Analytical determination of specific 4,4'-methylene diphenyl diisocyanate hemoglobin adducts in human blood.

4,4'-Methylene diphenyl diisocyanate (MDI) is one of the most important isocyanates in the industrial production of polyurethane and other MDI-based synthetics. Because of its high reactivity, it is known as a sensitizing agent, caused by protein adducts. Analysis of MDI is routinely done by determination of the nonspecific 4,4'-methylenedianiline as a marker for MDI exposure in urine and blood. Since several publications have reported specific adducts of MDI and albumin or hemoglobin, more information about their existence in humans is necessary. Specific adducts of MDI and hemoglobin were only reported in rats after high-dose MDI inhalation. The aim of this investigation was to detect the hemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood for the first time. We found values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. Because there was no information available about possible amounts of this specific MDI marker, the analytical method focused on optimal sensitivity and selectivity. Using gas chromatography-high-resolution mass spectrometry with negative chemical ionization, we achieved a detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g). The robustness of the method was confirmed by relative standard deviations between 3.0 and 9.8 %. Combined with a linear detection range up to 10 ng ABP-Val-Hyd/g globin (31 pmol/g), the enhanced precision parameter demonstrates that the method described is optimized for screening studies of the human population.

Biological monitoring of pesticide exposures in residents living near agricultural land.

BACKGROUND: There is currently a lack of reliable information on the exposures of residents and bystanders to pesticides in the UK. Previous research has shown that the methods currently used for assessing pesticide exposure for regulatory purposes are appropriate for farm workers 1. However, there were indications that the exposures of bystanders may sometimes be underestimated. The previous study did not collect data for residents. Therefore, this study aims to collect measurements to determine if the current methods and tools are appropriate for assessing pesticide exposure for residents living near agricultural fields. METHODS/DESIGN: The study will recruit owners of farms and orchards (hereafter both will be referred to as farms) that spray their agricultural crops with certain specified pesticides, and which have residential areas in close proximity to these fields. Recruited farms will be asked to provide details of their pesticide usage throughout the spray season. Informed consenting residents (adults (18 years and over) and children (aged 4-12 years)) will be asked to provide urine samples and accompanying activity diaries during the spraying season and in addition for a limited number of weeks before/after the spray season to allow background pesticide metabolite levels to be determined. Selected urine samples will be analysed for the pesticide metabolites of interest. Statistical analysis and mathematical modelling will use the laboratory results, along with the additional data collected from the farmers and residents, to determine systemic exposure levels amongst residents. Surveys will be carried out in selected areas of the United Kingdom over two years (2011 and 2012), covering two spraying seasons and the time between the spraying seasons. DISCUSSION: The described study protocol was implemented for the sample and data collection procedures carried out in 2011. Based on experience to date, no major changes to the protocol are anticipated for the 2012 spray season although the pesticides and regional areas for inclusion in 2012 are still to be confirmed.

Development of an immunochromatographic assay for rapid detection of 1-Aminohydantoin in urine specimens.

A rapid immunochromatographic assay was developed and validated for detection of 1-aminohydantoin (AHD) in urine specimens. Colloidal gold-labeled polyclonal antibody specific to AHD derivative was used as the marker; based on the competitive reactivity theory, the metabolite of nitrofurantoin after derivatization with benzaldehyde would compete with carboxyphenyl AHD derivative-conjugated ovalbumin. The test strip could efficaciously detect the novel analyte with a visual detection limit of 10 ng mL(-1) and high specificity. The reliability of the assay was determined by testing 80 standard samples comparing with enzyme-linked immunosorbent assay. The semi-quantitative detection was accomplished in less than 15 min with low cost, especially for requirements of rapid and simple screening. This is the first publication of an immunochromatographic assay for detection of nitrofuran residues.

Determination of benzimidazole- and bicyclic hydantoin-derived selective androgen receptor antagonists and agonists in human urine using LC-MS/MS.

