Simultaneous determination of triclosan, triclocarban, triclocarban metabolites and byproducts in urine and serum by ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

RATIONALE: Triclosan (TCS) and triclocarban (TCC) are ubiquitous antimicrobial agents incorporated in consumer and personal care products. Due to their human health risks, it is essential to develop a sensitive and accurate analytical method to simultaneously quantify TCS, TCC, as well as their metabolites and byproducts in urine and serum samples. METHODS: The quantitative parameters of TCS, TCC, TCC metabolites and byproducts (2'-OH-TCC, 3'-OH-TCC, 6-OH-TCC, DHC, DCC, NCC) were optimized by using ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UHPLC/ESI-MS/MS). Enzymatic hydrolysis of the samples was optimized based on enzyme dosage and incubation time. The efficiencies of solid-phase extraction (SPE) and liquid-liquid extraction (LLE) were compared. The effectiveness of the established method was evaluated, and method application was validated using real urine and serum samples. RESULTS: The conjugates were sufficiently hydrolyzed under 500 U/mL ß-glucuronidase and 80 U/mL sulfatase at 37°C for 4 h. Compared with the LLE method, SPE achieved higher extraction efficiency in both urine and serum samples. The optimized SPE-UHPLC/ESI-MS/MS method showed low limits of detection (LODs) in the range 0.001-0.3 ng/mL and good linearity (R2 > 0.99) at 0.01-150 ng/mL in both matrices. Excellent recoveries of 82.0%-120.7% (urine) and 76.7%-113.9% (serum) were obtained with low relative standard deviation (RSD, <7.6%) for inter-day and intra-day injections. This method was applicable to quantify target compounds in multiple biological urine and serum samples. Notably, TCS and TCC were detected with average concentrations of 8.37 and 10.46 ng/mL, respectively, in 15 Chinese female urine samples, with the simultaneous detection of TCC metabolites and byproducts. CONCLUSIONS: A reliable method was established to simultaneously determine TCS, TCC, TCC metabolites and byproducts in urine and serum samples by using UHPLC/ESI-MS/MS. This sensitive methodology provides the basis for the evaluation of TCS and TCC exposure at the metabolic level.

Human fetal exposure to triclosan and triclocarban in an urban population from Brooklyn, New York.

Triclosan (TCS) and triclocarban (TCC) are antimicrobial agents formulated in a wide variety of consumer products (including soaps, toothpaste, medical devices, plastics, and fabrics) that are regulated by the U.S. Food and Drug Administration (FDA) and U.S. Environmental Protection Agency. In late 2014, the FDA will consider regulating the use of both chemicals, which are under scrutiny regarding lack of effectiveness, potential for endocrine disruption, and potential contribution to bacterial resistance to antibiotics. Here, we report on body burdens of TCS and TCC resulting from real-world exposures during pregnancy. Using liquid chromatography tandem mass spectrometry, we determined the concentrations of TCS, TCC, and its human metabolites (2'-hydroxy-TCC and 3'-hydroxy-TCC) as well as the manufacturing byproduct (3'-chloro-TCC) as total concentrations (Σ-) after conjugate hydrolysis in maternal urine and cord blood plasma from a cohort of 181 expecting mother/infant pairs in an urban multiethnic population from Brooklyn, NY recruited in 2007-09. TCS was detected in 100% of urine and 51% of cord blood samples after conjugate hydrolysis. The interquartile range (IQR) of detected TCS concentrations in urine was highly similar to the IQR reported previously for the age-matched population of the National Health and Nutrition Examination Survey (NHANES) from 2003 to 2004, but typically higher than the IQR reported previously for the general population (detection frequency = 74.6%). Urinary levels of TCC are reported here for the first time from real-world exposures during pregnancy, showing a median concentration of 0.21 µg/L. Urinary concentrations of TCC correlated well with its phase-I metabolite ∑-2'-hydroxy-TCC (r = 0.49) and the manufacturing byproduct ∑-3'-chloro-TCC C (r = 0.79), and ∑-2'-hydroxy-TCC correlated strongly with ∑-3'-hydroxy-TCC (r = 0.99). This human biomonitoring study presents the first body burden data for TCC from exposures occurring during pregnancy and provides additional data on composite exposure to TCS (i.e., from both consumer-product use and environmental sources) in the maternal-fetal unit for an urban population in the United States.

