High cytotoxicity and membrane permeability of Et3Pb+ in mammalian and plant cells

Cells of mammalian origin as well as those of higher plants appear to be very sensitive to triethyllead ion (Et₃Pb⁺). Neuroblastoma cells kept in the presence of 1 μM Et₃Pb⁺ lost their viability within 6 h. Growth of suspension culture cells of soybean (G. max(L.)Merr.) was inhibited by 1 μM Et₃Pb⁺, and finally the cells died. Morphologically, Et₃Pb⁺ caused the complete breakdown of microtubular structures in neuroblastoma cells; thus microtubules appeared to be the main target for the toxin. While in a previous study the effect of Et₃Pb⁺ on microtubules has been well documented at concentrations of 50–200 μM ¹, the present study demonstrates that the formation of microtubules from pig brain tubulin is disturbed at concentrations of Et₃Pb⁺ as low as 0.5 to 1 μM . We conclude from these data that Et₃Pb⁺ freely permeates the plasma membranes of mammalian as well as plant cells.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.     

Simultaneous determination of nefiracetam and its metabolites by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic assay was developed to simultaneously quantitate nefiracetam (NEF), a novel nootropic agent, and its three known oxidized metabolites (N-[(2,6-dimethylphenylcarbamoyl)methyl]succinamic acid (5-COOH-NEF), 4-hydroxy-NEF and 5-hydroxy-NEF) in human serum and urine. The quantitative procedure was based on solid-phase extraction with Sep-Pak C₁₈ and ultraviolet detection at 210 nm. The calibration curves of NEF and the metabolites were linear over a wide range of concentrations (0.5–21.5 nmol/ml for NEF and 0.4–9.5 nmol/ml for metabolites in serum and 4–86 nmol/ml for NEF and 8–190 nmol/ml for metabolites in urine). Intra- and inter-day assay coefficients of variation for the compounds were less than 10%. The limit of detection was 0.1 nmol/ml for NEF, 5-COOH-NEF and 4-hydroxy-NEF, and 0.2 nmol/ml for 5-hydroxy-NEF in both serum and urine. This method is applicable for the determination of NEF and its metabolites in human serum and urine with satisfactory accuracy and precision.     

Comparison of initial wetting pattern,nutrient concentrations in soil solution and the fate of15N-labelled urine in sheep and cattle urine patch areas of pasture soil

Three field experiments were carried out to compare cattle and sheep urine patches in relation to (i) initial wetting pattern and volume of soil affected, (ii) soil solution ionic composition and (iii) the fate of¹⁵N-labelled urine in the soil over the winter period. The distribution of Br^– (used as a urine tracer) across the soil surface and down the profile was irregular in all the patches. The pasture area covered by Br^– in the sheep patches was 0.04–0.06 m² and Br^– was detected to a depth of 150 mm. Cattle patches were significantly larger covering a surface area of 0.38–0.42 m² and penetrating to a depth of 400 mm. The rapid downward movement of urine occurred through macropore flow but even so, over half of the applied Br^– was detected in the 0–50 mm soil layer in both sheep and cattle patches. Due to the larger volume of urine added to the cattle patches (2000 mL for cattle and 200 mL for sheep) the effective application rate was about 5 L m^–2 compared with 4 L m^–2 for sheep. Concentrations of extractable mineral N and ionic concentrations in soil solution were higher in cattle than sheep patches particularly near the soil surface. In both sheep and cattle patches, urea was rapidly hydrolysed to NH ₄ ⁺ and nitrification occurred between 14 and 29 days after urine application. Initially the major anions and cations in the soil solution were HCO ₃ ^– , SO ₄ ⁼ , Cl^–, NH ₄ ⁺ , Mg⁺⁺, K⁺ and Na⁺, which were derived from the urine application. Ionic concentrations in the soil solution decreased appreciably over time due to plant uptake and possibly some leaching. As nitrification proceeded, NO ₃ ^– became the dominant anion in soil solution and the major accompanying cation was Ca⁺⁺. The fate of¹⁵N-labelled urine-urea was followed during a 5 month period beginning in late autumn. Greater leaching losses of NO ₃ ^– occurred below cattle patches (equivalent to 60 kg N ha^–1 below 300 mm and 37 kg N ha^–1 below 600 mm) compared with sheep patches (10 kg N ha^–1 below 300 mm and 1 kg N ha^– below 600 mm). While 6% of the applied¹⁵N was leached the amount of N leached was equivalent to 11% of the applied urine-N in cattle patches. This suggests that there was significant immobilsation-mineralisation turnover in urine patch soil with the release of mineral N from native soil organic matter. In both sheep and cattle patches 60% of the¹⁵N was accounted for in plant uptake, remaining in the soil and leaching. About 40% of the applied N was therefore lost through gaseous emission.     

