Temporal stability of eight phthalate metabolites and their glucuronide conjugates in human urine.

Humans are exposed to phthalates due to the ubiquitous use of these chemicals in consumer products. In the body, phthalates metabolize quickly to form hydrolytic and oxidative monoesters which, in turn, can be glucuronidated before urinary excretion. Exposure assessment studies typically report the total urinary concentrations of phthalate metabolites (i.e., free plus glucuronidated species). Nevertheless, because conjugation may potentially reduce the bioactivity of the metabolites by reducing their bioavailability, measuring the concentrations of free species may be of interest. An accurate, quantitative measurement of phthalate monoesters and their conjugated species requires data on the stability of these species in urine after sample collection and before analysis. We studied the stability of eight phthalate metabolites and their glucuronide conjugates at 25, 4, and -70 degrees C. Interestingly, the total concentrations of phthalate metabolites decreased over time at 25 and 4 degrees C, but not at -70 degrees C for up to 1 year and despite several freeze-thaw cycles. We further observed a considerable decrease in the concentrations of the glucuronides of some phthalate metabolites 1 day and 3 days after collection when the samples were stored at 25 and 4 degrees C, respectively. By contrast, the concentrations of the glucuronide conjugates at -70 degrees C remained unchanged for the whole duration of the study (1 year). Based on these findings, we recommend transferring urine specimens to a cooler or a refrigerator immediately after collection followed by permanent storage at subfreezing temperatures within hours of sample collection.

Exposure of the U.S. population to bisphenol A and 4-tertiary-octylphenol: 2003-2004.

BACKGROUND: Bisphenol A (BPA) and 4-tertiary-octylphenol (tOP) are industrial chemicals used in the manufacture of polycarbonate plastics and epoxy resins (BPA) and nonionic surfactants (tOP). These products are in widespread use in the United States. OBJECTIVES: We aimed to assess exposure to BPA and tOP in the U.S. general population. METHODS: We measured the total (free plus conjugated) urinary concentrations of BPA and tOP in 2,517 participants > or = 6 years of age in the 2003-2004 National Health and Nutrition Examination Survey using automated solid-phase extraction coupled to isotope dilution-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: BPA and tOP were detected in 92.6% and 57.4% of the persons, respectively. Least square geometric mean (LSGM) concentrations of BPA were significantly lower in Mexican Americans than in non-Hispanic blacks (p = 0.006) and non-Hispanic whites (p = 0.007); LSGM concentrations for non-Hispanic blacks and non-Hispanic whites were not statistically different (p = 0.21). Females had statistically higher BPA LSGM concentrations than males (p = 0.043). Children had higher concentrations than adolescents (p < 0.001), who in turn had higher concentrations than adults (p = 0.003). LSGM concentrations were lowest for participants in the high household income category (> $45,000/year). CONCLUSIONS: Urine concentrations of total BPA differed by race/ethnicity, age, sex, and household income. These first U.S. population representative concentration data for urinary BPA and tOP should help guide public health research priorities, including studies of exposure pathways, potential health effects, and risk assessment.

Urinary concentrations of triclosan in the U.S. population: 2003-2004.

BACKGROUND: Triclosan is a synthetic chemical with broad antimicrobial activity that has been used extensively in consumer products, including personal care products, textiles, and plastic kitchenware. OBJECTIVES: This study was designed to assess exposure to triclosan in a representative sample > or = 6 years of age of the U.S. general population from the 2003-2004 National Health and Nutrition Examination Survey (NHANES). METHODS: We analyzed 2,517 urine samples using automated solid-phase extraction coupled to isotope dilution-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: We detected concentrations of total (free plus conjugated) triclosan in 74.6% of samples at concentrations of 2.4-3,790 microg/L. The geometric mean and 95th percentile concentrations were 13.0 microg/L (12.7 microg/g creatinine) and 459.0 microg/L (363.8 mug/g creatinine), respectively. We observed a curvilinear relation between age and adjusted least square geometric mean (LSGM) concentrations of triclosan. LSGM concentrations of triclosan were higher in people in the high household income than in people in low (p < 0.01) and medium (p = 0.04) income categories. CONCLUSIONS: In about three-quarters of urine samples analyzed as part of NHANES 2003-2004, we detected concentrations of triclosan. Concentrations differed by age and socioeconomic status but not by race/ethnicity and sex. Specifically, the concentrations of triclosan appeared to be highest during the third decade of life and among people with the highest household incomes.

