4-Methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol as a urinary biomarker for monitoring of metofluthrin, a fluorine-containing pyrethroid, in exposed rats.

CONTEXT: A fluorine-containing pyrethroid metofluthrin is widely used recently in mosquito repellents. OBJECTIVE: The urinary excretion kinetics of its metabolites was evaluated in rats to establish an optimal biomarker for monitoring metofluthrin exposure of the general population. METHODS AND RESULTS: After metofluthrin had been administered intraperitoneally to rats, the urinary excretion kinetics of the major metofluthrin metabolites was evaluated by moment analysis. The urinary excretion amounts of 4-methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol were estimated to be proportional to the absorption amounts over a wide exposure range. CONCLUSION: Urinary 4-methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol was considered to be an optimal biomarker for metofluthrin exposure.

Cyphenothrin Flea and Tick Squeeze-On for Dogs: Evaluation of Potential Health Risks Based on the Results of Observational Biological Monitoring.

An observational biomonitoring study was conducted involving adults and children in households that purchased and applied a cyphenothrin-containing spot-on product for dogs as part of their normal pet care practices. The 3- to 6-yr-old children had greater exposure than the adult applicators in the same house, 3.8 and 0.6 µg/kg body weight, respectively. The mean measured values in children were 13-fold lower than those estimated using the U.S. Environmental Protection Agency (EPA) current standard operating procedures (SOP) for pet products (assuming 5% dermal absorption), although the maximum absorbed dosage of one child on one day was equivalent to the default value derived from the SOPs. With regard to potential human health risks, it can be concluded that despite the inherent conservatism in both the exposure and toxicology data, the margins of exposure (MOE) were consistently greater than 100 for average, 95th percentile, and maximum exposures. More specifically, the results of this study demonstrated that the MOE were consistently greater than 1,000 for mean exposures and exceeded 100 for 95th percentile and maximum measured exposures, which clearly indicates a reasonable certainty of no harm when using the cyphenothrin spot-on products. It is also noteworthy that Sergeant's spot-on products containing cyphenothrin currently sold in the United States have lower weight percentages of active ingredient and lower applied amounts than those used by all but two of the participant households in this study.

Analysis of the metabolic profile of parishin by ultra-performance liquid chromatography/quadrupole-time of flight mass spectrometry.

Parishin is a dominant active ingredient originating from Gastrodia elata Blume, and has good neuroprotective effects against brain disorders. In the present study, the metabolic profile of parishin by in vitro and in vivo experiments was investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC/Q-TOF MS) combined with an automated MS(E) technique. By comparison with reference compounds, accurate mass measurement, the characteristic fragmentation patterns of the parent drug parishin and gastrodin and relevant bio-transformation knowledge, 14 metabolites (seven hydrolyzates and seven derivatives of gastrodin) were detected and identified in rat plasma and urine after intragastric administration of parishin, including processes of hydrolyzation, oxidation, sulfation and glucuronidation. According to the proposed metabolic pathways of parishin, in vitro hydrolytic experiments and metabolic study of gastrodin in rat plasma, it can be inferred that parishin mainly functions as a prodrug and undergoes hydrolysis before being absorbed into the blood. The hydrolyzate, mainly gastrodin, was involved in further metabolism, which was responsible for pharmacological activities of parishin. In conclusion, this work provides valuable information on parishin metabolism using a rapid and reliable UHPLC/Q-TOF MS method, which could be widely used for the metabolic investigation of natural product.

Development of sensitive immunoassays for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative human urinary biomarker for pyrethroid exposure.

