Biotransformation of various substituted aromatic compounds to chiral dihydrodihydroxy derivatives.

The biotransformation of four different classes of aromatic compounds by the Escherichia coli strain DH5alpha(pTCB 144), which contained the chlorobenzene dioxygenase (CDO) from Pseudomonas sp. strain P51, was examined. CDO oxidized biphenyl as well as monochlorobiphenyls to the corresponding cis-2,3-dihydro-2,3-dihydroxy derivatives, whereby oxidation occurred on the unsubstituted ring. No higher substituted biphenyls were oxidized. The absolute configurations of several monosubstituted cis-benzene dihydrodiols formed by CDO were determined. All had an S configuration at the carbon atom in meta position to the substituent on the benzene nucleus. With one exception, the enantiomeric excess of several 1,4-disubstituted cis-benzene dihydrodiols formed by CDO was higher than that of the products formed by two toluene dioxygenases. Naphthalene was oxidized to enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. All absolute configurations were identical to those of the products formed by toluene dioxygenases of Pseudomonas putida UV4 and P. putida F39/D. The formation rate of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene was significantly higher (about 45 to 200%) than those of several monosubstituted cis-benzene dihydrodiols and more than four times higher than the formation rate of cis-benzene dihydrodiol. A new gas chromatographic method was developed to determine the enantiomeric excess of the oxidation products.

Photodegradation of the pharmaceutical drug diclofenac in a lake: pathway, field measurements, and mathematical modeling.

Vertical concentration profiles of diclofenac were measured in Lake Greifensee (Switzerland) under mixed (February/December) and stratified (July) lake conditions. The concentrations ranged from 1 to 12 ng/L and were lower in summer than in winter, especially near the lake surface, pointing to an efficient elimination of diclofenac by photodegradation in the lake. Laboratory experiments confirmed the rapid photodegradation of diclofenac in water when exposed to sunlight. First-order reaction rates varied seasonally according to actual solar radiation (half-lives, tau = 0.2-1.7 h). The initial photoproduct was 8-chlorocarbazole-1-acetic acid, which photodegraded even faster than the parent compound. Carbazole-1-acetic acid, previously reported as the main photoproduct, was only formed in the presence of a H-source, such as methanol. In the absence of a H-source and of air, hydroxycarbazole-1-acetic acid was formed. However, hydroxycarbazole-1-acetic acid was not observed in the presence of air and, thus, under conditions similar to those in a lake, likely because of its rapid further photooxidation. Computer simulations using a one-dimensional lake model taking actual solar radiation and flushing data of the lake into account confirmed that photolysis is the predominant elimination pathway for diclofenac in Lake Greifensee. These calculations further showed that the expected concentrations of the photoproduct 8-chlorocarbazole-1-acetic acid are less than the current detection limits of approximately 3 ng/L.

Isolation and identification of the metolachlor stereoisomers using high-performance liquid chromatography, polarimetric measurements, and enantioselective gas chromatography.

Because of the presence of two chiral elements (an asymmetrically substituted carbon and a chiral axis), the herbicide metolachlor consists of four stereoisomers stable at ambient temperature with aSS-, aRS-, aSR-, and aRR-configurations (aSS, the isomer with aS,1'S-configuration, etc.). Metolachlor, initially introduced into the market as the racemic product containing all four stereoisomers, is currently being replaced worldwide by S-metolachlor, the product enantiomerically enriched with the herbicidally active 1'S-isomers (aSS, aRS). The isomer-specific analysis of metolachlor requires not only enantioselective ("chiral") analytical techniques but also suitable reference compounds. In this study, two of the four metolachlor isomers were isolated from rac-metolachlor in enantio- (ee > 98%) and diastereomerically pure forms by a combination of achiral and chiral high-performance liquid chromatography (HPLC). The two isomers were identified as the aSS- and the aRR-isomers by polarimetric measurements, in reference to previous data. The two isomers were then thermally equilibrated to 1:1 mixtures of the aSS/aRS and aRR/aSR diastereomers, respectively, so that analytical data of all four metolachlor isomers became available; they were then used to identify these isomers in technical products by chiral high-resolution gas chromatography (HRGC). The kinetics of the thermally induced interconversion of the atropisomers was studied and the consequences, such as for GC analysis, are discussed. A comparison of on-column and split/splitless injection indicated that the latter technique results in significant isomerization prior to separation and, therefore, cannot be used for accurate isomer analysis.

