A high performance liquid chromatography (HPLC) method for the simultaneous analysis of the seven benzodiazepines (BZDs), oxazolam, nitrazepam, oxazepam, tofisopam, triazolam, clotiazepam and diazepam (DIA) is presented for application in the screening examination of dietary supplements as adulterants. Samples were analyzed after extraction with methanol. HPLC analysis was performed with on a column of Wakosil 5C18 (4.6 x 150 mm, 5 mu m) with the mobile phase consisting of 1-heptanesulfonic acid sodium salt 5 mM dissolved in water/acetonitrile 1000 ml (13 : 7, v/v), adjusted with phosphoric acid to pH 2.4. The column eluent was monitored with a photodiode array detector, and the quantitative analysis of BZDs was performed at 225 nm. The calibration curves of the seven BZDs showed good linearity and the correlation coefficients were better than 0.999 in all cases. When this procedure was applied to commercial supplements, an DIA-adulterated capsule-type supplement was detected. Finally, DIA was identified and determined using a combination of three different analytical methods: HPLC/photodiode array, thin-layer chromatography (TLC), and LC/MS. DIA was present at a concentration of 1.9 mg/capsule. The procedure described here can provide a broad analysis in a single run within 17 min and is available for the screening of seven BZDs in adulterated supplements with minimal sample preparation.

Thyroxine (T4), one of the thyroid hormones, in adulterated dietary supplements was analyzed using two different methods; enzyme-linked immunosorbent assay (ELISA) and LC/MS. To release T4 from thyroglobulin, samples were first hydrolyzed with proteolytic enzyme, and then the supernatant was diluted and directly analyzed using a commercial free T4 ELISA kit for diagnostic discrimination. In contrast, T4 was extracted with ethyl acetate from the supernatant, and then ethyl acetate layer was evaporated. The residue was dissolved in the mobile phase and analyzed by LC/MS with electrospray ionization (ESI) interface under positive ion mode. These methods were applied to the analyses of 13 dietary supplements advertised as weight reducers. T4 was detected in four of the samples and the analytical results by ELISA agreed well with those obtained by LC/MS. The ELISA technology described here is available for the screening of T4 in adulterated supplements.

A simple and rapid analytical method of oil-soluble coal tar dyes in cosmetics was established using reversed-phase TLC/scanning densitometry. Eleven kinds of oil-soluble coal tar dyes were able to be separated completely on reversed-phase TLC plates by the complementary use of 2 solvent systems. The solvent systems were A; n-hexane/2-butanone solution (5 : 1, v/v), solvent system B; acetonitrile/methanol solution (5 : 1, v/v). Then we measured visible absorption spectra of spots developed on the reversed-phase TLC plates by scanning densitometer to identify these coal tar dyes. The proposed method was successfully applied to the identification of oil-soluble coal tar dyes in commercial cosmetics.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of dehydroacetic acid (DHA), benzoic acid (BA), sorbic acid (SOA) and salicylic acid (SA) was developed for application to cosmetic products. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-n-butylammonium (TBA) hydroxide as an ion-pair reagent. Cosmetic samples were purified by solid-phase extraction using Bond-Elut SI cartridges. Four acidic preservatives were eluted with methanol from cartridges. The HPLC assay was carried out using TSK gel ODS-80TM column (5 microm, 150 x 4.6 mm I.D.). The mobile phase consisted of a mixture of water and methanol (65:35, v/v) containing 2.5 mM TBA hydroxide adjusted with phosphoric acid to pH 7.0. The calibration curves of these preservatives showed good linearity with UV detection (235 nm). The correlation coefficients were better than 0.999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2.5 ng for DHA, 4.0 ng for BA, 2.0 ng for SOA and 5.5 ng for SA. The procedure described here is simple, selective and is suitable for quality control of finished cosmetic products.