Selective androgen receptor modulators (SARMs) represent a novel class of drugs with tissue-specific agonistic and antagonistic properties, which are prohibited in sports from January 2008 according to the World Anti-Doping Agency. Preventive approaches to restrict the use of SARMs include early implementation of target analytes into doping control screening assays. Five model SARMs were synthesized, four of which are analogs to prostate-specific androgen receptor antagonists with a 5,6-dichloro-benzimidazole nucleus. The fifth SARM is a muscle-tissue specific agonist with a bicyclic hydantoin structure (BMS-564929). Dissociation pathways after negative electrospray ionization were studied using an LTQ-Orbitrap mass analyzer, and diagnostic product ions and common fragmentation patterns were employed to establish a screening procedure that target the intact SARMs as well as putative metabolic products. Sample preparation based on solid-phase extraction and subsequent LC-MS/MS measurement allowed for detection limits of 1-20 ng/mL, intra- and interday precisions of between 2.4 and 13.2% and between 6.5 and 24.2%, respectively. Recoveries varied from 89 to 106%, and tests for ion suppression or enhancement effects were negative for all analytes. [figure: see text]

Analysis of 3,5-dichloroaniline as a biomarker of vinclozolin and iprodione in human urine using liquid chromatography/triple quadrupole mass spectrometry.

The fungicides vinclozolin and iprodione are widely used in agriculture. These pesticides are dicarboximide fungicides containing the common moiety 3,5-dichloroaniline (3,5-DCA). It has been suggested that low-level exposures to such compounds may be associated with adverse health effects such as endocrine disruption. In this study a method using liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) was developed for the analysis of 3,5-DCA as a biomarker of exposure to these fungicides in human urine. The urine samples were treated by basic hydrolysis to degrade the fungicides, their metabolites and conjugates to 3,5-DCA. The 3,5-DCA was then extracted using toluene and derivatized using pentafluoropropionic anhydride (PFPA). Analysis of the derivative was carried out using selected reaction monitoring (SRM) in the negative ion mode. Quantification of the derivative was performed using [(13)C(6)]-labeled 3,4-DCA as an internal standard with good precision and linearity in the range 0.1-200 ng/mL urine. The limit of detection was determined to be 0.1 ng/mL. The metabolites in urine were found to be stable during storage at -20 degrees C. To validate 3,5-DCA as a biomarker the method was applied in a human experimental exposure to iprodione and vinclozolin. Two healthy volunteers received 200 microg single oral doses of each pesticide followed by urine sampling during 72-120 h post-exposure. Between 78-107% of the dose was recovered as 3,5-DCA in the urine after exposure.

Determination and validation of a simple high-performance liquid chromatographic method for simultaneous assay of iprodione and vinclozolin in human urine.

A method based on solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) was developed for the simultaneous determination of 3-(3,5-diclorophenyl)-5-ethenyl-5-methyl-2,4-oxazolidinedione (vinclozolin) and 3-(3,5-diclorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide (iprodione) in human urine. Urine samples containing vinclozolin and iprodione were collected by solid phase extraction using C(18) cartridges. The chromatographic separation was achieved on a Spherisorb ODS2 (250 mm x 4.6 mm, 5 microm) column with an isocratic mobile phase of acetonitrile-water (60:40, v/v). Detection was UV absorbance at 220 nm. The calibration graphs were linear from 30 to 1000 ng/mL for the two fungicides. Intra- and inter-day R.S.D. did not exceed 2.9%. The quantitation limit was 50 ng/mL for vinclozolin and 30 ng/mL for iprodione, respectively.

Application of 19F-n.m.r. spectroscopy to the identification of dog urinary metabolites of imirestat, a spirohydantoin aldose reductase inhibitor.

1. Urine from a dog dosed orally at 20 mg/kg with 14C-imirestat, a spirohydantoin aldose reductase inhibitor, contained 17.7 and 12.5% of the administered radioactivity at 0-48 and 48-72 h respectively. 2. Radio-h.p.l.c. of the 0-48 h urine revealed a complex mixture of metabolites and a small proportion of parent drug (1.6% of dose). Direct 19F-n.m.r. spectroscopy of this urine showed the fluoride ion, numerous metabolites which were predominantly glucuronide conjugates and, as a minor component, the parent drug. 3. After incubation with beta-glucuronidase the 0-48 h urine gave a 19F-n.m.r. spectrum showing fewer signals. This finding is consistent with aromatic ring hydroxylation followed by glucuronidation being the major metabolite pathways. 4. Deconjugated urine was analysed by proton-coupled 19F-n.m.r. and two-dimensional 19F-19F correlated spectroscopy. Results indicate that major components included three monohydroxy metabolites, a diphenol with both phenolic functions in the same ring, and a phenolic metabolite containing only one fluorine atom. 5. Semi-preparative h.p.l.c. of 0-48 h dog urine gave individual glucuronides isolated as mixtures of C-9 epimers. These fractions were hydrolysed and purified a second time by h.p.l.c. to give aglycones which were analysed by multi-nuclear n.m.r. and g.l.c.-mass spectrometry. The 3- and 4-hydroxy derivatives of imirestat were identified, as was the 2-hydroxy product obtained during or following defluorination. The other major aglycone was postulated to be the 3-fluoro-2-hydroxy metabolite. This represents a novel 'NIH-shift' type pathway for the metabolism of fluorobenzenes.