An immunoassay to evaluate human/environmental exposure to the antimicrobial triclocarban.

A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum 1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC(50) and detection range for TCC in buffer were 0.70 and 0.13-3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4'-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N'-glucuronide. TCC concentrations of biosolids measured by the ELISA were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid, inexpensive, and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects.

Triclosan/triclocarban levels in maternal and umbilical blood samples and their association with fetal malformation.

Triclosan (TCS) and triclocarban (TCC) are widely used as antimicrobial compounds in consumer products. TCS and TCC are frequently found in waste water and sewage. In this study, we investigate the potential impact of exposure to triclosan (TCS) and triclocarban (TCC) on fetal abnormalities. We measured TCS and TCC levels in maternal and umbilical cord blood samples from 39 pregnant women diagnosed with fetal or post-birth abnormalities at Beijing Obstetrics and Gynecology Hospital. 52 pregnant women who gave birth to healthy neonates during the same period of time were included as controls. Applying ultra-performance liquid chromatography-tandem mass spectrometry, TCS and TCC concentrations were measured in maternal and fetal sera. Significantly increased levels of TCS were detected in maternal sera from mothers with abnormal births. Similar levels of TCS or TCC were found in maternal and cord sera in control group. The concentrations of TCS or TCC in maternal sera correlated with those in umbilical cord sera (r=0.649, P<0.01). These observations suggest that maternal blood test could be a useful assay for detecting fetal exposure to TCS and TCC, and high exposure to TCS may be potentially associated with increased risk for fetal malformations.

Association of birth outcomes with fetal exposure to parabens, triclosan and triclocarban in an immigrant population in Brooklyn, New York.

BACKGROUND: Prior studies suggest associations between fetal exposure to antimicrobial and paraben compounds with adverse reproductive outcomes, mainly in animal models. We have previously reported elevated levels of these compounds for a cohort of mothers and neonates. OBJECTIVE: We examined the relationship between human exposure to parabens and antimicrobial compounds and birth outcomes including birth weight, body length and head size, and gestational age at birth. METHODS: Maternal third trimester urinary and umbilical cord blood plasma concentrations of methylparaben (MePB), ethylparaben (EtPB), propylparaben (PrPB), butylparaben (BuPB), benzylparaben (BePB), triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether or TCS) and triclocarban (1-(4-chlorophenyl)-3-(3,4-dichlorophenyl) urea or TCC), were measured in 185 mothers and 34 paired singleton neonates in New York, 2007-2009. RESULTS: In regression models adjusting for confounders, adverse exposure-outcome associations observed included increased odds of PTB (BuPB), decreased gestational age at birth (BuPB and TCC) and birth weight (BuPB), decreased body length (PrPB) and protective effects on PTB (BePB) and LBW (3'-Cl-TCC) (p<0.05). No associations were observed for MePB, EtPB, or TCS. CONCLUSIONS: This study provides the first evidence of associations between antimicrobials and potential adverse birth outcomes in neonates. Findings are consistent with animal data suggesting endocrine-disrupting potential resulting in developmental and reproductive toxicity.

Effect of method of delivering nicarbazin to mallards on plasma 4,4'-dinitrocarbanilide levels and reproduction.