A high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of triamterene, trichlormethiazide, furosemide and spironolactone is presented for application in the examination of health food supplements advertising weight reduction and in the analysis of pharmaceuticals. The HPLC assay was performed under gradient conditions using a Wakosil ODS 5C18 column (5 microns, 150 x 4.6 mm i.d.). The mobile phase consisted of a gradient program with a mixture of water and acetonitrile containing 0.1% triethylamine adjusted with phosphoric acid to pH 3.0: from 0 to 6 min, 15% acetonitrile; from 6 to 20 min, linear gradient from 15 to 50% acetonitrile; and from 20 to 40 min, 50% acetonitrile. The column effluent was monitored from 0 to 20 min at 260 nm and from 20 to 40 min at 235 nm. The calibration curves of the four drugs showed good linearity and the correlation coefficients were better than 0.999 in all cases. The lower limits of detection were approximately 40 ng for each drug. Commercially available health food supplements and pharmaceuticals were analyzed after extraction with a mixture of methanol and acetic acid (99:1). The procedure described here is suitable for the screening of four diuretic drugs in adulterated supplements and for the quality control of pharmaceuticals with minimal sample preparation.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of mefenamic acid (MFA), flufenamic acid (FFA) and tolfenamic acid (TFA) is presented for application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using tetra-pentylammonium bromide (TPAB) as an ion-pair reagent. Urine samples were purified by solid-phase extraction using a silica-based strong anion-exchanger, Bond-Elut SAX cartridge. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 μm, 150×4.6 mm I.D.). The mobile phase consisted of 1.9 g of TPAB dissolved in 1 l of a mixture of acetic acid–sodium acetate buffer solution, pH 5.0, and acetonitrile (11:9, v/v). The calibration curves of MFA, FFA and TFA showed good linearity in the concentration range of 33–167 μg/ml with a wavelength of 280 nm for pharmaceuticals, and in the low concentration range (1.7–30.1 μg/ml) with a wavelength of 230 nm for biological fluids. The correlation coefficients were better than 0.9999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 2 ng for MFA, 3.5 ng for FFA and 2.5 ng for TFA. The procedure described here is rapid, simple, selective and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

A high-performance liquid chromatographic (HPLC) method for simultaneous determination of naproxen (NAP), nabumetone (NAB) and its major metabolite, 6-methoxy-2-naphthylacetic acid (6-MNA), was developed for the application to pharmaceuticals and human urine. Isocratic reversed-phase HPLC was employed for quantitative analysis using triethylamine and 1-heptanesulfonic acid sodium salt (HSA) as ion-pair reagents. Urine samples were purified by solid-phase extraction using Bond-Elut Certify II cartridges containing reversed-phase and anion exchange functionalities. The HPLC assay was carried out using a Wakosil ODS 5C18 column (5 microm, 150 x 4.6 mm, i.d.). The mobile phase consisted of 0.5 g of HSA dissolved in 1,000 ml of a mixture of acetonitrile, water and triethylamine (500:500:1, v/v) adjusted with phosphoric acid to pH 3. The calibration curves of NAP and NAB showed good linearity in the concentration range 32-160 microg/ml with UV detection (270 nm) for pharmaceuticals. In the low concentration ranges (8-96 ng of NAP per ml, 24-288 ng of NAB per ml and 5.6-67.2 ng of 6-MNA per ml), the calibration curves were also obtained with fluorimetric detection (excitation 280 nm, emission 350 nm) for biological fluids. The correlation coefficients were better than 0.999 in all cases. The lower limits of detection (defined as a signal-to-noise ratio of about 3) were approximately 0.3 ng for NAP, 1.5 ng for NAB and 0.2 ng for 6-MNA. The procedure described here is rapid, simple, selective, and is suitable for routine analysis of pharmaceuticals and pharmacokinetic studies in human urine samples.

