Demographic and dietary risk factors in relation to urinary metabolites of organophosphate flame retardants in toddlers.

Organophosphate flame retardants (OPFRs), including Tris (1,3-dichloro-isopropyl) phosphate (TDCPP), triphenyl phosphate (TPP), and isopropylated triphenyl phosphate (ITP), are increasingly used in consumer products because of the recent phase out of polybrominated diphenyl ether (PBDE) flame retardants. OPFRs have been widely detected in adults and have been linked to reproductive and endocrine changes in adult males. Carcinogenicity and damage to immunologic, neurologic and developmental systems have been observed in human cell lines. Young children are especially vulnerable to OPFR exposure, but little is known about exposure levels or exposure risk factors in this population. We examined parent-reported demographic and dietary survey data in relation to OPFR urinary metabolite concentrations in 15- to 18-month old toddlers (n = 41). OPFR metabolites were detected in 100% of subjects. The metabolite of TPP, diphenyl phosphate (DPP) was detected most commonly (100%), with TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), detected in 85-95% of samples, and ITP metabolite, monoisopropylphenyl phenyl phosphate (ip-DPP), detected in 81% of samples (n = 21). Toddlers of mothers earning <$10,000 annually had geometric mean DPP concentrations 66% higher (p = 0.05) than toddlers of mothers earning >$10,000/year (7.8 ng/mL, 95% CI 5.03, 12.11 and 4.69 ng/mL, 95% CI 3.65-6.04, respectively). While no dietary factors were significantly associated with OPFR metabolite concentrations, results suggested meat and fish consumption may be associated with higher DPP and BDCPP levels while increased dairy and fresh food consumption may be associated with lower DPP, BDCPP, and ip-DPP levels. Research with larger sample sizes and more detailed dietary data is required to confirm these preliminary findings.

A physiologically based toxicokinetic model of inhalation exposure to xylenes in Caucasian men.

Widespread exposure to the volatile aromatic hydrocarbons, ortho-, meta-, and para-xylene occurs in many industries including the manufacture of plastics, pharmaceuticals, and synthetic fibers. This paper describes the development of a physiologically based toxicokinetic model using biomonitoring data to quantify the kinetics of ortho-, meta-, and para-xylenes. Serial blood concentrations of deuterium-labeled xylene isomers were obtained over 4 days after 37 controlled, 2h inhalation exposures to different concentrations of the isomers. Peak toxicant concentrations in blood occurred in all subjects at the termination of exposure. Systemic clearance averaged 116 L/h+/-34 L/h, 117 L/h+/-23 L/h, and 129 L/h+/-33 L/h for ortho-, para-, and meta-xylene, respectively. The half-life of each toxicant in the terminal phase (>90 h post-exposure) was fit by the model, yielding values of 30.3+/-10.2 h for para-xylene, 33.0+/-11.7 h for meta-xylene and 38.5+/-18.2 h for ortho-xylene. Significant isomeric differences were found (p<0.05) for toxicant half-life, clearance and extrahepatic metabolism. Inter-individual variability seen in this study suggests that airborne concentration guidelines may not protect all workers. A Biological Exposure Index is preferred for this purpose since it is integrative and reflective of inter-individual kinetic variability.

Evaluation of media and derivatization chemistry for six aldehydes in a passive sampler.

We evaluated the GMD passive sampler for its suitability to measure six aldehydes over a 7-d period in population exposure studies. The six target aldehydes were formaldehyde, acetaldehyde, acrolein, crotonaldehyde, glyoxal, and methylglyoxal. The GMD sampler contains a silica gel-impregnated cellulose pad coated with 2,4-dinitrophenylhydrazine (DNPH) hydrochloride. This agent reacts with formaldehyde to form a hydrazone that is quantified with a high-performance liquid chromatograph. The GMD sampler was tested for background contamination and aldehyde recoveries after 0, 1, and 7 d of storage. Results indicated that the GMD monitor, as currently manufactured, is suitable for shorter-term sampling (up to 24 h) of formaldehyde and acetaldehyde. It is however not acceptable for sampling of acetaldehyde, acrolein, crotonaldehyde, glyoxal, and methylglyoxal over a 7-d exposure period due to the chemical reactions on the silica gel-impregnated cellulose pad. Glyoxal- and methylglyoxal-DNPH derivatives formed on the cellulose and Teflon-coated glass fiber pads that had been prepared with glycerol under acidic and oxidative conditions. Acrolein- and crotonaldehyde-DNPH derivatives diminish through the reverse reaction of the DNPH derivatives to form free aldehydes under acidic conditions. We showed that the unknown reaction products of acrolein and crotonaldehyde derivatives were not pyrazolines but probably resulted from E/Zisomerization. These conversion reactions are favored in acidic conditions present in either the derivatization solution or the collection medium. The most consistent recovery was obtained on glass fiber pads. In particular, recoveries of crotonaldehyde- and acrolein-DNPH derivatives were increased through the use of a pH 4 buffered derivatization solution. These chemical instability problems were overcome by using a pH 4 buffer (citric acid/sodium citrate) and an alternative hygroscopic agent (1,3-butanediol) in the DNPH derivatization solution. Results with DNPH derivatives from these spiking experiments were further confirmed with gas-phase spiking experiments. We determined the optimal acidity, buffer solution, and concentrations of the buffer solution and 1,3-butanediol for the DNPH derivatization solution. This new formulation of the DNPH derivatization solution can be used for collection of the six target aldehydes over a 7-d sampling period.