Discrimination and thermal degradation of toxaphene compounds in capillary gas chromatography when using split/splitless and on-column injection.

Technical toxaphene and a 22-component Reference Mixture were analyzed using capillary gas chromatography with split/splitless injection (SSL) and on-column injection (OC). In both techniques, electron-capture, negative ionization mass spectrometry (ECNI-MS) was used for detection of chlorobornanes, chlorocamphenes and related compounds. Significant discrimination of highly chlorinated congeners was observed as a result of incomplete transfer of these compounds from the vaporizer to the analytical column when using SSL. This resulted in a much lower response for nona- and decachloro congeners than when using OC. In addition, several toxaphene components, especially the chlorobornanes with gem dichloro substitution on the six-member carbon ring, undergo thermal degradation when using SSL. Some of these congeners are major components of technical toxaphene, but generally are not present, except at low concentrations, in environmental and biological samples. Therefore, technical toxaphene may be discriminated and/or degraded differently than toxaphene compounds in environmental samples when using SSL. This results in significant bias of the quantitative data when using the technical material as a reference. OC suffers much less from these deficiencies and, therefore, is a preferable technique for toxaphene analysis.

Rapid anaerobic degradation of toxaphene in sewage sludge.

We studied the degradation of technical toxaphene in anaerobic sewage sludge from a municipal waste water treatment plant. Chlorobornanes, chlorocamphenes and related compounds were rapidly degraded, with degradation rates in the order of decachloro>nonachloro>octochloro>heptachloro approximately = hexachloro compounds. The half-lives of individual congeners ranged from <1 day to several days. We also studied the degradation of technical toxaphene in previously sterilized sludge (control), and found it was slower than in the anaerobic sludge. The chlorobornanes that degraded most rapidly in the non-sterilized anaerobic sludge were those with gem chloro substitution on the 6-member carbon-ring, including the toxic congeners, Toxicant A and B. Non-gem chloro substituted congeners, like the biologically persistent P26 and P50, also degraded, but less rapidly. Toxaphene degradation in sewage sludge proceeded primarily via reductive dechlorination, leading to HxSed, HpSed, TC2 and other persistent metabolites. Enantioselective determinations indicated little, if any, enantioselectivity in the formation and/or degradation of these compounds. The isomer and enantiomer profiles of the hexa-, hepta-, and octachlorobornanes are similar to those observed in sediment from the Baltic Sea, suggesting that technical toxaphene is the source of these compounds and that its composition was changed via similar anaerobic degradation pathways.

Fate of the herbicides mecoprop, dichlorprop, and 2,4-D in aerobic and anaerobic sewage sludge as determined by laboratory batch studies and enantiomer-specific analysis.

Aerobic degradation experiments with the racemic mixtures of mecoprop and dichlorprop revealed that activated sludge collected from the aeration tank of a municipal waste water treatment plant degraded both enantiomers of mecoprop and dichlorprop within 7 days, albeit in an enantioselective manner; the (S) enantiomers were preferentially degraded. Mecoprop, dichlorprop, and 2,4-D were completely metabolized under aerobic conditions, as shown by the 86-98% elimination of dissolved organic carbon. Under anaerobic conditions, the concentration of 2,4-D decreased exponentially with a first-order reaction rate constant of 0.24 per day and without a lag-phase. After an incubation time of 17 days, 2,4-D was completely removed. 2,4-Dichlorophenol was the main metabolite of anaerobic 2,4-D degradation; only traces of 4-chlorophenol were detected. In contrast, the chiral phenoxypropionic acid herbicides mecoprop and dichlorprop persisted under anaerobic conditions during 49 days of incubation.

