A sensitive GC-MS/MS method for the quantification of benzo[a]pyrene tetrol in urine.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants formed during the incomplete combustion of organic matter such as tobacco. Among these, benzo[a]pyrene (BaP) has been classified as a known carcinogen to humans. It unfolds its effect through metabolic activation to BaP-(7R,8S)-diol-(9S,10R)-epoxide (BPDE), the ultimate carcinogen of BaP. In this article, we describe a simple and highly sensitive GC-NICI-MS/MS method for the quantification of urinary BaP-(7R,8S,9R,10S)-tetrol (( +)-BPT I-1), the hydrolysis product of BPDE. The method was validated and showed excellent results in terms of accuracy, precision, and sensitivity (lower limit of quantification (LLOQ): 50 pg/L). In urine samples derived from users of tobacco/nicotine products and non-users, only consumption of combustible cigarettes was associated with a significant increase in BPT I-1 concentrations (0.023 ± 0.016 nmol/mol creatinine, p < 0.001). Levels of users of potentially reduced-risk products as well as non-users were all below the LLOQ. In addition, the urine levels of six occupationally exposed workers were analyzed and showed the highest overall concentrations of BPT I-1 (844.2 ± 336.7 pg/L). Moreover, comparison with concentrations of 3-hydroxybenzo[a]pyrene (3-OH-BaP), the major detoxification product of BaP oxidation, revealed higher levels of 3-OH-BaP than BPT I-1 in almost all study subjects. Despite the lower levels, BPT I-1 can provide more relevant information on an individual's cancers susceptibility since BPDE is generated by the metabolic activation of BaP. In conclusion, BPT I-1 is a suitable biomarker to distinguish not only cigarette smokers from non-smokers but also from users of potentially reduced-risk products.

The potential of new nicotine and tobacco products as tools for people who smoke to quit combustible cigarettes - a systematic review of common practices and guidance towards a robust study protocol to measure cessation efficacy.

New types of nicotine and tobacco products like electronic cigarettes (ECs), heated tobacco products or nicotine pouches have been discussed as less harmful alternatives to combustible cigarettes and other toxic forms of tobacco products. Their harm reduction potential lay in the efficient transition away from smoking to those new products. Numerous studies addressing the cessation efficacy of ECs have been published with contradictory outcomes. Yet, a comprehensive Cochrane review concluded with high certainty on the cessation efficacy of ECs. This prompted us to perform a review to identify weaknesses in common study designs and to summarize best practices for the study design on the potential of new nicotine products as cessation aids. 120 articles retrieved from Medline were found to be eligible. Most of the studies in the field were interventional trials while observational studies played a minor role in the evaluation of smoking cessation. Efficacy was predominantly assessed for ECs in 77% of the reports while heated tobacco (17%) and non-combustible products (11%) were less frequently investigated up to now. Measures to determine the efficacy were questionnaire-based assessments as well as use documentation/prevalence and abstinence rates. Studies varied largely in their duration and sample size with medians of 3 months and 156.5 participants, respectively.With the help of this review, we identified several weaknesses in the common study designs. One major limitation in longitudinal trials was the lack of compliance measures suited to verify the use status over longer time periods, relying solely on self-reports. Moreover, the motivation of the participants to quit was rarely defined and a profound familiarization period was not taken into account for the majority of the studies. To what extent such weaknesses influence the outcome of the studies was beyond the scope of this review. We encourage researchers to consider the recommendations which resulted from this review in order to determine the abuse liability and cessation efficacy of the products in a more robust manner. Finally, we like to call attention to the missing data for low- and middle-income countries which would require quitting strategies most urgently to combat the tobacco smoking epidemic.

Time trend of exposure to secondhand tobacco smoke and polycyclic aromatic hydrocarbons between 1995 and 2019 in Germany - Showcases for successful European legislation.

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.

Human metabolism and excretion kinetics of the surfactant 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) after oral and dermal administration.

