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Polycyclic aromatic hydrocarbons (PAHs) were included as priority substances for human biomonitoring (HBM) in the European Human Biomonitoring Initiative (HBM4EU), which intended to harmonise and advance HBM across Europe. For this project, a specific Quality Assurance and Quality Control (QA/QC) programme applying Inter-laboratory Comparison Investigations (ICIs) and External Quality Assurance Schemes (EQUASs) was developed to ensure the comparability and accuracy of participating analytical laboratories. This paper presents the results of four ICI/EQUAS rounds for the determination of 13 PAH metabolites in urine, i.e. 1-naphthol, 2-naphthol, 1,2-dihydroxynaphthalene, 2-, 3- and 9-hydroxyfluorene, 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene, 1-hydroxypyrene and 3-hydroxybenzo(a)pyrene. However, 4 PAH metabolites could not be evaluated as the analytical capacity of participating laboratories was too low. Across all rounds and biomarkers, 86% of the participants achieved satisfactory results, although low limits of quantification were required to quantify the urinary metabolites at exposure levels of the general population. Using high-performance liquid or gas chromatography coupled with mass spectrometry (HPLC-MS; GC-MS) and isotope dilution for calibration as well as performing an enzymatic deconjugation step proved to be favourable for the accurate determination of PAHs in urine. Finally, the HBM4EU QA/QC programme identified an international network of laboratories providing comparable results in the analysis of urinary PAH biomarkers, although covering all parameters initially selected was still too challenging.

Summary With increasing use of new generation tobacco/nicotine products (TNPs) instead or in addition to conventional cigarettes (CCs), the question arises, whether the user of these new TNPs and CCs can be distinguished on the basis of their exposure in terms of intake and uptake of specific chemicals. For this purpose, the exposure to chemicals for users of 5 product types including CCs, HNB (heat-not-burn products), electronic cigarettes (ECs), oral tobacco products (OT, with the focus on snus), nicotine replacement therapy (NRT) products (only nicotine gum (NG) was considered in this study) was evaluated on the basis of published data. For both intake and biomarker-based uptake, 4 relative exposure levels with the assigned values from 1 (lowest exposure) to 4 (highest exposure) were defined resulting in exposure level patterns. Unique single-biomarker-based exposure levels were found for smokers (11 chemicals), vapers (1 chemical) and non-users (NU, 1 chemical). A few selected biomarkers (usually with relative levels of 3–4) were found to be sufficient for unequivocal differentiation of one user/NU group from the other 5 groups. The impact of dual-product use is also briefly discussed. [Contrib. Tob. Nicotine Res. 30 (2021) 167–198]

Urinary mercapturic acids (MAs) are detoxification products for electrophiles occurring in the human body. They are suitable biomarkers of exposure to directly acting electrophilic chemicals or to chemicals which generate the electrophile during its metabolism. We determined the urinary excretion of 19 MAs in habitual users of combustible cigarettes (CCs), electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy (NRT) products, and nonusers (NUs) of any tobacco/nicotine products. The 19 MAs are assumed to be physiologically formed primarily from 15 toxicants with three of them belonging to IARC Group 1 (human carcinogen), seven to Group 2A (probable human carcinogen), four to Group 2B (possible human carcinogen), and one to Group 3 (not classifiable as carcinogen). Smoking (CC) was found to be associated with significantly elevated exposure to ethylene oxide (or ethylene), 1,3-butadiene, benzene, dimethylformamide, acrolein, acrylamide, styrene, propylene oxide, acrylonitrile, crotonaldehyde, and isoprene compared with the other user groups and NU. Users of HTPs revealed slight elevation in the MAs related to acrolein, acrylamide, and crotonaldehyde compared with the other non-CC groups. Vaping (EC) was not found to be associated with any of the MAs studied. In conclusion, the determination of urinary MAs is a useful tool for assessing the exposure to toxicants (mainly potential carcinogens) in users of various tobacco/nicotine products. Our data also give cause to clarify the role of vaping (EC) in urinary excretion of DHPMA (precursor: glycidol).

The formation of carbonyls and epoxides in e-cigarette (EC) aerosol is possible due to heating of the liquid constituents. However, high background levels of these compounds have inhibited a clear assessment of exposure during use of ECs. An EC containing an e-liquid replaced with 10% of 13C-labeled propylene glycol and glycerol was used in a controlled use clinical study with 20 EC users. In addition, five smokers smoked cigarettes spiked with the described e-liquid. Seven carbonyls (formaldehyde, acetaldehyde, acrolein, acetone, crotonaldehyde, methacrolein, propionaldehyde) were measured in the aerosol and the mainstream smoke. Corresponding biomarkers of exposure were determined in the user’s urine samples. 13C-labeled formaldehyde, acetaldehyde and acrolein were found in EC aerosol, while all seven labeled carbonyls were detected in smoke. The labeled biomarkers of exposure to formaldehyde (13C-thiazolidine carboxylic acid and 13C-N-(1,3-thiazolidine-4-carbonyl)glycine), acrolein (13C3-3-hydroxypropylmercapturic acid) and glycidol (13C3-dihydroxypropylmercapturic acid) were present in the urine of vapers indicating an EC use-specific exposure to these toxicants. However, other sources than vaping contribute to a much higher extent by several orders of magnitude to the overall exposure of these toxicants. Comparing data for the native (unlabeled) and the labeled (exposure-specific) biomarkers revealed vaping as a minor source of user’s exposure to these toxicants while other carbonyls and epoxides were not detectable in the EC aerosol.

