Biodegradation of neutralized sarin.

This research investigated the biotransformation of IMPA, the neutralization product of the nerve agent Sarin, by a microbial consortia. As mandated by the Chemical Weapons Convention signed by 132 countries in 1993, all chemical warfare agents are to be destroyed within ten years of ratification. Technologies must be developed to satisfy this commitment. This paper presents data from a biodegradation kinetics study and background information on the biological transformation of IMPA. Microbial transformation of organophosphate nerve agents and organophosphate pesticide intermediates can be incorporated into a treatment process for the fast and efficient destruction of these similar compounds. Sarin (isopropyl methylphosphonofluoridate), also known as GB, is one of several highly neurotoxic chemical warfare agents that have been developed over the past 50 to 60 years. Four mixed cultures were acclimated to the Sarin hydrolysis product, isopropyl methylphosphonic acid (IMPA). Two of these cultures, APG microorganisms and SX microorganisms, used IMPA as the sole phosphorus source. Extended exposure to IMPA improved the cultures' abilities to degrade IMPA to form methylphosphonic acid (MPA) and inorganic phosphate. The presence of free phosphate in the reactor suppressed the degradation of IMPA. IMPA did not inhibit either cultural consortia within the tested concentration range (0 to 1250 mg/L). The numax was 120.9 mg/L/day for the SX microorganisms and 118.3 mg/L/day for the APG microorganisms. Initial IMPA concentrations of 85 to 90 mg/L were degraded to nondetectable levels within 75 h. These results demonstrate the potential for biodegradation to serve as a complementary treatment process for the destruction of stockpiled Sarin.     

Sequencing Batch Reactor Biodegradation of Hydrolyzed Sarin as Sole Carbon Source

The chemical nerve agent sarin (o-isopropyl methylphosphonofluoridate) was hydrolyzed at 7.18 weight percent in aqueous sodium hydroxide yielding primarily o-isopropyl methylphosphonic acid (IMPA). This hydrolysate was diluted and fed as sole carbon source to activated sludge in an aerobic sequencing batch reactor. Feed chemical oxygen demand (COD) concentrations ranged from approximately 2500 mg/L (initial) to 5000 mg/L (final). The reactor was operated essentially on a 15-day hydraulic residence time. Overall COD removal efficiency was 86.2% and the IMPA in the feed was converted to methylphosphonic acid. MICROTOX® and Daphnia magna aquatic toxicity testing showed the effluent to be of very low toxicity to aquatic test organisms. The final MPA product was effectively absorbed by Phoslock?, which is a lanthanide modified clay.     

Sequencing Batch Reactor Biodegradation of Hydrolyzed Sarin as Sole Carbon Source

The chemical nerve agent sarin (o-isopropyl methylphosphonofluoridate) was hydrolyzed at 7.18 weight percent in aqueous sodium hydroxide yielding primarily o-isopropyl methylphosphonic acid (IMPA). This hydrolysate was diluted and fed as sole carbon source to activated sludge in an aerobic sequencing batch reactor. Feed chemical oxygen demand (COD) concentrations ranged from approximately 2500 mg/L (initial) to 5000 mg/L (final). The reactor was operated essentially on a 15-day hydraulic residence time. Overall COD removal efficiency was 86.2% and the IMPA in the feed was converted to methylphosphonic acid. MICROTOX® and Daphnia magna aquatic toxicity testing showed the effluent to be of very low toxicity to aquatic test organisms. The final MPA product was effectively absorbed by Phoslock™, which is a lanthanide modified clay.     

Determination of metabolites of pyrethroids in human urine using solid-phase extraction and gas chromatography–mass spectrometry

The described method permits the determination of the five most important metabolites of the pyrethroids permethrin, cypermethrin, deltamethrin, λ-cyhalothrin, fenvalerate, phenothrin and β-cyfluthrin in human urine in one run. The major urinary metabolites of these substances are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl₂CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br₂CA), fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA). After acidic hydrolysis to release the conjugated carboxylic acid metabolites, the analytes were separated from the matrix by means of solid-phase extraction using a reversed-phase column. The components of the eluate were converted to their methyl esters and extracted in hexane. Separation and quantitative analysis of the pyrethroid metabolites was carried out by capillary gas chromatography and mass selective detection. 2-Phenoxybenzoic acid served as an internal standard. The detection limits lay between 0.3 and 0.5 μg per litre urine. The relative standard deviations of the within-series imprecision were between 1% and 6%. The relative recovery rates ranged between 90% and 98%. Using this method we determined the elimination of pyrethroid metabolites in 24-h urine samples from eight pest controllers after indoor application of permethrin. The detected concentrations ranged from 1 to 70 μg g⁻¹ creatinine.     

