Determination of trace levels of pyrethroid metabolites in human urine by capillary gas chromatography-high resolution mass spectrometry with negative chemical ionization

Summary Applications of high-resolution gas chromatography and high-resolution mass spectrometry (GC-MS) for identification and quantitation of trace amounts of pyrethroid metabolites in human urine samples are demonstrated. The method covers the pyrethroid metabolitescis- andtrans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane carboxylic acid (cis- andtrans-DCCA),cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DBCA), 4-fluoro-3-phenoxybenzoic acid (FPBA), and 3-phenoxybenzoic acid (3-PBA). After acid-induced hydrolysis of urine samples and exhaustive solvent extraction, a carbodiimide-coupled esterification of the free carboxylic acids with hexafluoroisopropanol (HFIP) is applied. Identification of the derivatives formed is achieved by low-resolution electron-impact mass spectrometry (EIMS) using an ion-trap detector. Quantitation was by capillary gas chromatography—high-resolution mass spectrometry using negative chemical ionization (GC-NCIMS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard. The limits of detection forcis- andtrans-DCCA,cis-DBCA, FPBA and 3-PBA were 0.03 μg L⁻¹ or below. The applicability of the presented method was tested on urine samples of persons exposed to low levels of pyrethroids.     

Fast Analysis of Synthetic Pyrethroid Metabolites in Water Samples Using In-Syringe Derivatization Coupled Hollow Fiber Mediated Liquid Phase Microextraction with GC-ECD

A fast and efficient method has been demonstrated for the trace determination of six important metabolites of synthetic pyrethroids including cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis- and trans-Cl₂CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Br₂CA), 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA), 3-phenoxybenzoic acid (3-PBA), and 2-phenoxybenzoic acid (2-PBA) in environmental water samples using hollow fiber (HF)-mediated liquid-phase microextraction (LPME) coupled with in-syringe derivatization (ISD) followed by gas chromatography (GC) with electron capture detector (ECD) analysis. This method utilizes a HF membrane segment impregnated with extraction solvent as the LPME sampling probe, which was connected to a microsyringe pre-filled with derivatizing agents, and it was immersed into sample solution for extraction. After extraction, the extracting solution was subjected to derivatization reaction that was performed inside the syringe barrel followed by GC-ECD analysis. Under optimal conditions, the best extraction efficiency was obtained using sampling probe (2.0 cm hollow fiber) impregnated with 1-octanol immersed into water sample (5.0 mL, adjusted pH below 1.0) and stirring (1,250 rpm) for 10 min at 70 °C and diisopropylcarbodiimide (2 μL) and 1,1,1,3,3,3-hexafluoro-2-propanol (1 μL) were the derivatizing agents used. The detection limits of 3 ng mL^?1 for cis- and trans-Cl₂CA, 2 ng mL^?1 for cis-Br₂CA, 6 ng mL^?1 for 4-F-3-PBA, and 0.6 ng mL^?1 for 3-PBA and 2-PBA. The method showed good linearity (R ² = 0.973?0.998), repeatability from 4.0 to 13 % (n = 5), recovery from 79.2 to 95.7 %, and enrichment factors ranged between 109 and 159 for target analytes spiked in water samples. The proposed method and conventional methods were compared. Results suggested that the proposed HF-LPME-ISD/GC-ECD method was a rapid, simple, inexpensive, and eco-friendly technique for the analysis of metabolites of pyrethroids.     

Immunochemical analysis of 3-phenoxybenzoic acid,a biomarker of forestry worker exposure to pyrethroid insecticides

Pyrethroid insecticides widely used in forestry, agricultural, industrial, and residential applications have potential for human exposure. Short sample preparation time and sensitive, economical high-throughput assays are needed for biomonitoring studies that analyze a large number of samples. An enzyme-linked immunosorbent assay (ELISA) was used for determining 3-phenoxybenzoic acid (3-PBA), a general urinary biomarker of exposure to some pyrethroid insecticides. A mixed-mode solid-phase extraction reduced interferences from acid hydrolyzed urine and gave 110 ± 6% recoveries from spiked samples. The method limit of quantification was 2 μg/L. Urine samples were collected from forestry workers that harvest pine cone seeds where pyrethroid insecticides were applied at ten different orchards. At least four samples for each worker were collected in a 1-week period. The 3-PBA in workers classified as high, low, or no exposure based on job analysis over all sampling days was 6.40 ± 9.60 (n = 200), 5.27 ± 5.39 (n = 52), and 3.56 ± 2.64 ng/mL (n = 34), respectively. Pair-wise comparison of the differences in least squares means of 3-PBA concentrations among groups only showed a significant difference between high and no exposure. Although this difference was not significant when 3-PBA excretion was normalized by creatinine excretion, the general trend was still apparent. No significant differences were observed among days or orchards. This ELISA method using a 96-well plate was performed as a high-throughput tool for analyzing around 300 urine samples measured in triplicate to provide data for workers exposure assessment.     

