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Streptococcus pyogenes (group A streptococcus; GAS) is an important gram-positive human pathogen capable of causing diseases ranging from mild superficial skin and pharyngeal infections to more severe invasive diseases, including streptococcal toxic shock syndrome (STSS). GAS produces a T protein, and T serotyping has considerable discriminatory power for epidemiological characterization of GAS. To clarify the relationship between STSS and pharyngitis in Japan, we examined the T serotypes of GAS strains isolated from clinical specimens of streptococcal infections (STSS, 951 isolates; pharyngitis, 16268 isolates) from 2005 to 2017. The most prevalent T serotype from pharyngitis isolates was T12, followed by T1, T4, and TB3264. The most prevalent T serotype from STSS isolates was T1, followed by TB3264. Trend of increase and decrease in the frequency of T1 or TB3264 isolation from pharyngitis was correlated with that of STSS patients. The increase of T1 or TB3264 strain-infection in pharyngitis patients may increase the probability of causing STSS, indicating that careful monitoring of GAS serotypes is essential for the prediction of rapid increase of STSS in time to develop effective management strategies.

Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes emm 89 strains has been increasing in several countries and reported to be linked with a recently emerged clade of emm89 strains, designated clade 3. In Japan, epidemiological and genetic information for emm89 strains remains elusive. In this study, we utilized emm89 strains isolated from both STSS (89 isolates) and non-STSS (72 isolates) infections in Japan from 2011 to 2019, and conducted whole-genome sequencing and comparative analysis, which resulted in classification of a large majority into clade 3 regardless of disease severity. In addition, STSS-associated genes and SNPs were found in clade 3 strains, including mutations of streptokinase (Ska), control of virulence sensor (CovS), serum opacity factor (SOF), sortase (SrtB), and fibronectin-binding protein F1 (PrtF1), and absence of the hylP1 gene encoding hyaluronidase. These findings provide insights into notable genetic features of emm89 strains.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification is a popular analytical method. Strain Solution proteotyping software available for MALDI-TOF MS has great potential for the precise and detailed discrimination of microorganisms at serotype- or strain-level, beyond the conventional mass fingerprinting approaches. Here, we constructed a theoretically calculated mass database of Salmonella enterica subspecies enterica consisting of 12 biomarker proteins: ribosomal proteins S8, L15, L17, L21, L25, and S7, Mn-cofactor-containing superoxide dismutase (SodA), peptidyl-prolyl cis-trans isomerase C (PPIase C), and protein Gns, and uncharacterized proteins YibT, YaiA, and YciF, that can allow serotyping of Salmonella. Strain Solution ver. 2 software with the novel database constructed in this study demonstrated that 109 strains (94%), including the major outbreak-associated serotypes, Enteritidis, Typhimurium, and Infantis, could be correctly identified from others by colony-directed MALDI-TOF MS using 116 strains belonging to 23 kinds of typed and untyped serotypes of S. enterica from culture collections, patients, and foods. We conclude that Strain Solution ver. 2 software integrated with the accurate mass database will be useful for the bacterial proteotyping by MALDI-TOF MS-based microbial classification in the clinical and food safety fields.

Subtypes of stx1 and stx2 in 45 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle were investigated by PCR. Only subtype stx1a was detected among all the stx1-positive strains. The major stx2 subtype was stx2a followed by stx2d, stx2c, stx2b, and stx2g in decreasing order of frequency. stx2c was found in strains of serotypes O157 and O174. stx2d was found in 11 strains. These strains were confirmed by DNA sequencing to carry both the activatable tail and the END motif; all were eae-negative, and 3 contained stx2d as the only stx. stx2g was found in 2 strains in association with stx2a, estA1, and astA. In addition, 7 hybrid strains of shigatoxigenic and enterotoxigenic E. coli (STEC/ETEC) were found to harbor one or both of stx1a and stx2a (stx1a/stx2a) and estA1. Among 27 serotypes of STEC strains isolated from cattle, O157:H7 and O109:H- strains were eae-positive. Other putative adhesin genes, such as saa, iha, espP, and lpfAO113 were detected in more than 12 serotypes.

