Masahiko Takino

Determination of chloramphenicol residues in fish meats by liquid chromatography-atmospheric pressure photoionization mass spectrometry

A liquid chromatography-atmospheric pressure photoionization (APPI) mass spectrometry method was developed for the determination of chloramphenicol (CAP) in fish meats (young yellowtail and flatfish). For the optimization of APPI, several APPI ion source parameters and mobile phases were investigated. CAP with APPI using the optimized parameters gave simple mass spectra and a strong signal corresponding to [M-H]- was observed. Further, APPI was compared with atmospheric pressure chemical ionization (APCI) and APPI gave similar results in terms of structural information and a better signal-to-noise ratio. The samples were extracted with acetonitrile and evaporated to dryness followed by a clean-up step using the liquid-liquid distribution between acetonitrile and n-hexane. The mean recoveries of chloramphenicol from a young yellowtail meat and a flatfish meat spiked at 0.1-2 ng/g were 89.3-102.5 and 87.4-94.8%, respectively. The limit of detection (signal-to-noise ratio = 3) of the young yellowtail meat and the flatfish meat were 0.1 and 0.27 ng/g.

Determination of nivalenol and deoxynivalenol by liquid chromatography/atmospheric pressure photoionization mass spectrometry

Fusarium species, a plant pathogenic fungus of wheat and other cereals, produces toxic metabolites such as nivalenol (NIV) and deoxynivalenol (DON). Control of contamination by these toxins is very difficult, and a continuous survey of the occurrence is necessary for these toxins. Thus, the accurate and convenient determination of the cereals contaminated with these toxins is important for the supply of safe foods. A selective analytical method based on high-performance liquid chromatography, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for simultaneous determination of NIV and DON. The parameters investigated for the optimization of APPI were the ion source parameters fragmentor voltage, capillary voltage, and vaporizer temperature, and also mobile phase composition and flow rate. Furthermore, chemical noise and signal suppression of analyte signals due to sample matrix interference were investigated for APPI. The results indicated that APPI provides lower matrix effect and the correlation coefficient of NIV and DON in the range 0.2-100 ng x mL(-1) was above 0.999. Recoveries of NIV and DON in wheat ranged from 86 to 107% and limits of detection of NIV and DON were 0.20 ng x g(-1) and 0.39 ng x g(-1), respectively. In addition, the proposed method was applied for the analysis of naturally contaminated wheat samples. APPI was found to offer lower matrix effect and was a convenient technique for routine analysis of NIV and DON residues in wheat at trace levels.

Quantitative liquid chromatography-mass spectrometry determination of catechins in human plasma by automated on-line extraction using turbulent flow chromatography.

A simple, fast and sensitive liquid chromatography-mass spectrometry (LC-MS) method with automated on-line extraction using turbulent flow chromatography (TFC) for the determination of five catechins in human plasma was developed. In this method, after on-line extraction by its injection onto an extractor column at turbulent flow, five catechins were backwashed onto a reversed phase column via on-line column switching and separated chromatographically at a laminar flow of 1 ml min(-1). Using this tandem LC-LC-MS system, the extraction, the separation and the quantitation of five catechins in human plasma could be achieved with satisfactory selectivity and sensitivity. The limit of detection (S/N = 3) ranged from 0.6 to 2 ng ml(-1). The described procedure was very simple and rapid since no off-line sample preparation was required, total analysis time being 18.5 min.

Determination of Fusarium mycotoxins by liquid chromatography/tandem mass spectrometry coupled with immunoaffinity extraction.

A method for the simultaneous quantitative determination of deoxynivalenol (DON), T-2 toxin (T-2), HT-2 toxin (HT-2) and zearalenone (ZEN) in wheat and biscuit by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) coupled with immunoaffinity extraction is described. A clean-up was carried out using a DZT MS-PREP immunoaffinity column (IAC), and the effect of the sample dilution rate and sample loading was investigated. Furthermore, the effects of ion suppression of a multifunctional column (MFC) and the IAC in the clean-up were compared. The results with the DZT MS-PREP IAC showed that it is possible to make the sample dilution rate low, and indicated a higher solvent-tolerance than usual with an IAC. Sample loading was optimized at 0.25 g. Ion suppression was lowered by purification of the toxins using the DZT MS-PREP IAC. Recoveries of each mycotoxin from wheat and biscuit samples spiked at two levels ranged from 78 to 109%. The limits of detection in wheat and biscuit was in the range of 0.03-0.33 ng x g(-1). From these studies, it is suggested that use of an IAC is effective in the clean-up of each mycotoxin, and, when combined with LC/ESI-MS/MS, it is good for the determination of mycotoxins in foodstuffs due to its rapidity and high sensitivity.

GliA in Aspergillus fumigatus is required for its tolerance to gliotoxin and affects the amount of extracellular and intracellular gliotoxin.

Gliotoxin is an important virulence factor of Aspergillus fumigatus. Although GliA putatively belongs to the major facilitator superfamily in the gliotoxin biosynthesis cluster, its roles remain unclear. To determine the function of GliA, we disrupted gliA in A. fumigatus. gliA disruption increased the susceptibility of A. fumigatus to gliotoxin. The gliT and gliA double-disrupted mutant had even higher susceptibility to gliotoxin than each individual disruptant. The extracellular release of gliotoxin was greatly decreased in the gliA disruptant. Mice infected with the gliA disruptant of A. fumigatus showed higher survival rates than those infected with the parent strain. These results strongly indicate that GliA, in addition to GliT, plays a significant role in the tolerance to gliotoxin and protection from extracellular gliotoxin in A. fumigatus by exporting the toxin. This also allows the fungus to evade the harmful effect of its own gliotoxin production. Moreover, GliA contributes to the virulence of A. fumigatus through gliotoxin secretion.