Measurement of urinary methoxyphenols and their use for biological monitoring of wood smoke exposure.

A urinary assay for methoxyphenols was developed for the biological monitoring of wood smoke exposure. Methoxyphenols in 10-ml samples of urine were extracted after acid hydrolysis using XAD in a solid-phase extraction cartridge. The methoxyphenols were eluted with ethyl acetate and then analyzed by gas chromatography/mass spectrometry. Specific chemicals quantified were guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-propylguaiacol, syringol, 4-methylsyringol, 4-ethylsyringol, vanillin, eugenol, and syringaldehyde. Recoveries ranged from 60 to 90%, with coefficients of variation of < or =20%. Background levels of the compounds were measured in 21 nonsmoking adults. Guaiacol, 4-methylguaiacol, eugenol, and vanillin were detected in all subjects. An experimental feeding of a commercial wood smoke flavoring demonstrated that methoxyphenols were rapidly and efficiently eliminated in urine. Preliminary field studies demonstrated that urinary excretion rates of some methoxyphenols increased after inhalation exposure to wood smoke.

A comparison of 1H8- and 2H8-toluene toxicokinetics in men.

1. To examine the bioequivalence of an isotope-labelled tracer to study toxicant disposition, we conducted 33 controlled human exposures to a mixture of 50 ppm 1H8-toluene and 50 ppm 2H8-toluene for 2 h, and measured concentrations in blood and breath, and metabolite levels in urine for 100 h post-exposure. 2. A physiologically based kinetic (PBK) model found that compared with 1H8-toluene, 2H8-toluene had a 6.4+/-13% (mean+/-SD) lower AUC, a 6.5+/-13% higher systemic clearance (1.46+/-0.27 versus 1.38+/-0.25 l/h-kg), a 17+/-22% larger terminal volume of distribution (66.4+/-14 versus 57.2+/-10 l/kg) and a 9.7+/-26% longer terminal half-life (38+/-12 versus 34+/-10 h) (p < 0.05 for all comparisons). 3. The higher 2H8-toluene clearance may have been due to an increased rate of ring oxidation, consistent with the 17% higher observed fraction of 2H5- versus 1H5-cresol metabolites in urine. 4. The larger terminal volume and half-lives for 2H8-toluene suggested a higher adipose tissue/blood partition coefficient. 5. Observed isotope differences were small compared with interindividual differences in 1H8-toluene kinetics from previous studies. 6. The PBK model allowed us to ascribe observed isotope differences in solvent toxicokinetics to underlying physiologic mechanisms.

Individual prior information in a physiological model of 2H8-toluene kinetics: an empirical Bayes estimation strategy.