Concentrations of the sunscreen agent benzophenone-3 in residents of the United States: National Health and Nutrition Examination Survey 2003--2004.

BACKGROUND: The capability of benzophenone-3 (BP-3) to absorb and dissipate ultraviolet radiation facilitates its use as a sunscreen agent. BP-3 has other uses in many consumer products (e.g., as fragrance and flavor enhancer, photoinitiator, ultraviolet curing agent, polymerization inhibitor). OBJECTIVES: Our goal was to assess exposure to BP-3 in a representative sample of the U.S. general population > or = 6 years of age. METHODS: Using automated solid-phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometry, we analyzed 2,517 urine samples collected as part of the 2003--2004 National Health and Nutrition Examination Survey. RESULTS: We detected BP-3 in 96.8% of the samples. The geometric mean and 95th percentile concentrations were 22.9 microg/L (22.2 microg/g creatinine) and 1,040 microg/L (1,070 microg/g creatinine), respectively. Least-square geometric mean (LSGM) concentrations were significantly higher (p < or = 0.04) for females than for males, regardless of age. LSGM concentrations were significantly higher for non-Hispanic whites than for non-Hispanic blacks (p < or = 0.01), regardless of age. Females were more likely than males [adjusted odds ratio (OR) = 3.5; 95% confidence interval (95% CI), 1.9-6.5], and non-Hispanic whites were more likely than non-Hispanic blacks (adjusted OR = 6.8; 95% CI, 2.9-16.2) to have concentrations above the 95th percentile. CONCLUSIONS: Exposure to BP-3 was prevalent in the general U.S. population during 2003--2004. Differences by sex and race/ethnicity probably reflect differences in use of personal care products containing BP-3.

Polyfluoroalkyl chemicals in the U.S. population: data from the National Health and Nutrition Examination Survey (NHANES) 2003-2004 and comparisons with NHANES 1999-2000.

BACKGROUND: Polyfluoroalkyl chemicals (PFCs) have been used since the 1950s in numerous commercial applications. Exposure of the general U.S. population to PFCs is widespread. Since 2002, the manufacturing practices for PFCs in the United States have changed considerably. OBJECTIVES: We aimed to assess exposure to perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonic acid (PFHxS), perfluorononanoic acid (PFNA), and eight other PFCs in a representative 2003-2004 sample of the general U.S. population >or= 12 years of age and to determine whether serum concentrations have changed since the 1999-2000 National Health and Nutrition Examination Survey (NHANES). METHODS: By using automated solid-phase extraction coupled to isotope dilution-high-performance liquid chromatography-tandem mass spectrometry, we analyzed 2,094 serum samples collected from NHANES 2003-2004 participants. RESULTS: We detected PFOS, PFOA, PFHxS, and PFNA in > 98% of the samples. Concentrations differed by race/ethnicity and sex. Geometric mean concentrations were significantly lower (approximately 32% for PFOS, 25% for PFOA, 10% for PFHxS) and higher (100%, PFNA) than the concentrations reported in NHANES 1999-2000 (p < 0.001). CONCLUSIONS: In the general U.S. population in 2003-2004, PFOS, PFOA, PFHxS, and PFNA serum concentrations were measurable in each demographic population group studied. Geometric mean concentrations of PFOS, PFOA, and PFHxS in 2003-2004 were lower than in 1999-2000. The apparent reductions in concentrations of PFOS, PFOA, and PFHxS most likely are related to discontinuation in 2002 of industrial production by electrochemical fluorination of PFOS and related perfluorooctanesulfonyl fluoride compounds.

Quantification of 22 phthalate metabolites in human urine.

