Methyl, ethyl, n-propyl, n-butyl, and benzyl paraben, 4-methylumbelliferyl glucuronide, 4-methylumbelliferyl sulfate, and β-glucuronidase/sulfatase (Helix pomatia, H1) were purchased from Sigma Chemical Co. (St. Louis, MO). ¹³C₄-4-Methylumbelliferone was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). d₄-Methyl paraben was purchased from C/D/N Isotopes Inc. (Quebec, Canada). d₄-Ethyl, d₄-n-propyl, and d₄-n-butyl parabens were obtained from CanSyn Chem Corp. (Toronto, Canada). Because d₄-benzyl parabens was not available, d₄-n-butyl paraben was used as the internal standard for benzyl paraben. The urine samples analyzed for this study were collected anonymously from a demographically diverse group of 100 U.S. male and female adults with no known exposure to parabens. The samples were collected from 2003 to 2005 at different times throughout the day—not necessarily first morning voids. The Centers for Disease Control and Prevention Human Subjects Institutional Review Board reviewed and approved the study protocol. A waiver of informed consent was requested under the Code of Federal Regulations dealing with human subjects [45 CFR 46.116(d)].

We measured the total concentrations of methyl, ethyl, n-propyl, butyl (n- and iso-), and benzyl parabens with a modification of a method for measuring other environmental phenols in urine (Ye et al. 2005a) using online solid-phase extraction high-performance liquid chromatography–tandem mass spectrometry (SPE-HPLC-MS/MS) (Ye et al. 2006). Briefly, 100 μL urine was spiked with 50 μL internal standard solution, 50 μL enzyme solution, and, to monitor the completion of the deconjugation reaction, 10 μL 4-methylumbelliferyl glucuronide/4-methylumbelliferyl sulfate/¹³C₄-4-methylumbelliferone standard solution (Ye et al. 2006). Samples were incubated for 4 hr at 37°C and then acidified with 0.1 M formic acid. The online SPE-HPLC-MS/MS system consisted of several Agilent 1100 modules (Agilent Technologies, Wilmington, DE)—two binary HPLC pumps, an autosampler with a 900-μL injection loop, and one column compartment with a 10-port switching valve, and an API 4000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) equipped with an atmospheric pressure chemical ionization interface. The procedure for extracting the deconjugated parabens from the urine involved concurrent online SPE-HPLC operation with peak focusing. While the autosampler and one of the HPLC pumps were used for the SPE cleanup of one sample, the 10-port switching valve, the second HPLC pump, and the mass spectrometer were used to collect data from the previous sample (Ye et al. 2006). The limits of detection (LODs), calculated as 3S₀, where S₀ is the standard deviation as the concentration approaches zero (Taylor 1987), were 0.13 ng/mL (methyl paraben), 0.18 ng/mL (n-propyl paraben), and 0.1 ng/mL (ethyl, butyl, and benzyl parabens) (Ye et al. 2006).

To determine the concentrations of the free species (C_free), we followed the above procedure without the enzymatic hydrolysis treatment. We used β-glucuronidase (Escherichia coli-K12; Roche Applied Science, Penzberg, Germany) to selectively deconjugate glucuronide metabolites and determine the concentrations of free plus glucuronidated species (C_free+glu). Similarly, we used β-glucuronidase/sulfatase (H. pomatia, 463 000U/g solid) to deconjugate both glucuronide and sulfate metabolites and to determine the concentrations of free plus glucuronidated plus sulfated species (C_free+glu+sul). We calculated the concentration of glucuronide conjugates by subtracting C_free from C_glu+free. Similarly, we calculated the concentration of sulfate metabolites by subtracting C_free+glu+sul from C_free+glu (Ye et al. 2005b).

To ensure accuracy and precision of the data, we analyzed quality control (QC) materials along with the samples. Low-concentration (QCL, 2–5 ng/mL) and high-concentration (QCH, 10–50 ng/mL) QC materials were prepared from a base urine pool obtained from multiple anonymous donors (Ye et al. 2006). The materials were dispensed in 1-mL aliquots and stored at −70°C. Each QC material was characterized to define the mean and the 95% and 99% control limits of parabens concentrations using a minimum of 40 repeated measurements during a 2-week period.

We performed the statistical analyses using the Statistical Analysis System (SAS Institute, Cary, NC) software. For concentrations below the LOD, we used a value equal to the LOD divided by the square root of 2 (Hornung and Reed 1990). Statistical significance was set at p < 0.05.