2,4,7,9-Tetramethyl-5-decyne-4,7-diol (TMDD) is a non-ionic surfactant with a wide range of applications. TMDD is considered a high-production chemical and, due to its low biodegradation rate, possesses a potentially high prevalence in the environment. However, despite its widespread use, toxicokinetic data and data on internal exposure to TMDD in the general population are completely lacking. Hence, we developed a human biomonitoring (HBM) method for TMDD. Our approach included a metabolism study with four subjects, who were administered an oral dose of 75 µg TMDD/kg body weight and a dermal dose of 750 µg/kg body weight. Terminal methyl-hydroxylated TMDD (1-OH-TMDD) was previously identified as the main urinary metabolite in our lab. The results of the oral and dermal applications were used to determine the toxicokinetic parameters of 1-OH-TMDD as a biomarker of exposure. Finally, the method was applied to 50 urine samples from non-occupationally exposed volunteers. Results show that TMDD was rapidly metabolized, with an average tmax of 1.7 h and a rapid and almost complete (96%) excretion of 1-OH-TMDD until 12 h after oral dosage. Elimination was bi-phasic, with half-lives of 0.75-1.6 h and 3.4-3.6 h for phases 1 and 2, respectively. The dermal application resulted in a delayed urinary excretion of this metabolite with a tmax of 12 h and complete excretion after about 48 h. The excreted amounts of 1-OH-TMDD represented 18% of the orally administered TMDD dose. The data of the metabolism study demonstrated a fast oral as well as substantial dermal resorption of TMDD. Moreover, the results indicated an effective metabolism of 1-OH-TMDD, which is excreted rapidly and completely via urine. Application of the method to 50 urine samples revealed a quantification rate of 90%, with an average concentration of 0.19 ng/mL (0.97 nmol/g creatinine). With the urinary excretion factor (Fue) derived from the metabolism study, we estimated an average daily intake of 1.65 µg TMDD from environmental and dietary sources. In conclusion, 1-OH-TMDD in urine is a suitable biomarker of exposure to TMDD and can be applied for biomonitoring of the general population.

Assessment of the Exposure to Tobacco-Specific Nitrosamines and Minor Tobacco Alkaloids in Users of Various Tobacco/Nicotine Products.

Tobacco-specific nitrosamines (TSNAs), in particular, the human carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN), are important toxicants in tobacco and also (as contaminants) in nicotine products. In a clinical study comprising a period of 74 h under confinement, we investigated the exposure to NNK, NNN, N'-nitrosoanabasine (NAB), and N'-nitrosoanatabine (NAT) as well as to the minor tobacco alkaloids anabasine (AB) and anatabine (AT) by measuring suitable biomarkers in habitual users of combustible cigarettes (CCs), electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy products (NRTs). Non-users (NU) of any tobacco/nicotine products served as the (negative) control group. Smokers exhibited the highest levels for all biomarkers measured, except for AB in urine, which was found to be highest in OT users. Somewhat elevated levels compared to NU, EC, and NRT groups were also observed in the users of HTPs. In the users of tobacco-containing products (CC, HTP, and OT), most frequently the biomarkers significantly correlated with the dose markers such as daily consumption, urinary nicotine equivalents (Nequ), and plasma cotinine (CotP). In conclusion, except for smokers (CC) and OT users, exposure of users of ECs, HTPs, and NRTs to TSNAs as well as the minor tobacco alkaloids AB and AT is marginal and statistically not distinguishable from that of NU. Finally, our results for NNN in the saliva provide preliminary evidence for the formation of NNN from the precursor nornicotine in the presence of thiocyanate as a catalyst. The latter hypothesis requires experimental verification.

Assessment of the Exposure to NNN in the Plasma of Smokeless Tobacco Users.

N-Nitrosonornicotine (NNN) is a human carcinogen present in cigarette smoke and smokeless tobacco. Urinary NNN is usually measured in order to assess the exposure to this toxicant for tobacco users. NNN excretion in urine can be highly biased due to the formation of NNN by nitrosation of nornicotine under acidic conditions, both endogenously and exogenously. Hence, urinary NNN levels may not necessarily correctly reflect the product-specific exposure. Measurement of plasma NNN may be less prone to endogenous formation due to the stable pH (7.4) of blood. We developed an LC-MS/MS method for the quantification of NNN using 1 mL of human plasma. Validation according to FDA guidelines proved that the method is selective and highly sensitive with an LLOQ of 0.3 pg/mL. Accuracy and precision averaged to 98.7 and 7.5% (CV), respectively. The assay was applied to plasma samples collected from 10 experienced moist smokeless tobacco users during and after a single use of 2 g of the product for 40 min under controlled use conditions. Blood was drawn at 15 time points over a 6 h time course. The maximum NNN concentration (Cmax) ranged from 3.5 to 10 pg/mL (mean: 7.1 pg/mL) at a tmax of 32 min. Plasma NNN and nicotine were found to have similar time courses. In conclusion, the determination of NNN in plasma may be fit-for-purpose to evaluate the product-use-specific exposure to this carcinogen.

