Non-parametric estimation of low-concentration benzene metabolism.

Two apparently contradictory findings in the literature on low-dose human metabolism of benzene are as follows. First, metabolism is approximately linear at low concentrations, e.g., below 10 ppm. This is consistent with decades of quantitative modeling of benzene pharmacokinetics and dose-dependent metabolism. Second, measured benzene exposure and metabolite concentrations for occupationally exposed benzene workers in Tianjin, China show that dose-specific metabolism (DSM) ratios of metabolite concentrations per ppm of benzene in air decrease steadily with benzene concentration, with the steepest decreases below 3 ppm. This has been interpreted as indicating that metabolism at low concentrations of benzene is highly nonlinear. We reexamine the data using non-parametric methods. Our main conclusion is that both findings are correct; they are not contradictory. Low-concentration metabolism can be linear, with metabolite concentrations proportional to benzene concentrations in air, and yet DSM ratios can still decrease with benzene concentrations. This is because a ratio of random variables can be negatively correlated with its own denominator even if the mean of the numerator is proportional to the denominator. Interpreting DSM ratios that decrease with air benzene concentrations as evidence of nonlinear metabolism is therefore unwarranted when plots of metabolite concentrations against benzene ppm in air show approximately straight-line relationships between them, as in the Tianjin data. Thus, an apparent contradiction that has fueled heated discussions in the recent literature can be resolved by recognizing that highly nonlinear, decreasing DSM ratios are consistent with linear metabolism.

Evidence for non-linear metabolism at low benzene exposures? A reanalysis of data.

The presence of a high-affinity metabolic pathway for low level benzene exposures of less than one part per million (ppm) has been proposed although a pathway has not been identified. The variation of metabolite molar fractions with increasing air benzene concentrations was suggested as evidence of significantly more efficient benzene metabolism at concentrations <0.1 ppm The evidence for this pathway is predicated on a rich data set from a study of Chinese shoe workers exposed to a wide range of benzene concentrations (not just "low level"). In this work we undertake a further independent re-analysis of this data with a focus on the evidence for an increase in the rate of metabolism of benzene exposures of less than 1 ppm. The analysis dataset consisted of measurements of benzene and toluene from personal air samplers, and measurements of unmetabolised benzene and toluene and five metabolites (phenol hydroquinone, catechol, trans, trans-muconic acid and s-phenylmercapturic acid) from post-shift urine samples for 213 workers with an occupational exposure to benzene (and toluene) and 139 controls. Measurements from control subjects were used to estimate metabolite concentrations resulting from non-occupational sources, including environmental sources of benzene. Data from occupationally exposed subjects were used to estimate metabolite concentrations as a function of benzene exposure. Correction for background (environmental exposure) sources of metabolites was achieved through a comparison of geometric means in occupationally exposed and control populations. The molar fractions of the five metabolites as a function of benzene exposure were computed. A supra-linear relationship between metabolite concentrations and benzene exposure was observed over the range 0.1-10 ppm benzene, however over the range benzene exposures of between 0.1 and 1 ppm only a modest departure from linearity was observed. The molar fractions estimated in this work were near constant over the range 0.1-10 ppm. No evidence of high affinity metabolism at these low level exposures was observed. Our reanalysis brings in to question the appropriateness of the dataset for commenting on low dose exposures and the use of a purely statistical approach to the analysis.

Sensitive determination of positional isomers of benzenediols in human urine by boronate affinity capillary electrophoresis with chemiluminescence detection.

A boronate ACE coupled with chemiluminescence (CL) detection was developed for sensitive determination of three isomeric benzenediols, which was based on the principle of an inhibited effect of borate complexation on the CL reaction between luminol and potassium hexacyanoferrate (K3 Fe(CN)6 ) in alkaline solution. The effects of some important factors on CE separation and CL intensity were systemically investigated. Baseline separation of isomeric benzenediols including o-benzenediol, m-benzenediol, and p-benzenediol was achieved by using a mobile phase of 40 mmol/L glycine-NaOH buffer at pH 9.4 containing 0.8 mmol/L luminol and 0.4 mol/L 4-iodophenylboronic acid. The calibration curves of the analytes by plotting the peak height against corresponding concentration were linear over the range of 4.5 × 10(-8) ∼ 4.5 × 10(-5) mol/L for p-benzenediol, 6.8 × 10(-8) ∼ 2.7 × 10(-5) mol/L for m-benzenediol, and 9.0 × 10(-8) ∼ 4.5 × 10(-5) mol/L for o-benzenediol. The corresponding detection limits for p-, m-, and o-benzenediols were 2.8 × 10(-8) mol/L (68 amol), 3.2 × 10(-8) mol/L (108.4 amol), and 3.7 × 10(-8) mol/L (125.8 amol; S/N = 3), respectively. The proposed method has been successfully applied to the analysis of trace benzenediols in spiked human urine sample and the recoveries were >97.2%. Our primary result demonstrated the proposed CE-CL method has great potential for biomarker determination in clinical diagnosis.

