Rapid determination of nitroimidazole residues in honey by liquid chromatography/tandem mass spectrometry.

We developed a rapid and efficient means of determining residues of four nitroimidazoles-i.e., dimetridazole, ipronidazole, metronidazole, and ronidazole-and three hydrophilic metabolites- i.e., 2-hydroxymethyl-1-methyl-5-nitroimidazole, 1 -methyl-2-(2'-hydroxyisopropyl)-5-nitroimidazole, and 1-(2-hydroxyethyl)-2-hydroxymethyl-nitroimidazole--in honey. We applied a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) procedure improved to suit a nitroimidazole analysis, which is fast (approximately 30 min) and uses less organic solvent. The procedure involves initial single-phase extraction of 5 g of honey with acetonitrile containing 1% acetic acid, followed by liquid-liquid partitioning involving the addition of 5 g sodium chloride, 1.5 g trisodium citrate dihydrate, and 4 g magnesium sulfate. Moreover, matrix from honey was reduced by an SPE method with an alumina-N cartridge. The samples were analyzed using LC/MS/MS. Chromatographic separation of these nitroimidazoles and metabolites was performed in the gradient mode on a pentafluorophenylpropyl-bonded silica column (150x2.0 mm, 3 pm particle size) at 40 degrees C. The mobile phase consisted of a 0.01% acetic acid solution and acetonitrile, and the flow rate was 0.2 mL/min. The method was validated using honey spiked with these nitroimidazoles from 0.1 to 0.5 microg/kg. The overall recovery of the seven nitroimidazoles ranged from 76.1 to 98.5%; intra- and interassay CV values were <9.5 and <14.2%, respectively. The LOQ ranged from 0.1 to 0.5 microg/kg. LC/MS/MS coupled with the QuEChERS method showed good potential as a method for determining nitroimidazole residues in honey.

Rapid determination of fumagillin residues in honey by liquid chromatography-tandem mass spectrometry using the QuEChERS method.

A new, rapid, and efficient method for determining the fumagillin residues in honey was developed. The samples extracted were analyzed using LC/MS/MS. Chromatographic separation of fumagillin was performed in gradient mode on a C8 column (100 x 2.0 mm, 5 microm) at 40 degrees C. The mobile phase consisted of a mixture of 2 mM ammonium formate-0.01% formic acid solution and methanol; the flow rate was set to 0.2 mL/min. Under these conditions, it was possible to measure fumagillin and its isomers as a single peak. The sample preparation procedure used is based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, which is fast (approximately 30 min) and uses less organic solvent. The fumagillin was extracted with acetonitrile containing 0.1% formic acid, then purified using a solid-phase extraction method with an Oasis mixed-mode weak anion-exchange cartridge. The overall recovery of fumagillin ranged from 88.1 to 99.4%; the intra- and interassay CVs were <4.5% and <4.9%, respectively. The LOQ was 0.1 microg/kg. LC/MS/MS coupled with the QuEChERS method showed strong potential as a method for determining fumagillin residues in honey.

Determination of bithionol, bromophen, nitroxynil, oxyclozanide, and tribromsalan in milk with liquid chromatography coupled with tandem mass spectrometry.

LC/MS/MS was developed to determine the residues of bithionol (BTN), bromofen (BMF), nitroxynil (NTX), oxyclozanide (OCZ), and tribromsalan (TBS) in milk. Samples were extracted with ethyl acetate and cleaned up by liquid-liquid separation with acetonitrile and n-hexane. The compounds were determined by RP-LC using a C18 column with 0.1% formic acid-methanol. Mass spectral acquisition was performed in the negative mode by applying selected-reaction monitoring. The method was validated in milk spiked with these compounds at 5-600 microg/kg; average recoveries were in the range 83.8-97.1%, with RSD values of 1.4-8.0%. The interassay RSDs were less than 11%. The LODs of these compounds in milk were 0.1 microg/kg. The method was applied to 24 raw milk samples. The concentration of these compounds in all samples was lower than the Japanese maximum residue limits. The method is rapid, sensitive, and specific for monitoring residues of BTN, BMF, NTX, OCZ, and TBS in milk.

[Rapid determination of residues of 4 tetracyclines in meat by a microbiological screening, HPLC and LC/MS/MS].