Phthalates are ubiquitous industrial chemicals with high potential for human exposure. Validated analytical methods to measure trace concentrations of phthalate metabolites in humans are essential for assessing exposure to phthalates. Previously, we developed a sensitive and accurate automated analytical method for measuring up to 16 phthalate metabolites in human urine by using on-line solid phase extraction coupled with isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry. To include the measurement of seven additional analytes, including oxidative metabolites of diisononyl and diisodecyl phthalates, two chemicals used extensively in numerous consumer products, we used a novel nontraditional HPLC solvent gradient program. With this approach, we achieved adequate resolution and sensitivity for all 22 analytes with limits of detection in the low ng/mL range, without increasing the analytical run time. The method also has high accuracy with automatic recovery correction, high precision, and excellent sample throughput with minimal matrix effects. Although it is possible to measure these 22 phthalate metabolites with adequate precision and accuracy at sub-parts-per-billion levels, additional information, including toxicokinetic data, is needed to demonstrate the usefulness of these phthalate metabolites for exposure assessment purposes.

Oxidative metabolites of diisononyl phthalate as biomarkers for human exposure assessment.

Diisononyl phthalate (DINP) is a complex mixture of predominantly nine-carbon branched-chain dialkyl phthalate isomers. Similar to di(2-ethylhexyl) phthalate, a widely used phthalate, DINP causes antiandrogenic effects on developing rodent male fetuses. Traditionally, assessment of human exposure to DINP has been done using monoisononyl phthalate (MINP) , the hydrolytic metabolite of DINP, as a biomarker. However, MINP is only a minor urinary metabolite of DINP. Oxidative metabolites, including mono(carboxyisooctyl) phthalate (MCIOP) , mono(oxoisononyl) phthalate (MOINP) , and mono(hydroxyisononyl) phthalate (MHINP) are the major urinary metabolites in DINP-dosed rats. The urinary concentrations of MINP, MCIOP, MOINP, and MHINP were measured in 129 adult anonymous human volunteers with no known exposure to DINP. Although MINP was not present at detectable levels in any of the samples analyzed, MCIOP, MHINP, and MOINP were detected in 97, 100, and 87% of the urine samples at geometric mean levels equal to 8.6, 11.4, and 1.2 ng/mL, respectively. The concentrations of all three oxidative metabolites were highly correlated with each other (p<0.0001), which confirms a common precursor. MCIOP was excreted predominantly as a free species, whereas MOINP was excreted mostly in its glucuronidated form. The percentage of MHINP excreted either glucuronidated or in its free form was similar. The significantly higher frequency of detection and urinary concentrations of oxidative metabolites than of MINP suggest that these oxidative metabolites are better biomarkers of exposure assessment of DINP than is MINP. Therefore, we concluded that the prevalence of human exposure to DINP is underestimated by using MINP as the sole DINP urinary biomarker.

Parabens as urinary biomarkers of exposure in humans.

BACKGROUND: Parabens appear frequently as antimicrobial preservatives in cosmetic products, in pharmaceuticals, and in food and beverage processing. In vivo and in vitro studies have revealed weak estrogenic activity of some parabens. Widespread use has raised concerns about the potential human health risks associated with paraben exposure. OBJECTIVES: Assessing human exposure to parabens usually involves measuring in urine the conjugated or free species of parabens or their metabolites. In animals, parabens are mostly hydrolyzed to p-hydroxybenzoic acid and excreted in the urine as conjugates. Still, monitoring urinary concentrations of p-hydroxybenzoic acid is not necessarily the best way to assess exposure to parabens. p-hydroxybenzoic acid is a nonspecific biomarker, and the varying estrogenic bioactivities of parabens require specific biomarkers. Therefore, we evaluated the use of free and conjugated parent parabens as new biomarkers for human exposure to these compounds. RESULTS: We measured the urinary concentrations of methyl, ethyl, n-propyl, butyl (n- and iso-), and benzyl parabens in a demographically diverse group of 100 anonymous adults. We detected methyl and n-propyl parabens at the highest median concentrations (43.9 ng/mL and 9.05 ng/mL, respectively) in nearly all (> 96%) of the samples. We also detected other parabens in more than half of the samples (ethyl, 58%; butyl, 69%). Most important, however, we found that parabens in urine appear predominantly in their conjugated forms. CONCLUSIONS: The results, demonstrating the presence of urinary conjugates of parabens in humans, suggest that such conjugated parabens could be used as exposure biomarkers. Additionally, the fact that conjugates appear to be the main urinary products of parabens may be important for risk assessment.

Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate.