Analysis of N-methylhistamine by gas chromatography–mass spectrometry

A gas chromatography–electron capture mass spectrometry assay has been developed for the histamine H₃ receptor agonist, N^α-methylhistamine (N^α-MH). The assay is linear from 50 pg–10 ng, with a limit of detection of 50 pg/ml for gastric juice and plasma, and 50 pg/sample for bacteria (10⁷–10⁸ CFU) and gastric tissue (5–10 mg wet weight). The limits of quantification are 100 pg/ml for gastric juice (%RSD=1.4) and plasma (%RSD=9.4), and 100 pg/sample for bacteria (%RSD=3.9) and tissue (%RSD=5.8). N^α-MH was not present in human plasma, but low levels (1.4 ng/ml and 0.4 ng/ml) were detected in two samples of human gastric juice obtained from patients infected with Helicobacter pylori.     

Systematic analysis of acid, neutral and basic drugs in horse plasma by combination of solid-phase extraction, non-aqueous partitioning and gas chromatography–mass spectrometry

A sample preparation method for mass chromatographic detection of doping drugs from horse plasma is described. Bond Elut Certify (1 g/6 ml) is used for the extraction of 4 ml of horse plasma. Fractionation is performed with 6 ml of CHCl₃–Me₂CO (8:2) and 5 ml of 1% TEA–MeOH according to its property. Simple and effective clean-up based on non-aqueous partitioning is adopted to remove co-eluted contaminants in both acid and basic fractions. Two kinds of 1-(N,N-diisopropylamino)-n-alkanes are co-injected with the sample into the GC–MS system for the calculation of the retention index. Total recoveries of 107 drugs are examined. Some data of post administration plasma are presented. This procedure achieves sufficient recoveries and clean extracts for GC–MS analysis. The method is able to detect ng/ml drug levels in horse plasma.     

Determination of diphenylmethane antihistaminic drugs and their analogues in body fluids by gas chromatography with surface ionization detection

Eleven diphenylmethane antihistaminic drugs and their analogues were tested for their detection by capillary gas chromatography (GC) with surface ionization detection (SID). The GC—SID response was highest for doxylamine, diphenhydramine and orphenadrine and lowest for terodiline, clemastine and pipethanate. The detection limits for drugs with the highest response were 2–5 pg (ca. 6–20 fmol) on-column (100–250 pg/ml of body fluid). The detection limits with GC—SID were 10–100 times higher than those with GC with nitrogen—phosphorus detection. A detailed procedure for the isolation of the antihistaminics from human whole blood and urine by the use of Sep-Pak C₁₈ cartridges, prior to GC—SID, is also presented. The recoveries of the drugs (50 or 500 pmol), which had been added to 1 ml of body fluids, were>60%. The baselines remained steady as the column temperature was increased and the background was clean, especially for whole blood extracts.     

Trimethyl lead degradation by free and immobilized cells of an Arthrobacter sp. and by the wood decay fungus Phaeolus schweinitzii

Summary The continuing production of leaded petrol generates liquid wastes containing recalcitrant trialkyl lead, for which no suitable chemical treatment has been formulated. This investigation explores the feasibility of using microorganisms to catalyse the rate-limiting step of trimethyl lead degradation to dialkyl lead; this disproportionates chemically to give, ultimately, Pb²⁺ which is treatable by classical methods. An Arthrobacter sp. and a wood decay macrofungus, Phaeolus schweinitzii provide novel evidence for metabolic trimethyl lead (Me₃Pb⁺) degradation. The retention of this activity in immobilized cell column reactors challenged with Me₃Pb⁺-supplemented flows suggests that a future biotreatment process may be possible. Offprint requests to: M. E. Macaskie     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

Determination of ephedrines in urine by gas chromatography–mass spectrometry

A selective gas–liquid chromatographic method with mass spectrometry (GC–MS) for the simultaneous confirmation and quantification of ephedrine, pseudo-ephedrine, nor-ephedrine, nor-pseudoephedrine, which are pairs of diastereoisomeric sympathomimetic amines, and methyl-ephedrine was developed for doping control analysis in urine samples. O-Trimethylsilylated and N-mono-trifluoroacetylated derivatives of ephedrines — one derivative was formed for each ephedrine — were prepared and analyzed by GC–MS, after alkaline extraction of urine and evaporation of the organic phase, using d₃-ephedrine as internal standard. Calibration curves, with r²>0.98, ranged from 3.0 to 50 μg/ml depending on the analyte. Validation data (specificity, % RSD, accuracy, and recovery) are also presented.     