Pyrethroids are widely used in agriculture as insecticides. This study describes a sensitive enzyme-linked immunosorbent assay for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Four antisera were elicited against two different immunizing haptens. Antisera were characterized in combination with several coating haptens. The lowest IC50 value (0.5 ng/mL) was obtained with antiserum 1891 and 3-phenoxybenzoic acid-BSA conjugate as the coating antigen. Antiserum 1891 was highly selective for the target compound with an overall cross-reactivity of <0.3% to structurally related compounds. The assay sensitivity was negligibly affected by pH 4-9. A 5-fold improvement in IC50 was observed using a 10-fold concentrated phosphate-fuffered saline as the assay buffer. Compared to assays conducted in normal phosphate-fuffered saline, the maximal absorbance was almost identical. A good correlation (r 2 = 0.99 and 0.97 for urine samples A and B, respectively) was observed between spiked levels and the levels detected by the immunoassay.

Olodaterol and vilanterol detection in sport drug testing.

The possibility of the detection of olodaterol and vilanterol, two novel ß2 -agonists, in human urine for the purpose of sport drug testing was investigated. Compounds of interest were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) employing methods commonly used in the World Anti-Doping Agency (WADA) accredited laboratories. For both substances, the respective parent compound was found to be a suitable target analyte for monitoring therapeutic dose administration.

An NMR study of the metabolic fate of 2-, 3- and 4-fluorobenzyl alcohols in the rat: detection of N-acetylcysteinyl conjugates as minor metabolites in urine.

Following the administration of 2-, 3- and 4-fluorobenzyl alcohols, the major metabolites detected in urine corresponded to the glycine conjugates of the corresponding benzoic acids. Little, or no, unchanged parent compound was detected in the samples. In addition to glycine-conjugated benzoic acids, a small proportion of the urinary metabolites for each of the fluorobenzyl alcohols was found to correspond to N-acetylcysteinyl conjugate. These were probably formed as the result of the production of a reactive sulphate ester during metabolism. The overall urinary recoveries of metabolites for the 2- and 3-fluorobenzyl alcohols were lower than that observed for the corresponding benzoic acids whilst that for 4-fluorobenzyl alcohol was similar.

Metabolism of inhaled ethylbenzene in rats.

Male Wistar rats were exposed to ethylbenzene (0, 300, or 600 ppm for 6 h), and the metabolic fate of the compound was elucidated on the basis of the biotransformation products found in the urine. Fourteen different compounds thought to originate from ethylbenzene were identified. The main metabolites were 1-phenylethanol, mandelic acid, and benzoic acid. The metabolic conversion proceeded mainly through oxidation of the side chain, whereas ring oxidation seemed to be of minor importance. At the exposure level of 600 ppm, only 6% of the amount absorbed was eliminated in the urine during exposure. During the period of 48 h from the onset of exposure, the total urinary elimination was 59%. The corresponding values at 300 ppm were 13 and 83%.

Urinalysis of minor metabolites of ethylbenzene and m-xylene.

Gas chromatographic methods have been developed for the urinalysis of metabolic products of ethylbenzene. "Minor" metabolites were emphasized in this process. The methods were worked out so that simultaneous determination of several compounds could be achieved with one method. On the whole, four assays are described, which together allow 12 different compounds to be measured. Since one of the methods presented can be applied to determine "minor" metabolites of xylene as well, methodological data for these are also reported. The methods described have successfully been used in metabolic studies of ethylbenzene in rat and man.

Simultaneous determination of p-hydroxybenzaldehyde, p-hydroxybenzyl alcohol, 4-(beta-D-glucopyranosyloxy)-benzyl alcohol, and sugars in Gastrodia elata blume measured as their acetylated derivatives by GC-MS.

A method for the simultaneous separation and determination of the active constituents and three sugars in the roots of Gastrodia elata Blume (GE), which is used as a famous Chinese traditional herbal medicine, by gas chromatography-mass spectrometry is established. The samples are acetylated with pyridine-acetic anhydride. The contents of 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol (HA), fructose, glucose, 4-(beta-D-glucopyranosyloxy)-benzyl alcohol (GA), and sucrose in GE are 0.004%, 0.03%, 1.36%, 1.12%, 1.97%, and 4.25%, respectively, and the detection limits are 1.5, 3.0, 11.0, 5.0, 33.0, and 35.0 pg, respectively. The contents of HA and GA in the urine and brain of a mouse are also determined. This method is simple, reliable, and quick for the simultaneous determination of the active constituents and sugars in GE.

