Fast automated extraction and clean-up of biological fluids for polychlorinated dibenzo-p-dioxins,dibenzofurans and coplanar polychlorinated biphenyls analysis

A fast automated extraction and clean-up procedure for low-level analysis of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (cPCBs) in biological fluids is presented. Online extraction of prepared fluids is carried out using disposable octadecyl bonded (C(18)) solid-phase extraction columns. Extracts are then cleaned up through disposable multi-layer silica (acidic, basic and neutral) and dispersed PX-21 carbon columns. This new methodology is compared with classical Soxhlet extraction and manual solid-phase extraction in terms of repeatability, reproducibility, accuracy and recovery rates for reference and certified materials. Robustness is evaluated on different matrices, such as cow's milk, breast milk and human serum. As a consequence of the reduced number of reusable glassware used, as well as lowering of solvent consumption, recorded blank levels are decreased in favor of limits of detection (LODs). Total analysis time and cost are further reduced using simultaneous sample preparation units and the sample throughput is increased compared to classical methods. As a result, this new approach appears to be suitable for the fast sample preparation often required for such fluids in case of emergency foodstuffs analysis or during large epidemiological studies.     

Degradation of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans by the white rot fungus Phanerochaete sordida YK-624.

A method for the degradation of dioxins by white rot fungi was developed. Degradation of a mixture of 10 kinds of tetra- to octachlorodibenzo-p-dioxins (polychlorinated dibenzo-p-dioxins [PCDDs]) and tetra- to octachlorodibenzofurans (polychlorinated dibenzofurans [PCDFs]), which were chlorinated at 2-, 3-, 7-, and 8-positions of the molecules, by the white rot fungus Phanerochaete sordida YK-624 was studied in a stationary low-nitrogen medium. The percent degradation values of PCDDs and PCDFs were approximately 40 (tetra-chloro-) to 76% (hexachloro-) and 45 (tetrachloro-) to 70% (hexachloro-), respectively. Metabolites of 2,3,7,8-tetra- and octaCDD formed by P. sordida YK-624 included 4,5-dichlorocatechol and tetrachlorocatechol, respectively. These results suggest that white rot fungus is able to substantially degrade both PCDDs and PCDFs. This is the first report of the degradation of highly chlorinated PCDDs and PCDFs by a microorganism.     

土壤环境多氯二苯并二噁英/呋喃(PCDD/Fs)污染及其修复研究进展

多氯二苯并二噁英/呋喃(PCDD/Fs)是持久性有毒有机污染物,易沉积在土壤环境中,通过生物放大和生物富集最终输入到人和动物体内,对生态系统和人体健康有极大的威胁.本文在对PCDD/Fs环境行为与污染来源进行阐述的基础上,概述了PCDD/Fs污染的地域性特点以及土壤中PCDD/Fs的背景值和污染控制参考值;分析了我国土壤PC-DD/Fs污染状况和潜在污染源,江西、广州贵屿、长江三角洲、台湾南部、浙江台州、天津某化工厂等6个区域污染较严重,主要污染源有含氯化学品的生产与使用,垃圾焚烧和纸浆生产.最后,介绍了PCDD/Fs污染土壤的处置方法与修复技术,主要有物理化学处置方法、微生物修复方法、植物修复方法、植物-微生物联合修复方法,并对今后该研究方向进行了展望.     

The application of hybrid mass spectrometry/mass spectrometry and high-resolution mass spectrometry to the analysis of fish samples for polychlorinated dibenzo-p-dioxins and dibenzofurans

A hybrid mass spectrometer operated in low-resolution selected decomposition monitoring (SDM) mode was used for the analysis of whole fish samples for the 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and poly-chlorinated dibenzofurans (PCDD/Fs). These fish samples were previously analyzed for PCDD/Fs using a high-resolution mass spectrometer following EPA protocols. The hybrid tandem mass spectrometric method using loss of COCl gave similar quantification results to those obtained by high-resolution mass spectrometry and eliminated the interferences attributed to polychlorinated biphenyls that were encountered in the high-resolution mass spectrometric analysis. Comparison of the two methods shows that the high-resolution mass spectrometric method surpasses the tandem mass spectrometric method in other analytical figures of merit, such as detection limit, linearity and reproducibility.     