Assessment of the exposure to polycyclic aromatic hydrocarbons in users of various tobacco/nicotine products by suitable urinary biomarkers.

Polycyclic aromatic hydrocarbons (PAHs) occur naturally (bitumen and oils) and are formed during all incomplete combustions of organic materials. PAH exposure sources are manifold and include specific workplaces, ambient air, various foodstuffs, tobacco smoke and some medications. At least four members of this class of chemicals have been classified as proven or probable human carcinogens. Assessment of the exposure to PAHs with suitable methods is of importance, particularly in users of new-generation tobacco/nicotine products, which are intended to replace combustible cigarettes (CCs), a major source of non-occupational exposure to PAHs. In a clinical study comprising a period of 74 h under confinement, we investigated the exposure to naphthalene (Nap), fluorene (Flu), phenanthrene (Phe), pyrene (Pyr) and benzo[a]pyrene (BaP) by measuring urinary monohydroxy-PAH (OH-PAH) derived from these parent compounds in habitual users of CCs, electronic cigarettes (ECs), heated tobacco products (HTPs), oral tobacco (OT), and nicotine replacement therapy products (NRTs). Non-users (NU) of any tobacco/nicotine products served as (negative) control group. Smokers exhibited the highest levels for all PAH biomarkers measured, almost all of which were significantly different from the NU and user groups of all other products investigated. CC smokers were the only group which showed a significant relationship between almost all PAH biomarkers and dose markers such as daily consumption, urinary nicotine equivalents (Nequ) and plasma cotinine (CotP). The ratios in urinary OH-PAH between CC and all other groups were dependent on the biomarker and range from < 2 to > 10. These ratios could at least partly be explained by the enzymes involved, their region-selectivity and inducibility by smoking. In conclusion, cigarette smokers (CC) were the only group, which showed product use dependent exposure to PAHs, whereas users of EC, HTP, NRT and OT were not distinguishable from NU of any tobacco/nicotine products.

Benzene metabolite SPMA and acrylamide metabolites AAMA and GAMA in urine of children and adolescents in Germany - human biomonitoring results of the German Environmental Survey 2014-2017 (GerES V).

Benzene and acrylamide are carcinogenic substances contained inter alia in tobacco smoke. The mercapturic acid metabolites of benzene, N-acetyl-S-phenyl-L-cysteine (SPMA), and of acrylamide, N-acetyl-S-(3-amino-3-oxopropyl)-cysteine (AAMA) and N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)-cysteine (GAMA), were analysed in 2260 first-morning void urine samples from children and adolescents aged 3-17 years, participating in the population-representative German Environmental Survey on Children and Adolescents, GerES V 2014-2017. SPMA was detected in 98% of the participants with a geometric mean (GM) of 0.097 µg/L urine. Smokers had about 10-fold higher levels of the benzene metabolite SPMA than non-smokers. The sample comprises of 48 self-reported smokers, mainly in the oldest age group (14-17-year-olds). Second-hand smoke exposure, living near busy or very busy roads, and using domestic fuels for heating were additionally associated with higher benzene metabolite levels. SPMA levels in GerES V were lower compared to levels found in other countries, which in part however may reflect different proportions of smokers. The acrylamide metabolites AAMA and GAMA were detected in 100% of the participants with a GM of 72.6 µg/L urine for AAMA and 15.0 µg/L urine for GAMA. Smoking children and adolescents had about 2.5-fold higher AAMA levels than non-smoking ones. The frequency of consumption of french-fried potatoes and potato crisps consumption was also positively associated with urinary AAMA and GAMA levels. Compared to the urinary AAMA and GAMA levels in Germany and other countries, levels in GerES V tended to be higher than in the few studies reported. The urinary levels of the benzene biomarker SPMA, and the acrylamide biomarkers AAMA and GAMA build the basis to derive reference values for the exposure of children and adolescents in Germany. The results reveal options for exposure reduction mainly in personal choices regarding smoking and diet, but also requiring policy to maintain efforts in non-smoking regulations and improving ambient air quality. Providing these results also to the European HBM Initiative HBM4EU will contribute to gain knowledge on the exposure of the European population, the health impact of carcinogens and thus providing support for substantiated exposure assessment.