Development of HPLC-MS/MS method for the simultaneous determination of metabolites of organophosphate pesticides,synthetic pyrethroids,herbicides and DEET in human urine

We developed an isotopic dilution high-performance liquid chromatography (HPLC)/tandem mass spectrometer (MS/MS) method to rapidly and accurately quantify nine metabolites of several classes of pesticide in 1 mL human urine specimens. The analytes covered in the method are two organophosphate (OP) pesticide metabolites: 3,5,6-trichloro-2-pyridinol (TCPy), 2-isopropyl-6-methyl-4-pyrimidinol (IMPY); three synthetic pyrethroid metabolites: 3-phenoxy benzoic acid (3-PBA), 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA) and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-1(1-cyclopropane) carboxylic acid (t-DCCA); three herbicide metabolites: 2,4-dichlorophenoxyacetic acid (DCPAA), 2,4,5-trichlorophenoxyacetic acid (TCPAA) and atrazine mercapturate; and one insect repellent: N,N-diethyl-meta-toluamide (DEET). The analytes are first deconjugated by incubating with acetate/β-glucuronidase buffer at 37°C for 17 h. The deconjugated analytes are extracted and concentrated from the urine matrix using solid-phase extraction cartridges, separated through C18 reversed phase HPLC, and analysed on MS/MS. The MS/MS was operated in positive and negative electrospray ionisation switch mode. Two ions from each analyte and one from each labelled internal standard are monitored for quantification and confirmation. The limit of detections (LODs) for all the analytes are in the low parts-per-trillion (0.05 ng/mL) except TCPy where it was 0.5 ng/mL) with a wide linear range (0.05 up to 40 ng/mL) and provides high accuracy (recoveries: 90–118%) and high precision (coefficient of variation <15%). The method accuracy was also verified by the analysis of proficiency testing urine samples. We analysed 101 urine samples for a recent California study cohort, and detection frequencies ranged from ~100% to 0%: 3-PBA (98%), IMPY (91%), TCPy, (89%), DCPAA (66%), 4-F-3-PBA (11%), TCPAA (0%).     

Gas-chromatography mass-spectrometry determination of phthalic acid in human urine as a biomarker of folpet exposure

Agricultural workers are exposed to folpet, but biomonitoring data are limited. Phthalimide (PI), phthalamic acid (PAA), and phthalic acid (PA) are the ring metabolites of this fungicide according to animal studies, but they have not yet been measured in human urine as metabolites of folpet, only PA as a metabolite of phthalates. The objective of this study was thus to develop a reliable gas chromatography–tandem mass spectrometry (GC–MS) method to quantify the sum of PI, PAA, and PA ring-metabolites of folpet in human urine. Briefly, the method consisted of adding p-methylhippuric acid as an internal standard, performing an acid hydrolysis at 100 °C to convert ring-metabolites into PA, purifying samples by ethyl acetate extraction, and derivatizing with N,O-bis(trimethylsilyl)trifluoro acetamide prior to GC–MS analysis. The method had a detection limit of 60.2 nmol/L (10 ng/mL); it was found to be accurate (mean recovery, 97%), precise (inter- and intra-day percentage relative standard deviations <13%), and with a good linearity (R ² > 0.98). Validation was conducted using unexposed peoples urine spiked at concentrations ranging from 4.0 to 16.1 μmol/L, along with urine samples of volunteers dosed with folpet, and of exposed workers. The method proved to be (1) suitable and accurate to determine the kinetic profile of PA equivalents in the urine of volunteers orally and dermally administered folpet and (2) relevant for the biomonitoring of exposure in workers.     

