in milk being less than 10%. Investigation of nitroxynil residues in milk (raw, liquid whole, processed) and dairy products (processed cheese, natural cheese, butter, sweetened condensed milk, evaporated skimmed milk, skimmed milk powder, formulated milk powder) was performed during 1976-1979. Nitroxynil was detected in 20% of milk samples at a maximum level of 39 ng/ml, with one formulated milk powder at level of 0.34 ng/g. Confirmation of nitroxynil was performed by GCMS. The results of this investigation were promptly reported to the government. Since then administrative guidance was provided leading to appropriated use of nitroxynil thereafter. The ordinance controlling dairy production amended in 1979 revised the withdrawal time for medicines administrated to cows from "three days" to "the period when medicine remains in the milk". There was no mutagenicity of nitroxynil evident by the Ames test.

Determination of phenol in honey by liquid chromatography with amperometric detection.

A sensitive, selective analytical method has been developed for determination of phenol in honey by liquid chromotography (LC) with amperometric detection (AMD). Phenol is extracted with benzene from the distillate of honey. The benzene extract is washed with 1% sodium bicarbonate solution and then reextracted with 0.1N sodium hydroxide followed by cleanup on a C18 cartridge. Phenol is determined by reverse-phase LC with amperometric detection. An Inertsil ODS column (150 X 4.6 mm, 5 microns) is used in the determination. The mobile phase is a mixture (20 + 80 v/v) of acetonitrile and 0.01M sodium dihydrogen phosphate containing 2mM ethylenediaminetetraacetic acid, disodium salt (EDTA) with the pH adjusted to 5.0. The flow rate is 1 mL/min under ambient conditions. The applied potential of the AMD using a glassy carbon electrode is 0.7 V vs an Ag/AgCl reference electrode. Average recoveries of phenol added to honey were 79.8% at 0.01 ppm spiking level, 90.4% at 0.1 ppm, and 91.0% at 1.0 ppm. Repeatabilities were 3.4, 1.3, and 1.8%, respectively. The detection limit of phenol in honey was 0.002 ppm. For analysis of 112 commercial honey samples, the range and average values of 32 detected samples were 0.05-5.88 ppm and 0.71 ppm, respectively.