Assessment of the potential vaping-related exposure to carbonyls and epoxides using stable isotope-labeled precursors in the e-liquid.

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.

A novel quantification method for sulfur-containing biomarkers of formaldehyde and acetaldehyde exposure in human urine and plasma samples.

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.

Development of a human biomonitoring method for assessing the exposure to ethoxyquin in the general population.

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 validated UPLC-MS/MS method for the determination of urinary metabolites of Uvinul® A plus.

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.

Biomarkers of Exposure Specific to E-vapor Products Based on Stable-Isotope Labeled Ingredients.

INTRODUCTION: An important basis for risk estimation for e-cigarette (e-cig) users is a well-founded dosimetry. The objective of this study was to assess the applicability of stable-isotope e-liquid ingredients for exposure studies in vapers. METHODS: E-cigs with 10% of labeled propylene glycol (PG), glycerol (G), and nicotine was used by 20 experienced vapers under controlled (Part A) and free (Part B) conditions. In Part A, 10 subjects vaped at 10 W and another 10 subjects at 18 W power setting of the e-cig. In Part B, the same subjects used the same product ad libitum in their usual environment. Five smokers, smoking 10 non-filter cigarettes, spiked with labeled PG, G, and nicotine, served as positive control during Part A. PG, G, nicotine and its metabolites were measured in plasma, urine, and saliva. RESULTS: Peak nicotine levels (sum of measured labeled and unlabeled) in plasma were lower in vapers (15.8 to 19.6 ng/mL) than in smokers (36 ng/mL). The labeled plasma nicotine levels were ten times lower than the unlabeled, reflecting the ratio in the e-liquid. PG levels in plasma and urine also reflected the vaping activities in Part A, while G in these body fluids showed no association with vaping. CONCLUSIONS: This proof of concept study shows that the application of labeled e-liquid ingredients allows the accurate quantification of the dose of nicotine and PG when other nicotine and tobacco products were used simultaneously. Unchanged G was not assessable by this approach. IMPLICATIONS: This approach allows the investigations of the absorption of potential PG-, G-, and nicotine-derived vapor constituents (eg, aldehydes and epoxides) by vaping. Appropriate studies are in progress in our laboratory.

Analysis of Urinary Eicosanoids by LC-MS/MS Reveals Alterations in the Metabolic Profile after Smoking Cessation.

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.

Review of biomarkers to assess the effects of switching from cigarettes to modified risk tobacco products.

Context: One approach to reducing the harm caused by cigarette smoking, at both individual and population level, is to develop, assess and commercialize modified risk alternatives that adult smokers can switch to. Studies to demonstrate the exposure and risk reduction potential of such products generally involve the measuring of biomarkers, of both exposure and effect, sampled in various biological matrices.Objective: In this review, we detail the pros and cons for using several biomarkers as indicators of effects of changing from conventional cigarettes to modified risk products.Materials and methods: English language publications between 2008 and 2017 were retrieved from PubMed using the same search criteria for each of the 25 assessed biomarkers. Nine exclusion criteria were applied to exclude non-relevant publications.Results: A total of 8876 articles were retrieved (of which 7476 were excluded according to the exclusion criteria). The literature indicates that not all assessed biomarkers return to baseline levels following smoking cessation during the study periods but that nine had potential for use in medium to long-term studies.Discussion and conclusion: In clinical studies, it is important to choose biomarkers that show the biological effect of cessation within the duration of the study.

Suitability of biomarkers of biological effects (BOBEs) for assessing the likelihood of reducing the tobacco related disease risk by new and innovative tobacco products: A literature review.