Physiologically-based toxicokinetic (PBTK) models are widely used to quantify whole-body kinetics of various substances. However, since they attempt to reproduce anatomical structures and physiological events, they have a high number of parameters. Their identification from kinetic data alone is often impossible, and other information about the parameters is needed to render the model identifiable. The most commonly used approach consists of independently measuring, or taking fom literature sources, some of the parameters, fixing them in the kinetic model, and then performing model identification on a reduced number of less certain parameters. This results in a substantial reduction of the degrees of freedom of the model. In this study, we show that this method results in final estimates of the free parameters whose precision is overestimated. We then compared this approach with an empirical Bayes approach, which takes into account not only the mean value, but also the error associated with the independently determined parameters. Blood and breath 2H8-toluene washout curves, obtained in 17 subjects, were analyzed with a previously presented PBTK model suitable for person-specific dosimetry. Model parameters with the greatest effect on predicted levels were alveolar ventilation rate QPC, fat tissue fraction VFC, blood-air partition coefficient Kb, fraction of cardiac output to fat Qa/co and rate of extrahepatic metabolism Vmax-p. Differences in the measured and Bayesian-fitted values of QPC, VFC and Kb were significant (p < 0.05), and the precision of the fitted values Vmax-p and Qa/co went from 11 +/- 5% to 75 +/- 170% (NS) and from 8 +/- 2% to 9 +/- 2% (p < 0.05) respectively. The empirical Bayes approach did not result in less reliable parameter estimates: rather, it pointed out that the precision of parameter estimates can be overly optimistic when other parameters in the model, either directly measured or taken from literature sources, are treated as known without error. In conclusion, an empirical Bayes approach to parameter estimation resulted in a better model fit, different final parameter estimates, and more realistic parameter precisions.

Biological monitoring of controlled toluene exposure.

OBJECTIVES: Widespread exposure to toluene occurs in the printing, painting, automotive, shoemaking, and speaker-manufacturing industries. The relationship between air concentrations and the absorbed dose is confounded by dermal exposure, personal protective devices, movement throughout the workplace, and interindividual differences in toluene uptake and elimination. METHODS: To determine the best biological indicator of exposure we examined the blood and alveolar breath concentrations of toluene as well as the urinary excretion rates of hippuric acid and of o-, m-, and p-cresols from 33 controlled human inhalation exposures to 50 ppm for 2 h. RESULTS: Among the metabolites, o-cresol was least influenced by background contributions, whereas the p-cresol and hippuric acid rates were obscured by endogenous and dietary sources. Toluene levels in alveolar breath proved to be the most accurate and noninvasive indicator of the absorbed dose. A physiologic model described blood and breath data using four measured anthropometric parameters and the fit values of extrahepatic metabolism and adipose-tissue blood flow. CONCLUSIONS: After breathing rate and extrahepatic metabolism had been set to conservative (protective) values (the 97.5th and 2.5th percentiles, respectively) the model predicted that pre-final-shift breath levels of < or =10 micromol/m3 and post-final-shift levels of < or =150 micromol/m3 corresponded to average workplace exposure levels of < or =50 ppm toluene. Alternately, we used the distributions and covariances of the measured and fit model parameters to yield conservative pre-final-shift levels of < or =7.3 micromol/m3 and post-final-shift breath levels of < or =120 micromol/m3 that were reflective of workplace exposure levels of < or =50 ppm toluene.

Methods to reduce background interferences in electron-capture gas chromatographic analysis of valproic acid and its unsaturated metabolites after derivatization with pentafluorobenzyl bromide.

Analysis of the branched, medium-chain fatty acid anticonvulsant, valproic acid, and its unsaturated metabolites by gas chromatography with electron-capture detection suffered from background interference caused by the derivatizing reagent pentafluorobenzyl bromide. Background was reduced by keeping the derivatization anhydrous, using an inert solvent, minimizing the amount of pentafluorobenzyl bromide, using hypernucleophilic bases and displacing the derivatization solvent with isooctane. However, these strategies proved difficult to reproduce. Post-derivatization clean-up with HPLC was much more reliable and provided sufficient sensitivity for the analysis of extracts of plasma and brain homogenate. The assay was validated for plasma and brain samples from humans, rats and mice.

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

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

Interindividual differences in 2H8-toluene toxicokinetics assessed by semiempirical physiologically based model.

Recent applications of physiologically band toxicokinetic (PBTK) models have used animal to human scaling, the hypothetical average man, and Monte Carlo techniques to estimate human exposure to toxicants. Our study built a PBTK model suitable for person-specific dosimetry. Individual measurements of age, ventilation rate, blood/air partition coefficient, body weight, and adipose tissue fraction were made on 26 male subjects exposed to 50 ppm 2H8-toluene and 50 ppm toluene for 2 hr at rest, with collection of venous blood samples for 120 hr postexposure. Fitted lung metabolism was a novel feature of the PBTK model, used to explain a systemic clearance of 2H8-toluene well in excess of hepatic blood flow. A 10-fold interindividual range in venous concentrations was found. Subject-specific modeling explained 91% of the observed data variability, compared to 53% using literature values for model parameters. Body weight, adipose tissue fraction, and blood/air partition coefficient were correlated with terminal half-life, steady-state volume of distribution, and terminal volume of distribution. Lung metabolism was correlated with systemic clearance and terminal half-life. Interindividual differences in lung metabolism resulted in divergent predicted fractions of 2H8-toluene in the body at 2 and 100 hr. An increased adipose fraction led to lower blood concentrations up to 8 hr postexposure, and simulations showed that at 98 hr, adipose tissue contained 97-99% of 2H8-toluene in the body. Use of subject-specific model parameters greatly improved model fit and demonstrated interindividual differences in toxicokinetics.