Postnatal development of urea- and ammonia-excretion in urine of very-low-birth-weight infants small for gestational age.

In 12 very-low-birth-weight (VLBW) infants with intrauterine growth retardation and in 14 VLBW-infants appropriate for gestational age (AGA) fed a human milk (HM) formula (HM enriched with 6 g freeze dried HM per 100 ml) the renal excretion of urea and ammonia was studied on the 10th, 21st and 42nd days of life. The lowest excretion of urea was found in both groups on the 10th day of life. Up to the 42nd day of life the excretion raised significantly more in the AGA- than in the small for gestational age (SGA)-infants. In contrast to the urea excretion the excretion of ammonia was highest on the 10th day of life in both groups, but the excretion was significantly higher in the SGA-infants if compared to the AGA-infants. In the AGA-infants excretion of ammonia decreased with postnatal age whereas in the SGA-infants the high excretion remained up to the 42nd day of life. The data suggest that in VLBW-infants the urea synthesizing capacity is decreased and develops within the first weeks of postnatal life. The postnatal development is delayed in SGA-infants when compared to AGA-infants. The differences are more pronounced with increasing degree of intrauterine growth retardation.

Influence of postnatal age on nitrogen metabolism in very low birth weight infants appropriate for gestational age.

In 14 very low birth weight (VLBW) infants appropriate for gestational age the absorption and retention of nitrogen and the energy balance were studied on the 8th day and the 6th week of postnatal age. All infants were fed with a human milk formula composed by fresh mother's milk enriched with 6 g freeze-dried human milk/100 ml. The absorption rates of nitrogen and fat increase significantly with postnatal age but even on the 8th day of life they are on high levels (nitrogen absorption: 86.9 +/- 3.2% of intake; fat absorption: 85.7 +/- 4.3% of intake). Despite the higher nitrogen absorption during the 6th week of life the excretion of total nitrogen is lower in comparison to the 8th day of life. Similarly, the percentages of alpha-amino-nitrogen and of ammonium-nitrogen of the total nitrogen excretion in the urine decrease whereas the percentage of urea-nitrogen increases with postnatal age. The results indicate a postnatal development of both the digestive, as well as the metabolic capacity. The postnatal adaptation of the metabolic capacity to utilize nitrogen seems to be the stronger limiting factor for nutrition than the functional capacity of digestion and absorption. Thus, metabolic monitoring is essential for the individual nutrition of VLBW-infants during the first weeks of postnatal life. Urine analysis data are very sensitive for evaluating the metabolic state of these infants.

Enantiomer resolution and assay of propionic acid-derived herbicides in formulations by using chiral liquid chromatography and achiral gas chromatography.

Enantiomers of 6 propionic acid-derived herbicides in the form of their esters were resolved using liquid chromatography with a chiral column. Free acids are converted to methyl esters by means of a BF3-catalyzed reaction. Chromatographic resolutions for 6 of 8 herbicides investigated were in the range of 2 to 4. The method was applied for the simultaneous determination of mecoprop and 2,4-D content and individual mecoprop enantiomers in 2 formulations containing racemic and R-mecoprop in mixture with 2,4-D. Precision and accuracy of content determination was comparable to standard methods, and enantiomer contents were in good agreement with declared values. The enantiomers of dichlorprop and mecoprop were also resolved as diastereomeric menthyl esters by achiral high resolution gas chromatography (HRGC). HRGC data on enantiomer composition were in good agreement with those from the LC method and other data.

Trace level detection of chlorinated paraffins in biological and environmental samples, using gas chromatography/mass spectrometry with negative-ion chemical ionization.

A method is described for detection of chlorinated paraffins (CPs) in biological and environmental samples. Sample preparation includes sulfuric acid treatment followed by adsorption chromatography on alumina, which yields the CPs in one fraction that is almost free of interfering material. Using gas chromatography/mass spectrometry with negative-ion chemical ionization, the limit of detection is 5 ng (corresponding to the lower ppb range). CP levels of 30 ppm, 200 ppb, and 5 ppb were found in sewage sludge, human fat, and sediment, respectively.