Nicarbazin (NCZ), a coccidiostat used in the poultry industry, has been developed as a contraceptive for resident Canada geese. We tested the efficacy of NCZ as a contraceptive using mallards (Anas platyrhynchos) as a model for Canada geese. Nicarbazin-treated corn was fed ad libitum for 14 d at 0, 750, 1,000, or 1,500 ppm. Plasma and egg levels of 4,4'-dinitrocarbanilide (DNC), the active anticoccidial component of NCZ, differed among treatment groups in a dose-response relationship, but plasma levels did not differ between sexes. Nicarbazin caused a decrease in egg weight, but there was no effect of NCZ on the numbers of eggs laid per female per day. Nicarbazin did not significantly impact bird health. An additional trial tested the effect of the method of NCZ delivery on plasma DNC levels. Mallards were given NCZ daily for 12 d either by gavage with a corn oil suspension, gavage with a water suspension, peroral administration of a capsule, or feeding 500 mg of NCZ/kg of pelleted feed ad libitum. The method of delivery significantly affected plasma DNC levels, with the highest levels in the corn oil suspension group and the lowest levels in the pelleted feed group. This is likely due to decreased availability of NCZ in a pellet compared with gavage with a suspension or capsule. Mallards receiving 34.2 mg of NCZ/kg of BW when fed cracked corn coated with NCZ daily for 14 d had higher plasma DNC levels than those obtained by liquid gavage, capsule, or pelleted NCZ feed. For maximum effect in the field, NCZ should be coated onto corn. A higher concentration of NCZ is needed in pelleted feed to obtain comparable plasma DNC levels to allow for the decreased absorption of DNC.

Comparison of nicarbazin absorption in chickens, mallards, and Canada geese.

Nicarbazin (NCZ), a coccidiostat commonly used in the poultry industry, causes reduced hatchability and egg quality in layer hens at a concentration of 125 ppm (8.4 mg/kg) in the feed. Although this effect is undesirable in the poultry industry, NCZ could provide a useful wildlife contraception tool for waterfowl, particularly urban geese. We tested the absorption of NCZ in chickens (Gallus gallus), mallards (Anas platyrhynchos), and Canada geese (Branta canadensis) gavaged with 8.4 mg of NCZ/kg per bird each day for 8 d. Plasma levels of 4,4'-dinitrocarbanilide (DNC) differed significantly among species. Peak plasma DNC levels were 2.87 +/- 0.15 microg/mL, 2.39 +/- 0.15 microg/mL, and 1.53 +/- 0.15 microg/ mL in chickens, mallards, and Canada geese respectively. It took 6 d to obtain peak DNC levels in chickens as opposed to 8 d in mallards and Canada geese. The half life of DNC in plasma was 1.43 d in chickens, 0.72 d in mallards, and 1.26 d in Canada geese. Mallards eliminated 100% of plasma DNC 4 d post-treatment, whereas Canada geese eliminated 100% of plasma DNC 8 d post-treatment. Chickens had only eliminated 99% of plasma DNC 8 d post-treatment. Mallard plasma DNC levels were highly correlated with Canada goose plasma DNC levels. This research showed mallards are an ideal model species for the Canada goose for future reproductive studies on NCZ in a laboratory setting. However, levels higher than 8.4 mg/kg must be fed to waterfowl in order to obtain a plasma level comparable to chickens.

Early life triclocarban exposure during lactation affects neonate rat survival.

Triclocarban (3,4,4'-trichlorocarbanilide; TCC), an antimicrobial used in bar soaps, affects endocrine function in vitro and in vivo. This study investigates whether TCC exposure during early life affects the trajectory of fetal and/or neonatal development. Sprague Dawley rats were provided control, 0.2% weight/weight (w/w), or 0.5% w/w TCC-supplemented chow through a series of 3 experiments that limited exposure to critical growth periods: gestation, gestation and lactation, or lactation only (cross-fostering) to determine the susceptible windows of exposure for developmental consequences. Reduced offspring survival occurred when offspring were exposed to TCC at concentrations of 0.2% w/w and 0.5% w/w during lactation, in which only 13% of offspring raised by 0.2% w/w TCC dams survived beyond weaning and no offspring raised by 0.5% w/w TCC dams survived to this period. In utero exposure status had no effect on survival, as all pups nursed by control dams survived regardless of their in utero exposure status. Microscopic evaluation of dam mammary tissue revealed involution to be a secondary outcome of TCC exposure rather than a primary effect of compound administration. The average concentration of TCC in the milk was almost 4 times that of the corresponding maternal serum levels. The results demonstrate that gestational TCC exposure does not affect the ability of dams to carry offspring to term but TCC exposure during lactation has adverse consequences on the survival of offspring although the mechanism of reduced survival is currently unknown. This information highlights the importance of evaluating the safety of TCC application in personal care products and the impacts during early life exposure.