Rapid, accurate and reliable high performance liquid chromatographic methods for the determination of non-steroidal anti-inflammatory drugs epirizole and tiaramide in pharmaceutical preparations were developed using triethylamine, a modifying agent to the mobile phase. Extraction was carried out with methanol-water (1:1,v/v) after grounding tablet and granule preparations. The methods utilized reversed phase C18 column, UV monitoring at 250 nm, ethyl p-hydroxybenzoate as an internal standard for epirizole, and at 295 nm, methyl p-aminobenzoate as an internal standard for tiaramide. Regression analyses of three standard plots in concentration ranges of 0.05-0.4 mg/mL for epirizole and 0.18-1.44 mg/mL for tiaramide gave respective correlation coefficients > 0.99998 and > 0.99997. Relative standard deviations of the slopes were 0.180% and 0.888%, respectively. Percentage recoveries of these compounds for four commercially available drugs ranged between 99.06 and 103.14 and between 99.15 and 99.71 of the labeled amounts of epirizole and tiaramide, respectively. The methods were successfully applied to determine contents of epirizole and tiaramide in marketed drugs.

The purity test of the Japanese Pharmacopoeia Absorbent Gauze for fluorescent whitening agents is regulated to perform by a method of irradiating the gauze with ultraviolet rays in a dark place. This method has been reported to have a difficulty in detecting the existence of fluorescent whitening agents in several cases. We, therefore, tried to establish a method to examine the existence of fluorescent whitening agents using physical and chemical methodologies. After cutting the Japanese Pharmacopoeia Absorbent Gauze samples in small pieces, fluorescent whitening agents were extracted by 30% pyridine solution at 100°C for 30 min. We analysed the fluorescent agents using reversed phase TLC, spectrofluorophotometer and HPLC with a fluorescence detector using 8 standard fluorescent whitening agents as controls. Conditions of reversed phase TLC : plate, RP-18W (Merck) ; solvent system, A) acetonitrile-water (1 : 4), B) acetonitrile-methanol-water (3 : 3 : 10), C) methyl ethyl ketone-methanol-water (1 : 1 : 1). Conditions of HPLC with fluorescence detection : column, Wakosil 5C18 (i.d. 4.6×150 mm) ; mobile phase, 0.01M ammonium acetate-acetonitrile (7 : 3) ; flow rate, 1.0 ml/min ; injection volume, 20 μl ; detections, fluorescence detector (excitation 345 nm and emission 435 nm).

When the content of methanol in certain types of waterproofing aerosol products was determined according to the official method for household products containing harmful substances, the existence of an interfering substance in the products was found. The interfering substance was identified to be tert-butanol by GC-MS,In order to avoid this interference, we developed a simple headspace (HS)-gas chromatographic (GC) method. For this method, one gram of the sample sprayed into a 200 mi flask under ice cooling was weighed into a centrifugal tube. Each 10 mi of water and chloroform was added to the centrifugal tube. After shaking vigorously and:centrifuging (3500 rpm, 5 min), 0.5 g of the aqueous layer was weighed into a 24 mi bottle. The bottle was incubated for 30 minutes at 25 degrees C and then 50 mu l of the HS gas was injected into a GC with a fused silica capillary column coated with methyl phenyl cyanopropyl silicone.The proposed method was successfully applied to the screening analysis of methanol in 70 kinds of commercial household aerosol products.

Ninety-five commercially available items were analyzed for a small-scale survey of organotin compounds (OTCs) in household commodities such as textile products, shoe polish, adhesives, paint, and wax. Neither tributyltin nor triphenyltin species were found in any items; however, di- and trioctyltin and/or dibutyltin species (DOT, TOT, and DBT, respectively) were detected in 15 items; DOT was found in 14 items, TOT in 5 items, and DBT in 4 items. DOT was found in 2 diaper covers at about 0.1%. Detection frequencies of OTC were 6/10 for diaper covers, 3/10 for bibs, 2/10 for sanitary panties, 1/5 for outer-garments, 1/5 for stockings, 1/5 for socks, and 1/10 for shoe polish.