Biostimulation and bioaugmentation to enhance dechlorination of polychlorinated dibenzo-p-dioxins in contaminated sediments

Dechlorination of spiked 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) was investigated in sediment microcosms from three polychlorinated dibenzo-p-dioxin and dibenzofuran (CDD/F)-contaminated sites: River Kymijoki, Finland; Gulf Island Pond, Maine; and Lake Roosevelt, Washington. Dechlorination was stimulated by addition of electron donor and halogenated priming compounds, and bioaugmentation by a mixed culture containing Dehalococcoides ethenogenes strain 195. Amendment with 1,2,3,4-tetrachlorobenzene (1,2,3,4-TeCB) promoted rapid dechlorination of 1,2,3,4-TeCDD to 2-monochlorodibenzo-p-dioxin (2MCDD) in Gulf Island Pond and River Kymijoki sediments, however, only slow dechlorination to 1,4-dichlorodibenzo-p-dioxin was observed in Lake Roosevelt sediments. The dechlorination pathway in 1,2,3,4-TeCB-amended microcosms proceeded mainly via 1,3-dichlorodibenzo-p-dioxin, with less production of 2,3-dichlorodibenzo-p-dioxin in comparison with other treatments. Microbial community analyses indicated that Dehalococcoides-like bacteria were enriched with 1,2,3,4-TeCB. Quantitative real-time PCR analysis of Dehalococcoides-specific 16S rRNA genes and the D. ethenogenes strain 195 dehalogenase gene, tceA, showed at least an order of magnitude higher gene copy numbers in the bioaugmented than in the nonbioaugmented microcosms. An active-dechlorinating population is present in the River Kymijoki and biostimulation may enhance both native Dehalococcoides spp. and the bioaugmented D. ethenogenes strain 195.     

Simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry

Methionine sulfoxide is an oxidation product of methionine with reactive oxygen species via 2-electron-dependent mechanism. Such oxidants can be generated from activated neutrophils; therefore, methionine sulfoxide can be regarded as a biomarker of oxidative stress in vivo. We describe here a method for the simultaneous determination of methionine sulfoxide and methionine in blood plasma using gas chromatography-mass spectrometry with isotopically labeled compounds as internal standards. This method comprises the inclusion of [Me-13C, Me-2H(3)]methionine sulfoxide and [Me-13C, Me-2H(3)]methionine into plasma, the removal of plasma proteins using acetonitrile, the purification of amino acids with cation-exchange chromatography, and the derivatization of methionine sulfoxide and methionine to their corresponding tert-butyldimethylsilyl derivatives using N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. Quantitation was performed by electron impact mode. The levels of methionine sulfoxide in healthy human blood plasma were 4.0 +/- 1.0 microM (means +/- SD, n = 8), indicating that approximately 10% of methionine is detected as the oxidized form in healthy human plasma. The ratio of methionine sulfoxide in total methionine increased with treatment of human blood with phorbol 12-myristate 13-acetate, while this ratio remained constant in plasma from alloxan-induced hyperglycemic rabbits. These results indicate that this method is applicable for plasma samples and methionine sulfoxide can represent oxidative stress caused by nonradical oxidation in vivo.     

Kinetics and organotropy of some polyfluorinated dibenzo-p-dioxins and dibenzofurans (PFDD/PFDF) in rats

While polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF) and the corresponding polybrominated congeners must be considered as animal teratogens and carcinogens, little information is available on corresponding polyfluorinated compounds (PFDD/PFDF). Kinetic studies on a few fluorinated dibenzo-p-dioxins and dibenzofurans revealed a rapid elimination, suggesting a much lower toxicity than the corresponding polychlorinated and polybrominated congeners. In order to obtain further clues on the possible toxicity, the kinetics and organ distribution (in liver, thymus and adipose tissue) of a PFDD/PFDF-mixture were studied in Wistar rats after intravenous application. The congeners investigated included four of the 2,3,7,8-substituted, and four of the not-2,3,7,8-substituted dibenzo-p-dioxins, as well as two dibenzofurans. The main result of our studies is the finding that the concentration in the thymus of several of the 2,3,7,8-substituted PFDD/PFDF greatly exceeded that in hepatic tissue. An organotropy quite different from that of the other polyhalogenated congeners must be expected, immunosuppressive effects presumably being the predominant ones. Overall, the elimination half-life of all the PFDD/PFDF studied is considerably shorter than that of the corresponding polychlorinated or polybrominated congeners, in the rat, suggesting a much lower toxicity in this species. No information is available for other species, e.g. nonhuman primates or humans.     