Partition coefficients between human blood or adipose tissue and air for aromatic solvents.

OBJECTIVES: The partitioning of lipophilic toxicants into blood and into adipose tissue plays an important role in the physiological distribution and toxicology of these substances. The partition coefficients between blood and air and adipose tissue and air were determined for widely used aromatic solvents in an in vitro test system using human tissue samples. METHODS: Samples of whole venous blood (N = 35) were drawn from 10 subjects. In addition, samples of perirenal and epididymal adipose tissue were obtained from F344 rats, along with subcutaneous, omental, or inguinal adipose tissue from 43 patients who had undergone surgery. Portions of each tissue were injected into vials for equilibration with atmospheres containing deuterated and nondeuterated organic solvents. Gas chromatographic headspace analysis was then used to determine the partition coefficients between blood and air and adipose tissue and air. RESULTS: The mean partition coefficients between human blood and air or adipose tissue and air were 334 (SE 11) (adipose tissue) for benzene; 1764 (SE 49) (adipose tissue) for ethylbenzene; 3184 (SE 84) (adipose tissue) for styrene; 18.3 (SE 0.24) (blood) and 962 (SE 32) (adipose tissue) for toluene; 35.2 (SE 0.45) (blood) and 2460 (SE 63) (adipose tissue) for O-xylene; 31.9 (SE 0.45) (blood) and 1919 (SE 53) (adipose tissue) for m-xylene; and 39.0 (SE 0.70) (blood) and 2019 (SE 102) for p-xylene. Regression analyses revealed coefficients of determination of 0.88 (human) and 0.98 (rat) between blood and air and log tissue and air. A value of 0.98 was found for partition coefficients between rat and human adipose tissue. CONCLUSIONS: The partition coefficients between blood and air and adipose tissue and air were strongly correlated. The partitioning of aromatic solvents into rat adipose tissue is predictive of partitioning into human adipose tissue.

Distribution of unsaturated metabolites of valproate in human and rat brain--pharmacologic relevance?

The concentrations of valproate (VPA) and six of its pharmacologically active, unsaturated metabolites (E-delta 2-VPA, Z-delta 3-VPA, E-delta 3-VPA, E,E-delta 2,3'-VPA, delta 4-VPA, and E-delta 2,4-VPA) were measured in serum and cortical brain samples from 24 patients undergoing epilepsy surgery. Collectively, the six metabolites were present at concentrations < 13% of VPA brain concentrations. Because the six unsaturated metabolites were present at such low brain concentrations, we concluded that these metabolites probably did not contribute significantly to the anticonvulsant effect of VPA. Results from a parallel pharmacodynamic study in rats in which VPA was administered three times daily for 8 weeks supported this conclusion. Only three unsaturated metabolites (E-delta 2-VPA, delta 3-VPA, E,E-delta 2,3'-VPA) were detected in rat brain. No correlation was observed between the time course of anticonvulsant effect [as measured by the timed intravenous pentylenetetrazol (PTZ) test] and the time course of VPA or metabolite concentrations in rat brain. Despite the structural similarity of VPA and its metabolites, striking differences were observed in their serum protein binding and blood-brain distribution properties. In the human brain, VPA and delta 4-VPA exhibited brain-to-free serum concentration ratios that were less than unity. In contrast, compounds with the double bond at the 2- or 3-position had brain:free concentration ratios that were much higher than unity. The structure-distribution relationship observed with VPA and its unsaturated metabolites suggested that these branched-chain fatty acids differ in their asymmetric transport across the blood-brain barrier (BBB).

Inter-individual variability in blood/air partitioning of volatile organic compounds and correlation with blood chemistry.