Pathogenic genes such as stx1, stx2, STh gene, STp gene, LT gene, invE, eae, aggR, afaD, astA, cdt and cnf were investigated in Escherichia coli isolated from cattle during Nov. 2012 and Aug. 2013. Plural pathogenic genes were concurrently detected by multiplex PCR, and screen-positive genes were confirmed and sub-classified by PCR. Among 100 cattle investigated, 180 E. coli strains with diarrheic genes (DEC) were detected in 79 cattle, and 45 of them, isolated from 32 cattle, were Shiga toxin-producing E. coli (STEC). More than 30% of cattle carried astA, cdt, cnf and stx2 in descending order. STh gene, LT gene, invE, aggR and afaD were not detected in this study. Both stx1 and stx2 were concurrently detected from 6 of 45 STEC strains and stx2 alone was detected from 19. Seventeen STEC strains carried STp gene, astA, or cdt along with stx1 or stx2. Additionally, 135 remaining DEC were classified into 18 enterotoxigenic E. coli with STp gene, 25 enteropathogenic E. coli with eae, and 92 other DEC with astA, cdt and cnf. Both O and H serotypes were identified in 48 strains, including O157 : H7, O1H7 and so on. O157 : H7 were identified in 3 strains that carried stx2 and eae together, as found in human pathogenic strains isolated from patients with gastroenteritis and hemolytic-uremic syndrome.

A combined additives study of lead-free KNbO3 (KN) ceramics in the solid-state reaction was performed to improve the mechanical quality factor (Q m) for high power piezoelectric applications. Improved Q m values of 1137 and 1241 were confirmed in KN specimens with addition of 1.0 mol% K4CuNb8O23 (KCN) and 1.0 mol% KCN + 0.5 wt% GeO2, respectively. It is important for achievement of the higher Q m in KN systems to control the composition of the Cu segregated phase. In this study, the Cu segregated phase which was necessary to improve the Q m resulted from decomposition of the KCN compound during the sintering. Co-doping of KCN and GeO2 to KN specimens has a remarkable effect in suppressing the deliquescence accompanying KCN addition and improvement of the Q m without significant deterioration of the electromechanical coupling factor k 31.

Subtypes of stx1 and stx2 in 45 Shiga toxin-producing Escherichia coli (STEC) strains isolated from cattle were investigated by PCR. Only subtype stx1a was detected among all the stx1-positive strains. The major stx2 subtype was stx2a followed by stx2d, stx2c, stx2b, and stx2g in decreasing order of frequency. stx2c was found in strains of serotypes O157 and O174. stx2d was found in 11 strains. These strains were confirmed by DNA sequencing to carry both the activatable tail and the END motif; all were eae-negative, and 3 contained stx2d as the only stx. stx2g was found in 2 strains in association with stx2a, estA1, and astA. In addition, 7 hybrid strains of shigatoxigenic and enterotoxigenic E. coli (STEC/ETEC) were found to harbor one or both of stx1a and stx2a (stx1a/stx2a) and estA1. Among 27 serotypes of STEC strains isolated from cattle, O157:H7 and O109:H- strains were eae-positive. Other putative adhesin genes, such as saa, iha, espP, and lpfA

We developed a rapid and sensitive liquid chromatography/time-of-flight mass spectrometry (LC/TOF–MS) method using atmospheric pressure photoionization (APPI) for the determination of paraquat and diquat in human serum. Serum samples were extracted with aqueous formic acid and acetonitrile. After vortexing and centrifugation, the supernatants were filtered with ultrafiltration cartridges followed by APPI-LC/TOF–MS analysis using a hydrophilic interaction chromatography analytical column. The total amount of time required for analyzing serum samples was ≤20 min. Calibration curves using solvent standards were linear in the range of 0.0005–1.25 μg/ml. The intraday recoveries (n = 6) for paraquat and diquat added to human serum at 0.1 μg/ml were 99.0 and 91.9 %, respectively, with relative standard deviations of 5.8 and 6.5 %. The limits of detection calculated at a signal-to-noise ratio of 3 were 0.005 μg/ml for paraquat and 0.006 μg/ml for diquat. This method was applied in the analysis of four autopsy cases involving suspected paraquat intoxications. The concentration of paraquat and diquat in serum ranged from

