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.

Survey of residual tetracyclines in kidneys of diseased animals in Aichi Prefecture, Japan (1985-1997).

A survey was conducted to determine the incidence of tetracycline antibiotic (TCAs) residues in the kidneys of slaughtered animals that did not pass inspection for human consumption by the Japanese Food Sanitation Law and the Meat Inspection Law at the slaughterhouses in Aichi Prefecture, Japan, from April 1985 to March 1998. The kidneys were analyzed by the AOAC Official Method 995.09. Among 424 animals (147 cattle and 277 pigs), 131 (30.9%) were contaminated with TCAs, including 69 (16.3%) with chlortetracycline (CTC), 61 (14.4%) with oxytetracycline (OTC), 3 (0.7%) with tetracycline (TC), and 1 (0.2%) with doxycycline (DC). One sample (cattle kidney) was contaminated with both OTC and DC. The frequencies of OTC and TC residues were significantly higher (p < 0.05) in cattle than in pigs, whereas, the frequency of CTC was significantly higher (p < 0.01) in pigs. Pig kidney samples collected in 1991-1997 had significantly higher incidences of TCAs and CTC (p < 0.01) residues than those in 1985-1986.

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.

Application of ion-exchange cartridge clean-up in food analysis. V. Simultaneous determination of sulphonamide antibacterials in animal liver and kidney using high-performance liquid chromatography with ultraviolet and mass spectrometric detection.

A simple, rapid, and reliable method for the determination of residual sulphonamide antibacterials (SAs) (sulfadiazine, sulfamerazine, sulfadimidine, sulfamethoxypiridazine, sulfisozole, sulfamonomethoxine, sulfamethoxazole, sulfisoxazole, sulfadimethoxine, and sulfaquinoxaline) in animal liver and kidney was developed using a combination of clean-up on a Bond Elut PSA cartridge and HPLC with UV detection. The SAs were extracted with ethyl acetate and then dissolved in 5 ml of 50 v/v% ethyl acetate-n-hexane after being evaporated to dryness. For clean-up of the crude sample, the resuspended extract was applied to a Bond Elut PAS (primary/secondary amine cartridge), and then SAs were eluted from the cartridge using 5 ml of 20 v/v% acetonitrile-0.05 M ammonium formate before being analysed by HPLC. Recoveries of the SAs at the levels of 0.5 and 0.1 microg/g were 70.8-98.2%, the rerative standard deviation were less than 7.0%, and the detection limits were 0.03 microg/g. The present analysis method of SAs in animal kidney and liver using HPLC with a clean-up procedure was demonstrated to be highly applicable to the direct LC-MS-MS analysis without any modification.

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.

Application of ion-exchange cartridge clean-up in food analysis. I. Simultaneous determination of thiabendazole and imazalil in citrus fruit and banana using high-performance liquid chromatography with ultraviolet detection.

A simple, rapid and reproducible analytical method for thiabendazole (TBZ) and imazalil (IMA) in citrus fruit and banana has been developed. 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 TBZ and IMA from citrus fruits spiked at levels of 10 microgram/g and 5 microgram/g were in the range of 94-98% and 93-98% with coefficients of variation of 0.5-2.2% and 1.6-2.7%, respectively. The recoveries of TBZ and IMA from banana spiked at levels of 3 microgram/g and 2 microgram/g were 94% and 94% with coefficients of variation 1.1% and 4.9%, respectively. The detection limits for TBZ and IMA were 0.1 microgram/g in citrus fruit and 0.05 microgram in banana.

Mass spectrometric analysis of tetracycline antibiotics in foods.

We will review recent developments in mass spectrometric analysis of tetracycline antibiotics (TCs) in foods. The mass spectrometric techniques discussed are as follows: the collision-activated decomposition mass-analysed ion kinetic energy spectrometry (CAD MIKES), thin-layer chromatography (TLC)- fast atom bombardment (FAB) mass spectrometry (MS), particle beam (PB) liquid chromatography (LC)-MS, LC-fit FAB MS, thermospray (TSP) LC-MS, atmospheric chemical ionization (APCI) LC-MS and tandem electrospray (ESI) LC-MS. Their advantages and limitations are described in the confirmation of TCs in foods: CAD MIKES can confirm TCs with high sensitivity; however, its practical application is questionable because of uncommon instrumentation. TSP has a problem in reproducibility of the mass spectrum. Although TLC-FAB-MS can be applied to any kind of samples, it cannot be used for the quantitative analysis. LC-frit FAB-MS is a useful technique for the confirmation of TCs in honey, but it cannot be applied to animal tissues because of a lack of sensitivity. PB negative chemical ionization, APCI, and ESI-MS-MS can reliably confirm TCs in foods with good reproducibility.

Total structures of colistin minor components.

Structural characterization of the colistin (CL) components were carried out using Frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS), tandem mass spectrometry (MS/MS) and the amino acid analysis proposed by MARFEY, and the total structures of 4 minor components including the absolute configuration of the constituent amino acids were proposed. The structures of the minor components were the same as those of the main component colistin A or B except that L-leucine is replaced by L-valine or L-isoleucine.