Quantitative determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine by high-performance liquid chromatography with amperometric detection

A rapid and simple high-performance liquid chromatographic method with amperometric detection has been developed for the quantitation of cilazapril and its active metabolite and degradation product cilazaprilat in urine and tablets. The chromatographic system consisted of a μBondapak C₁₈ column, using a mixture of methanol–5 mM phosphoric acid (50:50, v/v) as mobile phase, which was pumped at a flow-rate of 1.0 ml/min. The column was kept at a constant temperature of (40±0.2)°C. Detection was performed using a glassy carbon electrode at a potential of 1350 mV. Sample preparation for urine consisted of a solid-phase extraction using C₈ cartridges. This procedure allowed recoveries greater than 85% for both compounds. The method proved to be accurate, precise and sensitive enough to be applied to pharmacokinetic studies and it has been applied to urine samples obtained from four hypertensive patients (detection limit of 50 ng/ml for cilazapril and 40 ng/ml for cilazaprilat in urine). Results were in good agreement with pharmacokinetic data.     

Characteristics of 3-O-methylfluorescein phosphate hydrolysis by the (Na+ + K+)-ATPase

With 3-O-methylfluorescein phosphate (3-OMFP) as substrate for the phosphatase reaction catalyzed by the (Na⁺ + K⁺)-ATPase, a number of properties of that reaction differ from those with the common substratep-nitrophenyl phosphate (NPP): theK _m is 2 orders of magnitude less and the V_max is two times greater, and dimethyl sulfoxide (Me₂SO) inhibits rather than stimulates. In addition, reducing the incubation pH decreases both theK _m and V_max for K⁺-activated 3-OMFP hydrolysis as well as theK _0.5 for K⁺ activation. However, reducing the incubation pH increases inhibition by P_i and the V_max for 3-OMFP hydrolysis in the absence of K⁺. When choline chloride is varied reciprocally with NaCl to maintain the ionic strength constant, NaCl inhibits K⁺-activated 3-OMFP hydrolysis modestly with 10 mM KCl, but stimulates (in the range 5–30 mM NaCl) with suboptimal (0.35 mM) KCl. In the absence of K⁺, however, NaCl stimulates increasingly over the range 5–100 mM when the ionic strength is held constant. These observations are interpreted in terms of (a) differential effects of the ligands on enzyme conformations; (b) alternative reaction pathways in the absence of Na⁺, with a faster, phosphorylating pathway more readily available to 3-OMFP than to NPP; and (c) a (Na⁺ + K⁺)-phosphatase pathway, most apparent at suboptimal K⁺ concentrations, that is also more readily available to 3-OMFP.Abbreviations Et₃N triethyl amine - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - MES 2-(N-morpholino)ethanesulfonate - Me₂SO dimethyl sulfoxide - NPP p-nitrophenyl phosphate - 3-OMFP 3-O-methylfluorescein phosphate - TNP-ATP 2, (or 3)-O-(2,4,6-trinitrophenyl)-ATP     

Study of the solid-phase extraction of diclofenac sodium, indomethacin and phenylbutazone for their analysis in human urine by liquid chromatography

A selective semi-automated solid-phase extraction (SPE) of the non-steroidal anti-inflammatory drugs diclofenac sodium, indomethacin and phenylbutazone from urine prior to high-performance liquid chromatography was investigated. The drugs were recovered from urine buffered at pH 5.0 using C₁₈ Bond-Elut cartridges as solid sorbent material and mixtures of methanol–aqueous buffer or acetonitrile–aqueous buffer as washing and elution solvents. The extracts were chromatographed on a reversed-phase ODS column using 10 mM acetate buffer (pH 4.0)–acetonitrile (58:42, v/v) as the mobile phase, and the effluent from the column was monitored at 210 nm with ultraviolet detection. Absolute recoveries of the anti-inflammatory drugs within the range 0.02–1.0 μg/ml were about 85% for diclofenac and indomethacin, and 50% for phenylbutazone without any interference from endogenous compounds of the urine. The within-day and between-day repeatabilities were in all cases less than 5% and 10%, respectively. Limits of detection were 0.007 μg/ml for diclofenac sodium and indomethacin and 0.035 μg/ml for phenylbutazone, whereas limits of quantitation were 0.02 μg/ml for diclofenac and indomethacin and 0.1 μg/ml for phenylbutazone.     