Analysis of hydroquinone and catechol in peripheral blood of benzene-exposed workers.

We have developed a gas chromatography-mass spectrometry method for analysis of benzene (BZ) metabolites in human urine and blood. Here we describe peripheral blood concentrations of hydroquinone (HQ(1)) and catechol (CAT(2)) in total, protein-bound, and unbound (free) forms obtained from BZ-exposed factory workers and controls. Total and unbound metabolites were directly measured in independent experiments, while bound forms were calculated as [total]-[unbound]. In this subset of a larger study, breathing zone benzene, toluene, and xylene were measured for the duration of a workshift, and end-shift blood samples taken from 143 subjects and controls. Potential lifestyle and environmental influences were assessed by questionnaire and bioassay, and single nucleotide polymorphisms in xenobiotic metabolizing enzymes NQO1, MPO, CYP2E1, and GSTT1 were also analyzed for potential contribution to differences in blood metabolite concentration. Total CAT, bound CAT, total HQ, and bound HQ correlated well with benzene exposure, while unbound CAT and HQ displayed no correlation. Nearly all of the metabolites found in blood were bound to protein (CAT 96-99+%, HQ 78-92+%), and when the ratio of bound to unbound metabolites were compared in subsets of exposed workers, the increase in blood metabolite concentration was nearly all due to an increase in the protein-bound molecule. These findings suggest that a threshold for conjugation does not exist within the exposure spectrum studied (0.01-78.8 mg/m(3)). This method demonstrates the feasibility of analyzing benzene metabolites in human blood, and should allow for further investigation of the health effects of benzene and its metabolites.

Human benzene metabolism following occupational and environmental exposures.

We previously reported evidence that humans metabolize benzene via two enzymes, including a hitherto unrecognized high-affinity enzyme that was responsible for an estimated 73% of total urinary metabolites [sum of phenol (PH), hydroquinone (HQ), catechol (CA), E,E-muconic acid (MA), and S-phenylmercapturic acid (SPMA)] in nonsmoking females exposed to benzene at sub-saturating (ppb) air concentrations. Here, we used the same Michaelis-Menten-like kinetic models to individually analyze urinary levels of PH, HQ, CA and MA from 263 nonsmoking Chinese women (179 benzene-exposed workers and 84 control workers) with estimated benzene air concentrations ranging from less than 0.001-299 ppm. One model depicted benzene metabolism as a single enzymatic process (1-enzyme model) and the other as two enzymatic processes which competed for access to benzene (2-enzyme model). We evaluated model fits based upon the difference in values of Akaike's Information Criterion (DeltaAIC), and we gauged the weights of evidence favoring the two models based upon the associated Akaike weights and Evidence Ratios. For each metabolite, the 2-enzyme model provided a better fit than the 1-enzyme model with DeltaAIC values decreasing in the order 9.511 for MA, 7.379 for PH, 1.417 for CA, and 0.193 for HQ. The corresponding weights of evidence favoring the 2-enzyme model (Evidence Ratios) were: 116.2:1 for MA, 40.0:1 for PH, 2.0:1 for CA and 1.1:1 for HQ. These results indicate that our earlier findings from models of total metabolites were driven largely by MA, representing the ring-opening pathway, and by PH, representing the ring-hydroxylation pathway. The predicted percentage of benzene metabolized by the putative high-affinity enzyme at an air concentration of 0.001 ppm was 88% based upon urinary MA and was 80% based upon urinary PH. As benzene concentrations increased, the respective percentages of benzene metabolized to MA and PH by the high-affinity enzyme decreased successively to 66 and 77% at 0.1 ppm, 20 and 58% at 1 ppm, and 2.7 and 17% at 10 ppm. This indicates that the putative high-affinity enzyme was active primarily below 1 ppm and favored the ring-opening pathway.

Urinary propofol metabolites in early life after single intravenous bolus.