Improvement of chemical analysis of antibiotics. XXIII. Identification of residual tetracyclines in bovine tissues by electrospray high-performance liquid chromatography-tandem mass spectrometry.

To reliably identify the residual tetracycline antibiotics (TCs), oxytetracycline (OTC), tetracycline, chlortetracycline (CTC) and doxycycline (DC), in bovine tissues, we have established a confirmation method using electrospray ionization liquid chromatography-tandem mass spectrometry (ESI LC-MS-MS) with daughter ion scan. All TCs gave [M+H-NH3]+ and [M+H-NH3-H2O]+ as the product ions, except for DC when [M+H]+ was selected as the precursor ion. The combination of C18 cartridge clean-up and the present ESI LC-MS-MS method can reliably identify TCs fortified at a concentration of 0.1 ppm in bovine tissues, including liver, kidney and muscle, and has been successfully applied to the identification of residual OTC in bovine liver and residual CTC in bovine muscle samples previously found at concentrations of 0.58 ppm and 0.38 ppm by LC, respectively.

Total structures and antimicrobial activity of bacitracin minor components.

Total structures of 13 minor components of bacitracin (BC) were proposed, and their antimicrobial activities were investigated. The components of BC including bacitracins A (BC-A) and F (BC-F) were isolated by preparative HPLC and were hydrolyzed under acidic conditions. The resulting amino aids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide and were separated by HPLC to determine their absolute configurations. It was found that the N-terminal amino acids of BC-A and its related components were epimerized during the hydrolysis to yield their enantiomers. The formation of these artifactual amino acids suggests that our previously proposed structures of the BC minor components are incorrect; therefore, the structures were corrected based on these results. The structures of the BC minor components were the same as that of BCs-A and -F except that one to three of the L-isoleucines, including the N-terminal one, were replaced by L-valines. These structures were confirmed by tandem mass spectrometry under fast atom bombardment (FAB) conditions and Frit-FAB liquid chromatography/mass spectrometry. Based on the UV spectra of the BC components determined by photodiode array detection-HPLC analysis, a new systematic nonmenclature was proposed for the minor components. The isolated components were also used for the determination of their minimal inhibition concentrations and it was found that BC-A is 2 approximately 8 times more potent than the other minor components against strains of Micrococcus luteus and Staphylococcus aureus.

Reliable and sensitive method for determination of microcystins in complicated matrices by frit-fast atom bombardment liquid chromatography/mass spectrometry.

A reliable and sensitive method for determination of hepatotoxic microcystins in complicated matrices by frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS) is described. Immonium ions of constituent amino acids, which were obtained together with molecular ion species by FAB mass spectral analysis of standard microcystins RR, YR, LR, and [D-Asp3] and [Dha7]microcystins LR using flow injection system composed of Frit-FAB probe, showed potential for reliable identification of microcystins by Frit-FAB LC/MS. Frit-FAB LC/MS using a microbore column provided not only the baseline separation of standard microcystins RR, YR, and LR but 200-fold higher sensitivity than that using conventional column. Furthermore, when a selected ion monitoring (SIM) technique was used, the detection limits of microcystins RR, YR, and LR were 300, 350, and 400 pg, respectively, at a signal-to-noise ratio of 5:1, and calibration curves of each microcystin showed a linear relationship from 2 ng to 50 ng. Finally, identification and quantitative analyses of microcystins in water samples were carried out.

Analysis of methyl isothiocyanate in wine by gas chromatography with dual detection.

Methyl isothiocyanate (MITC) was extracted from a 5 mL wine sample with 2 mL dichloromethane, and the extract was washed with 5% sodium chloride solution. MITC was determined by gas chromatography with a dual nitrogen-phosphorus detector (NPD) and a flame photometric detector (FPD, S mode) (DB-210 column, 30 m x 0.53 mm id, column temperature, 70 degrees C). The limit of detection and quantitation of MITC with NPD-GC were 0.003 and 0.01 ppm, respectively. Average recoveries of MITC added to wine at 1 and 0.1 ppm were over 95%. This method is simple and rapid for routine analysis of MITC in wine.

Single neurons in the ventral tegmental area that project to both the cerebral and cerebellar cortical areas by way of axon collaterals.

Recently, we have found that the ventral tegmental area of the rat sends dopaminergic and non-dopaminergic axons, respectively, to the cerebellar cortex and deep cerebellar nuclei [Ikai et al. (1992) Neuroscience 51, 719-728]. In the present study, employing fluorescent retrograde double labeling with Fast Blue and Diamidino Yellow, we examined whether individual neurons in the ventral tegmental area of the rat send their axons to both the cerebellum and other brain regions. The neurons projecting to the cerebellar cortex often issued axon collaterals to the cerebral cortical areas, including the prelimbic-anterior cingulate cortices and piriform-entorhinal cortices, but not so frequently to the subcortical regions, including the nucleus accumbens, lateral septum, amygdala, and lateral habenula. On the other hand, the neurons projecting to the deep cerebellar nuclei rarely sent axon collaterals to the cerebral cortical and subcortical regions.