Passive smoking is a preventable and significant cause of many serious health problems, with children being particularly at risk. In the fifth German Environmental Survey (GerES V), conducted from 2014 to 2017, information reflecting the extent of passive smoke exposure in children and adolescents was collected by interview-based questionnaires and human biomonitoring (HBM) analyses of cotinine in urine from 2260 participants, aged 3-17 years. Based on these population-representative data, we describe current passive smoke exposure stratified by different subgroups and identify specific exposure determinants using multivariate logistic regression. The questionnaire data revealed that 42% of children and adolescents lived with at least one smoker in the household. Quantifiable concentrations of cotinine could be detected in 56% of the participants. The overall median concentration of cotinine was 0.2 μg/L, with children and adolescents of low socioeconomic status found to be a group particularly affected by passive smoke with higher cotinine concentrations (median = 1.2 μg/L). In the multiple analysis, the most significant predictor of cotinine levels derived from the questionnaire was passive smoking at home (odds ratio (OR) 13.07 [95CI: 4.65, 36.70]). However, parental smoking and passive smoking among friends and relatives could also be identified as independent factors influencing elevated cotinine levels. The comparison between the previous cycle GerES IV (2003-2006) on 3-14-year-olds and GerES V shows that tobacco smoke exposure of children decreased significantly. This decrease is likely an effect of extensive non-smoker protection laws being enforced 2007-2008 on federal and state level. This is reflected by a halving of urinary cotinine concentrations. Nevertheless, our results indicate that passive smoke is still a relevant source of harmful pollutants for many children and adolescents in Germany, and thus support the need for further efforts to reduce passive smoke exposure, especially in the private environment.

Tobacco-specific nitrosamines (TSNAs), in particular, the human carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and

Ethoxyquin (EQ) is commonly used as an antioxidant in animal feeds. Although EQ is not permitted for usage in food products for humans within the EU, residues of EQ and its transformation products could be determined in food of animal origin. Despite its widespread use and concerns on its toxicological profile, no information about the systemic exposure to EQ in the general population is available. Hence, we developed a human biomonitoring (HBM) method for EQ. Our approach included a metabolism study with five subjects, who were administered an oral dose of 0.005 mg EQ/kg body weight. Unchanged EQ and the major metabolite 2,2,4-trimethyl-6(2H)-quinolinone (EQI) were identified as urinary excretion products of EQ. While small amounts of EQ could be determined in high concentrated samples from the metabolism study only, 28.5% of the orally applied EQ dose could be recovered as EQI. Toxicokinetic parameters were determined for EQI, the potential biomarker of exposure. In addition, an analytical method for EQI (LOQ = 0.03 µg/L) in urine based on UHPLC-MS/MS comprising enzymatic glucuronide hydrolysis and salt-assisted liquid-liquid extraction was developed, validated and applied to 53 urine samples from the general population. EQI could be quantified in 11 (21%) of the samples in levels up to 1.7 µg/L urine, proving the suitability of the developed method for the intended purpose.

A novel method for the quantification of the sulfur-containing metabolites of formaldehyde (thiazolidine carboxylic acid (TCA) and thiazolidine carbonyl glycine (TCG)) and acetaldehyde (methyl thiazolidine carboxylic acid (MTCA) and methyl thiazolidine carbonyl glycine (MTCG)) was developed and validated for human urine and plasma samples. Targeting the sulfur-containing metabolites of formaldehyde and acetaldehyde in contrast to the commonly used biomarkers formate and acetate overcomes the high intra- and inter-individual variance. Due to their involvement in various endogenous processes, formate and acetate lack the required specificity for assessing the exposure to formaldehyde and acetaldehyde, respectively. Validation was successfully performed according to FDA’s Guideline for Bioanalytical Method Validation (2018), showing excellent performance with regard to accuracy, precision, and limits of quantification (LLOQ). TCA, TCG, and MTCG proved to be stable under all investigated conditions, whereas MTCA showed a depletion after 21 months. The method was applied to a set of pilot samples derived from smokers who consumed unfiltered cigarettes spiked with 13C-labeled propylene glycol and 13C-labeled glycerol. These compounds were used as potential precursors for the formation of 13C-formaldehyde and 13C-acetaldehyde during combustion. Plasma concentrations were significantly lower as compared to urine, suggesting urine as suitable matrix for a biomonitoring. A smoking-related increase of unlabeled biomarker concentrations could not be shown due to the ubiquitous distribution in the environment. While the metabolites of 13C-acetaldehyde were not detected, the described method allowed for the quantification of 13C-formaldehyde uptake from cigarette smoking by targeting the biomarkers 13C-TCA and 13C-TCG in urine. Graphical abstract

Starting in 2002, regulations and legislative amendments in Germany focused on the non-smoker protection with several measures to reduce exposure to secondhand tobacco smoke (SHS). The present work aimed to evaluate the relationship between polycyclic aromatic hydrocarbons (PAHs) and SHS exposure and to determine to which extent enforced non-smoking regulations and smoking bans affected the exposure of the non-smoking population in Germany since their implementation in the early 2000s until today. For this purpose, cotinine and selected monohydroxylated PAHs (OH-PAHs) were analyzed by means of (UP)LC-MS/MS in 510 24-h-urine samples of the Environmental Specimen Bank collected over a time span of 24 years from 1995 to 2019. Median urinary cotinine levels were found to steadily and significantly decline by 82% from 1995 to 2019. A significant decrease of urinary 3-hydroxybenzo[a]pyrene (19%), 1-OH-pyrene (39%), 1-naphthol (66%), 1- (17%), 2- (25%), and 3-OH-phenanthrene (22%) was also observed throughout the same time span. The decline in urinary levels of cotinine and several OH-PAHs can most likely be attributed to smoking bans and regulations limiting SHS and PAH exposure. This study therefore emphasizes the relevance of human biomonitoring to investigate the exposure of humans to chemicals of concern, assess the effectiveness of regulatory measures, and help policies to enforce provisions to protect public health.