Percutaneous absorption of triclocarban in rat and man.

The route and rate of excretion by rats of the germicide (1 4 C) Triclocarban formerly called trichlorocarbanilide, given by parenteral injection has been investigated. Blood levels based on radioactivity and by chemical determination after parenteral injection have been compared with those obtained after topical application of (1 4 C) Triclocarban in soaps and in dimethylformamide (DMF) through occluded rat skin has been studied. Other soaps and a hand cleanser containing (1 4 C) Triclocarban have been applied to rat skin without occlusion and the effects of duration of contact, concentration and the use of a solubilizer have been investigated. In humans, absorption of Triclocarban through skin after bathing daily for 28 days has been investigated by chemical analysis of blood and urine. The data show that elimination by the rat is rapid and complete principally via the faeces. Blood levels after parenteral injection are low and comparison of the radioactivity and chemical determinations suggest rapid metabolism of the Triclocarban. After application to the skin, blood levels based on 1 4 C are very low. Absorption of (1 4 C) Triclocarban through occluded rat skin was greater from DMF than from soaps. With non-occluded rat skin, absorption from soaps was less and was dependent on concentration but independent of duration of contact. The use of a solubilizer did not increase absorption through skin. No measurable Triclocarban (less than 25 ppb) was present in blood and urine samples of volunteers during or shortly after a 28-day intensive bathing regimen.

Skin deposition and penetration of trichlorocarbanilide.

Studies are reported on the localization and quantitative distribution of 3,4,4'-trichloro[14C]carbanilide([14C]TCC) in guinea-pig and human skin, and on the percutaneous absorption of TCC following topical application to guinea pigs. [14C]TCC was applied to guinea-pig skin in various vehicles (conventional, superfatted, 10 percent non-soap detergent (NSD) and 30 percent NSD soap suspensions or in N,N-dimethylformamide [DMF]) and under various conditions (e.g. freshly prepared or equilibrated suspensions; single or multiple washes). Most of the amount of TCC remaining in the skin after rinsing was deposited on the skin surface and only relatively minute amounts actually penetrated through the epidermis into the dermis. Whereas conventional soap facilitated a greater deposition of TCC on the skin surface than NSD, the latter caused greater amounts of TCC to be deposited in the pilosebaceous system and lower dermis than conventional soap. The absence of TCC in the blood and tissues of guinea pigs given topical applications of TCC lends further support to the very low order of percutaneous absorption of TCC. The localisation of TCC followed a similar pattern in human skin as in guinea-pig skin, but the amount deposited was less and the rate of disappearance was more for human than for guinea-pig skin.

Quantitative analysis of triclocarban in blood.

A method is presented for quantitatively determining triclocarban in blood. Triclocarban is extracted from blood with ether, isolated by TLC, and measured through its UV absorption at 265 nm in methanol. This method is sensitive to 250 ng (50 ppb in 5 ml of blood) of free triclocarban with a relative standard deviation of 5.2%, correlated with a radiotracer analysis of 14C-labeled triclocarban. It has been applied successfully to the analysis of triclocarban in human and rabbit blood.

GLC determination of free triclocarban in blood.

