A novel method for the accurate quantification of two isomeric mercapturic acids of 1,3-dichlorobenzene in human urine using isotope dilution online-SPE-LC-MS/MS.

1,3-dichlorobenzene (1,3-DCB) is an aromatic solvent that might be formed during thermal decomposition of bis(2,4-dichlorobenzoyl)peroxide used as initiator in silicone rubber production with many workers exposed worldwide. During metabolism of 1,3-DCB, two isomeric mercapturic acids can be formed from ring oxidation of 1,3-DCB in the liver, namely 2,4-dichlorophenylmercapturic acid (24CPhMA) and 3,5-dichlorophenylmercapturic acid (35CPhMA). These urinary mercapturic acids might serve as biomarkers of the toxicologically relevant absorbed dose of 1,3-DCB and have not been determined so far. Thus, we were aimed to develop an analytical method for quantification of these biomarkers. Authentic standards of both mercapturic acids as well as deuterium-labelled analogues were self-synthesized. A method for the quantification of both CPhMAs in human urine using online-SPE LC/MS/MS was developed and validated with an LOQ of 0.1 ng mL-1 for both CPhMAs. The analytes were extracted from urine by online-SPE on a restricted access material phase, transferred to the analytical column and quantified by tandem mass spectrometry. Interday (n = 6) and Intraday (n = 10) precision for both CPhMAs ranged from 1.7 to 4.3 % with accuracies between 99.4 and 109.9 % at concentrations of 0.6 and 3 ng mL-1. We applied the method on post-shift urine samples of 16 workers of the silicone rubber industry with occupational exposure to 1,3-DCB. Both CPhMAs were above LOQ in 15 of 16 urine samples with median levels (range) for 24CPhMA and 35CPhMA of 1.64 ng mL-1 (<0.1 - 8.2 ng mL-1) and 3.98 ng mL-1 (0.36 - 24.1 ng mL-1), respectively. This is the first report on specific urinary mercapturic acids of 1,3-DCB in humans. Our results show that ring oxidation of 1,3-DCB is considered to be a toxicologically relevant metabolic pathway in humans. This might improve risk assessment of 1,3-DCB-emissions in silicone rubber industry.

PFAS levels and exposure determinants in sensitive population groups.

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants. The first exposure to PFAS occurs in utero, after birth it continues via breast milk, food intake, environment, and consumer products that contain these chemicals. Our aim was to identify determinants of PFAS concentrations in sensitive population subgroups- pregnant women and newborns. METHODS: Nine European birth cohorts provided exposure data on PFAS in pregnant women (INMA-Gipuzkoa, Sabadell, Valencia, ELFE and MoBa; total N = 5897) or newborns (3xG study, FLEHS 2, FLEHS 3 and PRENATAL; total N = 940). PFOS, PFOA, PFHxS and PFNA concentrations were measured in maternal or cord blood, depending on the cohort (FLEHS 2 measured only PFOS and PFOA). PFAS concentrations were analysed according to maternal characteristics (age, BMI, parity, previous breastfeeding, smoking, and food consumption during pregnancy) and parental educational level. The association between potential determinants and PFAS concentrations was evaluated using multiple linear regression models. RESULTS: We observed significant variations in PFAS concentrations among cohorts. Higher PFAS concentrations were associated with higher maternal age, primipara birth, and educational level, both for maternal blood and cord blood. Higher PFAS concentrations in maternal blood were associated with higher consumption of fish and seafood, meat, offal and eggs. In cord blood, higher PFHxS concentrations were associated with daily meat consumption and higher PFNA with offal consumption. Daily milk and dairy consumption were associated with lower concentrations of PFAS in both, pregnant women and newborns. CONCLUSION: High detection rates of the four most abundant PFAS demonstrate ubiquitous exposure of sensitive populations, which is of concern. This study identified several determinants of PFAS exposure in pregnant women and newborns, including dietary factors, and these findings can be used for proposing measures to reduce PFAS exposure, particularly from dietary sources.

Neuropsychological effects of occupational exposure to polychlorinated biphenyls.