Two studies were designed to examine amniotic fluid and maternal urine concentrations of the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethylhexyl) phthalate (MEHP) and the di-n-butyl phthalate (DBP) metabolite monobutyl phthalate (MBP) after administration of DEHP and DBP during pregnancy. In the first study, pregnant Sprague-Dawley rats were administered 0, 11, 33, 100, or 300 mg DEHP/kg/day by oral gavage starting on gestational day (GD) 7. In the second study, DBP was administered by oral gavage to pregnant Sprague-Dawley rats at doses of 0, 100, or 250 mg/kg/day starting on GD 13. Maternal urine and amniotic fluid were collected and analyzed to determine the free and glucuronidated levels of MEHP and MBP. In urine, MEHP and MBP were mostly glucuronidated. By contrast, free MEHP and free MBP predominated in amniotic fluid. Statistically significant correlations were found between maternal DEHP dose and total maternal urinary MEHP (p=0.0117), and between maternal DEHP dose and total amniotic fluid MEHP levels (p=0.0021). Total maternal urinary MEHP and total amniotic fluid MEHP levels were correlated (Pearson correlation coefficient=0.968). Statistically significant differences were found in amniotic MBP levels between animals within the same DBP dose treatment group (p<0.0001) and between animals in different dose treatment groups (p<0.0001). Amniotic fluid MBP levels increased with increasing DBP doses, and high variability in maternal urinary levels of MBP between rats was observed. Although no firm conclusions could be drawn from the urinary MBP data, the MEHP results suggest that maternal urinary MEHP levels may be useful surrogate markers for fetal exposure to DEHP.

Mono-(3-carboxypropyl) phthalate, a metabolite of di-n-octyl phthalate.

Di-n-octyl phthalate (DnOP) is found as a component of mixed C6-C10 linear-chain phthalates used as plasticizers in various polyvinyl chloride applications, including flooring and carpet tiles. Following exposure and absorption, DnOP is metabolized to its hydrolytic monoester, mono-n-octyl phthalate (MnOP), and other oxidative products. The urinary levels of one of these oxidative metabolites, mono-(3-carboxypropyl) phthalate (MCPP), were about 560-fold higher than MnOP in Sprague-Dawley rats dosed with DnOP by gavage. Furthermore, MCPP was also found in the urine of rats dosed with di-isooctyl phthalate (DiOP), di-isononyl phthalate (DiNP), di-isodecyl phthalate (DiDP), di-(2-ethylhexyl) phthalate, and di-n-butyl phthalate (DBP), although at concentrations considerably lower than in rats given similar concentrations of DnOP. The comparatively much higher urinary concentrations of MCPP than of the hydrolytic monoesters of the high-molecular-weight phthalates DiOP, DiNP, and DiDP in the exposed rats suggest that these monoesters may be poor biomarkers of exposure to their precursor phthalates and may explain the relatively low frequency of detection of these monoester metabolites in human populations. MCPP and MnOP were also measured in 267 human urine samples. The frequent detection and higher urinary concentrations of MCPP than MnOP suggest that exposure to DnOP might be higher than previously thought based on the measurements of MnOP alone. However, because MCPP is also a minor metabolite of DBP and other phthalates in rats, and the metabolism of phthalates in rodents and humans may differ, additional data on the absorption, distribution, metabolism, and elimination of MCPP are needed to completely understand the extent of human exposure to DnOP from the urinary concentrations of MCPP.

Perfluorinated chemicals in selected residents of the American continent.

Perfluorinated chemicals (PFCs) are used in multiple consumer products. Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), the most widely studied PFCs, may be potential developmental, reproductive, and systemic toxicants. Although PFCs seem to be ubiquitous contaminants found both in humans and animals, geographic differences may exist in human exposure patterns to PFCs. We measured 11 PFCs in 23 pooled serum samples collected in the United States from 1990 through 2002, and in serum samples collected in 2003 from 44 residents from Trujillo, Peru. PFOS and PFOA were detected in all the pooled samples; perfluorohexane sulfonic acid (PFHxS) was detected in 21. Median concentrations were 31.1 micrograms per liter (mug/l, PFOS), 11.6 microg/l (PFOA), and 2 microg/l (PFHxS). The 90th percentile concentrations of PFCs in the 44 Peruvian residents were 0.7 microg/l (PFOS), 0.1 microg/l (PFOA), and <0.3 microg/l (PFHxS). The frequencies of detection were 20% (PFOS), 25% (PFOA), and 9% (PFHxS). The frequent detection of selected PFCs in the pooled samples from the United States and the lack of clear concentration trends based on a year of collection suggest a sustained widespread exposure to these compounds among US residents, at least since the 1990s. By contrast, the much lower frequency of detection and concentration ranges of PFCs in Peru suggest a lower exposure of Peruvians to PFCs compared with North Americans. Genetic variability, diet, lifestyle, or a combination of all these may contribute to the different patterns of human exposure to PFCs in the United States and Peru.

Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population.

Bisphenol A (BPA) is used to manufacture polycarbonate plastic and epoxy resins, which are used in baby bottles, as protective coatings on food containers, and for composites and sealants in dentistry. 4-Nonylphenol (NP) is used to make nonylphenol ethoxylates, nonionic surfactants applied as emulsifying, wetting, dispersing, or stabilizing agents in industrial, agricultural, and domestic consumer products. The potential for human exposure to BPA and NP is high because of their widespread use. We measured BPA and NP in archived urine samples from a reference population of 394 adults in the United States using isotope-dilution gas chromatography/mass spectrometry. The concentration ranges of BPA and NP were similar to those observed in other human populations. BPA was detected in 95% of the samples examined at concentrations > or = 0.1 microg/L urine; the geometric mean and median concentrations were 1.33 microg/L (1.36 microg/g creatinine) and 1.28 microg/L (1.32 microg/g creatinine), respectively; the 95th percentile concentration was 5.18 microg/L (7.95 microg/g creatinine). NP was detected in 51% of the samples examined > or = 0.1 microg/L. The median and 95th percentile concentrations were < 0.1 microg/L and 1.57 microg/L (1.39 microg/g creatinine), respectively. The frequent detection of BPA suggests widespread exposure to this compound in residents of the United States. The lower frequency of detection of NP than of BPA could be explained by a lower exposure of humans to NP, by different pharmacokinetic factors (i.e., absorption, distribution, metabolism, elimination), by the fact that 4-n-nonylphenol--the measured NP isomer--represents a small percentage of the NP used in commercial mixtures, or a combination of all of the above. Additional research is needed to determine the best urinary biomarker(s) to assess exposure to NP. Despite the sample population's nonrepresentativeness of the U.S. population (although sample weights were used to improve the extent to which the results represent the U.S. population) and relatively small size, this study provides the first reference range of human internal dose levels of BPA and NP in a demographically diverse human population.

Automated solid-phase extraction and quantitative analysis of 14 phthalate metabolites in human serum using isotope dilution-high-performance liquid chromatography-tandem mass spectrometry.

Phthalates are industrial chemicals with many commercial applications. Because of their common usage, the general population is exposed to phthalates. A sensitive and selective analytical method is necessary to accurately determine the phthalate levels in serum. We improved our previously developed analytical method to measure nine phthalate metabolites in human serum by automating the solid-phase extraction (SPE) procedure and by including five additional phthalate metabolites: phthalic acid; mono-isobutyl phthalate, a metabolite of di-isobutyl phthalate; mono-(3-carboxypropyl) phthalate, a major oxidative metabolite of di-n-octyl phthalate; and mono-(2-ethyl-5-oxohexyl) phthalate and mono-(2-ethyl-5-hydroxyhexyl) phthalate, two oxidative metabolites of di-(2-ethylhexyl) phthalate. Automation of the SPE eliminated the human variation associated with the manual SPE, thus improving the reproducibility of the measurements. Additional wash steps during SPE produced cleaner extracts and resulted in higher recoveries (80-99%) than the manual SPE method. Furthermore, the automated SPE method allowed for the unattended extraction of samples, with a concomitant increase in sample throughput compared to the manual SPE method. The method is accurate, precise, and sensitive, with limits of detection in the low nanogram-per-milliliter range.

Mono(2-ethyl-5-hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate as biomarkers for human exposure assessment to di-(2-ethylhexyl) phthalate.