A rapid and precise determination residues of 4 tetracyclines (TCs) (oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DOXY)) in meat was developed by employing three analyses; a microbiological screening, HPLC and LC/MS/MS. TCs were extracted with pH 4.0 McIlvaine buffer containing 0.01 mol/L EDTA from a meat sample, and then purified using a mixed mode, reversed-phase and cation-exchange cartridge. The mean recoveries (n=5) of 0.2 microg/g OTC, TC and CTC, 0.05 microg/g DOXY spiked in meat samples were 76.6-99.0% (C.V. 1.6-5.4%). In 13 meat samples in which the microbiological screening indicated the presence of TCs, CTC (9 samples) and DOXY (4 samples) were identified with HPLC and LC/MS/MS.

[Microbiological method for the detection of antibiotic residues in meat using mixed-mode, reverse-phase and cation-exchange cartridge].

A microbiological method for screening of residual benzylpenicillin (PCG), oxytetracycline (OTC) and spiramycin (SPM) in meat using a single mixed mode, reversed-phase and cation-exchange cartridge was developed. A meat sample was extracted with 0.1 mol/mL pH 4.5 phosphate buffer and the extract was applied to a MCX cartridge. The cartridge was washed, and adsorbed antibiotic residues were eluted with acetonitrile for acidic fractions and acetonitrile containing 5% ammonia solution-0.1 mol/mL pH 4.5 phosphate buffer (9:1, v/v) for basic fractions. Each eluate was evaporated to dryness and the residue was dissolved in phosphate buffer to prepare test solutions for microbiological assay. When the diameter of the inhibition zone was more than 12 mm, the result was considered positive. In this method, the average recoveries of PCG at 0.05 microg/g, OTC at 0.1 microg/g and SPM at 0.2 microg/g were 70%, 92% and 84%, respectively. Tolerances of the three antibiotics were detected. All the results demonstrate that this method is simple, rapid and useful for screening of these three antibiotic residues in meat.

Screening assay of residual antibiotics in livestock samples by LC-MS/MS.

A LC-MS/MS screening assay of multi-class antibiotics was developed for 19 residual antibiotics in livestock samples. Sample preparation employed the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach using 0.5% formic acid in acetonitrile-methanol (8 : 2), with salting-out using magnesium sulfate, trisodium citrate and sodium chloride. Recovery values from 5 different livestock samples ranged from 45.5 to 121.6%, and the RSDs were under 18% at two concentration levels. The limit of quantification values of 19 analytes were under 10 µg/kg in all livestock samples, and the procedure can detect almost all analytes under the MRL. Screening capability was confirmed by employing spiked samples. This new screening assay for residual antibiotics in livestock samples is expected to be useful for routine laboratory tests.

[Determination of nequinate and buquinolate in livestock products using liquid chromatography-tandem mass spectrometry].

We studied the simultaneous determination of nequinate and buquinolate, which are used as feed additives to prevent coccidiosis, by means of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The sample was extracted with acetonitrile, then loaded onto an HLB mini-column with 20% methanol. After clean-up with 20% methanol, the analytes were eluted with acetonitrile-methanol (1 : 1). The coccidiostats in the purified samples were determined using ESI-MRM mode LC-MS/MS with a sample matrix calibration curve. Mean recoveries of nequinate and buquinolate from 8 kinds of livestocks samples (chicken muscle, chicken liver, chicken heart, swine muscle, swine heart, cattle muscle, sheep muscle, egg) were in the range of 89.5% to 108.6%, and the relative standard deviation values were <20% (n=10) at the levels of 0.01 µg/g and 0.05 µg/g, respectively. The limits of quantification of these compounds were 0.001 µg/g in each sample.

Determination of dimetridazole, metronidazole and ronidazole in salmon and honey by liquid chromatography coupled with tandem mass spectrometry.

A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed to determine the residues of dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ) in salmon and honey. These compounds were extracted with ethyl acetate from samples and cleaned up using a silica solid phase extraction (SPE) cartridge. These compounds were determined by reversed-phase LC using a C18 column with distilled water-methanol as the mobile phase, and MS detection in the positive mode by applying selected reaction monitoring (SRM). DMZ-d(3), MNZ-(13)C(2),(15)N(2) and RNZ-d(3) were used as internal standards. The method was validated in salmon and honey spiked with these compounds at 0.4-2 µg/kg, and average recoveries were in the range of 91.2-107.0%. Repeatability was 1.7-17.1% and intermediate precision was less than 20%. The detection limits of DMZ, MNZ and RNZ in salmon and honey were 0.05-0.2 µg/kg. The method was applied to 3 salmon and 20 honey samples. The concentrations of these compounds in all samples were lower than the detection limits established by the Ministry of Health, Labour and Welfare in Japan.