Urinary Concentrations of Diisoheptyl Phthalate Biomarkers in Convenience Samples of U.S. Adults in 2000 and 2018-2019.

We know little about the potential health risks from exposure to diisoheptyl phthalate (DiHpP), a plasticizer used in commercial applications. The production of DiHpP ended in the United States in 2010, but DiHpP may still be present in phthalate diester mixtures. To investigate human exposure to DiHpP, we used three oxidative metabolites of DiHpP: Monohydroxyheptyl phthalate (MHHpP), mono-oxoheptylphthalate (MOHpP), and monocarboxyhexyl phthalate (MCHxP) as exposure biomarkers. We analyzed urine collected anonymously in 2000 (N = 144) and 2018-2019 (N = 205) from convenience groups of U.S. adults using high-performance liquid chromatography coupled with isotope-dilution high-resolution mass spectrometry. We detected MCHxP in all the samples tested in 2000 (GM = 2.01 ng/mL) and 2018-2019 (GM = 1.31 ng/mL). MHHpP was also detected in 100% of the 2018-2019 samples (GM = 0.59 ng/mL) and 96% of the 2000 urine samples analyzed (GM = 0.38 ng/mL). MOHpP was detected in 57% (2018-2019, GM = 0.03 ng/mL) and 92% (2000, GM = 0.19 ng/mL) of samples. The presence of MHHpP, MOHpP, and MCHxP in the 2018-2019 samples suggests recent exposure to DiHpP. Intercorrelations between metabolite concentrations were more significant in samples collected in 2000 than in samples collected in 2018-2019. The differences in urinary metabolite profiles and intercorrelations from samples collected during 2000 and 2018-2019 likely reflects changes in the composition of commercial DiHpP formulations before and after 2010.

Exposure to di-2-ethylhexyl terephthalate in the U.S. general population from the 2015-2016 National Health and Nutrition Examination Survey.

BACKGROUND: Di-2-ethylhexyl terephthalate (DEHTP) is used as a replacement plasticizer for other phthalates, including di-2-ethylhexyl phthalate (DEHP). Use of consumer products containing DEHTP may result in human exposure to DEHTP. OBJECTIVE: To assess exposure to DEHTP in a nationally representative sample of the U.S. general population 3 years and older from the 2015-2016 National Health and Nutrition Examination Survey (NHANES). METHOD: We quantified two DEHTP metabolites, mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP) and mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) in 2970 urine samples by using online solid-phase extraction coupled with isotope dilution-high-performance liquid chromatography-tandem mass spectrometry. We used linear regression to examine associations between MEHHTP and MECTPP and several parameters including age, sex, race/ethnicity, and household income. We also compared the MEHHTP and MECPTP results to those of their corresponding DEHP metabolite analogs, namely mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP) and mono-2-ethyl-5-carboxypentyl phthalate (MECPP). RESULTS: The weighted detection frequencies were 96% (MEHHTP) and 99.9% (MECPTP); urinary concentrations of the two metabolites correlated significantly (Pearson correlation coefficient = 0.89, p < 0.0001). MECPTP concentrations were higher than MEHHTP in all age, sex, race/ethnicity groups examined. Furthermore, MECPTP adjusted geometric mean (GM) concentrations were significantly higher in samples collected in the evening than in the morning or afternoon. Females had significantly higher adjusted GM concentrations of MEHHTP and MECPTP than males. We observed no significant associations between the adjusted GM concentrations of the metabolites and race/ethnicity. Both metabolite adjusted GM concentrations increased significantly with household income, and decreased significantly with age. Only household income was significantly associated with the concentrations of MECPP, but not of MEHHP, the two DEHP metabolites. The adjusted GM of the [MEHHTP]:[MECPTP] molar concentrations ratio increased with age, and was significantly higher in samples collected in the morning than in those collected in the afternoon or evening. CONCLUSIONS: Exposure to DEHTP is widespread in the U.S. general population 3 years and older. These data represent the first U.S. population-based representative background exposure to DEHTP.

Exposure to di-2-ethylhexyl terephthalate in a convenience sample of U.S. adults from 2000 to 2016.