During a 15-year monitoring survey (April 1995–March 2010) of pesticide residues in agricultural products, 1542 domestic samples (cereals, vegetables, fruits, etc.) collected in Hyogo prefecture, Japan, were analyzed. The number of pesticides tested increased annually from 110 to 615, and 2198 pesticides, including trace levels, were detected in 844 samples. Annual detection rates of positive samples ranged from 36 to 77% during 15 years, and the average rate was 65% in the past 5 years. With the increase in pesticides tested, total detections in FY 2005–2009 markedly increased, trace level detections composed more than half of all detections, and the numbers of samples containing 4 or more residues increased two-fold. Procymidone and iprodione were frequently found in vegetables, and kresoxim-methyl, acetamiprid, iprodione and captan in fruits. In addition, the detection of carbendazim, dinotefuran and imidacloprid increased in the past 5 years. Violations of MRLs or the uniform limit (0.01 μg/g) were observed in 9 samples (0.6%), among which pesticides unregistered for application were detected in 8 samples. The residue levels of unregistered pesticides should be assessed, as the uniform limit was applied for their regulation in many cases.

The applicability of multi-residue analytical method for 496 pesticides (including metabolites) in frozen gyoza dumplings was verified. This method involved extraction using aqueous acetonitrile (around 70% of acetonitrile content) followed by cleanup using octadecylsilyl (ODS) and primary secondary amine (PSA) mini-column solid-phase extraction (SPE). The target compounds were determined by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Validation tests were performed on frozen gyoza dumplings fortified at 0.01 and 0.10 μg/g in accordance with the guideline issued by the Japanese Ministry of Health, Labour and Welfare. Among the 496 pesticides tested, 378 pesticides were found to conform to the guideline when the solvent standard was used, and 432 pesticides conformed when the matrix-matched standard was used, respectively. The limits of quantitation (S/N≥10) were set at 0.01 μg/g for all, except 20 of the pesticides that were tested. Although no significant matrix effects were observed for most of the pesticides, the use of the matrix-matched standard was preferable to the solvent standard for accurate quantitation. The method was applied to 11 commercial samples, and 14 different pesticides were detected from 8 samples at concentrations ranging from trace levels (

Liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) with atmospheric pressure photoionization (APPI) has been used for the determination of five post-harvest fungicides and metabolite, o-phenylphenol (OPP), diphenyl (DP), thiabendazole (TBZ), imazalil (IMZ), and its major metabolite R14821 (IMZ-M) in citrus fruits. Samples were extracted with diethyl ether and the extracts with minimal sample preparation were analyzed by LC/TOF-MS with APPI by measuring accurate mass. DP, IMZ, and IMZ-M were ionized in positive ion mode, and OPP and TBZ were in negative ion mode. The recoveries fortified to citrus fruits at 1 μg/g ranged from 70.5% to 101.4%. The detection limits (S/N = 3) were 0.002 μg/g (OPP, TBZ, and IMZ-M), 0.001 μg/g (IMZ), and 0.03 μg/g (DP).

A simple and rapid screening method of 95 acidic pesticides in agricultural products was developed. As acidic pesticides adsorb on the primary secondary amine mini-column with fatty acids which become interfering peaks during GC/MS analysis, they were not included in multi-residue analysis entailing PSA clean-up. In this study, the intermediate extracted solutions before PSA clean-up were analyzed by LC/TOF-MS. Accurate mass measurement of TOF-MS enabled the detection of molecular ions and fragment ions with high selectivity. Mean recoveries of 95 pesticides added to 6 agricultural products at 0.1 μg/g were 49–127% with RSD

A multiresidue screening method using GC/MS and LC/MS was investigated for analysis of 202 pesticides (including metabolites) in meat. Many target pesticides with a wide range of polarity were successfully extracted with ethyl acetate-cyclohexane (1 : 1) from meat samples rich in lipid components. Matrix components such as cholesterol, monoglycerides and fatty acids were effectively eliminated by means of GPC and 2 kinds of mini-columns (PSA and Silica). These procedures enabled analysis in the SCAN mode by GC/MS and LC/MS without any matrix interference. Among the pesticides tested, 17 had low recoveries (

