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.

Quantification of tetrabromo benzoic acid and tetrabromo phthalic acid in rats exposed to the flame retardant Uniplex FPR-45.

The first withdrawal of certain polybrominated diphenyl ethers flame retardants from the US market occurred in 2004. Since then, use of brominated non-PBDE compounds such as bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP) and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) in commercial formulations has increased. Assessing human exposure to these chemicals requires identifying metabolites that can potentially serve as their biomarkers of exposure. We administered by gavage a dose of 500 mg/Kg bw of Uniplex FRP-45 (>95 % BEH-TEBP) to nine adult female Sprague-Dawley rats. Using authentic standards and mass spectrometry, we positively identified and quantified 2,3,4,5-tetrabromo benzoic acid (TBBA) and 2,3,4,5-tetrabromo phthalic acid (TBPA) in 24-h urine samples collected 1 day after dosing the rats and in serum at necropsy, 2 days post-exposure. Interestingly, TBBA and TBPA concentrations correlated well (R (2) = 0.92). The levels of TBBA, a known metabolite of EH-TBB, were much higher than the levels of TBPA both in urine and serum. Because Uniplex FRP-45 was technical grade and EH-TBB was present in the formulation, TBBA likely resulted from the metabolism of EH-TBB. Taken together, our data suggest that TBBA and TBPA may serve as biomarkers of exposure to non-PBDE brominated flame retardant mixtures. Additional research can provide useful information to better understand the composition and in vivo toxicokinetics of these commercial mixtures.

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.

Variability over 1 week in the urinary concentrations of metabolites of diethyl phthalate and di(2-ethylhexyl) phthalate among eight adults: an observational study.

BACKGROUND: Phthalates are metabolized and eliminated in urine within hours after exposure. Several reports suggest that concentrations of phthalate metabolites in a spot urine sample can provide a reliable estimation of exposure to phthalates for up to several months. OBJECTIVES: We examined inter- and intraperson and inter- and intraday variability in the concentrations of monoethyl phthalate (MEP), the major metabolite of diethyl phthalate, commonly used in personal care products, and mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), a metabolite of di(2-ethylhexyl) phthalate (DEHP), a polyvinyl chloride plasticizer of which diet is the principal exposure source, among eight adults who collected all urine voids (average, 7.6 samples/person/day) for 1 week. METHODS: We analyzed the urine samples using online solid-phase extraction coupled to isotope dilution-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Regardless of the type of void (spot, first morning, 24-hr collection), for MEP, interperson variability in concentrations accounted for > 75% of the total variance. By contrast, for MEHHP, within-person variability was the main contributor (69-83%) of the total variance. Furthermore, we observed considerable intraday variability in the concentrations of spot samples for MEHHP (51%) and MEP (21%). CONCLUSIONS: MEP and MEHHP urinary concentrations varied considerably during 1 week, but the main contributors to the total variance differed (interday variability, MEHHP; interperson variability, MEP) regardless of the sampling strategy (spot, first morning, 24-hr collection). The nature of the exposure (diet vs. other lifestyle factors) and timing of urine sampling to evaluate exposure to phthalates should be considered. For DEHP and phthalates to which people are mostly exposed through diet, collecting 24-hr voids for only 1 day may not be advantageous compared with multiple spot collections. When collecting multiple spot urine samples, changing the time of collection may provide the most complete approach to assess exposure to diverse phthalates.

Cross validation and ruggedness testing of analytical methods used for the quantification of urinary phthalate metabolites.

Since the publication of our first analytical method in 2000 to detect and quantify phthalate metabolites in human urine, we have modified the method several times to improve performance, reduce the volume of matrix and solvents used, and to increase the number of analytes in one analytical run. We performed cross method validation and ruggedness testing after each modification to ensure that the analytical method adopted is robust and produces accurate and reproducible data when compared to the previously used method. Here, we present the results from the evaluation of the ruggedness of our analytical approach under variable experimental conditions, using the current analytical method. Minor deviations of the standard experimental conditions, i.e., pH, incubation time, amount of deconjugation enzyme, and incubation temperature, had no effect on final analyte concentrations. Furthermore, we validated the method to ensure accuracy at concentrations beyond the highest calibration standard. The concentrations obtained by using a lower volume of urine agreed well with original levels, suggesting broad linear calibration range as well as complete hydrolysis of the glucuronide conjugates with the standard amount of beta-glucuronidase used for deglucuronidation; also, the time of incubation (90 min) was adequate regardless of the amount of glucuronide present. We also summarize the precision of concentration data acquired by the five different analytical approaches we have used since 2000. The correlation plots of concentration data for each analyte obtained from split sample analysis, using three of these approaches, produced linear curves (R(2)>0.98) with slopes and intercepts that were not statistically different (p>0.05) from 1 and 0, respectively. These results suggest that the data are reproducible and accurate, regardless of the analytical method used. Furthermore, analysis of quality control urine samples made over the years confirmed the stability of the phthalate metabolites in urine at -70 degrees C for several years and the consistency of the analytical measurements obtained by using various methodological approaches over time.

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.

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.

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.

Comprehensive solid-phase extraction method for persistent organic pollutants. Validation and application to the analysis of persistent chlorinated pesticides.

The Centers for Disease Control and Prevention (CDC) is involved in many epidemiological studies regarding the measurement of chlorinated pesticides and polychlorinated biphenyls in specimens obtained from humans. In addition to these commonly determined analytes, there is a need to include additional persistent organic pollutants (POPs) in our analyses, which further stresses the analyses because sample volumes remain small. Thus, a single method of analysis for all POPs in human serum is needed. CDC has recently developed a semiautomated and comprehensive solid-phase extraction method for POPs. The method is comprehensive since it was optimized for the extraction of many different POP compound classes. We then developed a purification and fractionation scheme that allows (a) separation of different compound classes by particular functionalities and (b) purification of those fractions to remove coextracted interferences. This paper describes the first step in the semiautomated comprehensive extraction and multiple fractionation method developed by CDC for monitoring POPs. In this paper, we validate the analysis of the persistent chlorinated pesticides, a compound class difficult to examine because of their structural diversity, in human plasma. The method was validated against an existing CDC method by using a spiked quality-control serum pool. The concentrations determined for all analytes using both methods were within 2%-14% relative standard deviations. A multilevel (i.e., 3-4 point) matrix spike showed good linearity for the analytes tested (r2 = 0.978-0.999). The method was then applied to 40-year-old archived plasma samples for the quantitative analysis of selected chlorinated pesticides. Mean recoveries of the 13C-labeled internal quantification standards ranged from 64% to 123% for the 11 monitored pesticides. The overall method proved to be robust by handling old coagulated plasma samples. It allowed faster throughput of samples than our previous methods and provided cleaner samples with less frequent interferences or background as analyzed by high-resolution mass spectrometry. The method represents a preliminary step in establishing an automated, comprehensive multiresidue analysis method for POPs in human serum.