GC-ECD determination of cypermethrin and its major metabolites in soil, elm bark, and litter

An analytical method has been developed for the simultaneous determination of residues of four pairs of isomers of the pyrethroid insecticide cypermethrin, and their main metabolites, cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropanecarboxylic acid (CCA) and 3-phenoxybenzoic acid (PBA), in elm bark, litter, and soil in the control of elm bark beetles, the vector of the Dutch elm disease. The residues of cypermethrin isomers and their metabolites were extracted with methanol under acidic conditions from elm bark, litter, and soil, cleaned up with liquid-liquid partitioning, and chromatographed by GC-ECD together after the CCA and PBA in the sample had been derivatized with α-bromo-2,3,4,5,6-pentafluorotoluene. The average recoveries of cypermethrin isomer pairs were 82 to 112% with relative standard deviations (RSDs) of 2.3 to 10% at the fortification levels of 2, 10, 100 μg/g in elm bark, 2, 15, 150 μg/g in litter, and 0.2, 2, 20 μg/g in soil. The recoveries of cypermethrin metabolites were 83 to 107% with RSDs from 2 to 12% at the fortification levels of 0.5, 5, and 10 μg/g in elm bark, and litter, and 0.1, 1, and 10 μg/g in soil. Received: 8 April 1997 / Revised: 21 July 1997 / Accepted: 23 July 1997     

An immunoassay for a urinary metabolite as a biomarker of human exposure to the pyrethroid insecticide permethrin

Permethrin is the most popular synthetic pyrethroid insecticide used in agriculture and public health. For the assessment of human exposure to permethrin, a competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the glycine conjugate of a major metabolite, cis-/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was developed based on a polyclonal antibody. An assay based on an antibody with a high sensitivity was optimized and characterized. The IC₅₀ value and the detection range for trans-DCCA–glycine, in the assay buffer were 1.2 and 0.2−7.0 μg/L, respectively. The antibody recognized trans-DCCA–glycine and the mixture of cis-/trans-DCCA–glycine with an isomer range from 30:70 to 50:50 nearly equally. Little or no cross-reactivity to permethrin and its other free metabolites or glycine conjugates was measured. The integration of the ELISA and solid-phase extraction which was used to reduce the matrix effect from human urine samples provided for analysis of total cis-/trans-DCCA–glycine at low parts per billion levels in the samples. The limit of quantitation of the target analyte was 1.0 μg/L in urine with a limit of detection of 0.1 μg/L in buffer. This assay might be a useful tool for monitoring human exposure to permethrin.     

Determination of pyrethroid metabolites in human urine by capillary gas chromatography-mass spectrometry

Summary An analytical method for the simultaneous determination of the pyrethroid metabolites cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis 3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylic acid, 3-phenoxybenzoic acid and 4-fluoro-3-phenoxybenzoic acid in human urine samples is described. The urine is subjected to acid-induced hydrolysis followed by exhaustive solvent extraction, covering both conjugated and free acids, followed by a common derivatisation step yielding the corresponding methyl esters. Quantitation was by diastereomeric, capillary gas chromatography-mass spectrometry. It appears that 4-fluoro-3-phenoxybenzoic acid is a characteristic urinary marker for cyfluthrin exposure. The limits of determination are 0.5–1.0 g L^–1 urine depending on the metabolites concerned. The applicability of the method was tested on urine samples from pest control operators exposed occupationally to cypermethrin and cyfluthrin.     

Ibuprofen interference in the determination of 3-phenoxybenzoic acid in urine

Pyrethroid insecticides are widely used in agriculture and private households. Analysis of urine for pyrethroid metabolites is one way to detect human exposure to these insecticides and is carried out regularly as part of the Occupational and Environmental Medicine Monitoring Program recommended by the Deutsche Forschungsgemeinschaft (DFG). Samples are analyzed using GC-MS (selected ion monitoring) following acid hydrolysis, solid phase extraction, esterification with methanol/sulfuric acid, and liquid-liquid extraction. The metabolite, 3-phenoxybenzoic acid (3-PBA), can be derived from several pyrethroids and is, therefore, a useful diagnostic analyte; however, the presence of the over-the-counter drug, ibuprofen ((R,S)-2-(4-isobutylphenyl)propionic acid), interferes with this determination, even after the ingestion of only one 200-mg tablet. The interfering analyte is carboxy-ibuprofen which is not removed by the cleanup step. Experimental work shows that it takes two days for most of the ibuprofen to clear the body before 3-PBA can reliably be determined in urine.     