Exposure to di-(2-ethylhexyl) phthalate (DEHP) is prevalent based on the measurement of its hydrolytic metabolite mono-(2-ethylhexyl) phthalate (MEHP) in the urine of 78% of the general U.S. population studied in the 1999-2000 National Health and Nutrition Examination Survey (NHANES). However, despite the high level of production and use of DEHP, the urinary MEHP levels in the NHANES samples were lower than the monoester metabolites of phthalates less commonly used than DEHP, suggesting metabolic differences between phthalates. We measured MEHP and two oxidative DEHP metabolites, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) to verify whether these other metabolites account for a greater proportion of DEHP metabolic products in 127 paired human urine and serum samples. We found that the urinary levels of MEHHP and MEOHP were 10-fold higher than levels of MEHP; concentrations of urinary MEOHP and MEHHP were strongly correlated (r = 0.928). We also found that the serum levels of MEOHP and MEHHP were comparatively lower than those in urine. Furthermore, the glucuronide-bound conjugates of the oxidative metabolites were the predominant form in both urine and serum. MEOHP and MEHHP cannot be formed by serum enzymes from the hydrolysis of any contamination from DEHP potentially introduced during blood collection and storage. Therefore, concentrations of MEHHP and MEOHP in serum may be a more selective measure of DEHP exposure than is MEHP. However, additional data on the absorption, distribution, metabolism, and elimination of these oxidative metabolites are needed to completely understand the extent of DEHP exposure from the serum concentrations of oxidative DEHP metabolites.

Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers.

Phthalates are a group of industrial chemicals with many commercial uses, such as solvents, additives, and plasticizers. For example, di-(2-ethylhexyl) phthalate (DEHP) is added in varying amounts to certain plastics, such as polyvinyl chloride, to increase their flexibility. In humans, phthalates are metabolized to their respective monoesters, conjugated, and eliminated. However, despite the high production and use of DEHP, we have recently found that the urinary levels of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in 2,541 persons in the United States were lower than we anticipated, especially when compared with urinary metabolite levels of other commonly used phthalates. This finding raised questions about the sensitivity of this biomarker for assessing DEHP exposure. We explored the utility of two other DEHP metabolites, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), as additional DEHP biomarkers. These metabolites are formed by oxidative metabolism of MEHP. In urine from 62 people, both the range and the mean urinary levels of MEOHP and MEHHP were on average 4-fold higher than those of MEHP; the mean of the individual ratios of MEHHP/MEOHP, MEHHP/MEHP, and MEOHP/MEHP were 1.4, 8.2, and 5.9, respectively. These data suggest that MEOHP and MEHHP are more sensitive biomarkers of exposure to DEHP than is MEHP. These findings also suggest a predominant human metabolic route for DEHP hydrolysis to MEHP followed by oxidation of MEHP; they also imply that a similar mechanism may be relevant for other high-molecular-weight phthalates, such as di-n-octyl, di-isononyl, and di-isodecyl phthalates.

Urinary levels of seven phthalate metabolites in the U.S. population from the National Health and Nutrition Examination Survey (NHANES) 1999-2000.

We measured the urinary monoester metabolites of seven commonly used phthalates in approximately 2,540 samples collected from participants of the National Health and Nutrition Examination Survey (NHANES), 1999-2000, who were greater than or equal to 6 years of age. We found detectable levels of metabolites monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), and mono-(2-ethylhexyl) phthalate (MEHP) in > 75% of the samples, suggesting widespread exposure in the United States to diethyl phthalate, dibutyl phthalate or diisobutylphthalate, benzylbutyl phthalate, and di-(2-ethylhexyl) phthalate, respectively. We infrequently detected monoisononyl phthalate, mono-cyclohexyl phthalate, and mono-n-octyl phthalate, suggesting that human exposures to di-isononyl phthalate, dioctylphthalate, and dicyclohexyl phthalate, respectively, are lower than those listed above, or the pathways, routes of exposure, or pharmacokinetic factors such as absorption, distribution, metabolism, and elimination are different. Non-Hispanic blacks had significantly higher concentrations of MEP than did Mexican Americans and non-Hispanic whites. Compared with adolescents and adults, children had significantly higher levels of MBP, MBzP, and MEHP but had significantly lower concentrations of MEP. Females had significantly higher concentrations of MEP and MBzP than did males, but similar MEHP levels. Of particular interest, females of all ages had significantly higher concentrations of the reproductive toxicant MBP than did males of all ages; however, women of reproductive age (i.e., 20-39 years of age) had concentrations similar to adolescent girls and women 40 years of age. These population data on exposure to phthalates will serve an important role in public health by helping to set research priorities and by establishing a nationally representative baseline of exposure with which population levels can be compared.