Biogeochemistry of trimethyllead and lead in a forested ecosystem in Germany

Lead compounds, especially ionic organolead compounds (OLC), are highly toxic and mobile pollutants strongly affecting many ecosystems. Soil pools and fluxes with precipitation, litterfall and runoff of trimethyllead (TML), one of the dominant ionic OLC in the environment, and Pb_total were investigated in a forested ecosystem in NE-Bavaria, Germany. In addition, ad/desorption of TML to soils was studied in batch experiments and its degradation in soils was investigated using long term incubations. Total soil storage in the catchment was 11.56 mg Pb ha^–1 for TML and 222 kg Pb ha^–1 for Pb_total. More than 90% of the soil storage of TML was found in the wetland soils of the catchment representing only 30% of the area. Most Pb_total (>90%) was found in the upland soils. In upland soils, TML was only detectable in the forest floor. The annual total deposition from the atmosphere, estimated as throughfall + litterfall fluxes, amounted to 3.7 mg Pb ha^–1 year^–1 for TML and 52 g Pb ha^–1 year^–1 for Pb_total. The contribution of litterfall was 1.5 and 32%, respectively. The concentrations of TML and Pb_total in wet precipitation were: fog > throughfall > bulk precipitation. The annual fluxes with runoff from the catchment was 0.5 mg Pb ha^–1 year^–1 for TML and 2.8 g Pb ha^–1 year^–1 for Pb_total. TML degraded rapidly in the forest floor (Oa horizon) with a half-life (t _1/2) of 33.5 days. The degradation of TML in Fen (t _1/2 = 421 days) and in the mineral soil (Bw-C horizon, t _1/2 = 612 days) was much slower. Emission of tetramethyllead from wetland soils was not observed during the 1 year incubation. The adsorption affinity of TML to different soils was Fen > Oa > A Bw-C. The ratio of total soil storages to the present annual input were 3.6 years for TML. TML and Pb_total are still deposited in remote areas even after the use of tetraalkyllead as additives has been terminated for years. The rates of deposition are, however, much lower than in the past. Forest soils act as a sink for deposited TML and Pbtotal. TML is accumulated mostly in wetland soils and seems to be stable under anoxic conditions for a long time. In upland soils, TML decomposes rapidly. Only small amounts of TML are transferred from soils into runoff.     

Organic and inorganic nitrogen nutrition of western red cedar,western hemlock and salal in mineral N-limited cedar–hemlock forests

Western red cedar (Thuja plicata Donn.), western hemlock (Tsuga heterophylla Raf. Sarge) and salal (Gaultheria shallon Pursh) are the main species growing in cedar–hemlock forests on Vancouver Island, Canada. Based on the dominance of organic N in these systems, we tested the hypotheses that: (1) organic N can be utilized by the three plant species; and (2) salal, which is ericoid mycorrhizal and has high tannin concentration in its tissues, would absorb more N from the complex organic N compounds than the other two species. The abilities of cedar, hemlock and salal to take up ¹⁵N,¹³C-labelled glutamic acid were measured and the capacities of the three species to use nitrate (NO₃^–), ammonium (NH₄⁺), glutamic acid, protein and protein–tannin N were compared over a 20-day period. Based on ¹³C enrichment, all three species absorbed at least a portion of glutamic acid intact. Cedar, hemlock and salal also showed similar patterns of N uptake from the NO₃^–, NH₄⁺, glutamic acid, protein and protein–tannin treatments. The largest proportions of applied N were taken up from the NO₃^– and NH₄⁺ treatments while smaller amounts of N were absorbed from the organic N compounds. Thus organic N was accessed to a modest degree by all three species, and salal did not have a greater capacity to utilize protein and protein–tannin–N.     