Biomarkers for monitoring transfluthrin exposure: urinary excretion kinetics of transfluthrin metabolites in rats.

The urinary excretion kinetics of a fluorine-containing pyrethroid transfluthrin [(2,3,5,6-tetrafluorophenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropane-1-carboxylate], which is widely used recently as mosquito repellents, was examined in rats to search for urinary metabolites suitable as biomarkers for monitoring transfluthrin exposure of the general population. After a single dose of 26, 64, 160 or 400 mg/kg body weight of transfluthrin had been administered intraperitoneally to male Sprague-Dawley rats, their urine was collected periodically for one week. Three major urinary transfluthrin metabolites were measured: 2,3,5,6-tetrafluorobenzyl alcohol, 2,3,5,6-tetrafluorobenzoic acid and 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid. The kinetics was evaluated by moment analysis of the urinary excretion rate of the metabolites versus time curves. The urinary excretion amounts of these three metabolites were estimated to be proportional to the absorption amounts of transfluthrin over a wide exposure range. Urinary 2,3,5,6-tetrafluorobenzoic acid was considered to be an optimal biomarker for monitoring transfluthrin exposure.

Biomarkers for monitoring profluthrin exposure: urinary excretion kinetics of profluthrin metabolites in rats.

Recently, a pyrethroid profluthrin [(2,3,5,6-tetrafluoro-4-methylphenyl)methyl 2,2-dimethyl-3-(prop-1-enyl)cyclopropane-1-carboxylate] is widely used as mothproof repellents in indoors. The urinary excretion kinetics of its metabolites was examined in rats to search for urinary metabolites suitable as biomarkers of profluthrin exposure in the general population. A single dose (26-400 mg/kg body weight) of profluthrin was administered intraperitoneally to the rats, and then their urine was collected periodically. Four major profluthrin metabolites, 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol (CH3-FB-Al), 4-hydroxymethyl-2,3,5,6-tetrafluorobenzyl alcohol, 4-methyl-2,3,5,6-tetrafluorobenzoic acid and 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid (MCA) were determined in the urine samples by gas chromatography/mass spectrometry. The kinetic evaluation for each metabolite was achieved by moment analysis of the urinary excretion rate versus time curve. The urinary excretion amounts of the three metabolites, expect for MCA, were estimated to be proportional to the amounts of absorbed profluthrin over a wide exposure range. Urinary CH3-FB-Al was considered to be an optimal biomarker for monitoring of profluthrin.

Analytical method for urinary metabolites of the fluorine-containing pyrethroids metofluthrin, profluthrin and transfluthrin by gas chromatography/mass spectrometry.

An analytical method was developed for measurement of the major urinary metabolites in rats administered fluorine-containing pyrethroids (metofluthrin, profluthrin and transfluthrin) which are widely used recently as mosquito repellents or mothproof repellents. Eight metabolites, 2,3,5,6-tetrafluorobenzoic acid, 4-methyl-2,3,5,6-tetrafluorobenzoic acid, 2,2-dimethyl-3-(1-propenyl)-cyclopropanecarboxylic acid, 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (carboxylic metabolites), 2,3,5,6-tetrafluorobenzyl alcohol, 4-methyl-2,3,5,6-tetrafluorobenzyl alcohol, 4-methoxymethyl-2,3,5,6-tetrafluorobenzyl alcohol and 4-hydroxymethyl-2,3,5,6-tetrafluorobenzyl alcohol (alcoholic metabolites), were extracted from enzymatic hydrolyzed urine using toluene and then concentrated. After transformation to their tert-butyldimethylsilyl derivatives for carboxylic metabolites or their trimethylsilyl derivatives for alcoholic metabolites, analysis was conducted by gas chromatography/mass spectrometry in the electron impact ionization mode. The calibration curves for each metabolite were linear over the concentration range of 0-20µg/ml in urine, and the quantification limits were between 0.009 and 0.03µg/ml. The relative errors and the relative standard deviations on replicate assays were less than 6% and 5%, respectively, for all concentrations studied. The measurements were accurate and precise. The collected urine samples could be stored for up to 1 month at -20°C in a freezer. The proposed method was applied to the analysis of several urine samples collected from rats treated with these pyrethroids.

