A new analytical framework for multi-residue analysis of chemically diverse endocrine disruptors in complex environmental matrices utilising ultra-performance liquid chromatography coupled with high-resolution tandem quadrupole time-of-flight mass spectrometry.

This manuscript presents a comprehensive analytical framework for identification and quantification of chemically diverse endocrine disrupting chemicals (EDCs) used in personal care and consumer products in diverse solid and liquid environmental matrices with an ultimate goal of evaluating public exposure to EDCs via water fingerprinting. Liquid chromatography coupled with tandem quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS/MS) was used for targeted analysis of selected EDCs as well as to identify and quantify a few metabolites using post-acquisition data mining. Solid-phase extraction (SPE) was applied to liquid matrices in order to reduce matrix effects and provide required sample concentration and ultimately, high sensitivity and selectivity of measurements. SPE recoveries in liquid samples ranged from 49 to 140% with method quantification limits not exceeding 1 ng L-1 for the majority of EDCs. Microwave-assisted extraction (MAE) was applied to solid samples and when followed by SPE, it permitted the analysis of EDCs in digested sludge. MAE/SPE recoveries varied from 11 to 186% and MQLs between 0.03 and 8.1 ng g-1 with the majority of compounds showing MQLs below 2 ng g-1. Mass error for quantifier and qualifier ions was below 5 ppm when analysing river water and effluent wastewater and below 10 ppm when analysing influent wastewater and solid samples. The method was successfully applied to environmental samples, with 33 EDCs identified and quantified in wastewater and receiving waters. In addition, several EDCs were found in digested sludge, which confirms that for a more comprehensive understanding of exposure patterns and environmental impact, analysis of solids cannot be neglected. Finally, post-acquisition data mining permitted the identification and quantification of a metabolite of BPA and the identification of a metabolite of 4-Cl-3-methylphenol. Graphical abstract ᅟ.

New Analytical Framework for Verification of Biomarkers of Exposure to Chemicals Combining Human Biomonitoring and Water Fingerprinting.

Molecular epidemiology approaches in human biomonitoring are powerful tools that allow for verification of public exposure to chemical substances. Unfortunately, due to logistical difficulties and high cost, they tend to evaluate small study groups and as a result might not provide comprehensive large scale community-wide exposure data. Urban water fingerprinting provides a timely alternative to traditional approaches. It can revolutionize the human exposure studies as urban water represents collective community-wide exposure. Knowledge of characteristic biomarkers of exposure to specific chemicals is key to the successful application of water fingerprinting. This study aims to introduce a novel conceptual analytical framework for identification of biomarkers of public exposure to chemicals via combined human metabolism and urban water fingerprinting assay. This framework consists of the following steps: (1) in vitro HLM/S9 assay, (2) in vivo pooled urine assay, (3) in vivo wastewater fingerprinting assay, (4) analysis with HR-MSMS, (5) data processing, and (6) selection of biomarkers. The framework was applied and validated for PCMC (4-chloro-m-cresol), household derived antimicrobial agent with no known exposure and human metabolism data. Four new metabolites of PCMC (hydroxylated, sulfated/hydroxylated, sulfated PCMC, and glucuronidated PCMC) were identified using the in vitro HLM/S9 assay. But only one metabolite, sulfated PCMC, was confirmed in wastewater and in urine. Therefore, our study confirms that water fingerprinting is a promising tool for biomarker selection and that in vitro HLM/S9 studies alone, although informative, do not provide high accuracy results. Our work also confirms, for the first time, human internal exposure to PCMC.

The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs).

1. Selective androgen receptor modulators (SARMs) are a group of substances that have potential to be used as doping agents in sports. Being a relatively new group not available on the open market means that no reference materials are commercially available for the main metabolites. In the presented study, the in vitro metabolism of SARMs by the fungus Cunninghamella elegans has been investigated with the purpose of finding out if it can produce relevant human and equine metabolites. 2. Three different SARMs, S1, S4 and S24, were incubated for 5 days with C. elegans. The samples were analysed both with and without sample pretreatment using ultra performance liquid chromatography coupled to high resolution mass spectrometry. 3. All the important phase I and some phase II metabolites from human and horse were formed by the fungus. They were formed through reactions such as hydroxylation, deacetylation, O-dephenylation, nitro-reduction, acetylation and sulfonation. 4. The study showed that the fungus produced relevant metabolites of the SARMs and thus can be used to mimic mammalian metabolism. Furthermore, it has the potential to be used for future production of reference material.

Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry.

RATIONALE: Bupivacaine is a local anaesthetic prohibited in equine sports. It is highly metabolized in the horse but a thorough description of its metabolite profile is lacking. An administration study should find appropriate analytical targets for doping control. Furthermore, knowledge of an in vitro system for production of metabolites would be beneficial. METHODS: Marcain® (bupivacaine hydrochloride) was administered subcutaneously to a horse and urine samples were collected. In vitro metabolic systems consisting of the fungi Cunninghamella elegans and Cunninghamella blakesleeana were incubated with bupivacaine and bupivacaine-d(9). Samples were analyzed directly after dilution or cleaned up using liquid-liquid extraction. Separation was achieved with liquid chromatography. Mass spectrometric analysis was performed using positive electrospray ionization with both a tandem quadrupole and an ion trap instrument using MS(n) and hydrogen/deuterium exchange. RESULTS: In horse urine, seven phase I metabolites were found: 3'- and 4'-hydroxybupivacaine, N-desbutylbupivacaine, two aliphatically hydroxylated metabolites, one N-oxide, and dihydroxybupivacaine. Sulfated hydroxybupivacaine and glucuronides of 3'- and 4'-hydroxybupivacaine and of dihydroxybupivacaine were also detected. All these metabolites were previously undescribed in the horse, except for 3'-hydroxybupivacaine. 3'- and 4'-Hydroxybupivacaine were designated as appropriate targets for doping control. Interestingly, all the equine phase I metabolites were also detected in the samples from C. elegans and C. blakesleeana. CONCLUSIONS: The qualitative aspects of the metabolism of bupivacaine in the horse have been investigated with many novel metabolites described. The fungi C. elegans and C. blakesleeana have proven to be relevant models for mammalian metabolism of bupivacaine and they may in the future be used to produce analytical reference materials.

Publisher Correction: A new approach towards biomarker selection in estimation of human exposure to chiral chemicals: a case study of mephedrone.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Publisher Correction: A new approach towards biomarker selection in estimation of human exposure to chiral chemicals: a case study of mephedrone.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

A new approach towards biomarker selection in estimation of human exposure to chiral chemicals: a case study of mephedrone.

Wastewater-based epidemiology is an innovative approach to estimate public health status using biomarker analysis in wastewater. A new compound detected in wastewater can be a potential biomarker of an emerging trend in public health. However, it is currently difficult to select new biomarkers mainly due to limited human metabolism data. This manuscript presents a new framework, which enables the identification and selection of new biomarkers of human exposure to drugs with scarce or unknown human metabolism data. Mephedrone was targeted to elucidate the assessment of biomarkers for emerging drugs of abuse using a four-step analytical procedure. This framework consists of: (i) identification of possible metabolic biomarkers present in wastewater using an in-vivo study; (ii) verification of chiral signature of the target compound; (iii) confirmation of human metabolic residues in in-vivo/vitro studies and (iv) verification of stability of biomarkers in wastewater. Mephedrone was selected as a suitable biomarker due to its high stability profile in wastewater. Its enantiomeric profiling was studied for the first time in biological and environmental matrices, showing stereoselective metabolism of mephedrone in humans. Further biomarker candidates were also proposed for future investigation: 4'-carboxy-mephedrone, 4'-carboxy-normephedrone, 1-dihydro-mephedrone, 1-dihydro-normephedrone and 4'-hydroxy-normephedrone.

Measuring biomarkers in wastewater as a new source of epidemiological information: Current state and future perspectives.