We measured total (i.e., free plus glucuronidated and sulfated conjugates) concentrations of five parabens in 100 human urine samples collected between 2003 and 2005 from 100 adult anonymous volunteers with no known occupational exposure to these compounds. The median concentrations, selected percentiles, and frequency of detection of each paraben are all shown in Table 1.

Methyl and n-propyl paraben were detected in almost all of the samples (99% and 96%, respectively). Two other parabens were detected in more than half of the samples (ethyl, 58%; butyl, 69%); benzyl paraben was detected less frequently (39%) (Table 1). Median urinary concentrations were highest for methyl (43.9 ng/mL) and n-propyl (9.1 ng/mL) parabens. Methyl paraben was also the most abundant (mean value of 12.8 ng/g) among 6 parabens tested in human breast tumors (Darbre et al. 2004). The higher frequency of detection and median urinary concentrations of methyl and n-propyl parabens could be related to a) the fact that these are the most widely used parabens in cosmetics and food processing (Jackson 1992), b) differences in the absorption, distribution, metabolism, and excretion of the various parabens, or c) a combination of these factors. Furthermore, the high correlation (Pearson correlation coefficient r = 0.92, p < 0.0001) between total urinary concentrations of methyl and n-propyl parabens (Figure 1) suggests that human exposures to methyl and n-propyl parabens most likely share common sources.

Differences in metabolic profiles have been found depending on the exposure route (Bando et al. 1997; Daston 2005; Harvey 2005; Soni et al. 2005). After oral administration, parabens are most likely hydrolyzed by nonspecific esterases, widely distributed in the body, including the gut (Daston 2005). After dermal exposure, parabens can also be hydrolyzed by esterases present in human skin and subcutaneous fat tissue (Lobemeier et al. 1996). The presence of unchanged parabens in breast tumor tissues (Darbre et al. 2004) suggested, however, that at least a fraction of the parabens can be absorbed without hydrolysis. The high frequency of detection of some parabens in our study confirms that, after exposure, unhydrolyzed parabens may be excreted in urine.

The median concentrations of free methyl, ethyl, and n-propyl parabens were lower than their corresponding median total concentrations (Table 1). For methyl and n-propyl parabens, the total concentrations were about 50- and 10-fold higher, respectively, than the median concentrations of free species. This suggests that these parabens are mostly excreted in urine as conjugates (Table 1). Moreover, the correlations between the concentrations of free and total (free plus conjugated) species of methyl and n-propyl parabens were highly significant (p < 0.0001 and p = 0.0096, respectively; Figure 2). In other words, high concentrations of total methyl- or n-propyl parabens tended to be related to high concentrations of the corresponding free paraben. These findings suggest that parabens, like many xenobiotics, undergo phase II biotransformations to produce glucuronide or sulfate conjugates with increased water solubility that are more amenable to urinary excretion than are the free species (de Wildt et al. 1999; Wang and James 2006). If the biologically active compound were the free species (van den Berg et al. 2003), urinary excretion of the conjugated species would reduce the bioavailable concentration of the free species for target organs, thus minimizing the potential adverse effects related to parabens exposure.

We also examined the distribution of glucuronide and sulfate conjugates of parabens (Table 2). The sulfate conjugate represented 67% (methyl paraben) and 55% (n-propyl paraben) of the total amount of each paraben excreted in urine. The percentage of the glucuronide conjugate was 28% for methyl paraben, and 43% for n-propyl paraben; only a small amount of these parabens was excreted in free form (5% for methyl and 2% for n-propyl). The urinary concentrations of both glucuronide and sulfate conjugates correlated well with the concentrations of the total species (r ranged from 0.78 to 0.93) (Figure 3). The concentration of conjugated species increased with the concentration of total species, even at the highest concentrations of total species. These findings are in agreement with data on exposure of humans to phthalates, another class of environmental chemicals (Silva et al. 2006). The findings also suggest that saturation or inhibition of the enzymes catalyzing the glucuronidation or sulfatation reactions did not occur at environmental exposure levels.

In conclusion, our preliminary data suggest that parabens can be present in urine, mostly in their conjugated form. To our knowledge, this is the first comprehensive report on the urinary concentrations of individual parabens and their conjugates in humans. The high frequency of detection for methyl, ethyl, n-propyl, and butyl parabens suggests that parabens and their conjugates could be valid biomarkers to assess human exposures to these compounds. Additional information, including a better understanding of the metabolism of parabens in humans, is needed to link these biomarker measurements to exposure and to internal dose.