In the context of a health surveillance program for former PCB-exposed workers of a transformer and capacitor recycling company in Germany, their family members, employees of surrounding companies and area residents a broad range of cognitive functions covering attention, executive processing, reasoning, memory and motor performance was examined. The study aimed at identifying potential adverse effects of PCB load on cognitive functions. Detailed analysis of PCB burden of the participants revealed rather high correlations of lower and higher chlorinated as well as dioxin-like PCBs. Nearly one half of the participants exhibited increased burden in all three PCB classes whereas only 33 out of 237 participants did not show any increased PCB burden. Thus, data analysis followed a two-fold strategy: (1) Based on studies providing data on PCB exposure of the German general population the PCB burden of every participant was classified as normal (percentile rank PR <95) or increased (PR ≥95). Increased burden with respect to lower (LPCBs) and higher chlorinated (HPCBs) as well as dioxin-like (dlPCBs) PCBs was assumed if a participant showed at least one congener surpassing the PR95 criterion for the respective congener class and (2) Overall plasma PCB level per congener class was used as measure of PCB load. In a multivariate approach using structural equation modelling and multiple regression analysis we found a significant impact of PCBs on word fluency and sensorimotor processing irrespective of the measure of PCB burden (PR95 criterion or overall plasma level). However, no effect of PCB burden on memory, attention, and cognitive flexibility could be demonstrated. Particularly, an increase of LPCBs was associated with an overall reduction of verbal fluency of letter and semantic word generation as well as word production based on a single or two alternating criteria. In addition, participants with increased burden of LPCBs exhibited a time-on-task effect in terms of a stronger decline of performance with increasing duration of the verbal fluency task. Moreover, we found adverse effects of HPCBs on Aiming and of dlPCBs on Line Tracking. Results are discussed in terms of (1) a decrease of cerebral dopamine (DA) with non-coplanar PCBs resulting in an impact on fronto-striatal cerebral structures subserving verbal fluency and motor processing, (2) a PCB-induced reduction of norepinephrine leading to the time-on-task effect with verbal fluency, and (3) adverse effects of PCBs on dopaminergic receptors in the cerebellum resulting in impaired fine motor function.

Quantification of six potential unspecific human biomarkers of 1-vinyl-2-pyrrolidone exposure in Sprague-Dawley rat urine using gas chromatography coupled with triple mass spectrometry.

RATIONALE: The monomer 1-vinyl-2-pyrrolidone (VP) is a substance with excellent solvent features. It is used in a wide variety of pharmaceutical, cosmetic, food industrial or technical applications and produced on an industrial scale. Since VP has caused adenocarcinoma of the nasal cavity and liver cell carcinoma in long-term experiments with rats, a human biomarker would be appreciated for risk assessment. METHODS: A sensitive analytical electron ionization gas chromatography/tandem mass spectrometry (GC/MS/MS) method for the determination of six possible biomarkers for VP in urine was established and validated. Two isotope-labeled internal standards (ISTD) were used for quantification. A simple and easy to use freeze-drying step was performed prior to derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoracetamide (MTBSTFA) and following sample extraction for cleanup purposes. RESULTS: A calibration curve with six calibration standards ranging from 50 µg/L to 2000 µg/L (10-fold higher for H-OPAA) in urine was prepared. Validation results were satisfactory with recoveries ranging from 88.2 to 110.2 % with two exceptions for the lowest quality control for two substances without ISTD (126.4 % and 139.3 %). Three of the substances could be identified as VP metabolites in an exposure study with Sprague-Dawley (SD) rats. CONCLUSIONS: A quick and easy to use method has been established for six target molecules investigated for a better understanding of the metabolism of VP. Two of three substances identified as metabolites of VP could serve as a nonspecific human biomarker for VP exposure as shown with an excerpt of an exposure study performed in SD rats.

Quantification of N-methylmalonamic acid in urine as metabolite of the biocides methylisothiazolinone and chloromethylisothiazolinone using gas chromatography-tandem mass spectrometry.