A method is presented for the quantitative determination of free triclocarban in rat or human blood. The procedure involves the extraction from blood with acetone, a TLC cleanup, derivatization with N,O-bis (trimethylsily) acetonide, and GLC using an electron-capture detector. GLC-mass spectral analysis confirmed that the structure of the derivative was a bis (trimethylsilyl) molecule with one group on a nitrogen and the second group attached to the enol tautomer. The method is sensitive to 25 ng (12.5 microgram/liter of blood). Recoveries of added triclocarban in the 12.5--50-microgram/liter range weree between 80 and 90%.

Quantification of plasma and egg 4,4'-dinitrocarbanilide (DNC) residues for the efficient development of a nicarbazin-based contraceptive for pest waterfowl.

Urbanization and associated landscaping has increased the abundance of year-round habitat for waterfowl, resulting in vegetation damage, loss of recreational activities, air transportation mishaps and health hazards. As part of a research program to develop socially acceptable techniques for management of pest bird populations, we are evaluating nicarbazin as a contraceptive in pest and surrogate avian species. As reproductive studies with Canada Geese (Branta canadensis) are tedious due to the difficulty of conducting controlled field studies and/or breeding geese in captivity, we evaluated the effects of oral nicarbazin administration on the production and hatchability of chicken eggs. Blood plasma and egg DNC concentrations were correlated to contraceptive efficacy. Subsequent studies are being conducted with geese to determine the diet nicarbazin concentration required to produce the desired blood and plasma DNC concentrations. This approach permits the expeditious evaluation of formulations and dosing regimes by simply monitoring blood DNC concentrations in target species.

High-pressure liquid chromatographic studies of TCC and metabolites in experimental animals and man.

A reversed-phase high performance liquid chromatographic (HPLC) procedure for the analyses of 3,4,4'-trichlorocarbanilide (TCC) and its free and/or conjugated metabolic products in plasma/serum in described. A rapid, effective clean-up procedure, prior to chromatographic evaluation, involves a single-step combined protein removal and THF extraction. Detection of the TCC moiety after HPLC separation is by UV absorption at 265 nm; quantitation by peak height measurement. A detection limit of 10 ppb of TCC and/or metabolities has been demonstrated for this method. Verification of this method was by radiotracer counting of the appropriate HPLC peaks from plasma of animals adminstered 14C-TCC/TCC. The utility of this method was demonstrated in both animal pharmacological and toxicological studies and in human bathing studies.

Percutaneous penetration and disposition of triclocarban in man: body showering.

The percutaneous penetration and metabolic disposition of 14C-3-4,4'-trichlorocarbanilide (triclocarban, TCC) after body showering has been determined in man. Single intravenous doses of trace amounts 14C-triclocarban were given to subjects to determine the pharmacologic disposition of the compound before conducting the shower study. Radioactivity was rapidly cleared from blood after intravenous administrations of 14C-triclocarban in propylene glycol with a blood clearance half-life of 8.6 hours. About 54% of the dose was excreted in the feces and 21% of the dose in the urine with a urinary elimination half-life of ten hours. No radioactivity was detected in the saliva. Subjects took a single shower employing a whole body lather with approximately 7 gm of soap containing 2% 14C-triclocarban on a soap basis. Special blood withdrawal and urinary/fecal collection techniques were utilized that minimized contamination of samples by extraneous 14C-triclocarban. About 0.23% of the applied radioactive dose was recovered in the feces after six days and 0.16% of the dose in the urine after two days. At all sampling times blood levels of radioactivity were below the detection limit of the method (10 part per billion).

Whole blood is the sample matrix of choice for monitoring systemic triclocarban levels.

The antibacterial triclocarban (TCC) concentrates in the cellular fraction of blood. Consequently, plasma levels are at least two-fold lower than the TCC amount present in blood. Utilizing whole blood sampling, a low but significant absorption of TCC from soap during showering is demonstrated for a small group of human subjects.

Automated on-line column-switching HPLC-MS/MS method for the quantification of triclocarban and its oxidative metabolites in human urine and serum.