Metabolic fate of FCE 22716, a new antihypertensive agent, and of its N-oxide (FCE 24220) in the rat.

[3H]-FCE 22716 and [3H]-FCE 24220 were given both orally and intravenously to the rat. Radioactivity was mainly eliminated by the faecal route after oral administration in both cases. After intravenous administration, renal excretion was twice the faecal one in the case of FCE 22716, whereas for FCE 24220 the two routes were equal. In urine FCE 22716 was eliminated almost completely unchanged after both oral and intravenous administration. FCE 24220 was extensively reduced to FCE 22716 after oral administration, whereas after intravenous treatment, this reduction, although important, was not complete.

High-performance liquid chromatographic assay of the aldose reductase inhibitor spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567) in plasma and urine and its pharmacokinetics in humans.

Two selective high-performance liquid chromatographic (HPLC) methods have been developed for the quantitative determination of spiro-(2-fluoro-9H-fluorene-9,4'-imidazolidine)-2',5'-dione (AL01567; 1) in plasma and urine, with an assay sensitivity of 0.25 micrograms/mL for plasma and 0.13 micrograms/mL for urine. The plasma assay procedure involved precipitation of proteins with acetonitrile followed by dilution with water. The diluted supernatant was analyzed on an ODS column eluting with acetonitrile:0.5% phosphoric acid (30:70) adjusted to pH 7.2 with concentrated ammonium hydroxide. The urine assay procedure involved extraction of 1 with 10% n-butanol in hexane, followed by back extraction with 0.05 M sodium hydroxide. The basic extract was neutralized and analyzed on a phenyl column eluting with acetonitrile:10 mM potassium phosphate (30:70; monobasic, pH 5.6). The pharmacokinetics of 1 was investigated in humans following single and multiple oral doses. The elimination half-life from 12 normal subjects following single 100-400-mg oral doses was independent of dose, and the overall mean half-life was 66 +/- 9 h. The overall mean oral clearance (assuming a bioavailability of 100%) was 11 +/- 3 mL/min, and the mean apparent volume of distribution was 59 +/- 13 L. The mean urinary recovery of intact drug during the first 24 h after dosing was 1.2 +/- 0.4% of the administered dose. During once daily 100-mg oral dosing of 1 to five subjects for 21 d, plasma concentrations of 1 reached apparent steady-state by 7 d.(ABSTRACT TRUNCATED AT 250 WORDS)

GC and GC-MS procedures for simultaneous phenotyping with dextromethorphan and mephenytoin.

A genetic deficiency in the metabolism of dextromethorphan and mephenytoin may be revealed by the excretion pattern of dextromethorphan and its metabolite dextrorphan, and mephenytoin, 4-OH-mephenytoin, respectively, after a single dose of the test drugs. Existing methods were modified for determining the compounds in 0.1-0.5 ml urine samples. No prior derivatization of the compounds was necessary before their gaschromatographic or mass-spectrometric analysis by using crosslinked 5% phenylmethyl silicone fused silica columns. Seven healthy volunteers were phenotyped at weekly intervals with either 25 mg dextromethorphan or 100 mg mephenytoin, or both drugs. One subject was a poor metabolizer of mephenytoin, while all subjects were extensive metabolizers of dextromethorphan. Neither a pharmacokinetic nor an analytical interference was observed when the results of the single test were compared with those of the combined test. The results of the mephenytoin test were also tentatively given in form of metabolic ratios. The GC-MS assay was designed for clinical studies so that patients treated with other drugs could be phenotyped.

5-ethyl-5-phenylhydantoin N-glucuronide, the major urinary metabolite of 5-ethyl-5-phenylhydantoin (Nirvanol) in the dog.

A water-soluble metabolite, isolated from the urine of dogs given (S)-5-ethyl-5-phenylhydantoin [(S)-EPH], has been identified as 1-deoxy-1-[(5S)-5-ethyl-5-phenylhydantoin-3-yl] beta-D-glucopyranuronate [(S)-EPH N-glucuronide]. EPH N-glucuronide did not release the aglycone upon acid or beta-glucuronidase treatment, but incubation in alkaline solution (pH 12-13) readily formed 2-ethyl-2-phenylhydantoic acid (EPHA). The EPHA so formed could be quantitatively cyclized to EPH. With the knowledge of the conversion efficiency of EPH N-glucuronide to EPHA, a quantitative GLC assay for the metabolite was developed. EPH N-glucuronide was found to be the major urinary metabolite after administration to dogs of either (R)-, (S)-, or (RS)-EPH.