Methylisothiazolinone and the mixture of chloromethylisothiazolinone/methylisothiazolinone (MCI/MI, 3:1) are widespread biocides used in cosmetic and household products. Due to their skin permeability, they might be taken up by the general population via use of products containing these biocides. As both compounds are known skin sensitizers, the use of these products is under discussion by regulatory agencies. In order to evaluate the possible uptake of MI and/or MCI/MI by human biomonitoring, we have developed and validated a highly sensitive and specific GC/MS/MS-method for the quantification of N-methylmalonamic acid (NMMA), a known metabolite of MI and MCI in urine of rats. After freeze-drying of urine, the analyte is derivatised with pentafluorobenzyl bromide in anhydrous solution and the PFB-derivative is extracted into n-hexane. After concentration, the derivative is finally quantified by GC/MS/MS in EI-mode using 13C3-NMMA as internal standard. The limit of quantification for NMMA was 0.5ngmL-1 urine. Precision within and between-series was determined to range between 3.7-10.9% using native and spiked quality control samples. Accuracy ranged between 89 and 114%. In a pilot study we applied this method to spot urine samples of 63 persons not knowingly exposed to MI and/or MCI/MI. NMMA was quantifiable in every urine sample analysed, with no significant difference in urinary levels between male and female participants. The median (95th percentile) levels for urinary NMMA were 3.6 (7.4) ngmg-1 creatinine and 2.9 (9.1) ngmg-1 creatinine for males (n=32) and females (n=31), respectively. In a volunteer experiment, a relation of exposure to MI and/or MCI/MI and subsequent NMMA-excretion was shown. Our method is the first to report human urinary background levels of NMMA. However, the possibility of formation and urinary excretion of NMMA within physiological processes cannot be ruled out.

Isotope-dilution method for the determination of 1-vinyl-2-pyrrolidone-mercapturic acid as a potential human biomarker for 1-vinyl-2-pyrrolidone via online SPE ESI-LC/MS/MS in negative ionization mode.

We established and validated a specific and sensitive analytical method for the determination of 1-vinyl-2-pyrrolidone (VP) as 1-vinyl-2-pyrrolidone-mercapturic acid (VPMA) in urine using an electrospray liquid chromatography tandem mass spectrometry (ESI-LC/MS/MS) column switching method. An online solid phase extraction (SPE) for sample cleanup was performed by column switching to a restricted access material and back-flushing to the analytical column. A Phenomenex Luna C8 column was used for sample separation (150mm; ID 4,6mm; 3µm). D4-VPMA served as an isotope labeled internal standard and was detected in negative multiple-reaction monitoring (MRM) mode. The Limit of quantification (LOQ) for VPMA was 1.5µg/L, the intra-day precision of three concentrations (2µg/L, 75µg/L and 400µg/L) of spiked urine samples ranged from 2.7 to 7.3%, the inter-day precision from 3.4 to 14.4%. The accuracy ranged from 6.2 to 9.0%, for the intra-day experiments and from 0.3 to 6.9% for the inter-day experiments. The method was applied to urines of Sprague-Dawley rats exposed to VP as a proof of principle of VPMA as a potential biomarker.

Short-term health effects in the general population following a major train accident with acrylonitrile in Belgium.

BACKGROUND: Following a train derailment, several tons of acrylonitrile (ACN) exploded, inflamed and part of the ACN ended up in the sewage system of the village of Wetteren. More than 2000 residents living in the close vicinity of the accident and along the sewage system were evacuated. A human biomonitoring study of the adduct N-2-cyanoethylvaline (CEV) was carried out days 14-21 after the accident. OBJECTIVES: (1) To describe the short-term health effects that were reported by the evacuated residents following the train accident, and (2) to explore the association between the CEV concentrations, extrapolated at the time of the accident, and the self-reported short-term health effects. METHODS: Short-term health effects were reported in a questionnaire (n=191). An omnibus test of independence was used to investigate the association between the CEV concentrations and the symptoms. Dose-response relationships were quantified by Generalized Additive Models (GAMs). RESULTS: The most frequently reported symptoms were local symptoms of irritation. In non-smokers, dose-dependency was observed between the CEV levels and the self-reporting of irritation (p=0.007) and nausea (p=0.007). Almost all non-smokers with CEV concentrations above 100pmol/g globin reported irritation symptoms. Both absence and presence of symptoms was reported by non-smokers with CEV concentrations below the reference value and up to 10 times the reference value. Residents who visited the emergency services reported more symptoms. This trend was seen for the whole range of CEV concentrations, and thus independently of the dose. DISCUSSION AND CONCLUSION: The present study is one of the first to relate exposure levels to a chemical released during a chemical incident to short-term (self-reported) health effects. A dose-response relation was observed between the CEV concentrations and the reporting of short-term health effects in the non-smokers. Overall, the value of self-reported symptoms to assess exposure showed to be limited. The results of this study confirm that a critical view should be taken when considering self-reported health complaints and that ideally biomarkers are monitored to allow an objective assessment of exposure.