Beiträge zur Entsalzung mit Retardion 11A8

Zusammenfassung 1. Das Arbeiten mit dem Ionenverzögerungsverfahren wird eingehend beschrieben.2. Die optimalen Bedingungen für die Trennung organischer Testsubstanzen von anorganischen Salzen bei Raumtemperatur sind: zwei hintereinandergeschaltete Säulen mit einem Gesamtvolumen von 225 ml Retardion 11A8; 50 bis 100 ml Analysenprobe einer 3,5prozentigen oder 50 ml einer 7prozentigen Salzlösung; 60 ml/h Durchflußgeschwindigkeit; 4 bis 5 1 Wasser zum Eluieren und Regenerieren der Säulen.3. Aus der organischen Fraktion werden an zugesetzten Ionen entfernt: Cl^– 96,2%; So₄ ^–– 63,4%; Mg⁺⁺ 97,7%; Ca⁺⁺ 96,8%.4. Es werden Maßnahmen zur Wiederherstellung von gestörtem Gleichgewicht der Säulen beschrieben.5. Mit Retardion 11A8 läßt sich ein Salzgemisch in seine einzelnen Komponenten zerlegen. Die Reihenfolge der untersuchten Ionen im Säuleneluat ist: PO₄ ^–––, CO₃ ^–– und HCO₃ ^–, SO₄ ^––, Cl^–, Br^–; NH₄ ⁺, K⁺, Na⁺, Mg⁺⁺, Ca⁺⁺. Die Abtrennung der Erdalkali ist besonders scharf.6. Die Elutionskurven von vielen Aminosäuren und verwandten Verbindungen, Kohlenhydraten, anorganischen und organischen Phosphatverbindungen, organischen Säuren, Harnstoff und Gelbstoff werden beschrieben.7. Der Prozentsatz des Wiederauffindens zugesetzter organischer Testsubstanzen, die von Salzen getrennt werden, beträgt 95 bis 100 für die meisten Aminosäuren, Kreatin, Zucker, Zuckeralkohole und für einige Phosphorsäureester. Die Ausbeute von Asparaginsäure und basischen Aminosäuren, Histamin, Glucosamin, Glucosediphosphat und organischen Säuren ist geringer.8. Die Vor- und Nachteile sowie einige Möglichkeiten der Anwendung des Ionenverzögerungsverfahrens mit Retardion 11A8 in der Hydrobiologie, Biochemie und Meeresbiologie werden diskutiert.

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Contributions to desalting with Retardion 11A8

The isolation and determination of the soluble organic substances in sea water is very difficult because of the presence of large amounts of inorganic salts. In order to desalt the water, ion retardation with Retardion 11A8 seems to be a very efficient method. Since this procedure is at the present almost unknown, the optimal conditions of separation which have been found are described in this paper. The selectivity of the resin for different ions occurring in sea water is studied. The elution curves of a large number of organic substances (amino acids, carbohydrates, organic phosphorus compounds and organic acids) are described. Recovery of given concentrations of these substances, added to 50 ml of a salt solution similar to sea water, is 95 to 100% for most of the amino acids, carbohydrates and organic phosphorus compounds but less for other substances. Using ion retardation as a desalting means, more than 90% of the inorganic salts may be removed from the organic fraction by a one-step procedure. These experiments may be done on a larger scale in desalting larger volumes of sea water. Ion retardation has proved to be an excellent separation method for biochemistry and water chemistry. Work is in progress to use Retardion 11A8 as a desalting means in the isolation of organic matter from sea water.

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Herrn Professor Dr.Adolf Bückmann zum 65. Geburtstag in Verehrung gewidmet.

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Der Deutschen Forschungsgemeinschaft danke ich für die Unterstützung der Arbeit.     

Determination of loratadine in human plasma by high-performance liquid chromatographic method with ultraviolet detection

A HPLC–UV determination of loratadine in human plasma is presented. After simple liquid–liquid extraction with 2-methylbutane–hexane (2:1) and evaporation of organic phase the compounds were re-dissolved in 0.01 M HCl, evaporated again and finally separated on a Supelcosil LC-18-DB column. The analyses were done at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:480:80:1, v/v). UV detection was performed at 200 nm with a limit of quantification of 0.5 ng/ml. The precision was found to be satisfactory over the whole range tested (0.5–50 ng/ml) with relative standard deviations of 2.3–6.3 and 5.2–14.1% for intra- and inter-assays, respectively.     