Di-2-ethylhexyl terephthalate (DEHTP), a structural isomer of di-2-ethylhexyl phthalate (DEHP), is a plasticizer used in a variety of commercial applications, but data on Americans' exposure to DEHTP do not exist. We investigated the exposure to DEHTP in a convenience group of U.S. adults by analyzing urine collected anonymously in 2000 (N = 44), 2009 (N = 61), 2011 (N = 81), 2013 (N = 92), and 2016 (N = 149) for two major DEHTP oxidative metabolites: mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) and mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP). For comparison, we also quantified the analogous DEHP metabolites mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP) and mono-2-ethyl-5-carboxypentyl phthalate (MECPP). We detected MECPTP, MEHHP, and MECPP in all samples collected in 2016 with geometric means of 13.1, 4.1, and 6.7 ng/mL, respectively; we detected MEHHTP in 91% of the samples (geometric mean = 3.1 ng/mL). Concentrations of MECPTP correlated well with those of MEHHTP (R 2 = 0.8, p < 0.001), but did not significantly correlate with those of MEHHP (p > 0.05) suggesting different sources of exposure to DEHP and DEHTP. We also evaluated the fraction of the metabolites eliminated in their free (i.e., unconjugated) form. The median percent of unconjugated species was lower for the DEHP metabolites (MECPP [45.5%], MEHHP [1.9%]) compared to the DEHTP metabolites (MECPTP [98.8%], MEHHTP [21.2%]). Contrary to the downward trend from 2000 to 2016 in urinary concentrations of MEHHP and MECPP, we observed an upward trend for MEHHTP and MECPTP. These preliminary data suggest that exposure to DEHTP may be on the rise. Nevertheless, general population exposure data using MEHHTP and MECPTP as exposure biomarkers would increase our understanding of exposure to DEHTP, one of the known DEHP alternatives.

Identification of di-2-ethylhexyl terephthalate (DEHTP) metabolites using human liver microsomes for biomonitoring applications.

Di-2-ethylhexyl terephthalate (DEHTP), a structural isomer of the plasticizer di-2-ethylhexyl phthalate (DEHP), is used in food packaging and medical devices, among other applications, and is a potential replacement for DEHP and other ortho-phthalate plasticizers. Identifying sensitive and specific biomarkers of DEHTP is necessary to assess humans' background exposure to DEHTP. Using mass spectrometry, we investigated the metabolism of DEHTP by human liver microsomes to identify in vitro DEHTP metabolites. We unequivocally identified terephthalic acid (TPA) and mono-2-ethylhydroxyhexyl terephthalate (MEHHTP), using authentic standards, and tentatively identified mono-2-ethylhexyl terephthalate (MEHTP) and two other oxidative metabolites of DEHTP: mono-2-ethyloxohexyl terephthalate (MEOHTP), and mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) from their mass spectrometry fragmentation patterns. We also evaluated the formation of in vitro metabolites of DEHP. DEHTP and DEHP produced similar metabolites, but their metabolite profiles differed considerably. DEHTP metabolized to form TPA, a metabolite of several terephthalates, as the major in vitro metabolite, followed by MEHTP, MEHHTP, MEOHTP and MECPTP. MEHTP, MEHHTP, MEOHTP and MECPTP, which are specific metabolites of DEHTP, may be suitable biomarkers for assessing exposure to DEHTP. Nonetheless, data on the urinary excretion fraction and temporal stability of these metabolites, among other considerations, are needed to demonstrate their utility as exposure biomarkers.

In vitro metabolites of di-2-ethylhexyl adipate (DEHA) as biomarkers of exposure in human biomonitoring applications.