BACKGROUND: /st> Propofol clearance is lower in neonates than in adults and displays extensive interindividual variability, in part explained by postmenstrual age (PMA) and postnatal age (PNA). Since propofol is almost exclusively cleared metabolically, urinary propofol metabolites were determined in early life and compared with similar observations reported in adults. METHODS: /st> Twenty-four hours urine collections were sampled after a single i.v. bolus of propofol (3 mg kg(-1)) in neonates undergoing procedural sedation. Clinical characteristics (PMA, PNA, weight, and cardiopathy) were recorded. Urine metabolites [propofol glucuronide (PG), 1- and 4-quinol glucuronide (QG)] were quantified using high-pressure liquid chromatography. Urine recovery (% administered dose) and the contribution of PG and QG to urinary elimination were calculated. Data were reported by median and range, analysed by Mann-Whitney U or Spearman's rank. RESULTS: /st> Eleven neonates (median PNA 11 days, PMA 38 weeks) were included. Median propofol metabolite recovery was 64% (range 34-98%). PG contributed 34% (range 8-67%) and QG 65% (range 33-92%). There was no significant correlation between either PMA, PNA, or cardiopathy and propofol metabolites. Compared with adults, the contribution of PG (34% vs 77%) was lower and the contribution of QG (65% vs 22%) was higher in neonates. CONCLUSIONS: /st> Propofol metabolism in neonates differs from adults, reflecting the age-dependent limited glucuronidation capacity. Hydroxylation to quinol metabolites already contributes to propofol metabolism. These differences likely explain the PMA- and PNA-dependent reduced propofol clearance in neonates.

Recovery and long-term renal excretion of propofol, its glucuronide, and two di-isopropylquinol glucuronides after propofol infusion during surgery.

BACKGROUND: The metabolism of the short-acting anaesthetic agent propofol has been described over the first 24 h. However, the long-term disposition of propofol and its metabolites is unclear. We describe the pharmacokinetics (renal excretion rates and renal clearance) of propofol and its metabolites over 60 h. METHODS: Ten patients undergoing lung surgery were included in the study. They received anaesthesia with continuous i.v. propofol at an average rate of 10 mg min(-1). During surgery and 60 h thereafter, we sampled blood and urine. Propofol and its metabolites were measured using gradient high performance liquid chromatography (HPLC). RESULTS: In nine patients, propofol and its glucuronides were found in the plasma over the first 15 h. In the urine, however, even after 60 h, propofol and its quinol glucuronides were still detectable. One patient had a markedly different pharmacokinetic profile, showing a limited renal excretion or absorption of 12% of the dose. CONCLUSIONS: After an infusion of propofol, patients excrete propofol and its metabolites in the urine over a period in excess of 60 h. We hypothesize that (re)absorption of propofol and its metabolites by the kidney is a major process in elimination and that the reabsorbed compounds are gradually conjugated in the kidney and excreted in the urine. One patient showed a different pharmacokinetic profile for which we currently have no explanation.

Using urinary biomarkers to elucidate dose-related patterns of human benzene metabolism.

Although the toxicity of benzene has been linked to its metabolism, the dose-related production of metabolites is not well understood in humans, particularly at low levels of exposure. We investigated unmetabolized benzene in urine (UBz) and all major urinary metabolites [phenol (PH), E,E-muconic acid (MA), hydroquinone (HQ) and catechol (CA)] as well as the minor metabolite, S-phenylmercapturic acid (SPMA), in 250 benzene-exposed workers and 139 control workers in Tianjin, China. Median levels of benzene exposure were approximately 1.2 p.p.m. for exposed workers (interquartile range: 0.53-3.34 p.p.m.) and 0.004 p.p.m. for control workers (interquartile range: 0.002-0.007 p.p.m.). (Exposures of control workers to benzene were predicted from levels of benzene in their urine.) Metabolite production was investigated among groups of 30 workers aggregated by their benzene exposures. We found that the urine concentration of each metabolite was consistently elevated when the group's median benzene exposure was at or above the following air concentrations: 0.2 p.p.m. for MA and SPMA, 0.5 p.p.m. for PH and HQ, and 2 p.p.m. for CA. Dose-related production of the four major metabolites and total metabolites (micromol/l/p.p.m. benzene) declined between 2.5 and 26-fold as group median benzene exposures increased between 0.027 and 15.4 p.p.m. Reductions in metabolite production were most pronounced for CA and PH<1 p.p.m., indicating that metabolism favored production of the toxic metabolites, HQ and MA, at low exposures.