Method for the analysis of the methylphosphonic acid metabolites of sarin and its ethanol-substituted analogue in urine as applied to the victims of the Tokyo sarin disaster

An analysis method for the methylphosphonic acid metabolites of sarin in urine using trimethylsilyl derivatization and flame photometric detection is described in this report. Authentic reference standards of isopropyl methylphosphonic acid (IMPA) and ethyl methylphosphonic acid (EMPA) as well as methylphosphonic acid were employed to estimate the concentration in human urine. A sample pretreatment procedure was developed for urine using a column of cation-loaded ion-exchange resins (Ag⁺-, Ba²⁺- or H⁺-Dowex) and adjusting the pH of the eluate from the column to 3.75–3.85 improved recovery of the target compounds. The eluate was evaporated to dryness under vacuum prior to trimethylsilylation, to remove water and any hydroxy- or amino-carrying volatile substances. The sarin metabolites, because of their low volatility, were concentrated and could be derivatized for analysis. The use of synthesized authentic sarin and ethylsarin metabolites, i.e., IMPA and EMPA, made it possible to establish the necessary sample pretreatment procedures for derivatization and gas chromatography–flame photometric detection (GC–FPD) analysis. The detection limits were 0.025 ppm both for EMPA and IMPA, and 0.625 μM for MPA, respectively. This method can be useful for estimating the exposure level to sarin by assaying the metabolites in urine and it is applicable to a large numbers of samples.     

Quantitation of o-, m- and p-cresol and deuterated analogs in human urine by gas chromatography with electron capture detection

A gas chromatographic method for the analysis of cresol metabolites of toluene and [²H₈]toluene in urine was developed. Cresol glucuronides and sulfates in urine were hydrolyzed with β-glucuronidase and arylsulfatase. Following extraction with tert.-butyl methyl ether and solvent exchange into benzene, the cresols were derivatized with heptafluorobutyric anhydride to form the heptafluorobutyrate esters. The derivatives were analyzed by gas chromatography with electron capture detection. Chromatographic resolution was achieved between all cresol isomers and their ²H₇ analogs. Calibration ranged from 0.001 to 500 μg/ml. Recoveries were 55–97% and showed no trend with respect to analyte concentration. Within-day precision of analyses of benchmark urine samples had a coefficient of variation of less than 4%. The assay sensitivity was limited by chromatographic background but was sufficient for quantification of the unlabeled cresols in urine from men with only environmental exposure to toluene. Average levels in urine samples from 45 men were 0.023, 0.054 and 37 μg/ml for o-, m- and p-cresol, respectively.     

Fluxes and transformations of nitrogen in a high-elevation catchment,Sierra Nevada

The fluxes and transformations of nitrogen (N) were investigated from 1985 through 1987 at the Emerald Lake watershed (ELW), a 120 ha high-elevation catchment located in the southern Sierra Nevada, California, USA. Up to 90% of annual wet deposition of N was stored in the seasonal snowpack; NO ₃ ^– and NH ₄ ⁺ were released from storage in the form of an ionic pulse, where the first fraction of meltwater draining from the snowpack had concentrations of NO ₃ ^– and NH ₄ ⁺ as high as 28 eq L^–1 compared to bulk concentrations of <5 eq L^–1 in the snowpack. The soil reservoir of organic N (81 keq ha^–1) was about ten times the N storage in litter and biomass (12 keq ha^–1). Assimilation of N by vegetation was balanced by the release of N from soil mineralization, nitrification, and litter decay. Mineralization and nitrification processes produced 1.1 keq ha^–1 yr^–1 of inorganic N, about 3 1/2 times the loading of N from wet and dry deposition. Less than 1% of the NH ₄ ⁺ in wet and dry deposition was exported from the basin as NH ₄ ⁺ . Biological assimilation was primarily responsible for retention of NH ₄ ⁺ in the basin, releasing one mode of H⁺ for every mole of NH ₄ ⁺ retained and neutralizing about 25% of the annual acid neutralizing capacity produced by mineral weathering in the basin. Nitrate concentrations in stream waters reached an annual peak during the first part of snowmelt runoff, with maximum concentrations in stream water of 20 eq L^–1, more than 4 times the volume-weighted mean annual concentrations of NO ₃ ^– in wet deposition. This annual peak in stream water NO ₃ ^– was consistent with the release of NO ₃ ^– from the snowpack in the form of an ionic pulse; however soil processes occurring underneath the winter snowpack were another potential source of this NO ₃ ^– . Concentrations of stream water NO ₃ ^– during the summer growing season were always near or below detection limits (0.5 eq L^–1).