Di-2-ethylhexyl adipate (DEHA) is a common plasticizer used in food packaging. At high doses, DEHA can cause adverse health effects in rats. Although the potential for human exposure to DEHA is high, no DEHA specific biomarkers are identified for human biomonitoring. Using human liver microsomes, we investigated the in vitro phase I metabolism of DEHA and its hydrolytic metabolite mono-2-ethylhexyl adipate (MEHA) and, for comparison purposes, of the analogous di-2-ethylhexyl phthalate (DEHP) and its hydrolytic metabolite mono-2-ethylhexyl phthalate. We unequivocally identified MEHA, a DEHA specific biomarker, and adipic acid, a nonspecific biomarker, using authentic standards. On the basis of their mass spectrometric fragmentation patterns, we tentatively identified two other DEHA specific metabolites: mono-2-ethylhydroxyhexyl adipate (MEHHA) and mono-2-ethyloxohexyl adipate (MEOHA), analogous to the oxidative metabolites of DEHP. Interestingly, although adipic acid was the major in vitro metabolite of DEHA, the analogous phthalic acid was not the major in vitro metabolite of DEHP. Our preliminary data for 144 adults with no known exposure to DEHA suggests that adipic acid is also the main in vivo urinary metabolite, while MEHA, MEHHA, and MEOHA are only minor metabolites. Therefore, the use of these specific metabolites for assessing the exposure of DEHA may be limited to highly exposed populations.

Environmental exposure to the plasticizer 1,2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH) in U.S. adults (2000-2012).

1,2-Cyclohexane dicarboxylic acid, diisononyl ester (DINCH) is a complex mixture of nine carbon branched-chain isomers. It has been used in Europe since 2002 as a plasticizer to replace phthalates such as di(2-ethylhexyl)phthalate (DEHP) and diisononyl phthalate (DINP). Urinary concentrations of the oxidative metabolites of DINCH, namely cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (MCOCH); cyclohexane-1,2-dicarboxylic acid-mono(oxo-isononyl) ester (MONCH); and cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (MHNCH), can potentially be used as DINCH exposure biomarkers. The concentrations of MCOCH, MONCH and MHNCH were measured by online solid phase extraction-high performance liquid chromatography-tandem mass spectrometry in urine collected in 2000 (n=114), 2001 (n=57), 2007 (n=23), 2009 (n=118), 2011 (n=94) and 2012 (n=121) from convenience groups of anonymous U.S. adult volunteers with no known DINCH exposure. None of the DINCH metabolites were detected in samples collected in 2000 and 2001. Only one sample collected in 2007 had measureable concentrations of DINCH metabolites. The detection rate for all three metabolites increased from 2007 to 2012. The presence of oxidative metabolites of DINCH in urine suggests that these oxidative metabolites can be used as DINCH biomarkers for exposure assessment even at environmental exposure levels.

Identification of potential biomarkers of exposure to di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), an alternative for phthalate plasticizers.

Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) is used as an alternative for some phthalate plasticizers. In rats, DINCH mostly eliminates in feces as cyclohexane-1,2-dicarboxylic acid (CHDA), mono isononyl ester (MINCH) or in urine as CHDA. However, CHDA is not a specific biomarker of DINCH and measuring MINCH in feces is impractical. To identify additional potential biomarkers, we administered DINCH (500 mg/kg body weight) in a single subcutaneous (SC) or oral dose to four adult female Sprague-Dawley rats. We collected 24-h urine samples before dosing (to be used as controls) and 24-h and 48-h after dosing, and serum at necropsy after 48 h. We positively identified and accurately quantified CHDA and cyclohexane-1,2-dicarboxylic [corrected] acid, mono hydroxyisononyl ester (MHNCH) using authentic standards. Moreover, we tentatively identified MINCH and 12 oxidative metabolites, including 4 cyclohexane ring oxidation products, based on their mass spectrometric-fragmentation patterns. CHDA and MHNCH levels were higher in the urine collected 24 h after oral than SC administration. By contrast, 48-h after dosing, CHDA urinary levels were similar regardless of the exposure route. We detected all but two of the urine metabolites also in serum. Levels of CHDA and MHNCH in serum were lower than in the two post-dose urine collections. Our results suggest that several urinary oxidative metabolites, specifically CHDA, mono oxoisononyl ester and MHNCH may be used as specific biomarkers of DINCH exposure in humans.

Urinary and serum metabolites of di-n-pentyl phthalate in rats.