Analysis of the flame retardant metabolites bis(1,3-dichloro-2-propyl) phosphate (BDCPP) and diphenyl phosphate (DPP) in urine using liquid chromatography–tandem mass spectrometry

Organophosphate triesters tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate are widely used flame retardants (FRs) present in many products common to human environments, yet understanding of human exposure and health effects of these compounds is limited. Monitoring urinary metabolites as biomarkers of exposure can be a valuable aid for improving this understanding; however, no previously published method exists for the analysis of the primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), in human urine. Here, we present a method to extract the metabolites BDCPP and diphenyl phosphate (DPP) in human urine using mixed-mode anion exchange solid phase extraction and mass-labeled internal standards with analysis by atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry. The method detection limit was 8 pg mL⁻¹ urine for BDCPP and 204 pg mL⁻¹ for DPP. Recoveries of analytes spiked into urine ranged from 82 ± 10% to 91 ± 4% for BDCPP and from 72 ± 12% to 76 ± 8% for DPP. Analysis of a small number of urine samples (n = 9) randomly collected from non-occupationally exposed adults revealed the presence of both BDCPP and DPP in all samples. Non-normalized urinary concentrations ranged from 46–1,662 pg BDCPP mL⁻¹ to 287–7,443 pg DPP mL⁻¹, with geometric means of 147 pg BDCPP mL⁻¹ and 1,074 pg DPP mL⁻¹. Levels of DPP were higher than those of BDCPP in 89% of samples. The presented method is simple and sufficiently sensitive to detect these FR metabolites in humans and may be applied to future studies to increase our understanding of exposure to and potential health effects from FRs.     

Development and validation of a method for determination of residues of 15 pyrethroids and two metabolites of dithiocarbamates in foods by ultra-performance liquid chromatography-tandem mass spectrometry

This paper reports a novel approach for the detection, confirmation, and quantification of 15 selected pyrethroid pesticides, including pyrethins, and two metabolites of dithiocarbamates in foods by ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS–MS). The proposed method makes use of a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure that combines isolation of the pesticides and sample cleanup in a single step. Analysis of pyrethroids and dithiocarbamate metabolites was performed by UPLC–MS–MS operated with electrospray and atmospheric pressure chemical ionization, respectively. Two specific precursor–product ion transitions were acquired per target compound in multiple reaction monitoring (MRM) mode. Such acquisition achieved the minimum number of identification points according to European Commission (EC) document no. SANCO/10684/2009, thus fulfilling the EC point system requirement for identification of contaminants in samples. The method was validated with a variety of food samples. Calibration curves were linear and covered from 1 to 800 μg kg⁻¹ in the sample for all target compounds. Average recoveries, measured at mass fractions of 10 and 100 μg kg⁻¹ for pyrethroids and 5 and 50 μg kg⁻¹ for dithiocarbamate metabolites, were in the range of 70–120% for all target compounds with relative standard deviations below 20%. Method limits of quantification (MLOQ) were 10 μg kg⁻¹ and 5 μg kg⁻¹ for pyrethroids and dithiocarbamate metabolites, respectively. The method has been successfully applied to the analysis of 600 food samples in the course of the first Hong Kong total diet study with pyrethroids and metabolites of dithiocarbamates being the pesticides determined.     

Simultaneous quantitation of seven pyrethroid metabolites in human urine by capillary gas chromatography–mass spectrometry