Time-of-flight mass spectrometry (TOF-MS) exact mass database for benzodiazepine screening

Time-of-flight mass spectrometry coupled with liquid chromatography (TOF-MS) has been developed for screening and determination of benzodiazepines with an exact mass database. Benzodiazepines display similar chemical structures and molecular weights, and thus show similar mass spectra and protonated molecule ions. Discrimination of mass spectrometry at low resolving power using single liquid chromatography mass spectrometry (LC-MS) is commonly difficult. TOF-MS analysis was performed using a 1100 TOF (Agilent Technologies) equipped with a Zorbax C18 Extend column. Purine and fluorine compound solution was always introduced into the ion source, and real-time mass adjusting was performed. Specimens were prepared utilizing the liquid-liquid extraction procedure with 1-chlorobutane. Benzodiazepines are widely used in medical practice in Japan, and data acquired from TOF-MS measurements of 41 benzodiazepines, including active metabolites, were used to create an exact mass database. This database comprised molecular formulae, calculated exact masses and retention times. Calibrations were also included in a database. Precision for the 41 drugs was considered sufficient for quantitative analysis. In analysis of samples from patient who had taken > or =2 benzodiazepines, selectivity was improved using the TOF-MS exact mass database. TOF-MS is effective for forensic toxicology in discriminating between benzodiazepines with similar structure and metabolites.

Multi Mycotoxin Analysis in Food Products Using Immunoaffinity Extraction

We developed a method for the simultaneous determination of deoxynivalenol, T-2 toxin, HT-2 toxin and zearalenone in wheat and biscuit by liquid chromatography/electrospray ionization/tandem mass spectrometry coupled with immunoaffinity extraction. This chapter describes a method to extract, clean-up, and quantitate these mycotoxins and the effect of the ion suppression of multifunctional column and IAC in the clean-up were compared.

Analysis of Tolfenpyrad and its Metabolites in Plasma in a Tolfenpyrad Poisoning Case

Tolfenpyrad (TFP) is a pesticide that was first approved in 2002 in Japan under the trade name of Hachi-hachi. Analyses of TFP and its major metabolite, 4-[4-[(4-chloro-3-ethyl-1-methylpyrazol-5-yl)carbonylaminomethyl]phenoxy]benzoic acid (PTCA), in plasma obtained from a cadaver suspected to have died of TFP poisoning, were conducted by liquid chromatography-mass spectrometry. The existence of TFP and PTCA was confirmed by scan mode and quantitative analysis was performed by selected ion monitoring mode. Calibration curves showed good linearity over the range of 0.1-4 and 0.25-4 µg/mL, and concentrations were estimated to be 1.97 ± 0.02 and 2.88 ± 0.04 µg/mL for TFP and PTCA, respectively. The plasma extract was further examined to find other metabolites using quadrupole time-of-flight MS, and the results revealed three more metabolites, which were suggested to be hydroxy-TFP, dehydro-TFP and hydroxy-PTCA. Plausible metabolic pathways of TFP in humans are: (i) oxidation of the methyl group on the benzene ring, and (ii) hydroxylation followed by dehydration at the ethyl group on the pyrazole ring.

Occurrence of Penicillium brocae and Penicillium citreonigrum, which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin, Respectively, in Selected Commercially Available Rice Grains in Thailand

Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 μg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains.

Isolation of fungi from a fungivorous insect, the minute brown scavenger beetle ( Latridiidae ), and their potential ability for mycotoxin production

Fungivorous insects are serious hazard as potential contaminants in food. This study investigated fungi and their mycotoxins isolated from fungivorous insects (minute brown scavenger beetles [Latridiidae]) captured in a building located in Chiba, Narashino. We trapped 18 insects (heads) and isolated 780 colonies of fungi from them, which we classified into 3 genera: Penicillium (90.1%), Aspergillus (7.7%), Cladosporium (1.0%) and others (1.2%). The population of these fungi reflected that of fungi isolated from the environment inhabited by the insects. In the genus Aspergillus, sterigmatocystin and cyclopiazonic acid were detected by a simultaneous detection system of liquid chromatography-quadrupole time-of-flight mass spectrometry when the isolates were cultured in medium. These findings strongly suggest that Latridiidae are not only a physical but also a microbial hazard as a vector for the spread of mycotoxin-producing fungi in factories for food products and the storage of cereals. The contamination of food industrial factories and cereal storage areas by insects is a major problem threatening food safety management. Pest control programs are therefore a necessity at such sites. These pests have been known to be carriers of microbes, including fungi. The infestation of fungivorous insects is a particularly serious issue, since they have spores on their body surface and in their digestive tract1). The minute brown scavenger beetle (Latridiidae, Fig. 1) has a small size (1-3 mm) and an elongated oval body2). Most of the species are flightless and inhabit damp environments and storage areas for food products, where the insects spread fungal spores and contaminate stored food3),4). They have been reported to feed on fungi in the subdivisions Zygomycotina, Ascomycotina and Deuteromycotina3),5),6),7). In some latridiid species it has been reported that their food preference is the genus Penicillium, but in most of latridiid species the preference fungi is still unknown8). In Japan, the beetle has been found in clean rooms for sterile pharmaceutical products and the storage of cereals1),9), and Tani and Itou9) reported that the beetles carried 102–103 fungal spores in their bodies. Kawakami and Takahashi1) found that the Aspergillus ochraceus strains isolated from the Cigarette beetle (Lasioderma serricorne), a fungivorous insect, produced high levels of ochratoxin A. However, no study has described the fungi isolated from Latridiidae in Japan and their potential ability to produce mycotoxins. Therefore, in the present study, we surveyed fungi from Fig. 1 Latridiidae captured by a trap.