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated ¹⁴C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of ¹⁴C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

There is yet to be a reliable prediction of urolithiasis. To facilitate early diagnosis, a simple and rapid high performance liquid chromatography method with electrochemical detection using disposable copper-nanoparticle-plated electrodes (Cuⁿ-SPE) was developed for multiple detection of creatinine and 4 urolithic organic acids. A total of 206 normal and urolithic human and canine urines and urolith samples were collected for direct analysis of creatinine, cystine, uric acid, oxalic acid, and citric acid without sample cleanup and derivatization processes. Urinary organic acids were separated in 11 min and were devoid of ascorbic acid interference. The detection limits (S/N > 3) were at the nanomolar level with linear dynamic ranges spanning 2–3 orders of magnitude. Recoveries in urine ranged from 99.5% for creatinine to 86.5% for citric acid. The analytical variations (RSD) were less than 6.2% in phosphate buffer and 7.7% in urine. Important differences in organic acid levels/profiles between animal species and among normal and urolithic urines/urolith were unveiled and corresponded well (70–90%) with the urolithic risk in a retrospective assessment. The simplicity and reproducibility of this method using disposable Cuⁿ-SPE has made routine urine analysis possible and can be of great clinical and diagnostic potential in the screening of urolithiasis and abnormal states related to excess secretion of organic acids and amino acids in humans and animals.     

Metabolic products of arachidonic acid in rats

The pattern of eicosanoid metabolites appearing in urine and feces following oral administration of radioactive arachidonic acid was investigated using rats deficient in essential fatty acids. About 70–80% of the radioactivity in the urine during the first day after feeding was adsorbed to XAD-2 resin and he represented eicosanoid metabolites, whereas the rest of the radioactivity was mainly ³H₂O. The eicosanoid metabolites were fractioned into different polarity classes using reverse phase Sep-Pak C₁₈ cartridges. Gas chromatographic analysis of the urinary metabolites following their derivatization into methyl ester-methoxime- -butyl-dimethylsilyl ethers revealed that nearly one-half of the metabolites had ECL values less than 22 and represented metabolites more oxidized than commonly described. Only 30% of the metabolites had ECL values between 26 to 32, corresponding to the values for the metabolites that originate from exogenously infused prostaglandins. A large portion of the eicosanoid metabolites was also excreted with the feces. The isotropic patterns from the reverse phase chromatography indicated that many of the fecal metabolites may be similar to those in urine although some metabolites in feces were not present in urine. Based on the specific radioactivity of the administered arachidonic acid, it appeared that at least 6 to 8 mg of eicosanoid metabolites were excreted through urine and feces within 24 hrs following an oral bolus of 60 mg arachidonic acid. The rapid increase and subsequent decrease in eicosanoid metabolite excretion after oral administration of arachidonate indicates that the dietary intake of polyunsaturated fatty acids may have a more rapid effect upon the endogenous production of eicosanoids than is generally recognized.     

The trace analysis of alkyl alkylphosphonic acids in urine using gas chromatography-ion trap negative ion tandem mass spectrometry

A sensitive method has been developed for the trace analysis of alkyl alkylphosphonic acids, metabolites of nerve agents, in urine using a benchtop ion trap mass spectrometer. The acids were isolated from urine by simple solid phase extraction and converted to their pentafluorobenzyl esters. An ion trap mass spectrometer in selected reaction monitoring mode provided limits of detection of 0.1 ng/ml for isopropyl, isobutyl, pinacolyl and cyclohexyl methylphosphonic acids and for ethyl ethylphosphonic acid. The detection limit for ethyl methylphosphonic acid was higher (0.5 ng/ml) due to a lower recovery.     

Determination of the main hydrolysis product of O-ethylS-2-diisopropylaminoethyl methylphosphonothiolate, ethyl methylphosphonic acid, in human serum

For the unequivocal proof of the use of a nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX), a rapid, accurate and sensitive method which allows us to identify its main hydrolysis product ethyl methylphosphonic acid (EMPA) in human serum was explored by GC-MS. GC-MS analysis was performed after solvent extraction with acetonitrile in acidic conditions from the serum sample, which was previously deproteinized by micro-ultrafiltration, and subsequent tert.-butyldimethylsilyl derivatization with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) with 1% tert.-butyldimethylsilyl chloride (t-BDMSC). Linear calibration curves were obtained in the concentration range from 50 to 500 ng/ml for EMPA in the full-scan EI mode and from 5 to 50 ng/ml for EMPA in the SIM EI mode. The relative standard deviation obtained at a sample concentration of 50 ng/ml was 8.4% in the full-scan mode and 7.3% in the SIM mode. Upon applying the full-scan EI and CI mode, 40 ng/ml and 80 ng/ml were the detection limits. Using the SIM-EI mode, in which the ion at m/z 153 was chosen, the limit was 3 ng/ml.     

High-performance liquid chromatographic determination of N-methylformamide and N-methyl-N-(hydroxymethyl)-formamide in human urine

A reliable high-performance liquid chromatographic (HPLC) method which allows the determination in human urine of two important metabolites of N,N-dimethylformamide (DMF), namely N-methylformamide (MMF) and N-methyl-N-(hydroxymethyl)formamide (DMFOH), is reported. A single-step rapid purification of urine was performed on a C₁₈ solid-phase extraction column and the eluate was injected directly on to the HPLC column. HPLC was carried out isocratically on Aminex Ion Exclusion HPX-87H column using 7.5 · 10⁻⁴ M sulphuric acid as the mobile phase with ultraviolet detection at 196 nm. The method is specific, accurate, precise and sufficiently sensitive to be applied to the biological monitoring of MMF and DMFOH in workers exposed to DMF.     