Today various tobacco and nicotine products are available, many of them can be regarded as potentially risk-reduced products when compared to the most frequently used product, combustible cigarettes (CCs). A commonality of these products is that they deliver nicotine, although in quite different amounts and uptake routes, the most common of which are inhalation through the lung and absorption through the oral mucosa. Product-specific nicotine delivery as well as the subject-related use patterns are important factors which determine the pharmacokinetics and achieved internal dose levels of the alkaloid. The latter two parameters are highly relevant for the long-term product loyalty and, consequently, for the implicated health risks, since the risk-reduced products will replace CCs in the long-term only when users will experience a similar level of satisfaction. We measured nicotine and its major metabolites in plasma, saliva and urine samples collected in a controlled clinical study with habitual users (10 per group) of CCs, electronic cigarettes (ECs), heated tobacco products (HTP), oral tobacco (OT), and nicotine replacement therapy (NRT). Non-users (NU) of any tobacco/nicotine products served as (negative) control group. Moderate to strong correlations were observed between the daily consumption and the urinary nicotine equivalents (comprising nicotine and its 10 major metabolites, Nic + 10) or plasma and saliva cotinine concentrations. The average daily nicotine dose as measured by the urinary excretion of Nic + 10 (reflecting approximately 95 % of the absorbed nicotine) amounted to 17 and 22 mg/24 h for smokers (CC) and OT users, respectively, while it was in the range of 6-12 mg/24 h for users of ECs, HTP and NRT products, with high inter-individual variations in each user group. The individual daily nicotine intake, which was calculated by applying product-specific models, showed none to good agreement with the corresponding internal nicotine dose measured by Nic + 10 excretion. Possible reasons for the observed deviations between calculated and objectively measured nicotine doses are discussed.

Over the past decade, new emerging tobacco and nicotine-delivery products have changed the tobacco landscape. Especially, electronic cigarettes (ECs) have been suggested to be considered for tobacco harm reduction, reinforcing the need to identify novel biomarkers of exposure (BoE) specific to the EC use as this would complement exposure assessment and product compliance monitoring. Therefore, a sensitive LC-MS/MS method for the quantification of 1,2-propylene glycol (PG) and glycerol (G), the main e-liquid constituents, was established. PG and G were analyzed in plasma and urine samples from a clinical study comparing five nicotine product user groups, users of combustible cigarettes (CC), electronic cigarettes (EC), heated tobacco products (HTP), oral tobacco (OT), and oral/dermal nicotine delivery products (used for nicotine replacement therapy, NRT) with a control group of non-users (NU). Data demonstrate significantly elevated PG levels in urine and plasma in EC users compared to users of CC, HTP, NRT, OT as well as NU. In addition, PG in plasma and urine of vapers significantly correlated with nicotine (plasma) and total nicotine equivalents (urine), biomarkers reflecting product consumption, emphasizing the high specificity of PG as a BoE for EC consumption. We therefore suggest the use of PG as BoE in urine and/or plasma in order to monitor EC use compliance in exposure assessments.

Objective: To investigate the use of blood levels of N-(2 cyanoethyl)valine (CEVal), a haemoglobin adduct of acrylonitrile, to assess lack of compliance with smoking cessation. Methods: We report CEVal concentrations and percentiles over time for 2 cessation groups, compliance was established using NNAL concentrations. CEVal half-life was estimated. Optimal thresholds were calculated based on receiver operating characteristics analysis. Results: At 180 days, among 67 participants in the continued smoking group and 159 assigned to smoking cessation or sole THP use. CEVal half-life was estimated to be approximately 30 days and the optimal thresholds were for NNAL at 40 pg/mL and CEVal at 35 pmol/g globin (81% sensitivity and specificity). Conclusions: A new generation of biomarkers of compliance is required, specific to the new generation of nicotine products. Methodological validation and standardisation could allow robust assessment of effects across clinical and observational studies while promoting comparability between studies. CEVal could play an important role as biomarker of compliance for smoking cessation and switching studies.

Uvinul® A plus (DHHB) is a synthetic benzophenone derivative mainly used in sunscreens, and also in other skin care products. The compound is authorized by the EU as UV filter and a maximum concentration of 10% in consumer products is permitted. Despite its high production volume and usage in consumer products,to date, no information about the systemic exposure to Uvinul® A plus in humans is available. Therefore, we developed a human biomonitoring method which allows the simultaneous determination of three major metabolites of Uvinul® A plus in human urine samples. Furthermore, three minor metabolites of Uvinul® A plus were identified by ion trap experiments. Urine samples were enzymatically hydrolyzed, extracted via liquid-liquid extraction with ethyl acetate, and analyzed by means of UPLC-MS/MS. The final method was validated according to FDA guidelines and applied to 58 urine samples retrieved from the general German population. The three major and specific metabolites of Uvinul® A plus were found in about 36% of the samples, proving the suitability of the method for future human biomonitoring studies.

Background Assessing biomarker profiles in various body fluids is of large value to discern between the sole use of nicotine products. In particular, the assessment of the product compliance is required for long-term clinical studies. The objective of this study was the identification of biomarkers and biomarker patterns in body fluids, to distinguish between combustibles, heated tobacco products, electronic cigarettes, oral tobacco and oral/dermal nicotine products used for nicotine replacement therapy (NRT), as well as a control group of non-users. Methods A controlled, single-center study was conducted with 60 healthy subjects, divided into 6 groups (5 nicotine product user groups and one non-user group) based on their sole use of the products of choice. The subjects were confined for 76 h, during which, free and uncontrolled use of the products was provided. Sample collections were performed according to the study time schedule provided in Table 2. The primary outcome will be validated through analysis of the collected biospecimens (urine, blood, saliva, exhaled breath and exhaled breath condensate) by means of untargeted omics approaches (i.e. exposomics, breathomics and adductomics). Secondary outcome will include established biomarker quantification methods to allow for the identification of typical biomarker patterns. Statistical analysis tools will be used to specifically discriminate different product use categories. Results/Conclusions The clinical trial was successfully completed in May 2020, resulting in sample management and preparations for the quantitative and qualitative analyses. This work will serve as a solid basis to discern between biomarker profiles of different nicotine product user groups. The knowledge collected during this research will be required to develop prototype diagnostic tools that can reliably assess the differences and evaluate possible health risks of various nicotine products.