Improvement of chemical analysis of antibiotics. Part XIX: Determination of tetracycline antibiotics in milk by liquid chromatography and thin-layer chromatography/fast atom bombardment mass spectrometry.

A precise liquid chromatographic (LC) determination with high sensitivity and a reliable mass spectrometric (MS) confirmation were established for the determination of tetracycline antibiotics (TCs) in milk. The determination of the TCs was based on C18 cartridge cleanup followed by LC separation. Recoveries of TCs from milk fortified at 20 ppb were 73.0-81.8%, and coefficients of variation were 2.6-4.6%. With this method, concentrations of oxytetracycline and tetracycline as low as 10 ppb and chlortetracycline and doxycycline as low as 20 ppb were determined readily. Thin-layer chromatography (TLC)/fast atom bombardment (FAB) MS with a condensation technique was used to confirm TCs in milk. After the C18 cartridge cleanup, separation on a C8 TLC plate, and application of the sample condensation technique, FAB mass spectra were measured. TCs in milk at 50 ppb were reliably confirmed by TLC/FABMS.

Identification of unlawful food dyes by thin-layer chromatography-fast atom bombardment mass spectrometry.

A thin-layer chromatographic-fast atom bombardment mass spectrometric (TLC-FAB-MS) method incorporating an analyte condensation technique was established for the identification of the 27 food dyes consisting the twelve dyes permitted for use in foods and the fifteen unlawful dyes in Japan. The use of magic bullet [1,4-dithiothreitol-1,4-dithioerythritol (3:1)] as a matrix allowed the measurement of the FAB mass spectra of the food dyes except for Food Blue No. 2 (Indigo Carmine). The separation was performed on a C18-modified silica gel TLC plate using the following two solvent systems: methanol-acetonitrile-5% aqueous sodium sulphate solution (3:3:10) and methyl ethyl ketone-methanol-5% aqueous sodium sulphate solution (1:1:1). The condensation technique for concentration of a diffuse sample spot on the TLC plate improved the detection limit 4-20-fold with good reproducibility. The method was successfully applied to the identification of unlawful dyes in imported foods.

Direct projections from the entopeduncular nucleus to the lower brainstem in the rat.

The present study reports the existence of projection fibers from the entopeduncular nucleus to the superior colliculus and lateral parts of the pontobulbar tegmental regions (so-called lateral tegmental field) in the rat, suggesting that the entopeduncular nucleus may control eye-head and orofacial movements via these projection fibers. The anterograde axonal tracing with Phaseolus vulgaris-leucoagglutinin has revealed that the entopedunculotectal fibers terminate, bilaterally, with an ipsilateral predominance, in the deep layers of the superior colliculus through its rostral one-third level and that the entopedunculotegmental fibers terminate, bilaterally, with an ipsilateral predominance, in the parabrachial area, reticular formation surrounding the trigeminal motor nucleus, and parvicellular, dorsal, and ventral reticular nuclei. The cells of origin of the entopedunculotectal and entopedunculotegmental projections have been identified by retrograde axonal tracing with Fluoro-Gold and cholera toxin B subunit. The entopedunculotectal or entopedunculotegmental fibers originate, respectively, from the dorsal or ventral part of the entopeduncular nucleus. Additionally, a series of fluorescent retrograde double-labeling experiments with Fast Blue and Diamidino Yellow have indicated that single entopeduncular nucleus neurons projecting to the superior colliculus or lateral tegmental field often send their axon collaterals to the lateral habenular nucleus. The entopedunculotectal fibers are assumed to control head movements, which may be provoked via the tectospinal fibers, and further to participate in eye movements as the nigrotectal fibers that have been known to arise from the substantia nigra pars reticulata to end in the deep layers of the superior colliculus primarily through its caudal two-thirds level. The entopedunculotegmental fibers are presumed to be involved in control of orofacial movements, because the sites of termination of the entopedunculotegmental fibers correspond well with the reported areas of distribution of premotor interneurons for the trigeminal motor, facial, and hypoglossal nuclei.

Germinoma in siblings: case reports.

We present the first reported cases of germinoma occurring in siblings of different genders. One tumor occurred in the left basal ganglia of a 7-year-old boy and the other in the suprasellar region of his 9-year-old sister. Both tumors were diagnosed as germinoma of two-cell pattern based on light and electron microscopic studies. Possible genetic factors responsible for this phenomenon are discussed.

Direct projections from the deep layers of the superior colliculus to the subthalamic nucleus in the rat.

Direct projections from the superior colliculus to the subthalamic nucleus were found in the rat in antero- and retrograde tracing experiments; these projections arose mainly from the deep layers of the superior colliculus at its middle to caudal levels. The present findings suggest that signals from the deep layers of the superior colliculus may modulate the basal ganglia activity during orienting behavior.