In vitro blood/air partition coefficients (KB/A) for acetone, 1,1,1-trichloroethane, toluene, and styrene were measured in blood samples from 73 human subjects and correlated with blood chemistry parameters (hematocrit, total cholesterol, serum triglycerides, serum albumin, total plasma proteins, Na+, K+, Cl-, and HCO3-). Statistically significant inter-individual variation existed in KB/A between some subjects. Substitution of group or generic in vitro KB/A values for values determined in some individuals could introduce errors of up to 50%. However, most subjects could be well represented by group averages (mean +/- SD; acetone, 301 +/- 22; 1,1,1-trichloroethane, 6.0 +/- 0.8; toluene, 19 +/- 3; styrene, 62 +/- 10). The KB/A values for acetone, 1,1,1-trichloroethane and toluene were normally distributed. The data for styrene appeared to deviate from a normal distribution and may have been bimodal. The KB/A values for the two structurally related compounds, toluene and styrene, were strongly correlated within individuals, while the KB/A values for compounds with less structural similarity, such as acetone and styrene, were poorly correlated. At most, 15% of the variation in KB/A among individuals could be explained by variation in the measured blood chemistry parameters. When the entire sample group was considered, blood chemistry parameters were not significantly correlated with KB/A for any compound. The KB/A of 1,1,1-trichloroethane was significantly correlated with the concentration of cholesterol and triglycerides in females. Sex was a significant grouping variable for the correlation of albumin concentration with the KB/A of styrene. Age was not a significant correlation variable. Blood chemistry parameters which previously have been correlated with KB/A in small sample groups do not appear to be significantly correlated in our larger sample group.

Blood/air partition coefficient determinations: automation and improvement of the equilibrium partitioning in closed systems method.

The equilibrium partitioning in closed systems method has been frequently used for determining partition coefficients between liquid and gas phases. We developed several improvements in this method: preparation of test atmospheres with a dynamic solvent vapor generator, automation of gas phase analysis, and estimation of the precision of the partition coefficients using a statistical procedure that did not require pairing of test and reference measurements. Blood/air partition coefficients measured in a blood-bank blood sample for acetone, 1,1,1-trichloroethane, toluene, and styrene were: 309 +/- 2, 5.6 +/- 0.1, 16.2 +/- 0.5 and 49 +/- 2 (geometric mean +/- estimated SE, respectively). Water/air partition coefficients were: 361 +/- 7, 2.9 +/- 0.3, 4.6 +/- 0.5, and 6.9 +/- 0.9 for acetone, 1,1,1-trichloroethane, toluene, and styrene.

Evaluation of stable isotope-labeled probes in the study of solvent pharmacokinetics in human subjects.

The relationship between biomarkers of exposure (such as concentrations of toxicants in blood or breath, or metabolites in urine) and toxicant dose for individuals is influenced by many person- and episode-specific factors which contribute to overall variability in biomarker level for a given dose. This variability results in imprecise biological marker-based estimates of dose for individuals. We hypothesize that pharmacokinetic data from stable-isotope (deuterated) analogs can be used with a pharmacokinetic model to account for individual-related sources of variation, leading to more precise methods of dose estimation for individuals. To establish the degree of similarity in the pharmacokinetics of unlabeled (d0-) and fully deuterated (D8-) toluene, 21 men (ages 20-45) inhaled an equal molar mixture for 2h. Washout kinetics for both compounds were followed for 4 d in alveolar air and blood. Both compounds exhibited three-phase elimination kinetics in both fluids. The third phase was not always definable for d0-toluene because of concurrent uncontrolled environmental exposures. Considering data from only the first two phases, concentrations of d0- and d8-toluene in alveolar air and blood were well correlated for all subjects, even though pharmacokinetic parameters varied among individuals by 5-9 folds. Further experiments are needed to discern whether correlations between d0- and d8-toluene for the third phase are influenced by an isotope effect; present data support use of d8-toluene as a suitable probe for d0-kinetics.

Peripheral markers of neurochemical function among workers exposed to styrene.