The information obtained from the chemical analysis of specific human excretion products (biomarkers) in urban wastewater can be used to estimate the exposure or consumption of the population under investigation to a defined substance. A proper biomarker can provide relevant information about lifestyle habits, health and wellbeing, but its selection is not an easy task as it should fulfil several specific requirements in order to be successfully employed. This paper aims to summarize the current knowledge related to the most relevant biomarkers used so far. In addition, some potential wastewater biomarkers that could be used for future applications were evaluated. For this purpose, representative chemical classes have been chosen and grouped in four main categories: (i) those that provide estimates of lifestyle factors and substance use, (ii) those used to estimate the exposure to toxicants present in the environment and food, (iii) those that have the potential to provide information about public health and illness and (iv) those used to estimate the population size. To facilitate the evaluation of the eligibility of a compound as a biomarker, information, when available, on stability in urine and wastewater and pharmacokinetic data (i.e. metabolism and urinary excretion profile) has been reviewed. Finally, several needs and recommendations for future research are proposed.

A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry.

A new model is presented that can be used to screen for bioactivation of drugs. The evaluation of toxicity is an important step in the development of new drugs. One way to detect possible toxic metabolites is to use trapping agents such as glutathione. Often human liver microsomes are used as a metabolic model in initial studies. However, there is a need for alternatives that are easy to handle, cheap, and can produce large amounts of metabolites. In the presented study, paracetamol, mefenamic acid, and diclofenac, all known to form reactive metabolites in humans, were incubated with the fungus Cunninghamella elegans and the metabolites formed were characterized with ultra high performance liquid chromatography coupled to a quadrupole time of flight mass spectrometer. Interestingly, glutathione conjugates formed by the fungus were observed for all three drugs and their retention times and MS/MS spectra matched those obtained in a comparative experiment with human liver microsomes. These findings clearly demonstrated that the fungus is a suitable trapping model for toxic biotransformation products. Cysteine conjugates of all three test drugs were also observed with high signal intensities in the fungal incubates, giving the model a further indicator of drug bioactivation. To our knowledge, this is the first demonstration of the use of a fungal model for the formation and trapping of reactive drug metabolites. The investigated model is cheap, easy to handle, it does not involve experimental animals and it can be scaled up to produce large amounts of metabolites.

Isolation and characterization of a ß-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation.

In this study, using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, it has been confirmed that biotransformation with the fungus Cunninghamella elegans combined with chemical oxidation with the free radical tetramethylpiperidinyl-1-oxy (TEMPO) can produce drug glucuronides of ß-configuration. Glucuronic acid conjugates are a common type of metabolites formed by the human body. The detection of such conjugates in doping control and other kinds of forensic analysis would be beneficial owing to a decrease in analysis time as hydrolysis can be omitted. However the commercial availability of reference standards for drug glucuronides is poor. The selective androgen receptor modulator (SARM) SARM S1 was incubated with the fungus C. elegans. The sample was treated with the free radical TEMPO oxidizing agent and was thereafter purified by SPE. A glucuronic acid conjugate was isolated using a fraction collector connected to an ultra high performance liquid chromatographic (UHPLC) system. The isolated compound was characterized by NMR spectroscopy and mass spectrometry and its structure was confirmed as a glucuronic acid ß-conjugate of hydroxylated SARM S1 bearing the glucuronide moiety on carbon C-10.

Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO.

A new concept for the production of drug glucuronides is presented and the products formed were characterized using ultra high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). Glucuronic acid conjugates are important phase II metabolites of a wide range of drugs. There is a lack of commercially available glucuronides and classic synthetic methods are tedious and expensive. Thus, new methods of glucuronide synthesis are needed. Selective androgen receptor modulators (SARMs) of the aryl propionamide class were used as model compounds and were incubated with the fungus Cunninghamella elegans which was previously known to conjugate drugs with glucose. The resulting glucoside metabolites were then oxidized with tetramethylpiperidinyl-1-oxy (TEMPO). UPLC-HRMS analysis showed that the peaks corresponding to the glucosides had disappeared after the reaction and were replaced by peaks with m/z consistent with the corresponding glucuronic acid conjugates. The MS/MS spectra of the reaction products were investigated and the observed fragment ion pattern corroborated the suggested structural change. A comparison in terms of retention times and product ion spectra between the glucuronides formed by the new method and those produced by liver microsomes indicated that the conjugates from the two different sources were identical, thus demonstrating the human relevance of the presented technique. Furthermore, the glucuronides formed by the presented method were readily hydrolyzed by ß-glucuronidase which further gave evidence as to the fact that they were of ß configuration. The investigated method was easy to perform, required a low input of work and had a low cost.