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction.

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.

Improved quantitative detection of 11 urinary phthalate metabolites in humans using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry.

Phthalates are widely used as industrial solvents and plasticizers, with global use exceeding four million tons per year. We improved our previously developed high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC-APCI-MS/MS) method to measure urinary phthalate metabolites by increasing the selectivity and the sensitivity by better resolving them from the solvent front, adding three more phthalate metabolites, monomethyl phthalate (mMP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP); increasing the sample throughput; and reducing the solvent usage. Furthermore, this improved method enabled us to analyze free un-conjugated mono-2-ethylhexyl phthalate (mEHP) by eliminating interferences derived from coelution of the glucuronide-bound, or conjugated form, of the mEHP on measurements of the free mEHP. This method for measuring phthalate metabolites in urine involves solid-phase extraction followed by reversed-phase HPLC-APCI-MS/MS using isotope dilution with (13)C(4) internal standards. We further evaluated the ruggedness and the reliability of the method by comparing measurements made by multiple analysts at different extraction settings on multiple instruments. We observed mMP, monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mEHP, mEHHP and mEOHP in the majority of urine specimens analyzed with DEHP-metabolites mEHHP and mEOHP present in significantly higher amounts than mEHP.

Quantitative detection of nine phthalate metabolites in human serum using reversed-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

We developed a highly sensitive method for the quantitative detection of nine phthalate ester metabolites in human serum. This method requires denaturation of the serum enzymes immediately after blood collection to avoid the hydrolysis of the contaminant diester parent compounds introduced during blood collection and storage. Before analysis, the samples were subjected to an enzymatic deconjugation to hydrolyze the glucuronidated phthalate monoesters and a solid-phase extraction to isolate the monoesters from other serum components. The extracts were analyzed using reversed-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. The limits of detection of all nine phthalate monoesters in serum were in the low nanogram-per-milliliter range (0.6-1.3 ng/mL). Stable isotope-labeled internal standards for all analytes were used to improve precision and for recovery corrections. This highly selective method permits the analysis of phthalate monoesters without interferences resulting from the hydrolysis of the ubiquitous contaminant phthalate diesters by serum enzymes. In addition, it allows the direct measurement of the active phthalate monoester metabolites reportedly responsible for the reproductive and developmental toxicity of certain phthalates.

Metabolite profiles of di-n-butyl phthalate in humans and rats.

Di-n-butyl phthalate (DBP) is widely used in consumer products. In humans and in rats, DBP is metabolized to mono-n-butyl phthalate (MBP). MBP may also further oxidize to other metabolites of DBP. We studied the metabolic profiles of DBP in rats and humans to evaluate the similarities between the two species and between different exposure scenarios. In rats administered DBP by oral gavage, we identified MBP and three urinary oxidative metabolites of DBP: mono-3-oxo-n-butyl phthalate, mono-3-hydroxy-n-butyl phthalate (MHBP), and mono-3-carboxypropyl phthalate (MCPP). MBP, MHBP, and MCPP were also present in serum, albeit at lower levels than in urine. Statistically significant correlations (p < 0.01) existed between the concentrations of MBP and the concentrations of MHBP (Pearson correlation coefficient r = 0.82 [urine] and r = 0.96 [serum]) and MCPP (r = 0.77 [urine] and r = 0.97 [serum]). However, the concentrations of these metabolites in urine collected 6 h after dosing and in serum 24 h after dosing were not correlated, suggesting continuous metabolism of DBP and/or individual differences among rats. Serum DBP metabolite concentrations increased with the dose, whereas urinary concentrations did not. We also identified MBP, MHBP, and MCPP in the urine of four men exposed to DBP bytaking a prescription medication containing DBP, and MBP and MCPP in 94 adults with no documented exposure to DBP. In the human samples, we observed statistically significant correlations (p < 0.01) among the urinary concentrations of MBP and MCPP, although the correlation was stronger for the four exposed men (r = 0.99) than for the adults without a documented exposure to DBP (r = 0.70). Our results suggest that regardless of species and exposure scenario, MBP, the major DBP metabolite, is an optimal biomarker of exposure to DBP. In addition to MBP, MCPP and MHBP may be adequate biomarkers of exposure to DBP in occupational settings orin potential high-exposure scenarios.