Tobacco smoking contributes to tooth discoloration. Pigmented compounds in the smoke generated by combustion of tobacco can cause discoloration of dental hard tissues. However, aerosols from heated tobacco products cause less discoloration than cigarette smoke (CS) in vitro. The objective of the present study was to optimize a method for extracting the colored chemical compounds deposited on tooth enamel following exposure to total particulate matter (TPM) from CS or a heated tobacco product (Tobacco Heating System [THS] 2.2), analyze the extracts by gas chromatography coupled to time-of-flight mass spectrometry, and identify the key chemicals associated with tooth discoloration. Sixty bovine enamel blocks were exposed for 2 weeks to TPM from CS or THS 2.2 aerosol or to artificial saliva as a control. Brushing without toothpaste and color measurements were performed each week. Noticeable discoloration of enamel was observed following exposure to CS TPM. The discoloration following exposure to THS 2.2 aerosol TPM or artificial saliva was not distinguishable to the eye (ΔE 3.3). Carbon disulfide was used to extract surface-deposited chemicals. Untargeted analyses were followed by partial least squares correlation against discoloration scores (R

7-Hydroxy-3,7-dimethyl-1-octanal, also known as 7-hydroxycitronellal (7-HC, CAS No. 107-75-5) is a synthetic fragrance widely used in cosmetic and hygiene products. Because of its wide spread use and its known sensitizing properties, 7-HC was selected as one of 50 chemicals within the frame of the cooperation project between the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety (BMU) and the German Chemical Industry Association (VCI) to develop a suitable human biomonitoring (HBM) method in order to assess the exposure of the general population in Germany. Within this scope, the recently published analytical method for urinary 7-hydroxycitronellylic acid (7-HCA), the major metabolite of 7-HC, was applied to 329 24h-urine samples of young adults (20 to 29 years) collected between 2000 and 2018 and stored in the Environmental Specimen Bank (ESB). The widespread exposure to 7-HC as already observed in a pilot study with 40 volunteers could be confirmed with quantifiable concentrations of 7-HCA in all 329 study samples (mean: 14.9 ng/mL; median: 8.1 ng/mL). A significant, chronological decrease in 7-HCA levels was found for the monitored years (2000, 2004, 2008, 2012, 2015, 2018). The most pronounced decline occurred between 2000 and 2004 (means: 34.37 versus 23.31, medians: 20.97 versus 12.49 μg/24h; p 0.01). On average, females exhibited higher levels of urinary 7-HCA compared to males (29.34 versus 17.21 μg/24h, p 0.05). Based on the urinary 7-HCA excretion, the daily intake (DI) of 7-HC normalized for body weight (bw) was estimated. Over all sampling years, average DI in females was significantly higher compared to males (0.99 versus 0.46 μg/kg bw/d). Assuming dermal exposure as the main route of 7-HC intake, the mean DIs correspond to0.1% of the derived no effect level (DNEL) of 1,100 μg/kg bw/d defined by the European Chemical Agency (ECHA). The presented results for the exposure to the widely used fragrance 7-HC in Germany can be substantiated by applying the described methodology to the representative cohort of the launched German Environmental Survey in adults (GerES VI).

Geraniol (trans-3,7-dimethyl-2,6-octadiene-1-ol) is an acyclic isoprenoid monoterpene with a widespread use as fragrance in consumer products, agrochemicals and pharmaceuticals. The class of terpene chemicals has been associated with varying sensitizing potencies. A recently developed sensitive LC- MS/MS method for the analysis of geraniol metabolites was further improved and validated for the two metabolites, 8-carboxygeraniol and Hildebrandt acid. The successfully validated method was applied to 250 urine samples derived from the Environmental Specimen Bank (ESB) collected between 2004 and 2018. Both metabolites of this allergen of special concern were quantified in all urine samples of this study. Correlation analysis revealed that 8-carboxygeraniol appears to be the sole specific biomarker in urine for geraniol exposure. Overall, the excreted amounts of 8-carboxygeraniol remained unchanged in urine samples collected from 2004 to 2018. However, a significantly higher 8-carboxygeraniol excretion per 24 h was observed in females compared to males across the sampling years from 2004 to 2012. This trend equalized in the years 2015 and 2018. We could demonstrate that 8-carboxygeraniol may be a suited biomarker for assessing the geraniol exposure in the general population. Regardless of the fact that additional, preferably population representative studies combining HBM and health examination were helpful to further elucidate the risks of a geraniol exposure, the current study adds important data for identifying time trends and body burden of geraniol in the environment and shows the ubiquitous exposure towards mixtures of sensitizing chemicals.

Background: Biomarkers of tobacco exposure have a central role in studies of tobacco use and nicotine intake. The most significant exposure markers are nicotine itself and its metabolites in urine. Therefore, it is important to evaluate the performance of laboratories conducting these biomarker measurements. Methods: This report presents the results from a method performance study involving 11 laboratories from 6 countries that are currently active in this area. Each laboratory assayed blind replicates of seven human urine pools at various concentrations on three separate days. The samples included five pools blended from smoker and nonsmoker urine sources, and two additional blank urine samples fortified with pure nicotine, cotinine, and hydroxycotinine standards. All laboratories used their own methods, and all were based on some form of liquid chromatography/tandem mass spectrometry. Results: Overall, good agreement was found among the laboratories in this study. Intralaboratory precision was good, and in the fortified pools, the mean bias observed was < + 3.5% for nicotine, approximately 1.2% for hydroxycotinine, and less than 1% for cotinine (1 outlier excluded in each case). Both indirect and direct methods for analyzing the glucuronides gave comparable results. Conclusions: This evaluation indicates that the experienced laboratories participating in this study can produce reliable and comparable human urinary nicotine metabolic profiles in samples from people with significant recent exposure to nicotine. Impact: This work supports the reliability and agreement of an international group of established laboratories measuring nicotine and its metabolites in urine in support of nicotine exposure studies. Cancer Epidemiol Biomarkers Prev; 27(9); 1083–90. ©2018 AACR.