Pyrethroid insecticides are applied in the residential environment, as well as in agricultural crops, for insect control purpose. We developed and validated an accurate, sensitive, and reproducible analytical method to simultaneously detect seven pyrethroid metabolites, namely, 3‐(2,2‐dichlorovinyl)‐2,2‐dimethyl‐(1‐cyclopropane) carboxylic acid, 3‐(2,2‐dibromovinyl)‐2,2‐dimethyl‐(1‐cyclopropane) carboxylic acid, 3‐phenoxybenzoic acid, 4‐fluoro‐3‐phenoxybenzoic acid, 2‐methyl‐3‐phenylbenzoic acid, 4‐chloro‐α‐isoproply benzeneacetic acid, and 3‐(2‐chloro‐3,3,3‐trifluoroprop‐1‐enyl)‐2,2‐dimethylcyclopropanecarboxylic acid, in human urine. This method employs deconjugation with enzyme, SPE using Agilent C18 cartridges on a RapidTrace SPE workstation, derivatization using hexafluoro isopropanol and N,N′‐diisopropylcarbodiimide, and compounds separation and identification on GC–MS. The detection limits of seven metabolites were 0.02–0.08 ng/mL in urine. The recoveries of seven metabolites were 81–104%, 85–99%, and 83–99% in urine specimens fortified at 0.1, 0.4, and 3.2 ng/mL concentrations, respectively. The overall coefficient of variation was 4.3–10.8% in two quality control specimens which were repeatedly measured during a period of 2 months. This method was applied to urine samples collected from children living in Boston, MA. The median concentrations of six detected pyrethroid metabolites ranged from 0.06 to 0.86 ng/mL in urine.     

Identification of free and conjugated metabolites of mesocarb in human urine by LC-MS/MS

The objective of the present study was to investigate mesocarb metabolism in humans. Samples obtained after administration of mesocarb to healthy volunteers were studied. The samples were extracted at alkaline pH using ethyl acetate and salting-out effect to recover metabolites excreted free and conjugated with sulfate. A complementary procedure was applied to recover conjugates with glucuronic acid or with sulfate consisting of the extraction of the urines with XAD-2 columns previously conditioned with methanol and deionized water; the columns were then washed with water and finally eluted with methanol. In both cases, the dried extracts were reconstituted and analyzed by ultra-performance liquid chromatography–tandem mass spectrometry. Chromatographic separation was carried out using a C₁₈ column (100 mm × 2.1 mm i.d., 1.7 μm particle size) and a mobile phase consisting of water and acetonitrile with 0.01% formic acid with gradient elution. The chromatographic system was coupled to a mass spectrometer with an electrospray ionization source working in positive mode. Metabolic experiments were performed in multiple-reaction monitoring mode by monitoring one transition for each potential mesocarb metabolite. Mesocarb and 19 metabolites were identified in human urine, including mono-, di-, and trihydroxylated metabolites excreted free as well as conjugated with sulfate or glucuronic acid. All metabolites were detected up to 48 h after administration. The structures of most metabolites were proposed based on data from reference standards available and molecular mass and product ion mass spectra of the peaks detected. The direct detection of mesocarb metabolites conjugated with sulfate and glucuronic acid without previous hydrolysis has been described for the first time. Finally, a screening method to detect the administration of mesocarb in routine antidoping control analyses was proposed and validated based on the detection of the main mesocarb metabolites in human urine (p-hydroxymesocarb and p-hydroxymesocarb sulfate). After analysis of several blank urines, the method demonstrated to be specific. Extraction recoveries of 100.3 ± 0.8 and 105.9 ± 10.8 (n = 4), and limits of detection of 0.5 and 0.1 ng mL⁻¹ were obtained for p-hydroxymesocarb sulfate and p-hydroxymesocarb, respectively. The intra- and inter-assay precisions were estimated at two concentration levels, 50 and 250 ng mL⁻¹, and relative standard deviations were lower than 15% in all cases (n = 4).     

A solid phase extraction method for the screening and determination of pyrethroid metabolites and organochlorine pesticides in human urine.

A screening method has been developed for the determination of 23 organochlorine pesticides (OCPs) and 3-pyrethroid metabolities [cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid and 3-phenoxybenzoic acid] from human urine. OCPs were directly detected in urine samples while pyrethroid metabolites required acid-induced hydrolysis to convert their conjugates into free acids; all compounds were then cleaned-up/preconcentrated using solid phase extraction. Determination and quantitation was achieved by gas chromatography with a mass spectrometer detector operating in selected ion monitoring mode. Limits of detection varied between 0.1 and 0.3 ng/mL with linear ranges from 0.3 to 700 ng/mL; the precision of the method was high (4.3-7.2%). Recoveries of all analytes from urine samples fortified at levels of 30 ng/mL for each OCP and 15 ng/mL for each pyrethroid metabolite ranged from 88 to 101% (captan gave the lowest recovery). The results obtained from the analysis of real urine samples show the suitability of the proposed method for monitoring people exposed to organochlorine and pyrethroid pesticides.     