The health risk of tobacco smoking can best be avoided or reduced by not taking up or quitting the habit. The use of new and innovative tobacco (NTPs, e.g. electronic cigarettes) can either be an aid for smoking cessation or, for those who are not able or willing to quit, an alternative for smoking conventional tobacco products. Before the use of an NTP can be regarded as an effective approach in tobacco harm reduction (THR), the implicated risk has to be evaluated by suitable toxicological methods such as the analysis of the chemical composition as well as assessment of detrimental effects in animal and in vitro studies. In human (clinical) studies, the NTP-related exposure to toxicants and early biological effects can be assessed by the determination of suitable biomarkers. In this review, the suitability of established and newly developed biomarkers of biological effect (BOBEs) for the indicated purpose is evaluated according to five criteria, including the association to diseases, reported difference in BOBE levels between smokers and non-smokers, dose-response relationships, reversibility and kinetics after smoking cessation. Furthermore, the effect size and the resulting sample size required in clinical studies were estimated and considered in the BOBE evaluation process. It is concluded that the rating process presented is useful for selecting BOBEs suitable for risk evaluation of NTPs in clinical and other human studies.

Metabolomic Fingerprinting in Various Body Fluids of a Diet-Controlled Clinical Smoking Cessation Study Using a Validated GC-TOF-MS Metabolomics Platform.

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.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the human biomonitoring of non-occupational exposure to the fragrance 2-(4-tert-butylbenzyl)propionaldehyde (lysmeral).

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.

Analysis of 18 urinary mercapturic acids by two high-throughput multiplex-LC-MS/MS methods.

Mercapturic acids (MAs) are metabolic end products formed from conjugates between glutathione and electrophilic compounds. MAs are, therefore, suitable biomarkers of exposure to toxicants, which are either electrophiles by themselves or metabolized to electrophilic intermediates. We developed and validated two LC-MS/MS methods which allow the complementary, rapid, and sensitive determination of MAs derived from acrolein, acrylamide, acrylonitrile, benzene, 1,3-butadiene, crotonaldehyde, N,N-dimethylformamide, ethylene, ethylene oxide, vinyl chloride, propylene oxide, styrene, toluene as well as methylating and ethylating agents. Since separate determinations of single or small groups of MAs are time-consuming and expensive, we multiplexed several individual methods into two LC-MS/MS methods covering 18 individual mercapturic acids. Method validation according to FDA guidelines showed excellent results in terms of robustness, accuracy, and sensitivity of the methods. Moreover, the use of a minimal, simple, and straightforward sample cleanup procedure further accelerated the analytical workflow, which allows a time- and cost-efficient analysis of up to 18 MAs derived from various toxicants in environmental levels. The methods were applied to urine samples derived from a strictly diet-controlled clinical study, including 25 smoking and 25 non-smoking subjects. Significant increase in the urine concentrations in smokers as compared to non-smokers (p < 0.01; Student t test) was observed for 13 individual MAs. Moreover, a dose dependence was obtained for the majority of the analytes. In conclusion, the newly developed assays represent a powerful tool for the fast and reliable quantification of 18 MAs in clinical studies. A first method application suggests several suitable biomarkers for nine relevant toxicants in tobacco smoke.

Saliva as a matrix for human biomonitoring in occupational and environmental medicine.

PURPOSE: Human biomonitoring (HBM) implies the assessment of internal exposure to hazardous substances by measuring the substances, their metabolites or reaction products, as well as effect parameters in human body fluids. Along with blood, plasma and urine, saliva is of increasing interest as an alternative matrix for HBM. METHODS: This paper reviews studies that measure salivary background levels of hazardous substances, elevated levels after environmental or occupational exposure, as well as references which deal with physiological and toxicokinetic behaviour of saliva and salivary parameters, respectively. RESULTS: The studies revealed that the determination of biomarkers in saliva is a promising approach for HBM, even if only few substances showed a satisfying correlation with exposure data or established biomonitoring matrices such as blood, plasma and urine. Saliva has been proven to be particularly suitable for substances of low molecular weight such as organic solvents, selected pesticides, cotinine, and for some specific trace elements. Besides several advantages, serious problems and limitations were identified. Above all, the complex interactions between substance properties, sampling procedure, sample preparation, measurement techniques or individual factors, and the salivary analyte level are discussed. CONCLUSIONS: A major conclusion of the review is that more scientific studies are needed in order to systematically collect data on parameters, influencing salivary analyte levels. Crucially required is a harmonisation of the sampling as well as the sample preparation techniques and procedures, which is indispensable to achieve an overall comparability and interpretability of salivary biomarker levels.