A cross sectional study of biological markers of neurochemical function in peripheral blood cells, and self reported nervous system symptoms, was conducted among 60 workers exposed to styrene in three reinforced plastics plants and 18 reference workers not exposed to styrene or other solvents. Concentrations of styrene in the air at the plants ranged from less than 1 to 160 ppm. Biomarkers of neurochemical function measured were: sigma receptor binding in lymphocytes, monoamine oxidase type B (MAO-B) activity in platelets, and serotonin uptake by platelets. Blood styrene concentration was used as the exposure index to take account of the use of protective equipment and dermal uptake. Four blood styrene exposure groups were defined as: non-exposed (reference) and exposed to less than 0.05, 0.05-0.19, and greater than or equal to 0.20 micrograms/ml. The prevalences of headache, dizziness, light headedness, fatigue, irritability, memory loss, and feeling "drunk" at work increased with increasing blood styrene concentration. No effect on sigma receptor binding was seen. A slight positive correlation was found for uptake of serotonin, which has been used as an exposure related effect indicator in previous studies of workers exposed to solvents. The MAO-B activity decreased with increasing blood styrene concentration; the mean (SE) MAO-B values for the four groups were 34.2 (3.0), 28.1 (5.3), 20.1 (4.8), and 16.9 (7.7) pmol/10(7) cells/min. The MAO-B activity also correlated negatively with the number of reported nervous system symptoms, whereas no associations were seen between prevalence of symptoms and either serotonin uptake or sigma receptor binding. The findings for MAO-B activity are consistent with previously reported experimental data, and suggest that MAO-B may be a useful marker of styrene neurotoxicity.

Low and variable presence of valproic acid in human brain.

We studied the distribution of valproic acid (VPA) between brain (gray matter) and serum in 13 patients receiving chronic VPA therapy who underwent cortical resections for intractable seizures. Valproate concentration in cerebral cortex was remarkably low compared with either total or unbound valproate concentration in serum. The respective brain-to-serum partition ratios based on total and free drug in serum were 0.111 +/- 0.051 and 0.544 +/- 0.175. In comparison with other commonly used antiepileptic drugs, valproate has the distinction of exhibiting the lowest brain-to-blood partitioning. Moreover, the brain-to-serum concentration ratio varied over a four-fold range between patients. Some of this variability was related to variation in serum protein binding, as indicated by a modest correlation between the partition ratio and serum free fraction (r = +0.687). However, the brain-to-unbound concentration ratio still showed a three-fold variation. The variability in distribution of VPA between brain and blood is probably one of the underlying factors for the lack of a clearly definable therapeutic range of serum VPA concentration in epileptic patient populations.

Quantification of volatile solvents in blood by static headspace analysis.

A static headspace method for determination of volatile solvents in blood was developed. The solvents determined were 1,1,1-trichloroethane, toluene, xylene (o-, m- and p-), ethylbenzene, styrene, alpha-methylstyrene and 4-methylstyrene at concentrations ranging from 0.01 to 1 mug/ml. Internal standard calibration was used. Parameters affecting sensitivity and precision were determined and optimized.

Capillary gas chromatographic method for mandelic and phenylglyoxylic acids in urine.

In support of an occupational investigation of styrene exposure, a capillary gas chromatographic method was developed for the quantitation of the styrene metabolites mandelic and phenylglyoxylic acids. The method was based on that of Guillemin and Bauer, in which phenylglyoxylic acid was converted to mandelic acid by reduction before instrumental analysis. The earlier method had to be modified for use with capillary columns; the resulting method was sensitive, selective and reproducible. The detection limit was approximately 0.001 mg/ml urine. Approximately less than 5% relative precision was achieved in the range of 0.05-2 mg/ml urine. Mandelic acid was resolved from other components of urine and from by-products of derivatization.

3-Hydroxylation of salicylamide in mice.

Salicylamide is an important model compound for use in investigations concerning drug disposition. In this study the metabolic fate of salicylamide at high doses was evaluated in male mice using HPLC methodology. The concentrations of salicylamide and its metabolites were determined in urine and in blood at various times after the administration of 2 or 4 mmol kg-1 salicylamide. Salicylamide, gentisamide, and their glucuronide and sulfate conjugates were detected. 2,3-Dihydroxybenzamide, the 3-hydroxy metabolite of salicylamide, as well as its glucuronide and sulfate conjugates, were identified and quantitated for the first time by HPLC. 2,3-Dihydroxybenzamide had previously been detected only as a minor metabolite of salicylamide by paper chromatography. However, in the present study, 18% of the salicylamide metabolites appearing in urine after either dosage of salicylamide were 3-hydroxylation products. When a previously published HPLC method for salicylamide analysis was used, 2,3-dihydroxybenzamide glucuronide coeluted with salicylamide glucuronide. The possible formation of 3-hydroxy metabolites must be evaluated in any study of drug metabolism using salicylamide as a model compound.