Determination of dihydroxynaphthalenes in human urine by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method was developed for measuring 1,2-dihydroxynaphthalene (1,2-DHN) and 1,4-dihydroxynaphthalene (1,4-DHN) in urine. The method involves enzymatic digestion of urinary conjugates to release the DHNs which were then analyzed as trimethylsilyl derivatives by GC-MS. For 1,2-DHN and 1,4-DHN, respectively, the assay limits of detection were 0.21 and 0.15 microg/l, the assay limits of quantitation were 0.69 and 0.44 microg/l, and the coefficients of variation were 14.7 and 10.9%. This method was successfully applied to determine urinary levels of 1,2-DHN and 1,4-DHN in coke workers (14 top workers and 13 side-bottom workers) and 21 matching control workers from the steel industry of northern China. The geometric mean (GM) levels of 1,2-DHN were approximately 100 and 30 times higher than those of 1,4-DHN in exposed and control subjects, respectively. The GM levels 1,2-DHN and 1,4-DHN were significantly higher for coke workers (1,2-DHN: top workers--552 microg/l, side-bottom workers--260 microg/l; 1,4-DHN: top workers--3.42 microg/l, side-bottom workers--3.56 microg/l) than for controls (1,2-DHN: 38.8 microg/l; 1,4-DHN: 1.21 microg/l) (por=0.623; p<0.0001). Also, levels of 1,2-DHN were significantly correlated with those of serum albumin adducts of l,2-naphthoquinone (rs=0.492, p=0.0004). These results indicate that 1,2- and 1,4-DHN are good biomarkers for assessment of naphthalene exposure in coke workers. Since the DHNs are precursors of the naphthoquinones, which have been implicated as toxic products of naphthalene metabolism, measurements of urinary DHNs may have toxicological significance.

Metabolism and disposition of hydroquinone in Fischer 344 rats after oral or dermal administration.

Studies were conducted to determine the absorption, tissue distribution, excretion, and metabolism of 14C-hydroquinone (HQ) in male and female rats following single oral, repeated oral, or 24-h dermal administration. The concentration of parent compound in blood was also determined following a single 50-mg/kg gavage administration. Absorption into the blood was rapid after oral dosing; the maximum concentration of parent compound was attained within 20 min after dosing, and the maximum concentration of total 14C was attained within 30 min. Parent compound represented 1% of total 14C in blood, indicative of extensive first-pass metabolism. Excretion was primarily via the urine within the first 8h of gavage. Typically, 87-94% of the 14C was excreted in urine. Dermal application of 14C-HQ (20 microCi) as a 5.4% aqueous solution resulted in near background levels of 14C in blood; the maximum mean blood concentration was 0.65 microg HQ equivalents/g in females and not quantifiable in males. The majority (61-71%) of the 14C was recovered from the skin surface by washing at 24 h. HQ was extensively metabolized following oral dosing with typically <3% of the dose excreted as parent compound. The major urinary metabolites of HQ were glucuronide and O-sulfate conjugates, which represented 45-53% and 19-33%, respectively, of an oral dose. A <5% metabolite was identified as a mercapturic acid conjugate of HQ.

Use of stable isotopically labeled benzene to evaluate environmental exposures.

The use of stable, isotopically labeled compounds in controlled exposure experiments at environmentally relevant levels allows for the distinguishing of urinary metabolites associated with known exposure from background levels generally present in the urine. Exposures of volunteers to (13)C-benzene for 2 h at 40+/-10 p.p.b. were conducted after obtaining informed consent, and urinary phenol, catechol, hydroquinone and trans,trans- muconic acid were measured. Each isotopically labeled urinary metabolite was determined in the presence of significantly higher concentrations of the unlabeled metabolite. Following exposure, free and acid hydrolyzed phenol, acid hydrolyzed catechol and hydroquinone, and free trans,trans-muconic acid were determined by GC/MS. The percentage of trans,trans-muconic acid excreted was higher than reported following exposure at occupational levels. The use of isotopically labeled compounds has the potential to investigate the metabolism of common environmental contaminants for validation of toxicokinetic models and improve risk extrapolation from high concentration occupational exposures and animal studies to environmentally relevant pollutant levels.

Bacterial deconjugation of arbutin by Escherichia coli.