Pathogenic genes such as stx1, stx2, STh gene, STp gene, LT gene, invE, eae, aggR, afaD, astA, cdt and cnf were investigated in Escherichia coli isolated from cattle during Nov. 2012 and Aug. 2013. Plural pathogenic genes were concurrently detected by multiplex PCR, and screen-positive genes were confirmed and sub-classified by PCR. Among 100 cattle investigated, 180 E. coli strains with diarrheic genes (DEC) were detected in 79 cattle, and 45 of them, isolated from 32 cattle, were Shiga toxin-producing E. coli (STEC). More than 30% of cattle carried astA, cdt, cnf and stx2 in descending order. STh gene, LT gene, invE, aggR and afaD were not detected in this study. Both stx1 and stx2 were concurrently detected from 6 of 45 STEC strains and stx2 alone was detected from 19. Seventeen STEC strains carried STp gene, astA, or cdt along with stx1 or stx2. Additionally, 135 remaining DEC were classified into 18 enterotoxigenic E. coli with STp gene, 25 enteropathogenic E. coli with eae, and 92 other DEC with astA, cdt and cnf. Both O and H serotypes were identified in 48 strains, including O157 : H7, O1H7 and so on. O157 : H7 were identified in 3 strains that carried stx2 and eae together, as found in human pathogenic strains isolated from patients with gastroenteritis and hemolytic-uremic syndrome.

A simple and rapid simultaneous analytical method of four post-harvest fungicides, o-phenylphenol (OPP), diphenyl (DP), thiabendazole (TBZ), imazalil (IMZ) and its major metabolite R14821 (IMZ-M) in citrus fruits was developed. These compounds were extracted under basic conditions with diethyl ether. The organic layer was washed with water and evaporated at moderate pressure adding methanol. These compounds were determined by liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure photoionization (APPI). The recoveries of the five compounds added to citrus fruits at 1 microg/g ranged from 67 to 100%, with relative standard deviations (R.S.D.) ranging from 2 to 8%. The detection limits (S/N = 3) were 0.01 microg/g and 0.05 microg/g (DP).

A simultaneous screening method using conventional PCR was developed for the detection and discrimination of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii. A formulated multiprex method employing 4 kinds of paired primers on amplification of 4 corresponding different insertion sequences (IS481, IS1001, IS1002 and hIS1001) enabled rapid screening and identification. The detection limits of each DNA extracted from 3 kinds of Bordetella species were 5fg/microL for each. Obscure existences of B. pertussis and B. holmesii at low levels were confirmed with the LAMP method. This multiplex assay was applied to the clinical specimens obtained from patients with pertussis-like symptoms at sentinel clinics under the epidemiological surveillance of infectious diseases of Hyogo prefecture in FY2012. Among 42 nasopharyngeal swabs, B. pertussis was detected from 12 samples including 8 samples collected at outbreak in nursery school. The use of this method for the surveillance of infectious agents enabled us to search for 3 kinds of Bordetella species at once with low costs.

Solid-phase extraction cartridges, Silica, Florisil, ethylenediamine-N-propylsilane-bonded silica (PSA), trimethylaminopropylsilane-bonded silica (SAX) and graphitized nonporous carbon (ENVI-Carb) were investigated for cleanup of pesticides residues suspected as endocrine disruptors in food extracts. Elution patterns and recoveries were examined for 38 pesticides with 2 types of eluting solvents : acetone/n-hexane and diethyl ether/n-hexane. A 2% acetone-n-hexane eluant used with the Silica cartridge gave the best recovery for 33 pesticides and minimized interference from coextractants. The remaining 5 pesticides were eluted successively with 20% acetone-n-hexane from tandem connected Silica and PSA cartridges. Thirty-eight pesticides were analyzed in 30 duplicate meals with the limits of detection ranging from 0.01 to 0.5ppb. trans-Chlordane, trans-nonachlor and p, p'-dichlorodiphenyldichloroethylene (p, p'-DDE) were found frequently (Tr.-1.1ppb). Dietary daily intakes were 0.27μg/d for total chlordane, 0.85μg/d for total dichlorodiphenyltrichloroethane (DDT), which were significantly below the acceptable daily intake (ADI) values.