Simple and sensitive GC/MS-method for the quantification of urinary phenol, o- and m-cresol and ethylphenols as biomarkers of exposure to industrial solvents.

We have developed and validated a simple and sensitive method for the determination of urinary phenol as well as the urinary metabolites of toluene and ethylbenzene in one analytical run. After enzymatic hydrolysis for the cleavage of conjugates overnight, the analytes are extracted from the matrix with a liquid-liquid extraction procedure using toluene as solvent under acidic conditions. The analytes are then derivatised to volatile ethers using N,O-bis(trimethylsilyl) trifluoroacetamid (BSTFA) for cresols and ethylphenols as well as N-tert-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA) for the determination of phenol. Separation and detection was carried out using capillary gas chromatography with mass-selective detection (GC-MS). Deuterium-labeled o-cresol served as internal standard for the quantification of all metabolites and guaranteed good accuracy of the results. No matrix effects were observed in the quantification of the analytes. The limit of detection for o- and m-cresol and 2- and 4-ethylphenol was 10 and 20µg/l urine and linearity ranged up to 3 and 12mg/L urine, respectively. The limit of detection for urinary phenol was 0.5mg/L with a linear range up to 200mg/L. The relative standard deviation of the within-series imprecision ranged between 3.0 and 7.2% at two spiked concentrations of 60 and 400µg/l and the relative recovery was between 84 and 104%, depending on the analyte. The method was successfully applied in proficiency testing for urinary o-cresol and phenol. This method was used for the analysis of urine samples of 17 non-smoking and 13 smoking persons from the general population without known exposure to solvents. Smokers showed a significantly higher excretion of o-cresol (median: 23 vs. 33µg/l), m-cresol (median: 43 vs. 129µg/l) as well as 4-ethylphenol (median: 25 vs. 124µg/l). Especially excretion of 4-ethylphenol was significantly correlated to smoking habits. The method seems to be suitable for biological monitoring of low-level solvent exposures and allows determination of background values in the general population.

Effect of occupational polychlorinated biphenyls exposure on quality-adjusted life years over time at the HELPcB surveillance program.

HELPcB (Health Effects in High-Level Exposure to Polychlorinated Biphenyls [PCB]) is a surveillance program for former PCB-exposed workers of a capacitor recycling company and other concerned individuals. The aim of this study was to examine the influence of polychlorinated biphenyls (PCB) on the health-related quality of life (HRQL) and on quality-adjusted life years (QALY). The EQ-5D-3L questionnaire was used to determine the HRQL. After three cross-sectional examinations at intervals of 1 yr, the longitudinal development of QALY was compared by repeated-measurement analysis of covariance (ANCOVA). The cohort was split at the 95th percentile of the comparison group for each PCB congener; known confounders such as age were taken into account. A significant difference in height and development of QALY over time was shown for the higher chlorinated non-dioxin-like PCB (hcPCB) congeners. A significant between-groups effect was found on PCB 153, PCB 180, and the sum of hcPCB. It was found that QALY decreased in the high-burden group and QALY stabilized after yr 2 in the normal-burden group. Taking the dimensions of the EQ-5D into account, the between-groups effect seems to be based predominantly on the dimension anxiety. The development of the within-group effect, however, seems to be based on the dimension mobility. This study detected a significant influence of hcPCB on the development of HRQL and QALYs over time according to the level of internal PCB burden.

Acrylonitrile exposure in the general population following a major train accident in Belgium: a human biomonitoring study.