7-Hydroxy-3,7-dimethyl-1-octanal, also known as 7-hydroxycitronellal (7-HC, CAS No. 107-75-5) is a synthetic fragrance widely used in cosmetic and hygiene products. Due to its large scope, 7-HC was selected for the development of a biomonitoring method suitable for the general population within the frame of the cooperation project between the German Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI). In a human study with 5 healthy subjects who received single dermal and oral doses 7-HC, suitable metabolites and their urinary excretion kinetics was investigated. Two metabolites of 7-hydroxycitronellal were identified in urinary fractions after dermal and oral dosing: The alcohol 7-hydroxycitronellol (7-HCO) and the corresponding acid 7-hydroxycitronellylic acid (7-HCA). Only 7-HCA proved to be a suitable biomarker of exposure to 7-HC, since 7-HCO was quantifiable in only a minority of urine samples collected from the general population. Quantification of 7-HCA was conducted by means of a newly developed UPLC–MS/MS (ultra-high pressure liquid chromatography combined with tandem mass spectrometry) method. Peak excretion of 7-HCA occurred between 3 and 5 h after oral application and about 10 h after dermal administration. Due to the limited skin absorption of 7-HC, 7-HCA concentrations after dermal application were much lower than levels after oral application. After 24 h, about 9% and 50% of the dermally and orally applied dose, respectively, were excreted as 7- HCA. With the conversion factors derived from the controlled human study, we estimated median exposure doses in a group of 40 human volunteers from the general population of approximately 93 μg 7-HC per day. In conclusion, the 7-HC metabolite 7-HCA in urine is a suitable biomarker of exposure and can be applied for biomonitoring of the general population.

7-Hydroxycitronellal is a synthetic fragrance (CAS No. 107-75-5) which is used commonly in cosmetics, washing- and cleaning agents and as flavoring in foods. Due to its broad application in various fields, 7-hydroxycitronellal was selected for the development of a biomonitoring method for the quantitative exposure assessment within the frame of the cooperation project of the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI). For this purpose, an ultra performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) based method was developed for the determination of potential biomarkers of 7-hydroxycitronellal (7-HC) in human urine samples. 7-Hydroxycitronellylic acid (7-HCA) turned out to be the quantitatively most important metabolite of 7-HC in human urine, occurring in 1000 times higher amounts than 7-hydroxycitronellol (7-HCO) or other potential metabolites. Therefore, an analytical method for 7-HCA was developed using stable isotope-labeled 7-HCA as internal standard (IS). The method includes a cleavage step of possible metabolite conjugates with an enzyme mix of ß-glucuronidase and arylsulfatase. Subsequent sample cleanup was performed by liquid-liquid extraction (LLE) with dichloromethane. The method was calibrated by calculating the linear regression between the analyte/IS ratio and the nominal 7-HCA concentrations in water. The method was validated according to approved standard guidelines and proved to be robust, reliable and sensitive for the human biomonitoring of 7-HC. The method was applied to urine samples of 40 adult volunteers from the general population. 7-HCA was quantifiable in urine of all subjects. Thus the developed method proved to be suitable for assessing the background exposure to 7-HC in the general population.

Untargeted GC-TOF-MS analysis proved to be a suitable analytical platform to determine alterations in the metabolic profile. Several metabolic pathways were found to be altered in a first clinical study comparing smokers against nonsmokers. Subsequently, we conducted a clinical diet-controlled study to investigate alterations in the metabolic profile during the course of 3 months of smoking cessation. Sixty male subjects were included in the study, and plasma, saliva, and urine samples were collected during four 24 h stationary visits: at baseline, while still smoking, after 1 week, after 1 month, and after 3 months of cessation. Additionally, subjects were monitored for their compliance by measurements of CO in exhaled breath and salivary cotinine throughout the study. GC-TOF-MS fingerprinting was applied to plasma, saliva, and urine samples derived from 39 compliant subjects. In total, 52 metabolites were found to be significantly altered including 26 in plasma, 20 in saliva, and 12 in urine, respectively. In agreement with a previous study comparing smokers and nonsmokers, the fatty acid and amino acid metabolism showed significant alterations upon 3 months of smoking cessation. Thus these results may indicate a partial recovery of metabolic pathway perturbations, even after a relatively short period of smoking cessation.

2-(4-tert-Butylbenzyl)propionaldehyde, also known as lysmeral, lilial or lily-aldehyde (CAS No 80-54-6) is a synthetic fragrance used in a variety of consumer products like perfumes, after shave lotions, cosmetics and others. Due to its broad application, lysmeral was selected for the development of a biomonitoring method for the general population within the frame of the cooperation project of the Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI). The project also comprises the identification of suitable biomarkers of exposure in human urine as well as basic toxicokinetic data after defined, experimental exposure. For this purpose, 5 healthy subjects were orally dosed once with 5.26mg lysmeral. Urine was collected immediately before and for 48h after administration of the fragrance. The lysmeral metabolites lysmerol, lysmerylic acid, hydroxylated lysmerylic acid and 4-tert-butylbenzoic acid (TBBA) were determined in all urine samples by a newly developed UPLC-MS/MS (ultra-high pressure liquid chromatography combined with tandem mass spectrometry) method. Peak excretion for all metabolites occurred between 2 and 5h after oral application, with the primary metabolites (lysmerol and lysmerylic acid) being excreted about 1h earlier than the secondary metabolites (hydroxylated lysmerylic acid and TBBA). More than 90% of all measured lysmeral metabolites were excreted after 12h, with the renal excretion being virtually complete after 48h. After this time period, TBBA, lysmerol, lysmerylic acid and hydroxyl-lysmerylic acid represent on average 14.3, 1.82, 0.20 and 0.16%, respectively, of the dose administered. In total, the 4 metabolites determined represent about 16.5% of the dose. With the conversion factors derived from the controlled human study, we estimated median exposure doses for lysmeral in a group of 40 human volunteers from the general population of approximately 140-220μg per day. In conclusion, the lysmeral metabolites lysmerol, lysmerylic acid, hydroxyl-lysmerylic acid and TBBA in urine are suitable biomarkers of exposure and can be applied, either single or in any combination, for biomonitoring of the general population.

