Yoshitomo Ikai

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.

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 d,l-FDLA derivatization to determination of absolute configuration of constituent amino acids in peptide by advanced Marfey's method

Abstract A derivatization procedure including d,l -FDLA was developed for the advanced Marfeys method which is an unempirical method for the determination of the absolute configuration of an amino acid. The procedure was successfully applied to the determination of the absolute configuration of Ahp, one of constituent amino acids in aeruginopeptin 228-A ( 1 ).

Separation and identification of microcystins in cyanobacteria by frit fast atom bombardment liquid chromatography mass spectrometry

In order to separate and identify microcystins, a new analytical method was developed using a frit probe as an interface for fast atom bombardment mass spectral analysis of high performance liquid chromatographic (HPLC) effluents. Two types of HPLC conditions were designed for separation of standard microcystins RR, YR and LR. The HPLC conditions, for example, methanol:0.01% trifluoroacetic acid = 61:39 (containing 0.8% glycerol) as a mobile phase and 0.5 ml/min as a flow rate, provided a base line separation of standard microcystins RR, YR and LR. The HPLC conditions were also effective for separation of the non-toxic geometrical isomers of microcystins RR and LR. The total ion chromatogram of a mixture of standard microcystins showed excellent correlation with the HPLC separation using a u.v. detector. The method was subsequently applied to analysis of microcystins contained in both a culture strain and a field sample, and the procedure from toxin extraction to identification of microcystins was performed within 1 day. The mass chromatogram monitored at m/z 135 that is always observed with abundance in the FAB mass spectra of the purified microcystins, differentiated between microcystins and other types of compounds. This technique allowed the rapid identification of unknown microcystins without standard samples. Additionally, compounds other than microcystins were also found, which would not be seen by u.v. detection at 238 nm.

Improvement of chemical analysis of antibiotics : XV. Isocratic high-performance liquid chromatographic methods for the analysis and preparative separation of the components of bacitracin☆

Isocratic high-performance liquid chromatographic (HPLC) systems were established for analytical and preparative separation of the components of the antibiotic preparation bacitracin (BC). The best analytical results were obtained using a C18 modified silica gel column (Capcell Pak C18) with a solvent system of 0.04 M disodium hydrogenphosphate buffer and methanol (4:6), pH 9-10. The calibration graphs showed good linear relationships between 50 and 1000 ng for BC-A and between 65 and 1000 ng for BC-F. With respect to the preparative HPLC, a Capcell Pak C18 column with methanol-0.05 M aqueous sodium sulphate solution (6:4) as a mobile phase gave satisfactory results. The isolation of BC-A and -F was readily achieved without decomposition of the components by using the present preparative HPLC followed by desalting on a prepacked C18 cartridge.

Lysis of cyanobacteria with volatile organic compounds

One of bacteria collected from Lake Sagami, Japan, Brevibacillus sp., was found to have a lytic activity of cyanobacteria, but did not produce active compounds. Instead, the co-culturing of Microcystis with the Brevibacillus sp. enhanced the production of two volatile compounds, beta-cyclocitral and 3-methyl-1-butanol, and the former had a characteristic lytic activity. It was confirmed that these volatile compounds were derived from the cyanobacteria themselves. beta-Ionone, geosmin and 2-methylisoborneol derived from cyanobacteria and similar volatile compounds, terpenoids, produced by plants also had a lytic activity. The minimum inhibitory concentration values of the cyanobacterial metabolites were estimated to be higher than those of compounds from plants except for a few compounds. Among them, beta-cyclocitral only produced a characteristic color change of culture broth from green to blue. This color change is similar to the phenomenon observed when a sudden decline in growth of cyanobacteria begins in a natural environment.

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.

A method usingL/CMS for determination of absolute configuration of constituent amino acids in peptide --- advanced Marfey's method ---

A method including Marfeys method and frit-FAB or ESIL/CMS was developed for the determination of the absolute configuration of constituent amino acids in peptides. It was successfully applied to anabaenopeptins produced by the cyanobacteriumAnabaena flos-aquae NRC 525-17.

Simple method for the analysis of food dyes on reversed-phase thin-layer plates

A technique for the determination of food dyes using reversed-phase thin-layer chromatography on octadecyl-modified silica is described. A solvent system containing 5.0% aqueous sodium sulphate solution enables good separation of the food dyes. Their separation is dependent on the pH of the solvent system, good separation among all dyes being obtained between pH 6.0 and 7.0. The determination of thirteen dyes can be achieved by a combination of methanol-acetonitrile-5.0% aqueous sodium sulphate solution (3:3:10) (solvent system A) and methanol-methyl ethyl ketone-5.0% aqueous sodium sulphate solution (1:1:1) (solvent system B). Solvent system A was used for the determination of Tartrazine, Amaranth, Indigo Carmine, New Coccine, Sunset Yellow FCF, Allura Red AC, Fast Green FCF and Brilliant Blue FCF and for the screening of Acid Red (R-106), Eosin (R-103), Erythrosin (R-3), Rose Bengale (R-105) and Phloxine (R-104). When the spots of R-3, R-103, R-104, R-105 and R-106 appear on the plate, their determination can be achieved by using solvent system B. Each calibration graph was linear between 0.2 and 1.0 micrograms.

Further application of advanced Marfey's method for determination of absolute configuration of primary amino compound

Abstract The advanced Marfeys method was applied to the determination of the absolute configuration of primary amino compounds and its utility was extended from amino acids to primary amino compounds. This method was successfully applied to the characterization of not only constituent amino acids but also amino compounds in microginin and nostophycin produced by cyanobacteria.