BACKGROUND: On Saturday May 4, 2013, a train transporting chemicals derailed in the village of Wetteren (Belgium) and caused a leak of acrylonitrile (ACN). OBJECTIVES: To assess the human exposure to acrylonitrile in the local population with the highest suspected exposure. METHODS: Between May 18-25, 242 residents participated in the study. N-2-cyanoethylvaline (CEV), a biomarker that is highly specific for ACN exposure, was measured in the blood. To account for potential influence by smoking, cotinine was determined in the urine. Participants also filled in a short questionnaire. RESULTS: In the evacuated zone, 37.3% of the non-smokers and 40.0% of the smokers had CEV concentrations above the reference values of 10 and 200 pmol/g globin, respectively, at the time of the train accident. Spatial mapping of the CEV concentrations depending on the residential address showed a distribution pattern following the sewage system. DISCUSSION AND CONCLUSION: The train derailment resulted in a highly atypical sequence-of-events. In addition to exposure in the direct vicinity of the site of the train derailment, exposure also occurred via the sewage system, into which acrylonitrile had entered shortly after the accident.

Acrylonitrile exposure assessment in the emergency responders of a major train accident in Belgium: a human biomonitoring study.

BACKGROUND: On May 4, 2013, a train transporting chemicals derailed in Wetteren, Belgium. Several tanks loaded with acrylonitrile (ACN) exploded, resulting in a fire and a leakage of ACN. OBJECTIVES: To determine exposure to ACN and to assess discriminating factors for ACN exposure in the emergency responders involved in the on-site management of the train accident. METHODS: The study population consisted of 841 emergency responders. Between May 21 and June 28, they gave blood for the determination of N-2-cyanoethylvaline (CEV) hemoglobin adducts and urine for the measurement of cotinine. They also filled in a short questionnaire. RESULTS: 163 (26%) non-smokers and 55 (27%) smokers showed CEV concentrations above the reference values of 10 and 200 pmol/g globin, respectively. The 95th percentile in the non-smokers was 73 pmol/g globin and the maximum was 452 pmol/g globin. ACN exposure among the non-smokers was predicted by (1) the distance to the accident, (2) the duration of exposure, and (3) the occupational function. DISCUSSION AND CONCLUSION: Emergency responders involved in the on-site management of the train accident were clearly exposed to ACN from the accident. However, the extent of exposure remained relatively moderate with CEV concentrations staying within the ranges described in literature as background for a smoking population. Moreover, the exposure was less pronounced in the emergency responders as compared to that in the local population.

Assessment of the biological effects of welding fumes emitted from metal inert gas welding processes of aluminium and zinc-plated materials in humans.

The aim of this study was to investigate biological effects and potential health risks due to two different metal-inert-gas (MIG) welding fumes (MIG welding of aluminium and MIG soldering of zinc coated steel) in healthy humans. In a threefold cross-over design study 12 male subjects were exposed to three different exposure scenarios. Exposures were performed under controlled conditions in the Aachener Workplace Simulation Laboratory (AWSL). On three different days the subjects were either exposed to filtered ambient air, to welding fumes from MIG welding of aluminium, or to fumes from MIG soldering of zinc coated materials. Exposure was performed for 6 h and the average fume concentration was 2.5 mg m(-3). Before, directly after, 1 day after, and 7 days after exposure spirometric and impulse oscillometric measurements were performed, exhaled breath condensate (EBC) was collected and blood samples were taken and analyzed for inflammatory markers. During MIG welding of aluminium high ozone concentrations (up to 250 µg m(-3)) were observed, whereas ozone was negligible for MIG soldering. For MIG soldering, concentrations of high-sensitivity CRP (hsCRP) and factor VIII were significantly increased but remained mostly within the normal range. The concentration of neutrophils increased in tendency. For MIG welding of aluminium, the lung function showed significant decreases in Peak Expiratory Flow (PEF) and Mean Expiratory Flow at 75% vital capacity (MEF 75) 7 days after exposure. The concentration of ristocetin cofactor was increased. The observed increase of hsCRP during MIG-soldering can be understood as an indicator for asymptomatic systemic inflammation probably due to zinc (zinc concentration 1.5 mg m(-3)). The change in lung function observed after MIG welding of aluminium may be attributed to ozone inhalation, although the late response (7 days after exposure) is surprising.

Exposure of healthy subjects with emissions from a gas metal arc welding process: part 3--biological effect markers and lung function.