Absorption experiments of CO 2 and NH 3 in water were performed using a crossflow cascade packed column with 4, 8, and 12 stages. Packings used were 12-mm ceramic Raschig rings and 47-mm S-O Tellerettes. The flow behavior of the fluid in the cascade column was examined. The effect of baffle spacing arrangement on the absorption of CO 2 and NH 3 was investigated using the data on mass-transfer coefficients, gas-phase pressure drops, and mean residence times of the aqueous phase. The optimum operating conditions and baffle arrangements were identified for the design of crossflow cascade columns that could achieve efficiencies equivalent to those of conventional countercurrent packed beds

A simple and selective method is presented for the multiple residue determination of eight sulphonamides in consumers' milk. The drugs are sulphisomidine (ID), sulphadiazine (DZ), sulphamerazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine and sulphaquinoxaline (SQ). The milk sample was deproteinized with the same volume of 2 M hydrochloric acid and filtered. A 1-ml volume of the tiltrate was mixed with 1 ml each of 1.25 M sodium acetate solution and a buffer (pH 3.0) for derivatization with 0.6 ml of 0.02% fluorescamine solution in acetone. A high-performance liquid chromatographic analysis was carried out on a C 18 column with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55°C using a fluorescence detector at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. Average recoveries at fortification levels of 2, 5 and 10 ng/ml were 114%, 109% and 106%, respectively. Relative standard deviations were 1–4% at 10 ng/ml. The limit of determination was 10 ng/ml for ID, 5 ng/ml for DZ and SQ and 2.5 ng/ml for the other five sulphonamides. The method was applied to 25 milk samples and all appeared to be free from the drugs.

To assess the components and the contents of several health foods, especially processed by condensing, the contents of amygdalin (AM), benzoic acid (BA), levulinic acid (LVA), 5-hydroxymethylfurfural (HMF), 5 kinds of organic acids (citric acid, malic acid, succinic acid, tartaric acid and fumaric acid), potassium and sodium were determined for 15 Japanese apricot extracts (10 paste types, 5 pill types) and 7 prune extracts. AM was detected in all the samples of Japanese apricot extracts (0.067-4.64 mg/g), but was not detected in the prune extracts. The average contents of BA, LVA and HMF were 0.17 mg/g, 0.61 mg/g and 5.32 mg/g for the paste type of Japanese apricot extracts, 0.086 mg/g, 0.24 mg/g and 0.99 mg/g for the pill type, and 0.011 mg/g, 0.005 mg/g and 3.47 mg/g for the prune extracts. By the dry concentration of Japanese apricot juice at 95°C, HMF was produced in large quantities accompanied with a change in the state from liquid to semisolid, and then LVA was produced. Main organic acids in the components of Japanese apricot extracts were citric acid and malic acid. The average contents of citric acid and malic acid were 319 mg/g and 106 mg/g for the paste type of Japanese apricot extracts, and 94 mg/g and 29 mg/g for the pill type. In the prune extracts, the content of malic acid was detected to be 8 mg/g on the average. The contents of potassium were 24 mg/g for the paste type of Japanese apricot extracts, 16 mg/g for the pill type, and 9 mg/g for the prune extracts on the average. All these values were larger than those of respective contents of sodium in the three extracts.

During a 3-year monitoring survey (April 2002-March 2005) of pesticide residues in agricultural products, 592 samples (324 domestic; 268 imported) collected in Hyogo prefecture, Japan were analyzed. The number of pesticides tested increased from 232 in FY 2002 to 323 in FY 2004. The purpose of the study was to clarify the residue status by accumulating information about pesticides detected frequently, to allow effective and efficient regulation under the new "Positive List" legislation to be implemented in FY 2006. Overall, 47% of domestic and 61% of imported samples contained detectable residues and ca. 60% of positive samples contained multiple residues. The limit of quantitation was set at 0.01 microg/g and the limit of detection was 0.001-0.003 microg/ g. Most of the residues were present at low concentrations: 80% of the detections in samples excluding imported citrus fruits were0.05 microg/g. More than 5 different pesticides (0.01 microg/g) were detected simultaneously in 13 samples. The sum of the ratios of residues to MRLs was calculated as one of the indexes to represent the risk of multiple residues, and they exceeded 100% in 3 imported frozen vegetables; baby kidney bean, spinach, Welsh onion. Samples in violation of the Food Sanitation Law were not found in our survey, but 1.9% of the samples might be in conflict with the new "Positive List" legislation.