Effects of sample pretreatment and storage conditions in the determination of pyrethroids in water samples by solid-phase microextraction and gas chromatography–mass spectrometry

The aqueous instability of pyrethroids and other compounds usually found in commercial pesticide formulations has been demonstrated in this work. Several types of sample treatment have been studied to avoid analyte losses during sample manipulation and storage. Analysis was performed by SPME–GC–MS. Addition of sodium thiosulfate to tap water prevented pyrethroid degradation as a result of oxidation by free chlorine. The amount added was optimized to minimize the effect of the salt on the analytical results. Analysis of samples that had been stored at 4 °C for several days revealed loss of some of the pyrethroids in the first period of storage. The effect of freezing the samples was studied and it was confirmed that samples could be stabilized for at least one week by freezing. Finally, addition of a miscible organic solvent, for example acetone, led to improvement of the analytical precision. The quality of the SPME–GC–MS method was studied. Linearity (R > 0.993), repeatability (RSD < 15%), and sensitivity (detection limits between 0.9 and 35 pg mL⁻¹) were good. When the procedure was applied to real samples including run off and waste water some of the target compounds were identified and quantified.      

Validated method for the determination of ethylglucuronide and ethylsulfate in human urine

Detection of the alcohol metabolites ethylglucuronide (EtG) and ethylsulfate (EtS) has become routine in many forensic laboratories over the last few years. Most previously published methods using liquid chromatography coupled with electrospray tandem mass spectrometry require a post-chromatographic addition of solvent and/or extensive sample preparation prior to analysis. The aim of the study was to develop a simplified method. To 20 μL urine, internal standard containing EtG-d5 and EtS-d ₅ was added and the mixture was treated with elution buffer internal standard. EtG and EtS were separated using a Shimadzu Prominence high performance liquid chromatography (HPLC) system with a C18 separation column (Restek Ultra Aqueous C18, 4.6 × 150 mm, 5 μm), using isocratic elution with a mobile phase consisting of 10 mM ammonium acetate buffer pH 7 (total run time, 6 min). The compounds were detected using an Applied Biosystems API 5000 liquid chromatography tandem mass spectrometry system (atmospheric pressure chemical ionization, multiple-reaction monitoring mode). The method was fully validated according to international guidelines. The assay was found to be selective for the compounds of interest. It was linear from 0.1 to 10 mg/L for all analytes (R ² > 0.99). Matrix effects studies showed the presence of a slight but consistent ion enhancement (n = 10 different urine samples) at low concentrations and no effects at higher concentrations. Accuracy data were between 0.75% and 8.1% bias for EtG and between −5.0% and −11.3% bias for EtS. Precision data were between 4.3% and 6.9% relative standard deviations (RSD) for EtG and between 6.0% and 7.5% RSD for EtS. No instability was observed after repeated freezing and thawing. This fast, reliable, and accurate method enables the detection and quantification of alcohol metabolites in urine. The method is easier to use and more sensitive than previously published methods.     

Evaluation of a novel ELISA for serotonin: urinary serotonin as a potential biomarker for depression

Depression is a common disorder with physical and psychological manifestations often associated with low serotonin. Since noninvasive diagnostic tools for depression are sparse, we evaluated the clinical utility of a novel ELISA for the measurement of serotonin in urine from depressed subjects and from subjects under antidepressant therapy. We developed a competitive ELISA for direct measurement of serotonin in derivatized urine samples. Assay performance was evaluated and applied to clinical samples. The analytical range of the assay was from 6.7 to 425 μg serotonin/g creatinine (Cr). The limit of quantification was 4.7 μg/g Cr. The average recovery for spiked urine samples was 104.4%. Average intra-assay variation was 4.4%, and inter-assay variation was <20%. The serotonin analysis was very specific. No significant interferences were observed for 44 structurally and nonstructurally related urinary substances. Very good correlation was observed between urinary serotonin levels measured by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS; ELISA = 1.16 × LC-MS/MS − 53.8; r = 0.965; mean % bias = 11%; n = 18). Serotonin was stable in acidified urine for 30 days at room temperature and at −20 °C. The established reference range for serotonin was 54–366 μg/g Cr (n = 64). Serotonin levels detected in depressed patients (87.53 ± 4.89 μg/g Cr; n = 60) were significantly lower (p < 0.001) than in nondepressed subjects (153.38 ± 7.99 μg/g Cr). Urinary excretion of serotonin in depressed individuals significantly increased after antidepressant treatment by 5-hydroxy-tryptophane and/or selective serotonin re-uptake inhibitor (p < 0.01). The present ELISA provides a convenient and robust method for monitoring urinary serotonin. It is suitable to monitor serotonin imbalances and may be particularly helpful in evaluating antidepressant therapies.     