Di-n-pentyl phthalate (DPP) is used mainly as a plasticizer in nitrocellulose. At high doses, DPP acts as a potent testicular toxicant in rats. We administered a single oral dose of 500 mg kg(-1)bw of DPP to adult female Sprague-Dawley rats (N=9) and collected 24-h urine samples 1d before and 24- and 48-h after DPP was administered to tentatively identify DPP metabolites that could be used as exposure biomarkers. At necropsy, 48 h after dosing, we also collected serum. The metabolites were extracted from urine or serum, resolved with high performance liquid chromatography, and detected by mass spectrometry. Two DPP metabolites, phthalic acid (PA) and mono(3-carboxypropyl) phthalate (MCPP), were identified by using authentic standards, whereas mono-n-pentyl phthalate (MPP), mono(4-oxopentyl) phthalate (MOPP), mono(4-hydroxypentyl) phthalate (MHPP), mono(4-carboxybutyl) phthalate (MCBP), mono(2-carboxyethyl) phthalate (MCEP), and mono-n-pentenyl phthalate (MPeP) were identified based on their full scan mass spectrometric fragmentation pattern. The ω-1 oxidation product, MHPP, was the predominant urinary metabolite of DPP. The median urinary concentrations (µg mL(-1)) of the metabolites in the first 24h urine collection after DPP administration were 993 (MHPP), 168 (MCBP), 0.2 (MCEP), 222 (MPP), 47 (MOPP), 26 (PA), 16 (MPeP), and 9 (MCPP); the concentrations of metabolites in the second 24 h urine collection after DPP administration were significantly lower than in the first collection. We identified some urinary metabolic products in the serum, but at much lower levels than in urine. Because of the similarities in metabolism of phthalates between rats and humans, based on our results and the fact that MHPP can only be formed from the metabolism of DPP, MHPP would be the most adequate DPP exposure biomarker for human exposure assessment. Nonetheless, based on the urinary levels of MHPP, our preliminary data suggest that human exposure to DPP in the United States is rather limited.

Selecting adequate exposure biomarkers of diisononyl and diisodecyl phthalates: data from the 2005-2006 National Health and Nutrition Examination Survey.

BACKGROUND: High-molecular-weight phthalates, such as diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP), are used primarily as polyvinyl chloride plasticizers. OBJECTIVES: We assessed exposure to DINP and DIDP in a representative sample of persons ≥ 6 years of age in the U.S. general population from the 2005-2006 National Health and Nutrition Examination Survey (NHANES). METHODS: We analyzed 2,548 urine samples by using online solid-phase extraction coupled to isotope dilution high-performance liquid chromatography-tandem mass spectrometry. RESULTS: We detected monocarboxyisooctyl phthalate (MCOP), a metabolite of DINP, and monocarboxyisononyl phthalate (MCNP), a metabolite of DIDP, in 95.2% and 89.9% of the samples, respectively. We detected monoisononyl phthalate (MNP), a minor metabolite of DINP, much less frequently (12.9%) and at concentration ranges (> 0.8 µg/L-148.1 µg/L) much lower than MCOP (> 0.7 µg/L- 4,961 µg/L). Adjusted geometric mean concentrations of MCOP and MCNP were significantly higher (p < 0.01) among children than among adolescents and adults. CONCLUSIONS: The general U.S. population, including children, was exposed to DINP and DIDP. In previous NHANES cycles, the occurrence of human exposure to DINP by using MNP as the sole urinary biomarker has been underestimated, thus illustrating the importance of selecting the most adequate biomarkers for exposure assessment.

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.

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.

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.

Urinary biomarkers of di-isononyl phthalate in rats.

Commercial di-isononyl phthalate (DiNP) is a mixture of various branched-chain dialkyl phthalates mainly containing nine-carbon alkyl isomers. At high doses in rodents, DiNP is a carcinogen, and a developmental toxicant. After exposure, the diester isomers are de-esterified to form hydrolytic monoesters, monoisononyl phthalates (MiNP), which subsequently metabolize to form oxidative metabolites. These metabolites can be excreted in urine or feces. The urinary excretion of DiNP metabolites was monitored in adult female Sprague-Dawley rats after oral administration of a single dose (300 mg/kg) of commercial DiNP. The metabolites were extracted from urine, resolved with high performance liquid chromatography, analyzed by mass spectrometry, and tentatively identified based on their chromatographic separation and mass spectrometric fragmentation pattern. Because DiNP is an isomeric mixture, its metabolites were also isomeric mixtures that eluted from the HPLC column with close retention times. Mono(carboxy-isooctyl)phthalate (MCiOP) was identified as the major metabolite of DiNP; in addition, mono(hydroxy-isononyl)phthalate (MHiNP) and mono(oxo-isononyl)phthalate (MOiNP) were present. Furthermore, metabolites of di-isooctyl phthalate (DiOP) and di-isodecyl phthalate (DiDP) were also detected. Excretion toxicokinetics of the DiNP metabolites in urine followed a biphasic pattern with initial rapid decay in concentration. Despite potential differences in the metabolism of DiNP among species, MCiOP, MHiNP and MOiNP were detected in humans with no known exposure to DiNP at levels significantly higher than MiNP suggesting that these oxidative metabolites may be better urinary biomarkers of human exposure to DiNP than is MiNP.