BACKGROUND: Metal active gas welding (MAG) is a widely-used welding technique resulting in high emissions of welding fume particles. This study investigated whether short-term exposure to these fume particles results in changes in lung function and early stages of inflammatory reactions. METHODS: Twelve healthy, young male subjects were exposed to MAG fumes for 6 h with three different exposure concentrations in a three-fold cross-over study design. Exposure was performed in the "Aachen Workplace Simulation Laboratory" under controlled conditions with constant fume concentration. Fume concentrations were 0, 1, and 2.5 mg m(-3) in randomized order. Before and after each exposure, spirometry, and impulse oscillometry were performed and breath condensate samples were collected in order to quantify inflammatory markers like Nitrate, Nitrite, Nitrotyrosine, Hydroxyprolin and Malondialdehyde. RESULTS: A significant dependency on the exposure concentration could not be established for any of the endpoint parameters. CONCLUSION: In healthy, young subjects neither changes in spirometry nor changes in inflammatory markers measured in exhaled breath condensate could be detected after short-term exposure.

Accurate quantification of the mercapturic acids of acrylonitrile and its genotoxic metabolite cyanoethylene-epoxide in human urine by isotope-dilution LC-ESI/MS/MS.

Acrylonitrile is a highly important industrial chemical with a high production volume worldwide, especially in the plastics industry. It is classified as a possible human carcinogen by the International Agency for Research on Cancer (IARC group 2B). During metabolism of acrylonitrile, the genotoxic metabolite cyanoethylene-epoxide is formed. The urinary mercapturic acids of acrylonitrile, namely N-acetyl-S-(2-cyanoethyl)-L-cysteine (CEMA) and cyanoethylene-epoxide, namely N-acetyl-S-(1-cyano-2-hydroxyethyl)-L-cysteine (CHEMA) are specific biomarkers for the determination of individual internal exposure to acrylonitrile and its highly reactive metabolite. We have developed and validated a sensitive method for the accurate determination of CEMA and CHEMA in human urine with a multidimensional LC/MS/MS-method using deuterium-labelled analogues for both analytes as internal standards. Analytes were stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column and determined by tandem mass spectrometry. The limit of quantification (LOQ) for CEMA and CHEMA was 1 µg/L urine and allowed to quantify the background exposure of the (smoking) general population. Precision within and between series for CHEMA ranged from 2.6 to 8.0% at four concentrations ranging from 8.3 to 86 µg/L urine, mean accuracy was between 94 and 100%. For CEMA, precision within and between series ranged from 2.4 to 14.5% at four concentrations ranging from 15.1 to 196 µg/L urine, mean accuracy was between 91 and 104%. We applied the method to spot urine samples of 83 subjects of the general population with no known occupational exposure to acrylonitrile. Median levels (range) for CEMA and CHEMA in urine samples of non-smokers (n=47) were 1.9 µg/L (<1-16.4 µg/L) and<1 µg/L (<1-3 µg/L), while in urine samples of smokers (n=36), median levels were 184 µg/L (2-907 µg/L) and 29.3 µg/L (<1-147 µg/L), respectively. Smokers showed a significantly higher excretion of both acrylonitrile metabolites (p<0.001). Due to its automation and high sensitivity, our method is well suited for application in experimental studies on acrylonitrile metabolism or occupational studies.

Accurate quantification of mercapturic acids of styrene (PHEMAs) in human urine with direct sample injection using automated column-switching high-performance liquid chromatography coupled with tandem mass spectrometry.