Gas chromatographic-mass spectrometric studies on the metabolic fate of ethotoin in man.

Unchanged ethotoin and 11 metabolic products of ethotoin were detected in the urine of subjects (2 men and 1 woman) receiving ethotoin. Nine of these products were identified by comparison of their retention times and mass spectra with those of authentic synthetic samples. Hydroxylation of the hydantoin ring at the 5-position produced 5-hydroxyethotoin and 5-hydroxy-5-phenylhydantoin. Aryl hydroxylation resulted in the formation of p-hydroxyethotoin, o-hydroxyethotoin, m-hydroxyethotoin, 3-methoxy-4-hydroxyethotoin, and 3,4-dihydroxyethotoin. Most of these were excreted as the glucuronide conjugate. A dihydrodiol of ethotoin and 3-ethyl-5-hydroxy-5-(4-hydroxyphenyl)hydantoin were isolated along with unchanged ethotoin and 5-phenylhydantoin. 2-Phenylhydantoic acid was also isolated and was shown to have the (R)(-)-configuration.

Identification of mephenytoin metabolites in the dog.

In two dogs p-hydroxymephenytoin, m-hydroxymephenytoin, 5-ethyl-5-phenylhydantoin (nirvanol), and 5-ethyl-5-(p-hydroxyphenyl)hydantoin (p-hydroxynirvanol) were identified by use of synthetic metabolites as standards for comparison.

Characterization of mephenytoin metabolites in human urine by gas chromatography and mass spectrometry.

Metabolites of mephenytoin (5-ethyl-3-methyl-5-phenylhydantoin) were characterized in human urine following chromatography on XAD-2 resin, permethylation, and combined gas chromatography and mass spectrometry. Four glucuronide metabolites previously unidentified in man were characterized as their permethylated derivatives by chemical-ionization and electron-impact mass spectrometry. These metabolites included 5-ethyl-5-(hydroxyphenyl)-3-methylhydantoin O-glucuronide; 5-hydroxyethyl-3-methyl-5-phenyl-hydantoin O-glucuronide; 5-ethyl-5-(hydroxymethoxyphenyl)-3-methylhydantoin O-glucuronide; and a metabolite tentatively identified as 5-ethyl-5-phenylhydantoin N3-glucuronide in which both N-demethylation and glucuronide conjugation of the hydantoin ring have occurred. Mephenytoin, N-demethylmephenytoin, 5-ethyl-5-(hydroxyphenyl)-3-methylhydantoin, and 5-ethyl-5-(hydroxymethoxyphenyl)-3-methylhydantoin were characterized in extracts of enzymatically hydrolyzed urine.

Identification of 3-methyl-2-thiohydantoin, a metabolite of carbimazole, in man.

1. Classical and high-pressure liquid chromatographic separations were devised for the separation and isolation of the metabolite 3-methyl-2-thiohydantoin from the urine of patients receiving carbimazole orally. 2. 3-methyl-2-thiohydantoin was identified by comparing its absorption and mass spectral properties with authentic material. 3. 3-methyl-2-thiohydantoin was also detected in the plasma of patients receiving methimazole intravenously.

Mass fragmentographic quantitation of ethotoin and some of its metabolites in human urine.

A method for the determination of ethotoin and its p-hydroxylated and dealkylated metabolites in urine has been developed. Ethotoin and the metabolites were extracted from acidified urine with ethyl acetate and silylated before injection into a combined gas chromatograph--mass spectrometer. Four partly identified metabolites were recorded, but their exact quantitation was not possible as pure reference substances were not available. The limit of sensitivity was far below the amounts of ethotoin and of its metabolites found in urine from patients treated with therapeutic doses of ethotoin.

Saturable metabolic pathways for ethotoin in man.

1. The urinary excretion pattern of ethotoin and five metabolites were examined in three patients receiving continuous treatment with ethotoin at two dose levels, in order to investigate the mechanism behind the dose-dependent kinetics of this anticonvulsant drug. 2. The results suggest a partial saturation in the dealkylation process at high dose levels in three patients. 3. A rough approximation of the Michaelis-Menten constants for different enzymatic processes was attempted. On the basis of the results obtained, the p-hydroxylation may be a saturable process. 4. The dose-dependent kinetics of ethotoin in man seem to be explicable by the existence of partly saturable enzymatic pathways.