2-(4-tert-butylbenzyl)propionaldehyde (trade names, e.g. lysmeral or lilial) is a fragrance chemical frequently used in cosmetic products where it is labelled as Butylphenyl methylpropional. A recently developed LC-MS/MS method for the analysis of four lysmeral metabolites (tert-butylbenzoic acid (TBBA), lysmerol, lysmerylic acid, and hydroxy-lysmerylic acid) was applied to 329 urine samples from the Environmental Specimen Bank collected between 2000 and 2018. The two major metabolites TBBA and lysmerol were found in quantifiable concentrations in almost all samples in this study and correlated significantly. Hence, both analytes proved to be specific biomarkers indicating the broad exposure to lysmeral. A significant decline was found for TBBA and lysmerol for the monitored years with the most pronounced decrease from 2012 to 2015. The daily intake (DI) was used to evaluate potential health risks with respect to the derived no-effect level (DNEL) as a threshold for exposure of the general population. The median DI (1.63 μg/kg bw/d) and the 95th percentile (4.69 μg/kg bw/d) corresponded to 2.6% and 7.5% of the lowest DNEL (62.5 μg/kg bw/d for oral administration), respectively. Even though a decreasing trend in exposure was observed the data still calls for efforts to reduce the exposure towards lysmeral since metabolites of lysmeral were detected in nearly all samples and adverse effects cannot be excluded. Clearly, these results need to be substantiated by HBM campaigns in population representative samples like the German Environmental Survey in adults (GerES VI) to provide more robust data for the adult population.

The synthetic fragrance 2-(4-tert-butylbenzyl)propionaldehyde, also known as lysmeral, butylphenyl methylpropional, lilial, or lily aldehyde, is widely used in cosmetics, personal care products, laundry detergents, and air fresheners. It is classified as suspected to be harmful to fertility and possibly endocrine disrupting. Its maximum concentration in cosmetics is limited. First-morning void urine samples (N = 2133) were analysed for several metabolites of lysmeral (Chemical Abstract Service (CAS) No.: 80-54-6). Samples were collected in the population-representative German Environmental Survey for Children and Adolescents 2014–2017 (GerES V) from German residents aged 3–17 years. Four main metabolites tert-butylbenzoic acid, lysmerol, lysmerylic acid, and hydroxy-lysmerylic acid were found in quantifiable amounts in 100%, 99%, 40%, and 23% of the samples, respectively, with geometric mean concentrations of 10.21 μg/L (8.658 μg/gcrea) for tert-butylbenzoic acid, 1.528 μg/L (1.296 μg/gcrea) for lysmerol, and below the limit of quantification of 0.2 μg/L and 0.4 μg/L for lysmerylic acid and hydroxy-lysmerylic acid, respectively. Girls had higher urinary concentrations of lysmeral metabolites than boys. Usage of fragrances, fabric softener, and personal care products, especially perfume, was positively associated with urinary concentrations of lysmeral metabolites. Source identification builds a basis to derive proposals for reduction of exposure. These results can also provide the foundation for developing reference values for urinary metabolite concentrations of lysmeral in children and adolescents in Germany that will facilitate recognising future exposure trends.

Hexyl 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoate, better known under its trading name Uvinul A plus® is a UV filter mainly used in sunscreens, but also present in other cosmetic products with a maximum concentration of 10% (w/w) according to the EU directive. In this study we investigated the human metabolism after a single oral and a single dermal dose of Uvinul A plus®, respectively. Samples collected within 72 h of administration were analyzed with a newly developed UHPLC-MS/MS method. Results of the study revealed three major urinary metabolites, namely 2-(4-amino-2-hydroxybenzoyl)benzoic acid (AHB), 2-(4-(ethylamino)-2-hydroxybenzoyl)benzoic acid (EHB) and 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid (DHB), representing 52% of the administered oral dose. The three major metabolites are further converted into four minor metabolites with an additional hydroxyl group in the aniline moiety. Toxicokinetic parameters (amount excreted, tmax, elimination constant and half-life t1/2) and conversion factors were determined for the three major metabolites. The conversion factors were used to estimate the mean daily exposure to Uvinul A plus® in spot urine samples from 58 volunteers not intentionally exposed to Uvinul A plus® derived from a pilot study. The three major metabolites were quantifiable in 26% of the samples. In 35% of the samples, at least one major metabolite could be quantified. The daily systemic exposure to Uvinul A plus® was estimated to approximately 8.1–9.3 μg/d by applying the combined conversion factor for all three major metabolites. In conclusion, a very low systemic exposure to DHHB was observed with regard to the no observed adverse effect level (NOAEL) as an established threshold for chronic uptake.