Styrene is one of the most important industrial chemicals, with an enormously high production volume worldwide. The urinary mercapturic acids of its metabolite styrene-7,8-oxide, namely N-acetyl-S-(2-hydroxy-1-phenylethyl)-L-cysteine (PHEMA 1) and N-acetyl-S-(2-hydroxy-2-phenylethyl)-L-cysteine (PHEMA 2), are specific biomarkers for the determination of individual internal exposure to this highly reactive intermediate of styrene. We have developed and validated a fast, specific and very sensitive method for the accurate determination of the sum of phenylhydroxyethyl mercapturic acids (PHEMAs) in human urine with an automated multidimensional liquid chromatography-tandem mass spectrometry method using (13)C(6)-labelled PHEMAs as internal standards. Analytes were stripped from the urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry. The limit of quantification (LOQ) for the sum of PHEMAs was 0.3 microg/L urine and allowed us to quantify the background exposure of the (smoking) general population. Precision within series and between series ranged from 1.5 to 6.8% at three concentrations ranging from 3 to 30 microg/L urine; the mean accuracy was between 104 and 110%. We applied the method to spot urine samples from 40 subjects of the general population with no known occupational exposure to styrene. The median levels (range) for the sum of PHEMAs in urine of non-smokers (n = 22) were less than 0.3 microg/L (less than 0.3 to 1.1 microg/L), whereas in urine of smokers (n = 18), the median levels were 0.46 microg/L (less than 0.3 to 2.8 microg/L). Smokers showed a significantly higher excretion of the sum of PHEMAs (p = 0.02). Owing to its automation and high sensitivity, our method is well suited for application in occupational or environmental studies.

Simultaneous quantification of haemoglobin adducts of ethylene oxide, propylene oxide, acrylonitrile, acrylamide and glycidamide in human blood by isotope-dilution GC/NCI-MS/MS.

Haemoglobin adducts are highly valuable biomarkers of cumulative exposure to carcinogenic substances. We have developed and applied an analytical method for the simultaneous quantification of five haemoglobin adducts of important occupational and environmental carcinogens. The N-terminal adducts were determined with gas chromatography as pentafluorophenylthiohydantoine derivatives according to the modified Edman-procedure and subsequent acetonization of the glycidamide adduct N-(R,S)-2-hydroxy-2-carbamoylethylvaline (GAVal). The use of self-synthesized labelled internal standards in combination with tandem mass spectrometry using negative chemical ionisation guarantees both high accuracy and sensitivity of our determination. The limit of detection for N-2-hydroxyethylvaline (HEVal), N-(R,S)-2-hydroxypropylvaline (HPVal), N-2-carbamoylethylvaline (AAVal) and N-(R,S)-2-hydroxy-2-carbamoylethylvaline (GAVal) was 2 pmol/g globin, for N-2-cyanoethylvaline (CEVal) it was determined as 0.5 pmol/g globin, which was sufficient to determine the background levels of these adducts in the non-smoking general population. The between-day-precision for all analytes using a human blood sample as quality control material ranged from 4.7 to 12.3%. We investigated blood samples of a small group (n=104) of non-smoking persons of the general population for the background levels of these haemoglobin adducts. The median values for HEVal, HPVal, CEVal, AAVal and GAVal in a group of 92 non-smoking persons were 18.1, 4.1, <0.5, 29.9 and 35.2 pmol/g globin, respectively. The adduct levels in 12 persons reporting exposure to passive smoke at home were similar for most adducts with median values of 17.2, 4.1, 1.0, 24.9 and 29.7 pmol/g globin for HEVal, HPVal, CEVal, AAVal and GAVal, respectively. Our results point to an elevated uptake of acrylonitrile caused by passive smoking as indicated by higher levels of the corresponding haemoglobin adduct CEVal.

A method for the quantification of biomarkers of exposure to acrylonitrile and 1,3-butadiene in human urine by column-switching liquid chromatography-tandem mass spectrometry.

1,3-Butadiene and acrylonitrile are important industrial chemicals that have a high production volume and are ubiquitous environmental pollutants. The urinary mercapturic acids of 1,3-butadiene and acrylonitrile-N-acetyl-S-(3,4-dihydroxybutyl)cysteine (DHBMA) and MHBMA (an isomeric mixture of N-acetyl-S-((1-hydroxymethyl)-2-propenyl)cysteine and N-acetyl-S-((2-hydroxymethyl)-3-propenyl)cysteine) for the former and N-acetyl-S-2-cyanoethylcysteine (CEMA) for the latter-are specific biomarkers for the determination of individual internal exposure to these chemicals. We have developed and validated a fast, specific, and very sensitive method for the simultaneous determination of DHBMA, MHBMA, and CEMA in human urine using an automated multidimensional LC/MS/MS method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column, and subsequently determined by tandem mass spectrometry using labeled internal standards. The limits of quantification (LOQs) for DHBMA, MHBMA, and CEMA were 10 microg/L, 2 microg/L, and 1 microg/L urine, respectively, and were sufficient to quantify the background exposure of the general population. Precision within series and between series for all analytes ranged from 5.4 to 9.9%; mean accuracy was between 95 and 115%. We applied the method on spot urine samples from 210 subjects from the general population with no occupational exposure to 1,3-butadiene or acrylonitrile. A background exposure of the general population to acrylonitrile was discovered that is basically influenced by individual exposure to passive smoke as well as active smoking habits. Smokers showed a significantly higher excretion of MHBMA, whereas DHBMA levels did not differ significantly. Owing to its automation, our method is well suited for application in occupational or environmental studies.