Iridium(I)-Catalyzed Asymmetric Intermolecular Hydroarylation of Norbornene with Benzamide

 Starting from the dinuclear chloro-bridged Ir(I) complexes [IrCl(PP)]₂ (PP = (R)-(6,6′-dimethylbiphenyl-2,2′-diyl)-1,1′-bis-(diphenylphosphine), (R)-(6,6′-dimethoxy-biphenyl-2,2′-diyl)-1,1′-bis-(diphenylphosphine), and (R)-1-((S)-2-(diphenylphosphino-ferrocenyl))-ethyldicyclohexylphosphine), a new class of cyclopentadienyl Ir(I) complexes containing a chiral bisphosphine ([IrCp(PP)]) was prepared and characterized. These new complexes are suited precatalysts for the direct hydroarylation of norbornene with benzamide. 2-(exo-Norbornyl)-benzamide is formed with an enantiomeric excess of up to 94% by the use of 1 mol% iridium, albeit in low yield.     

Development of a monoclonal antibody-based, congener-specific and solvent-tolerable direct enzyme-linked immunosorbent assay for the detection of 2,2',4,4'-tetrabromodiphenyl ether in environmental samples

A sensitive direct enzyme-linked immunosorbent assay (ELISA) for the specific detection of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) in environmental samples was developed. A hapten mimicking BDE-47 was synthesized by introducing a butyric acid spacer into 5-hydroxy-BDE-47 and coupled to keyhole limpet hemocyanin to form an immunogen for the production of monoclonal antibodies (Mabs) against BDE-47. The most sensitive direct ELISA was formatted with a Mab, designated as 4F2, in combination with 5-(2,4-dibromophenoxy)pentanoic acid peroxidase as a tracer. The inhibition half-maximum concentrations and limit of detection of BDE-47 in phosphate buffered saline with 25% DMSO were 1.4 ± 0.05 and 0.1 ng mL⁻¹, respectively. Cross-reactivity values of the ELISA with the tested BDE congeners and metabolites were ≤5.8%. This assay was used to determine BDE-47 in soil, sediment and house dust samples after ultrasonic extraction, simple cleanup and concentration steps. The average recoveries, repeatabilities (intraday extractions and analyses), and intra-laboratory reproducibilities (interday extractions and analyses) were in a range of 92–126%, 8–19% and 9–25%, respectively. Applied to 44 real samples, the results of this assay displayed a statistically significant correlation with those of a gas chromatography–mass spectrometry method (R ² = 0.79-0.85), indicating this ELISA is a suitable tool for environmental analyses of BDE-47.     

Iridium(I)-Catalyzed Asymmetric Intermolecular Hydroarylation of Norbornene with Benzamide

Summary.  Starting from the dinuclear chloro-bridged Ir(I) complexes [IrCl(PP)]₂ (PP = (R)-(6,6′-dimethylbiphenyl-2,2′-diyl)-1,1′-bis-(diphenylphosphine), (R)-(6,6′-dimethoxy-biphenyl-2,2′-diyl)-1,1′-bis-(diphenylphosphine), and (R)-1-((S)-2-(diphenylphosphino-ferrocenyl))-ethyldicyclohexylphosphine), a new class of cyclopentadienyl Ir(I) complexes containing a chiral bisphosphine ([IrCp(PP)]) was prepared and characterized. These new complexes are suited precatalysts for the direct hydroarylation of norbornene with benzamide. 2-(exo-Norbornyl)-benzamide is formed with an enantiomeric excess of up to 94% by the use of 1 mol% iridium, albeit in low yield. Received July 10, 2000. Accepted August 27, 2000