3,4,4'-Trichlorocarbanilide (triclocarban, TCC) is widely used as an antimicrobial agent in a variety of consumer and personal care products. Because of its widespread use, the potential for human exposure to TCC is high. Human exposure to TCC may be assessed by measuring the concentrations of conjugated or free species of TCC and its two oxidative metabolites, 2'-hydroxy-TCC (2'-OH-TCC) and 3'-hydroxy-TCC (3'-OH-TCC), in urine or serum. To assess human exposure to TCC, we developed a method that uses restricted access materials (RAM) on-line solid phase extraction (SPE) coupled to high performance liquid chromatography-isotope dilution tandem mass spectrometry with peak focusing (HPLC-MS/MS). Sample clean-up by RAM relies on both size exclusion chromatography, to remove the high-molecular matrix components, and reversed phase partition, to extract and pre-concentrate the target analytes. TCC, 2'-OH-TCC and 3'-OH-TCC present in urine or serum were concentrated on the RAM SPE column, back-eluted from the SPE column, diluted through a mixing tee for peak focusing, separated by HPLC, and detected by isotope dilution-MS/MS. The method required a small amount of sample (50 µL) and minimal sample pretreatment. The limits of detection (LOD) ranged from 0.01 to 0.1 ng/mL. The method was applied to measure TCC and its metabolites in 158 urine and 16 serum samples collected from adults with no known exposure to TCC. TCC was detected in 35.4% of the urine samples (range:

Biomarkers of exposure to triclocarban in urine and serum.

3,4,4'-Trichlorocarbanilide (triclocarban, TCC) is widely used as an antimicrobial agent in a variety of consumer and personal care products. TCC is considered a potential endocrine disruptor, but its potential toxic effects in humans are still largely unknown. Because of its widespread uses, the potential for human exposure to TCC is high. In order to identify adequate exposure biomarkers of TCC, we investigated the metabolic profile of TCC in adult female Sprague Dawley rats after administering TCC once (500 mg/kg body weight) by oral gavage. Urine was collected 0-24 h before dosing, and 0-24 h and 24-48 h after dosing. Serum was collected at necropsy 48 h after dosing. We identified several metabolites of TCC in urine and serum by on-line solid phase extraction-high performance liquid chromatography-mass spectrometry. We unambiguously identified two major oxidative metabolites of TCC, 3'-hydroxy-TCC and 2'-hydroxy-TCC, by comparing their chromatographic behavior and mass spectral fragmentation patterns with those of authentic standards. By contrast, compared to these oxidative metabolites, we detected very low levels of TCC in the urine or serum. Taken together these data suggest that in rats, oxidation of TCC is a major metabolic pathway. We also measured TCC and its oxidative metabolites in 50 urine and 16 serum samples collected from adults in the United States. The results suggest differences in the metabolic profile of TCC in rats and in humans; oxidation appears to be a minor metabolic pathway in humans. Total (free plus conjugated) TCC could serve as a potential biomarker for human exposure to TCC.

Pharmacokinetics of imidocarb in normal dogs and goats.

The pharmacokinetics of imidocarb were studied in seven mongrel dogs and eight crossbred goats. An intravenous bolus dose (4 mg/kg) of 12% imidocarb dipropionate solution was injected into the cephalic vein in dogs and the jugular vein in goats. The plasma concentration of imidocarb was measured by spectrophotometry. The experimental data were analysed using a two-compartment open model. The apparent volume of the central compartment was significantly higher (P less than 0.01) in dogs than in goats. The significantly larger (P less than 0.05) apparent specific volume of distribution in goats than in dogs may be attributed to passive diffusion followed by ion trapping of the drug in rumen fluid. Neither the half-life nor body clearance differed significantly between dogs (t1/2, 207 +/- 45 min; ClB, 1.47 +/- 0.38 ml/min kg) and goats (t1/2, 251 +/- 94 min; ClB, 1.62 +/- 0.50 ml/min kg). While almost 80% of the dose had been eliminated at 8 h in both species, the high ratio of the imidocarb level in the peripheral-to-central compartment in goats suggests that a prolonged period may be required for complete elimination of the drug.