Stereoselective determination of the epimer mixtures of itraconazole in human blood plasma using HPLC and fluorescence detection

Itraconazole is an antifungal drug widely used in a variety of fungal infections, which have become a significant public-health problem in recent decades. Itraconazole is a chiral drug consisting of two diastereoisomeric racemates, i.e., four stereoisomers. Data in the literature suggests that stereochemistry may play a significant role in the action and disposition of the drug and therefore stereoselective analytical methods for the determination of the drug in biological fluids are needed for the elucidation of that role. We report a stereoselective HPLC method that incorporates solvent extraction, the use of an internal standard, two chiral stationary phases in series, and fluorescence detection. The procedure is enantioselective and partially diastereoselective and provides the concentrations in blood plasma of the two epimer mixtures 2R,4S,2'R/2R,4S2'S and 2S,4R,2'R/2S,4R,2'S, respectively, each of which is a combination of the two epimers that differ in the configuration at the sec-butyl group. The analytical method has suitable sensitivity, recovery, precision, and accuracy. Analysis of the plasma of a human subject six hours after the oral administration of a single 200-mg dose of itraconazole showed a 3.4-fold difference between the concentrations of the epimer mixtures. The method has certain advantages over the published alternative procedure that uses LC-MS.     

High-performance liquid chromatographic determination of penicillamine in whole blood, plasma, and urine

A high-performance liquid chromatographic method for the determination of penicillamine in plasma, whole blood, and urine samples is described. The method uses a commercially available electrochemical detector at a potential of +0.1 V versus the Ag/AgCl reference electrode. This method is selective and sensitive for sulfhydryl compounds. The chromatography separates penicillamine from other endogenous sulfhydryl compounds with a limit of detection for penicillamine in biological samples of ca. 10⁻⁷M.     

A specific method for the determination of threonine in rat blood plasma using aldehyde dehydrogenase.

A simple and sensitive method for the determination of threonine in rat blood plasma using aldehyde dehydrogenase after oxidation with periodate was developed. By the present method, threonine could be completely discriminated from serine and determined at the nanomole level. The amount of threonine in rat blood plasma obtained by the present method coincided well with the value determined on an amino acid analyzer.     

Degradation of chlorinated dibenzofurans and dibenzo-p-dioxins by two types of bacteria having angular dioxygenases with different features

Two kinds of bacteria having different-structured angular dioxygenases-a dibenzofuran (DF)-utilizing bacterium, Terrabacter sp. strain DBF63, and a carbazole (CAR)-utilizing bacterium, Pseudomonas sp. strain CA10-were investigated for their ability to degrade some chlorinated dibenzofurans (CDFs) and chlorinated dibenzo-p-dioxins (CDDs) (or, together, CDF/Ds) using either wild-type strains or recombinant Escherichia coli strains. First, it was shown that CAR 1,9a-dioxygenase (CARDO) catalyzed angular dioxygenation of all mono- to triCDF/Ds investigated in this study, but DF 4,4a-dioxygenase (DFDO) did not degrade 2,7-diCDD. Secondly, degradation of CDF/Ds by the sets of three enzymes (angular dioxygenase, extradiol dioxygenase, and meta-cleavage compound hydrolase) was examined, showing that these enzymes in both strains were able to convert 2-CDF to 5-chlorosalicylic acid but not other tested substrates to the corresponding chlorosalicylic acid (CSA) or chlorocatechol (CC). Finally, we tested the potential of both wild-type strains for cooxidation of CDF/Ds and demonstrated that both strains degraded 2-CDF, 2-CDD, and 2,3-diCDD to the corresponding CSA and CC. We investigated the sites for the attack of angular dioxygenases in each CDF/D congener, suggesting the possibility that the angular dioxygenation of 2-CDF, 2-CDD, 2,3-diCDD, and 1,2,3-triCDD (10 ppm each) by both DFDO and CARDO occurred mainly on the nonsubstituted aromatic nuclei.     

Rapid and sensitive gas-chromatographic determination of caffeine in blood plasma,saliva, and xanthine beverages

A gas chromatographic procedure is reported for the determination of caffeine in plasma, saliva, and xanthine beverages. Using a 75 cm column packed with OV-17, nitrogen-sensitive detection, and 1 ml samples, a suitable limit of analysis (coefficient of variation (CV)=10.2%) of 50 ng/ml was obtained in plasma. Within-day CVs at caffeine concentrations of 0.1–0.5–2.0–7.5–15.0 g/ml in plasma were 7.7–5.6–4.8–3.8–3.4%, respectively. The limit of detection, defined as the injected quantity of caffeine giving rise to a signal to noise ratio of 2, is 40 pg, corresponding to a plasma concentration of 1 ng/ml.The procedure involves addition of the internal standard 7-pentyl theophylline and alkaline extraction of the sample with dichloromethane. The method described rivals any gaschromatographic assay published so far in rapidness and accuracy.Plasma and saliva caffeine concentrations were determined in a healthy male volunteer after swallowing 400 ml of coffee. The calculated pharmacokinetic parameters, assuming complete absorption of caffeine from the G.I. tract, agree well with previously published values.     

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b]pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane—dichloromethane (7:3) and analysed on a C₁₈ column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 μg/ml for III and 0.05 μg/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.