Polycyclic aromatic hydrocarbons (PAH) mainly originate from incomplete combustion of organic materials and are, among other sources, found in traffic emissions, smoked or barbecued food, leafy vegetables, and tobacco smoke. Some PAH or their metabolites are hazardous for health and classified as carcinogenic, mutagenic, or toxic to reproduction. Urine samples from 3- to 17-year-old children and adolescents living in Germany were analysed for concentrations of metabolites of the PAH fluorene, naphthalene, phenanthrene, and pyrene in the population-representative German Environmental Survey for Children and Adolescents GerES V (2014–2017). PAH metabolites were analysed in urine samples of 516 participants and could be quantified in 88–100% of the samples. Geometric mean concentrations were: 0.785 μg/L (0.688 μg/gcreatinine) for 1-OH-naphthalene, 4.233 μg/L (3.706 μg/gcrea) for 2-OH-naphthalene, 0.139 μg/L (0.122 μg/gcrea) for 1-OH-phenanthrene, 0.085 μg/L (0.075 μg/gcrea) for 2-OH-phenanthrene, 0.131 μg/L (0.115 μg/gcrea) for 3-OH-phenanthrene, 0.045 μg/L (0.040 μg/gcrea) for 4-OH-phenanthrene, 0.058 μg/L (0.050 μg/gcrea) for 9-OH-phenanthrene, 0.511 μg/L (0.448 μg/gcrea) for Σ-OH-phenanthrene, and 0.099 μg/L (0.087 μg/gcrea) for 1-OH-pyrene. Analyses of subgroups revealed higher PAH metabolite concentrations in young children compared to adolescents, and also in residents of former East Germany compared to those living in former West Germany. Increased urinary PAH metabolite concentrations were found in participants using domestic fuel for heating or gas for cooking. Plastic objects were identified as another potential source of exposure. Urinary concentrations of naphthalene and fluorene metabolites were elevated in active smokers and to the same extent in non-smokers exposed to passive smoking. Comparison with previous cycles of GerES revealed a decrease over time and a further decline in the still significant differences in urinary PAH metabolite concentrations of participants living in former East versus West Germany.

A preceding untargeted metabolic fingerprinting approach in our lab followed by targeted fatty acid analysis revealed alterations in arachidonic acid metabolism in samples derived from a diet-controlled smoking cessation study in which compliant subjects ( N = 39) quit smoking at baseline and were followed over the course of 3 months. Consequently, urinary eicosanoids were evaluated by means of a validated LC-MS/MS method. A significant decrease was obtained for the prostaglandins PGF2α, 8-iso-PGF2α, thromboxane 2,3-d-TXB2, and leukotriene E4 upon quitting smoking. These findings indicate a partial recovery of smoking-induced alterations in the eicosanoid profile due to a reduction in oxidative stress and the inflammatory response.

cope : Research is limited on diet challenges to improve health. A short-term, vegan protein diet regimen nutritionally balanced in macronutrient composition compared to an omnivorous diet was hypothesized to improve metabolic measurements of blood sugar regulation, blood lipids, and amino acid metabolism. Methods and results : This randomized, cross-over, controlled vegan verses animal diet challenge was conducted on 21 (11 female,10 male) healthy participants. Fasting plasma was measured during a 3-day diet intervention for clinical biochemistry and metabonomics. Intervention diet plans met individual caloric needs. Meals were provided and supervised. Diet compliance was monitored. Conclusions : The vegan diet lowered triglycerides, insulin and homeostatic model assessment (HOMA-IR), bile acids, elevated magnesium levels, and changed branched-chain amino acids (BCAAs) metabolism (p

Mass spectrometry is an ideal tool for investigations of the metabolome in human plasma. To investigate the impact of smoking on the human metabolome, we performed an untargeted metabolic fingerprinting using GC-TOF-MS with EDTA-plasma samples from 25 smokers and 25 non-smokers. The observed elevated levels in the monounsaturated fatty acids (MUFAs) in smokers were verified by a targeted analysis using GC-FID, which revealed also significantly alterations in saturated and polyunsaturated fatty acids in smokers (p0.05, Mann-Whitney U test). Since the main fraction of fatty acids in plasma is esterified to phospholipids, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species composition in the plasma samples of the same subjects. The profiles of 39 PC and 40 PE species were analyzed with a newly developed and validated HILIC-ESI-MS/MS method. We were able to baseline separate the two lipid classes (PC from PE) by maintaining co-elution of individual lipid species of each class. The method shows a linear range from 0.5μM to 2000μM and an inter- and intraday coefficient of variation (CV)20% across all analytes. Application of the validated method to the plasma samples of smokers and non-smokers, derived from a diet-controlled smoking study, revealed significantly elevated levels of PC and PE species containing MUFAs in smokers. In summary, we could demonstrate that there is a significantly altered total fatty acid profile, with increased MUFAs, in the plasma of smokers compared to non-smokers. Results obtained with the new HILIC-MS/MS method indicate that the altered fatty acid profile is also reflected in the PC and PE profile of smokers.

Urinary determination of nicotine metabolites provides an ideal tool for the quantitative assessment of the tobacco use-related nicotine dose, provided that the considered metabolites comprise a large share of the amount taken up. A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the sensitive, fast and robust analysis of nicotine and 10 major nicotine metabolites ("Nic+10"), including cotinine, trans-3'-hydroxy-cotinine, nicotine-N-glucuronide, cotinine-N-glucuronide, trans-3'-hydroxy-cotinine-O-glucuronide, nornicotine, norcotinine, nicotine-N'-oxide, cotinine-N'-oxide and 4-hydroxy-(3-pyridyl)-butanoic acid. Corresponding deuterated internal standards were spiked prior to a simple and straightforward solid phase extraction (SPE) procedure. Liquid chromatography was performed on a reversed phase C8 column and mass-specific detection was conducted in scheduled-MRM mode. The method was validated according to FDA Guidelines, showing excellent selectivity, precision, accuracy and robustness. The limits of quantification were in the range 0.2-2.3ng/ml for all analytes. The novel method was applied to human urine samples derived from 25 smoking subjects. Quantitative results were correlated against a previously used LC-MS/MS method and compared to reports from the literature. The relative molar profile of nicotine and its 10 major metabolites was in good agreement with the literature. In addition, correlation amongst the two methods was excellent for almost all analytes, whereas the accordance between both methods was moderate for hydroxy-cotinine-O-glucuronide and norcotinine. These deviations, however, could be explained. The current method allows the simultaneous determination of nicotine and its 10 major metabolites (metabolite coverage about 95% of the absorbed dose) from a small sample volume and within a reasonable amount of time. Due to its wide dynamic range, high sensitivity and high throughput capabilities, this method could serve as a powerful tool for quantifying the nicotine dose of smokers, passive smokers as well as novel tobacco and nicotine product users in clinical and epidemiological studies.