UNLABELLED: Cystinol akut containing arbutin was developed as an antiseptic since it liberates hydroquinone in the urine. The in vivo release of hydroquinone from arbutin with or without addition of glusulase or an E. coli suspension was investigated in 4 volunteers. They ingested 6 dragees Cystinol akut (420 mg arbutin equivalent to 168 mg hydroquinone), urine was sampled and assayed by a validated HPLC method. RESULTS: In comparison to incubation with glusulase the E. coli-suspension resulted in a 2.3 fold higher increase in free hydroquinone. When separating bacteria from the urine, the hydroquinone concentration in bacteria was 20 fold higher than in the supernatant. CONSEQUENCES: Glucuronic acid or sulfuric acid conjugates of hydroquinones obviously are taken up, enriched and metabolized to hydroquinone by bacteria. Deconjugation of hydroquinone likely is catalyzed by intracellular enzymes presumably present in bacterial cytoplasm; comparable activities in eucaryotic cells usually are localized in lysosomes. Alkalization of the urine seems not to be a prerequisite to improve the antiseptic properties of hydroquinone released from arbutin.

Environmental benzene exposure assessment for parent-child pairs in Rouen, France.

There is a lack of data on environmental benzene exposure in children. In this study, we compared personal benzene exposure and inhalation uptake in a group of children to those of their parents. We also compared levels of urinary benzene metabolites, trans,trans-muconic acid (MA) and hydroquinone (HQ), for those two groups, and assessed the correlation between personal benzene exposure and urinary MA and HQ concentrations. The study was performed on 21, 2-3-year-old children and their parents recruited on a voluntary basis among non-smokers from the three largest day-care centers of the town of Rouen in France. Average benzene concentrations were measured over 5 consecutive days with diffusive samplers. The following simultaneous measurements were carried out: personal exposure of the parents, concentrations inside and outside the day care centers, and inside the volunteer's bedrooms. Morning and evening urine samples were collected during the same period. Benzene personal exposure levels were 14.4+/-7.7 microg/m(3) and 11.09+/-6.15 microg/m(3) in parents and children, respectively. Benzene inhalation uptake estimates were 2.51+/-1.23 microg/kg/day in the group of parents and 5.68+/-3.17 microg/kg/day in the group of children. Detectable levels of MA and HQ were found in 85% and 100% of the samples, respectively. Intra-individual variation of urinary MA and HQ concentrations expressed as a coefficient of variation (CV) ranged from 63 to 232% and from 13 to 144%, respectively. Mean values of MA and HQ (in mg/g creatinine) were 1.6- and 1.8-fold higher in the group of children than in the group of parents (P=0.008 and P<0.0001, respectively). Significant correlations between metabolites levels and benzene were not found.

Urinary excretion and metabolism of arbutin after oral administration of Arctostaphylos uvae ursi extract as film-coated tablets and aqueous solution in healthy humans.

Bearberry leaves and preparations made from them are traditionally used for urinary tract infections. The urinary excretion of arbutin metabolites was examined in a randomized crossover design in 16 healthy volunteers after the application of a single oral dose of bearberry leaves dry extract (BLDE). There were two groups of application using either film-coated tablets (FCT) or aqueous solution (AS). The urine sample analysis was performed by a validated HPLC coolarray method (hydroquinone) and a validated capillary electrophoresis method (hydroquinone-glucuronide, hydroquinone-sulfate). The total amounts of hydroquinone equivalents excreted in the urine from BLDE were similar in both groups. With FCT, 64.8% of the arbutin dose administered was excreted; with AS, 66.7% was excreted (p = 0.61). The maximum mean urinary concentration of hydroquinone equivalents was a little higher and peaked earlier in the AS group versus the FCT group, although this did not reach statistical significance (Cur max = 1.6893 micromol/ml vs. 1.1250 micromol/ml, p = 0.13; tmax (t midpoint) = 3.60 h vs. 4.40 h, p = 0.38). The relative bioavailability of FCT compared to AS was 103.3% for total hydroquinone equivalents. There was substantial intersubject variability. No significant differences between the two groups were found in the metabolite patterns detected (hydroquinone, hydroquinone-glucuronide, and hydroquinone-sulfate).

Validated method for the determination of hydroquinone in human urine by high-performance liquid chromatography-coulometric-array detection.