Fermentative degradation of alcohols and aldehydes in the absence of sulfate was investigated using a propionate-oxidizing, sulfate-reducing bacterium, Desulfobulbus propionicus strain MUD (DSM 6523). The organism converted ethanol plus CO2 to acetate and propionate. The conversion was not affected by the presence of hydrogen. Strain MUD converted propanol plus acetate to propionate. Acetaldehyde and propionaldehyde were also converted with a dismutation reaction in the absence of sulfate. The products were propionate and acetate from acetaldehyde, and propionate from propionaldehyde plus acetate.

A highly simplified anaerobic consortium which was able to degrade benzoate under mesophilic conditions was obtained from digested sludge acclimatized with benzoate. It converted 5 mM benzoate to methane quantitatively within 3 weeks in the absence of any organic nutrients under an N2/CO2 atmosphere. Degradation of benzoate was strictly inhibited by hydrogen. The consortium consisted of at least three microorganisms including an autofluorescent irregular coccus which was identified as Methanogenium sp., a short rod which did not autofluoresce and was considered to be a benzoate degrader, and a filamentous bacterium apparently classified as Methanothrix (= "Methanosaeta"). When sulfate was added to the medium, the methanogens were readily replaced by a sulfate-reducing bacterium, probably belonging to the genus Desulfovibrio, which had still remained in very low number in the consortium in the absence of sulfate, and benzoate was stoichiometrically converted to acetate without methanogenesis. Of various compounds which were expected to be intermediates in the benzoate degradation, only crotonate was degraded by concentrated cells of the consortium.

Three thermophilic Methanothrix ("Methanosaeta") strains, strains PTT (= DSM 6194T) (T = type strain), CALS-1 (= DSM 3870), and Z-517 (= DSM 4774), were characterized chemotaxonomically and compared with five mesophilic strains, Methanothrix soehngenii ("Methanosaeta concilii") GP6 (= DSM 3671), Opfikon (= DSM 2139), FE (= DSM 3013), UA, and PM. These methanogens were exclusively acetotrophic and had a characteristic sheathed structure. The DNA base compositions of the strains which we studied ranged from 50.3 to 54.3 mol% guanine plus cytosine. The thermophilic strains often had phase-refractive gas vesicles inside their cells. Denaturing electrophoresis of proteins showed that the mesophilic and thermophilic Methanothrix strains formed two distinct groups and that there were differences in protein patterns between the groups. The difference between the thermophiles and mesophiles was also verified by comparing partial 16S rRNA sequences (ca. 30 base differences in ca. 540 bases). On the basis of our results, we propose the name Methanothrix thermophila for the three thermophilic strains. The type strain of M. thermophila is strain PT (= DSM 6194). We also propose that the name Methanothrix thermoacetophila ("Methanosaeta thermoacetophila"), which was given to strain Z-517 (type strain), should be rejected because of its description, which was based on an enrichment culture, was inadequate.

A strictly anaerobic, homoacetogenic bacterium strain 69, which produces high amounts of intracellular vitamin B12, was isolated from sea sediment. Taxonomical and physiological studies revealed that the isolate should be classified in the genus Acetobacterium. The isolate grown on methanol produced ca. 11 mg of cyanocobalamin per gram dry cells after 7-d cultivation. Tetrachloromethane (TCM) resistant mutants, strain 67-7 and 69-23, were obtained from the isolate by ethylmethanesulfonate treatment, and produced ca. 23 mg of cyanocobalamin per gram dry cells for strain 69-7 and 20 mg for strain 69-23 in the presence of 10 μM of TCM.

Polyphosphate accumulating bacteria were isolated from activated sludge cultured in a fill and draw system with alternating anaerobic and aerobic conditions. One of the isolates, strain NM-1 tentatively identified with the genus Micrococcus accumulated a large amount of phosphorus and its content reached 166 mg of phosphorus per g of cells. This strain accumulated phosphorus rapidly under aerobic conditions in the absence of any organic substrates, and when glucose was supplied to this strain under anaerobic conditions, it released phosphate and took up glucose in a similar manner to activated sludge that has good phosphorus eliminating capacity. Micro-aerophilic growth conditions were required for the induction of phosphorus accumulating activity.