Absorption of cumene through the respiratory tract and excretion of dimethylphenylcarbinol in urine.

Experiments on the absorption of cumene and the excretion of dimethylphenylcarbinol were made on 10 healthy volunteers, five men and five women aged between 20 and 35 years. They were exposed to cumene vapours 240, 480, 720 mg/m3 under controlled conditions. It was found that the average retention of cumene vapours was about 50% which tended to diminish at the end of each exposure. Based on these results, an exposure test that allows one to calculate the absorbed cumene dose during eight hours' work with a precision of about +/- 13.5% was achieved.

Benzyl alcohol metabolism and elimination in neonates.

Reports on fatal benzyl alcohol poisoning in premature neonates implied that the toxicity may be due to larger doses per kilogram than for adults. It has been postulated that the load of benzoic acid (metabolite of benzyl alcohol) may exceed the capacity of the immature liver or kidney for detoxification through glycine conjugation to form hippuric acid. To test this hypothesis, 14 term and 9 preterm neonates receiving loading doses of phenobarbital containing benzyl alcohol were studied. Urine and serum benzoic and hippuric acid levels were measured by GC and HPLC methods, respectively. There was greater accumulation of benzoic acid in the serum of preterm compared to the term neonates which was reflected in higher normalized peak levels (2130.6 vs. 237.8 kg/l, p less than 0.001) and larger normalized AUCIV (1,253.2 vs. 483.0 kg.h/l, p less than 0.01). Furthermore, larger percentages of benzyl alcohol doses were found in urine as benzoic acid in preterm babies, while less hippuric acid appeared in their urine than term newborns. These results indicate that hippuric acid formation is deficient in preterm neonates. Although we did not encounter in our patients the specific toxic signs described as part of the benzyl alcohol toxicity syndrome, we cannot directly answer the issue of safety of 'low doses' of benzyl alcohol as found in some medications administered to neonates. This study confirms the immaturity of the benzoic acid detoxification process in premature newborns.

The metabolism of vanillin and isovanillin in the rat.

1. The metabolism of vanillin, isovanillin and the corresponding alcohols and acids in rats was investigated using t.l.c., g.l.c. and combined g.l.c.-mass spectrometry. 2. Oral dosage (100 mg/kg) of the aldehyde resulted in urinary excretion of most metabolites within 24 h, mainly as glucuronide and/or sulphate conjugates although the acids formed were also excreted free and as their glycine conjugates. In 48 h 94% of the dose of vanillin was accounted for as follows (%) : vanillin (7), vanillyl alcohol (19), vanillic acid (47), vanilloylglycine (10), catechol (8), 4-methylcatechol (2), guaiacol (0-5) and 4-methylguaiacol (0-6). Similarly, 89% of the dose of isovanillin was accounted for as follows: isovanillin (19), isovanillyl alcohol (10), isovanillic acid (22), vanillic acid (11), isovanilloylglycine (19), catechol(7) and 4-methylcatechol (1). Protocatechuic acid was also formed from both aldehydes. 3. By means of (a) investigation of biliary metabolites, (b) prevention of biliary excretion, (c) suppression of intestinal bacteria with neomycin sulphate and (d) inhibition of intestinal beta-glucuronidase with saccharo-1,4-lactone, it was found that glucuronides of the aldehydes and their respective alcohol and acid derivatives are excreted in the bile and that the conjugates are metabolized by the intestinal bacteria to toluene derivatives and decarboxylated products.