A liquid chromatographic (LC) method with diode array detection (DAD) was developed for screening of 10 sulfonyl urea herbicide residues in unpolished rice. The investigated herbicides were azimsulfuron, bensulfuron-methyl, chlorimuron-ethyl, chlorsulfuron, ethoxysulfuron, flazasulfuron, imazosulfuron, metsulfuron-methyl, pyrazosulfuron-ethyl and tribenuron-methyl. Acetonitrile-water (2:1) extracts of rice samples were cleaned up with solid-phase extraction cartridges (octadecylsilane-bonded silica (ODS) and graphitized carbon black (GCB)). Three fractions of the GCB eluate were taken for analysis using 3 separate injections in order to avoid interference in LC-DAD analysis and to reduce analyte coelution problems. Recoveries from rice samples fortified with the 10 herbicides at 0.05 and 0.2 microgram/g ranged from 46.6 to 119.6%, and coefficients of variation were 3.1-12.6%. The quantitation limits were 0.01-0.02 microgram/g.

A sensitive and rapid high-performance liquid chromatographic screening method is described for the determination of anabolic steroid trenbolone in bovine muscle and liver. Trenbolone was analyzed as alpha- and beta-trenbolone. Samples were extracted with ethyl acetate and cleaned up on a silica solid-phase extraction (SPE) cartridge. Liver samples were cleaned up on a multifunctional SPE cartridge before using a silica SPE cartridge. Analysis of alpha- and beta-trenbolone was performed by reversed-phase high-performance liquid chromatography (HPLC) with a fluorescence detector. The detection limits for this method were estimated to be 0.2 and 1.0 ng/g in bovine muscle and liver, respectively. The mean recoveries spiked in muscle at 2 ng/g and in liver at 10 ng/g were over 80%.

A rapid, sensitive and selective high-performance liquid chromatographic method is described for simultaneous determination of eight sulfa drugs in meat and meat products using pre-column derivatization with fluorescamine. The drugs are sulfisomidine, sulfadiazine, sulfamerazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline. The method includes blender extraction of 3-g samples with choloroform, partition with 3 M hydrochloric acid, derivatization with fluorescamine at pH 3.0 and subsequent high-performance liquid chromatographic analysis on a C 18 column with fluorescence detection at an excitation wavelength of 405 nm and an emission wavelength of 495 nm. The drugs were separated with a mobile phase of acetonitrile-2% acetic acid (3:5) at 55°C. The average recovery from samples fortified at 0.1 ng/g was 92.6% with a coefficient of variation of 6.2%. The detection limit was 0.01 ng/g for sulfaquinoxaline and 0.005 ng/g for the other seven drugs. The method was field-tested in a survey of 37 samples including beef (five), pork (seven), chicken (seven), ham (five), sausage (eight), bacon (two) and roast beef (three). Sulfadimidine was detected in one port sample at the level of 0.295 ng/g and in ham at 0.178 ng/g.

In order to apply the multiresidue screening method to degradation products of N-trihalomethylthio fungicides (captan, captafol, folpet, dichlofluanid, tolylfluanid) and oxidized organophosphorus insecticides generated during sample preparation or in plants, the recovery of sixteen of these degradation products and metabolites from 3 agricultural products was investigated. The recoveries ranged from 74 to 129%, and these data suggest that the degradation products can be determined simultaneously with the parent pesticides by this method.The degradation ratios of 5 N-trihalomethylthio fungicides, demeton-S-methyl and isofenphos were examined during sample preparation from 5 agricultural products and during GC injection. Cabbage, spinach and pumpkin readily caused the degradation of N-trihalomethylthio fungicides, and the degradation ratios of captan were 100, 100 and 81%, respectively. Decreases of the parent pesticides corresponded closely to the formation of the degradation products.