We developed and validated an efficient and robust method for the simultaneous quantification of 44 fatty acid species in human plasma via GC-TOF-MS. The method is characterized by its robustness, accuracy and precision covering a wide range of fatty acid species with various saturation degrees including short chain fatty acids (beginning with FA 4:0) and long chain fatty acids (up to FA 32:0). The fatty acids were methylated prior to analyses and subsequently detected as fatty acid methyl esters by means of GC-TOF-MS. A highly substituted polar column allowed the separation of geometrical and positional isomers of fatty acid species. The method was applied to plasma samples of a strictly diet controlled clinical smoking cessation study including 39 smokers followed over the course of three months after having quit. Statistical significant alterations within the fatty acid profile were observed when comparing the baseline (subjects still smoking) with one week, one month and three months of smoking cessation. After 3 months of smoking cessation, a partial recovery of alterations in the fatty acid profile evoked by smoking was observed. In conclusion, the developed fatty acid profiling method using GC-TOF-MS has proven as a reliable tool for the quantitative determination of 44 individual fatty acid species within clinical studies.

2-(4-tert-Butylbenzyl)propionaldehyde also known as lysmeral, lilial, or lily aldehyde (CAS No. 80-54-6) is a synthetic odorant mainly used as a fragrance in a variety of consumer products like cleaning agents, fine fragrances, cosmetics, and air fresheners. Due to its broad application in various fields, lysmeral was selected for the development of a biomonitoring method for the quantitative exposure assessment within the frame of the cooperation project of the Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI). A method based on ultra-high pressure liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of potential biomarkers of lysmeral in human urine samples. Sample cleanup was performed by liquid-liquid extraction (LLE). Quantification was achieved by standard addition using stable isotope-labeled, authentic reference standards. The method is characterized by its robustness, reliability, and excellent sensitivity as proven during method validation according to approved standard guidelines. The following five lysmeral metabolites were identified as potential biomarkers of exposure for lysmeral in human urine samples: lysmerol, lysmerylic acid, hydroxylated lysmerylic acid, tert-butylbenzoic acid (TBBA), and tert-butylhippuric acid (TBHA). The determination of lysmerol required derivatization with 3-nitrophthalic acid anhydride and showed the lowest limit of detection (LOD) and limit of quantification (LOQ) in urine (0.035 and 0.10 μg/L, respectively). LOD and LOQ for the other metabolites were in the range of 0.12–0.15 and 0.36–0.45 μg/L, respectively. Accuracy for all analytes was in the range of 90–110 %. Intra- and inter-day precision was in the range of 5–10 %, except for TBHA, for which the coefficient of variation was unacceptably high (>20 %) and therefore excluded from the method. The method was applied to urine samples of 40 adult volunteers. The four remaining lysmeral metabolites were detectable in most of the 40 urine samples in the following order according to quantity excreted: TBBA >> lysmerol ≈ lysmerylic acid > hydroxy-lysmerylic acid. In conclusion, we successfully developed a biomonitoring method for the assessment of the exposure to lysmeral in the general population. The method is characterized by its precision, robustness, and accuracy. The metabolites lysmerol, lysmerylic acid, hydroxylated lysmerylic acid, and TBBA turned out to be suitable biomarkers of exposure to lysmeral, either alone or in combination with one or more of the other metabolites. Sensitivity was found to be sufficient for assessing the background exposure to this chemical in the general population.

BACKGROUND: Stored platelet concentrates (PLCs) for transfusion develop a platelet storage lesion (PSL), resulting in decreased platelet (PLT) viability and function. The processes leading to PSL have not been described in detail and no data describe molecular changes occurring in all three components of stored PLCs: PLTs, PLC extracellular vesicles (PLC-EVs), and plasma. STUDY DESIGN AND METHODS: Fifty PLCs from healthy individuals were stored under standard blood banking conditions for 5 days. Changes in cholesterol, glycerophospholipid, and sphingolipid species were analyzed in PLTs, PLC-EVs, and plasma by mass spectrometry and metabolic labeling. Immunoblots were performed to compare PLT and PLC-EV protein expression. RESULTS: During 5 days, PLTs transferred glycerophospholipids, cholesterol, and sphingolipids to newly formed PLC-EVs, which increased corresponding lipids by 30%. Stored PLTs significantly increased ceramide (Cer; +53%) and decreased sphingosine-1-phosphate (−53%), shifting sphingolipid metabolism toward Cer. In contrast, plasma accumulated minor sphingolipids. Compared to PLTs, fresh PLC-EVs were enriched in lysophosphatidic acid (60-fold) and during storage showed significant increases in cholesterol, sphingomyelin, dihydrosphingomyelin, plasmalogen, and lysophosphatidylcholine species, as well as accumulation of apolipoproteins A-I, E, and J/clusterin. CONCLUSION: This is the first detailed analysis of lipid species in all PLC components during PLC storage, which might reflect mechanisms active during in vivo PLT senescence. Stored PLTs reduce minor sphingolipids and shift sphingolipid metabolism toward Cer, whereas in the plasma fraction minor sphingolipids increase. The composition of PLC-EVs resembles that of lipid rafts and confirms their role as carriers of bioactive molecules and master regulators in vascular disease.