The paper describes the computer aided method development and validation for the determination of hydroquinone in human urine from a clinical study on renal excretion of hydroquinone metabolites and the release of free hydroquinone in the urinary tract in order to evaluate the proposed urine disinfecting concept. The presented method uses high-performance liquid chromatography on reversed-phase material with a polar endcapping (Aqua-C18, 250x4.6 mm). Selective and sensitive determination (LOQ= 12.5 ng on-column) of the target compound was achieved by electrochemical array detection (CoulArray). Gradient and parameter optimization were supported by DryLab software in order to minimize efforts of the expensive and time-consuming method development. Specificity and selectivity were carried out by separation experiments involving the prodrug arbutin and the metabolites hydroquinone, hydroquinone glucuronide, and hydroquinone sulfate, respectively. Hydroquinone glucuronide reference standard was obtained from in vitro glucuronidation in a rat liver microsomes assay. The method was validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services, 1998.

Validated methods for direct determination of hydroquinone glucuronide and sulfate in human urine after oral intake of bearberry leaf extract by capillary zone electrophoresis.

Bearberry leaf extracts are used in herbal medicinal products for the treatment of lower urinary tract infections. Two metabolites of the major phenolic constituent in the extract, arbutin (hydroquinone-1-O-beta-D-glucoside), must be assumed to be precursors of the active disinfectant principle hydroquinone. In order to assay the renal elimination of these two metabolites. i.e., hydroquinone conjugates with glucuronic and sulfuric acid, two separate capillary electrophoresis methods have been developed. Both methods were validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services. 1998. As there is little sample preparation necessary, both methods are very suitable for urine analysis with large sample numbers as frequently coming up in the course of pharmaceutical bioavailability, bioequivalence and pharmacokinetic studies.

Validation of biomarkers in humans exposed to benzene: urine metabolites.

BACKGROUND: The present study was conducted among Chinese workers employed in glue- and shoe-making factories who had an average daily personal benzene exposure of 31+/-26 ppm (mean+/-SD). The metabolites monitored were S-phenylmercapturic acid (S-PMA), trans, trans-muconic acid (t,t-MA), hydroquinone (HQ), catechol (CAT), 1,2, 4-trihydroxybenzene (benzene triol, BT), and phenol. METHODS: S-PMA, t,t-MA, HQ, CAT, and BT were quantified by HPLC-tandem mass spectrometry. Phenol was measured by GC-MS. RESULTS: Levels of benzene metabolites (except BT) measured in urine samples collected from exposed workers at the end of workshift were significantly higher than those measured in unexposed subjects (P < 0.0001). The large increases in urinary metabolites from before to after work strongly correlated with benzene exposure. Concentrations of these metabolites in urine samples collected from exposed workers before work were also significantly higher than those from unexposed subjects. The half-lives of S-PMA, t,t-MA, HQ, CAT, and phenol were estimated from a time course study to be 12.8, 13.7, 12.7, 15.0, and 16.3 h, respectively. CONCLUSIONS: All metabolites, except BT, are good markers for benzene exposure at the observed levels; however, due to their high background, HQ, CAT, and phenol may not distinguish unexposed subjects from workers exposed to benzene at low ambient levels. S-PMA and t,t-MA are the most sensitive markers for low level benzene exposure.

Development of liquid chromatography-electrospray ionization-tandem mass spectrometry methods for determination of urinary metabolites of benzene in humans.

To investigate the ways in which different levels of exposure affect the metabolic activation pathways of benzene in humans, and to examine the relationship between urinary metabolites and other biological markers, we have developed two sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for quantitation of the benzene metabolites trans,transmuconic acid (t,t-MA), S-phenylmercapturic acid (S-PMA), hydroquinone (HQ), catechol (CAT), and for estimation of 1,2,4-trihydroxybenzene (BT). In our first assay, urinary S-PMA and t,t-MA were measured simultaneously by liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESI-MS/MS-SRM) in the negative ionization mode. In this assay, the metabolites [13C6]-S-PMA and [13C6]-t,t-MA were used as internal standards. The efficacy of this specific assay was evaluated in human urine specimens from 28 smokers and 18 nonsmokers serving as the benzene-exposed and nonexposed groups, respectively. The coefficient of variation (CV) of analyses on different days (n = 8) for S-PMA was 7% for samples containing 9.4 micrograms/L urine, and for t,t-MA was 10% for samples containing 0.07 mg/L. The mean levels of S-PMA and t,t-MA in smokers were 1.9-fold (p = 0.02) and 2.1-fold (p = 0.03) higher, respectively, than those in nonsmokers.