Urinary oxidative metabolites of di(2-ethylhexyl) phthalate in humans.

Di(2-ethylhexyl) phthalate (DEHP) is added to polyvinyl chloride (PVC) plastics used widely in medical devices and toys to impart flexibility and durability. DEHP produces reproductive and development toxicities in rodents. Initial metabolism of DEHP in animals and humans results in mono(2-ethylhexyl) phthalate (MEHP), which subsequently metabolizes to a wide range of oxidative metabolites before being excreted in urine and feces. We investigated the metabolism of DEHP in humans by identifying urinary oxidative metabolites of DEHP from individuals with urinary MEHP concentrations about 100 times higher than the median concentration in the general US population. In addition to the previously identified DEHP metabolites MEHP, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), and mono(2-carboxymethylhexyl) phthalate (MCMHP), we also identified for the first time in humans three additional oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), and mono(2-(1-oxoethyl)hexyl) phthalate (MOEHP) based on their chromatographic behavior and mass spectrometric fragmentation patterns. We also tentatively identified metabolites with two functional groups in the side alkyl chain as isomers of mono(2-hydroxyethyl-4-carboxybutyl) phthalate (MHECBP), mono(2-ethyl-4-oxo-5-carboxypentyl) phthalate (MEOCPP), and mono(2-ethyl-4-hydroxy-5-carboxypentyl) phthalate (MEHCPP). We report the presence of urinary DEHP metabolites in humans that have fewer than eight carbons in the alkyl chain. These metabolites were previously identified in rodents. Although quantitative information is not available, our findings suggest that, despite potential differences among species, the oxidative metabolism of DEHP in humans and rodents results in similar urinary metabolic products.

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.

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.

Detection of phthalate metabolites in human saliva.

In assessment of exposure to environmental contaminants, the use of unconventional matrices is becoming an increasingly important area of research. Saliva is one of the most promising alternative matrices because its collection is easy, noninvasive, and inexpensive. In this study, we measured the salivary concentrations of 14 phthalate metabolites in 39 anonymous adult volunteers using isotope-dilution, automated solid phase extraction-high performance liquid chromatography-tandem mass spectrometry. Seven phthalate metabolites were detected at the concentrations ranging from below the limit of detection (<1 ng/mL) to 10.6 ng/mL for phthalic acid, 3.1 ng/mL for monomethyl phthalate (MMP), 91.4 ng/mL for monoethyl phthalate (MEP), 65.8 ng/mL for mono-n-butyl phthalate (MBP), 17.9 ng/mL for mono-iso-butyl phthalate, 353.6 ng/mL for monobenzyl phthalate, and 6.8 ng/mL for mono-2-ethylhexyl phthalate (MEHP). The frequency of detection was highest for MBP (85%) and lowest for MMP (8%). The median salivary MBP level in this group of adults was higher than the median serum MBP level in another non-occupationally exposed human adult population in the United States, whereas, the median salivary levels of MEP and MEHP were lower than the corresponding median serum levels. The frequency of detection and the salivary levels of each phthalate monoester in this study population were lower than the frequency of detection and urinary level of the same monoester in the general US population. Although urine is preferred for exposure assessment to non-persistent chemicals such as phthalates, the similar levels in serum and saliva suggest that saliva could be used as a surrogate matrix for measuring the bioavailable dose of phthalates in biomonitoring studies.

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.