A multi-pesticide screening method for agricultural products was developed using an automated sample preparation instrument in combination with GC/MS by a modification of the previously reported method with two SPE columns. The instrument automatically conducts solid-phase extraction and evaporation. The sample extract (0.2mL) was applied to a PSA cartridge (100mg) in the system, followed by repeated elution with 0.8mL of n-hexane-acetone (1:1) and evaporation three times. The residue was dissolved in 0.2mL of n-hexane-acetone (4:1) for GC/MS. The automated system was tested for 164 pesticides, and recoveries of 56.1-129.5% (160 pesticides) were obtained with a mean CV of 4.1%. Four volatile pesticides gave poor recoveries (18.3-27.6%), but yields of 43.1-46.7% were available with the use of matrices. By using this automated sample preparation system, recoveries of 27 pesticides and 4 metabolites from 3 matrices fortified at 0.1ppm were 105.0% for brown rice, 102.0% for lemon, and 103.0% for spinach. This procedure could be employed as a screening method at the 0.01ppm level. The total time required for one sample preparation was ca. 1.3hr and about 10 samples could be analyzed automatically overnight.

A simple and selective HPLC method was developed for the systematic determination of bentazone (BEN), imazaril (IMZ), inabenfide (INA), pyrazoxyfen (PYR) and chinomethionate (CIN) in imported brown rice. A rice sample was extracted with 70% MeCN and divided into three fractions (acidic, BEN; basic, IMZ/INA; neutral, PYR/CIN). The acidic and basic fractions were purified by partition between n-hexane-dichloromethane (1:1) and either 0.01M borate buffer (pH 10.4) for BEN or 0.1M HCl for IMZ/INA, immediately before injection into the HPLC system. The PYR/CIN fraction was redissolved in isopropanol-MeCN (1:1) to remove lipid components. A sample was analyzed on an ODS column with a mobile phase of a mixture of MeCN-5mM K2HPO4 [BEN: (20:80), pH 2.5; IMZ/INA: (35:65), pH 4.5; PYR/CIN: (55:5)] at 45°C IMZ and INA were determined from the UV absorption at 220nm. Other pesticides were determined by using a postcolumn photolysis-fluorescence detection system at an excitation wavelength (Ex) of 329nm and emission wavelength (Em) of 415nm for BEN, Ex 333nm/Em 405nm for PYR, and Ex 377nm/Em 450nm for CIN. Average recoveries ranged from 80.9 to 89.1% at a fortification level of 0.025μg/g. The limit of determination was 0.005μg/g for INA and 0.01μg/g for other pesticides. The method was applied to 24 brown rice samples, and all appeared to be free from the five pesticides.

A simple and selective method for the determination of S-sulfo group has been developed. The principle of this method is as follows: S-sulfo group is reduced by dithiothreitol to thin) and sulfite; the latter is converted to sulfur dioxide by acidification and absorbed to monoethanolamine solution by aeration. Then an adduct is prepared by the reaction of sulfite with p-aminoazobenzene and formaldehyde and determined colorimetrically by high performance liquid chromatography. Calibration curves for S-sulfonates were linear in the range of 2.5 to 100μM. The present method permitted the determination of S-sulfo group in normal rabbit plasma in the range of 23.3 to 47.3μM.

A sensitive and specific method for the determination of N-acetylneuraminic acid and N-glycolylneuraminic acid was developed by using high performance liquid chromatography with fluorometric detection. Both neuraminic acids were derivatized with 4′-hydrazino-2-stilbazole, a fluorogenic reagent for α-keto acid. The derivatives were separated by reversed phase chromatography on a Radial PAK Cartridge C18 Column (Waters) with methanol-aqueous 1.5% triethylamine solution adjusted the pH to 2.3 with phosphoric acid (35:65 v/v) as a mobile phase at a flow rate of 1ml/min. The detection limits of both derivatives were 0.5ng in an injection volume of 5μl. This method was applied to the determination of NANA in human blood and urine.

ディスポ限外〓過膜器具による化粧水前処理法を用いた化粧水中のヒアルロン酸(HA)の定量法を検討した.化粧水中のHAをセルロース製のディスポ限外〓過膜器具を用いた前処理後,比色法,HPLCにより定量した.市販化粧水へのHAの添加回収率はほぼ100%と良好であり,コンドロイチン硫酸(chn S),アルギン酸などの妨害なく化粧水中のHAを定量できた.