Fast determination of urinary S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acid (S-BMA) by column-switching liquid chromatography-tandem mass spectrometry.

Benzene and toluene are important industrial chemicals and ubiquitous environmental pollutants. The urinary mercapturic acids of benzene and toluene, S-phenylmercapturic acid (S-PMA) and S-benzylmercapturic acids (S-BMA) are specific biomarkers for the determination of low-level exposures. We have developed and validated a fast, specific and very sensitive method for the simultaneous determination of S-PMA and S-BMA in human urine using an automated multidimensional LC-MS-MS-method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry using isotopically labelled S-PMA as internal standard. The lower limit of quantification (LLOQ) for both analytes was 0.05 microg/L urine and sufficient to quantify the background exposure of the general population. Precision within series and between series for S-PMA and S-BMA ranged from 1.0% to 12.2%, accuracy was 108% and 100%, respectively. We applied the method on spot urine samples of 30 subjects of the general population with no known exposure to benzene or toluene. Median levels (range) for S-PMA and S-BMA in non-smokers (n=15) were 0.14 microg/L (<0.05-0.26 microg/L) and 8.2 (1.6-77.4 microg/L), respectively. In smokers (n=15), median levels for S-PMA and S-BMA were 1.22 microg/L (0.17-5.75 microg/L) and 11.5 microg/L (0.9-51.2 microg/L), respectively. Due to its automation, our method is well suited for application in large environmental studies.

Trans-placental exposure of neonates to acrylamide--a pilot study.

OBJECTIVES: Acrylamide (AA) (CAS No 79-06-1) has most recently been identified as a food-borne toxicant generated during the heating process of starch-containing foods. It was the aim of the present study to investigate the trans-placental exposure of newborn infants to this possible human carcinogen by analysis of the specific haemoglobin adduct of AA ( N-2-carbamoylethylvaline, AAV) in the blood of mothers and the corresponding umbilical cord blood of neonates as a parameter of biochemical effects. METHODS: We investigated the blood of 11 women advanced in pregnancy (one smoker, ten non-smokers) and the corresponding umbilical cord blood of neonates for the N-terminal haemoglobin adducts of AA (AAV) and the smoking-specific adduct of acrylonitrile (CAS No 107-13-1) ( N-cyanoethylvaline, CEV). The limit of detection (LOD) was 5 pmol/g globin for AAV and 4 pmol/g globin for CEV. RESULTS: AAV could be determined in all blood samples of the mothers (median 21 pmol/g globin, range 18-104 pmol/g globin) as well as in the umbilical cord blood of neonates (median 10 pmol/g globin, range 6-43 pmol/g globin). The highest values were detected in the blood of the smoking mother and her child. CEV was detected only in the blood of the smoking mother (185 pmol/g globin) and the corresponding umbilical cord blood (69 pmol/g globin). DISCUSSION: AAV adduct levels in non-smoking mothers and neonates showed a good correlation (r=0.859). The concentration of AA adducts in the blood of neonates is approximately 50% of the adduct level found in the blood of the mother. In view of the shorter life span of neonatal erythrocytes and the lower body weight of newborn infants, the relative internal dose of AA in neonates (in microgrammes per kilogramme body weight) must be assumed to be at least equal to that of the mother. Because of the high cell-replication rates during foetal development, trans-placental exposure of neonates to AA might raise concerns. Neonates of smoking mothers take up much higher doses of AA than those of non-smoking mothers.