Two anaerobic rod-shaped bacteria, strains K-1 and ACR were isolated from mesophilic digester sludge acclimatized with 4-chlorobutyrate or acrylate as the main energy and carbon source. Both bacteria have tentatively been classified in the genus Clostridium because of their cell morphology and spore formation. Strain K-1 could utilize chlorinated compounds such as 4- and 3-chlorobutyrate and 3-chloropropionate, and the main products were butyrate and acetate, propionate and acetate, respectively. Crotonate and 3-hyflroxybutyrate could be also degraded by strain K-1 and the same products from the degradation of 4-chlorobutyrate were observed. Strain ACR could utilize acrylate to produce propionate and acetate. It could also utilize chlorinated fatty acids such as 4-chlorobutyrate and 3-chloropropionate. However, neither of the strains could utilize saturated fatty acids such as propionate and butyrate. Reductive dechlorination or saturation of unsaturated bond may thus possibly serve as a regeneration system for a reduced coenzyme, which may be formed in an oxidation pathway of such compounds to acetate.

A novel thermophilic acetotrophic Methanosaeta strain was isolated from a thermophilic anaerobic digest or by using acetate enrichment and serial dilution in the presence of vancomycin and neomycin. This isolate, designated Methanosaeta sp. strain PT, resembled Methanosaeta sp. strain CALS-1 morphologically; however, it occasionally formed filaments longer than 100 μm and exhibited autofluorescence. The content of coenzyme F420 was much higher than that of Methanosaeta reference strains, and coenzyme F420 with four glutamyl residues on the side chain was the predominant component. Furthermore, a comparative analysis of the antigenic fingerprint of strain PT with the fingerprints of reference organisms showed that this isolate was not related antigenically to the reference methanogens, including Methanosaeta sp. (“Methanothrix” sp.) strain CALS-1 and Methanosaeta concilii (“Methanothrix soehngenii”) Opfikon. Strain PT formed visible colonies in a deep agar medium when high cell concentrations were present. However, transfer of a colony into liquid medium resulted in no growth. Strain PT could utilize only acetate as a sole carbon and energy source. The optimum temperature and optimum pH for methanogenesis were near 55°C and 6.7, respectively. The specific methane formation rate μCH 4 under optimum conditions was 0.47 day−1, and the doubling time was 1.49 days. The DNA base composition was 52.7 mol% guanine plus cytosine.

Octyltin compounds were detected from diaper covers by thin layer chromatography using 0.1% pyrocatechol violet solution as a spray reagent, and identified as tri- and di-n-octyltin (TOT and DOT, respectively) species by gas chromatography-mass spectrometric analysis after derivatization to tetrasubstituted organotins with propyl magnesium bromide. TOT and DOT were determined by flame photometric detection-gas chromatography. Amounts of octyltin compounds found in three diaper covers were 67.1, 97.6, and 140.1-mu-g/g of TOT (as its chloride) and 405.5, 579.8, and 819.2-mu-g/g of DOT (as its dichloride).

Six strains of acetotrophic methanogens, identified as Methanothrix soehngenii, were highly purified from mesophilic anaerobic digesters and their properties were investigated in comparison with a type strain M. soehngenii Opfikon. All strains were filamentous, exclusively acetotrophic, electrophoretically identical and had the same values for guanine plus cytosine content. However, two of them, one of which originated from an up-flow anaerobic sludge blanket (UASB), were remarkably filamentous and a number of filaments aggregated to form a cotton-like floc. In contrast with these strains, the others formed shorter filaments which were dispersed homogeneously. These indicate that two types of Methanothrix exist in anaerobic digesters.