Determination of urinary 18β-glycyrrhetinic acid by gas chromatography and its clinical application in man

A sensitive and quantitative gas chromatographic assay for the determination of 18β-glycyrrhetinic acid (18β-GA), the main metabolite of glycyrrhizin after oral licorice consumption in human urine, has been developed and validated. For the extraction of 18β-GA from urine two Sep-Pak C₁₈ extractions, hydrolysis with Helix pomatia and three liquid–liquid extractions were performed, using 18α-glycyrrhetinic acid (18α-GA) as internal standard. Both 18β-GA and internal standard were converted into their pentafluorobenzyl-ester/trimethylsilyl-ether derivatives and detected by flame ionization detection using a WCOT-fused-silica capillary column. Good quality control data were obtained in precision and accuracy tests. The detection limit of the gas chromatographic method was 10 μg/l with a urine volume of 10 ml. A detection limit of 3 μg/l was obtained by performing GC–MS. The GC method was used to monitor the urinary excretion of 18β-GA after licorice consumption by two healthy volunteers and a patient suspected of licorice abuse. Furthermore, it was shown that this GC assay enables to detect other metabolites related to licorice consumption.     

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 sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Identification of a biomarker for propetamphos and development of a biological monitoring assay

This paper describes the identification of a human metabolite of propetamphos ((E-O-2-isopropylcarbonyl-1-methylvinyl-O-methylethylphosphoramidothioate), formed by the hydrolytic cleavage of the enol-vinyl-phosphate bond, and the development of an analytical method suitable for biological monitoring of propetamphos exposure. The metabolite has been detected in the urine of exposed workers but not in that of control subjects. The analytical method involves azeotropic distillation of the urine with acetonitrile, followed by derivatization with pentafluorobenzyl bromide and analysis using gas chromatography with flame photometric detection.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

In vitro and in vivo metabolism of myristicin in the rat

Myristicin [5-allyl-1-methoxy-2,3-(methylenedioxy)benzene] is a flavoring plant constituent and has been known to produce significant psychopharmacological responses as well as insecticidal activity. From in vitro and in vivo metabolism of myristicin, the two metabolites 5-allyl-1-methoxy-2,3-dihydroxybenzene and 1′-hydroxymyristicin were identified using GC–MS after derivatization of sample matrices with a mixture of BSTFA–TMCS. Those metabolites from in vitro study were also confirmed in urine after an oral administration of myrisitcin to rats, and enzymatic hydrolysis of urine suggested that these metabolites were excreted as conjugated forms as well.     

Simultaneous determination of catecholamines and dobutamine in human plasma and urine by high-performance liquid chromatography with fluorimetric detection

We report a reliable fluorimetric assay for the simultaneous determination of norepinephrine, epinephrine, dopamine and dobutamine in human plasma and urine, based on liquid—liquid extraction and derivatization with the fluorogenic agent 1,2-diphenylethylenediamine prior to chromatography. The method is sensitive (detection limit 0.3–0.8 pg injected) and reproducible (coefficients of variation 1–10%), and shows good accuracy (93–98%). The method should also be used when one only wants to measure the concentrations of the natural catecholamines, in order to avoid interference by metabolites of dobutamine and by the late-eluting dobutamine itself.     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

Determination of 5-S-cysteinyldopa in plasma and urine using a fully automated solid-phase extraction–high-performance liquid chromatographic method for an improvement of specificity and sensitivity of this prognostic marker of malignant melanoma

5-S-Cysteinyldopa (5-SCD) in plasma and urine was determined by means of a newly developed method. This method incorporates optimized conditions for blood collection and storage, as well as a new extraction and separation technique, required for the strong oxidation and light sensitive 5-SCD. The new aspects of the method are the following: immediate centrifugation and freezing of the samples after blood collection, fully automatical solid-phase extraction (SPE) with phenylboronic acid (PBA) cartridges and immediate HPLC injection of the eluate, nearly complete exclusion of light and air–oxygen during extraction, constant sample cooling, use of the more suitable internal standard 5-S- -cysteinyldopa and easy, sensitive and selective HPLC conditions (RP18-column with isocratic separation and electrochemical detection). The method has a linear range from 0.25 to 50 μg l⁻¹ and 25 to 5000 μg l⁻¹ for plasma and urine samples, respectively, a limit of detection of 0.17 μg l⁻¹, intra-assay variabilities from 1.7 to 3.6%, inter-assay variabilities from 4.0 to 18.3% and an average relative recovery of 103.5% for plasma and 105.4% for urine samples. In our study the measured 5-SCD concentrations of patients with melanomas at various stages correlated better with their clinical pictures than described in literature up to date. The results were obtained in comparison to patients with other skin tumors and in comparison to healthy control persons.