Assessment of exposure to pesticide mixtures in five European countries by a harmonized urinary suspect screening approach.

Humans are exposed to a mixture of pesticides through diet as well as through the environment. We conducted a suspect-screening based study to describe the probability of (concomitant) exposure to a set of pesticide profiles in five European countries (Latvia, Hungary, Czech Republic, Spain and the Netherlands). We explored whether living in an agricultural area (compared to living in a peri-urban area), being a a child (compared to being an adult), and the season in which the urine sample was collected had an impact on the probability of detection of pesticides (-metabolites). In total 2088 urine samples were collected from 1050 participants (525 parent-child pairs) and analyzed through harmonized suspect screening by five different laboratories. Fourty pesticide biomarkers (either pesticide metabolites or the parent pesticides as such) relating to 29 pesticides were identified at high levels of confidence in samples across all study sites. Most frequently detected were biomarkers related to the parent pesticides acetamiprid and chlorpropham. Other biomarkers with high detection rates in at least four countries related to the parent pesticides boscalid, fludioxonil, pirimiphos-methyl, pyrimethanil, clothianidin, fluazifop and propamocarb. In 84% of the samples at least two different pesticides were detected. The median number of detected pesticides in the urine samples was 3, and the maximum was 13 pesticides detected in a single sample. The most frequently co-occurring substances were acetamiprid with chlorpropham (in 62 urine samples), and acetamiprid with tebuconazole (30 samples). Some variation in the probability of detection of pesticides (-metabolites) was observed with living in an agricultural area or season of urine sampling, though no consistent patterns were observed. We did observe differences in the probability of detection of a pesticide (metabolite) among children compared to adults, suggesting a different exposure and/or elimination patterns between adults and children. This survey demonstrates the feasibility of conducting a harmonized pan-European sample collection, combined with suspect screening to provide insight in the presence of exposure to pesticide mixtures in the European population, including agricultural areas. Future improvements could come from improved (harmonized) quantification of pesticide levels.

A large scale multi-laboratory suspect screening of pesticide metabolites in human biomonitoring: From tentative annotations to verified occurrences.

Within the Human Biomonitoring for Europe initiative (HBM4EU), a study to determine new biomarkers of exposure to pesticides and to assess exposure patterns was conducted. Human urine samples (N = 2,088) were collected from five European regions in two different seasons. The objective of the study was to identify pesticides and their metabolites in collected urine samples with a harmonized suspect screening approach based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) applied in five laboratories. A combined data processing workflow included comprehensive data reduction, correction of mass error and retention time (RT) drifts, isotopic pattern analysis, adduct and elemental composition annotation, finalized by a mining of the elemental compositions for possible annotations of pesticide metabolites. The obtained tentative annotations (n = 498) were used for acquiring representative data-dependent tandem mass spectra (MS2) and verified by spectral comparison to reference spectra generated from commercially available reference standards or produced through human liver S9 in vitro incubation experiments. 14 parent pesticides and 71 metabolites (including 16 glucuronide and 11 sulfate conjugates) were detected. Collectively these related to 46 unique pesticides. For the remaining tentative annotations either (i) no data-dependent MS2 spectra could be acquired, (ii) the spectral purity was too low for sufficient matching, or (iii) RTs indicated a wrong annotation, leaving potential for more pesticides and/or their metabolites being confirmed in further studies. Thus, the reported results are reflecting only a part of the possible pesticide exposure.

Harmonized Quality Assurance/Quality Control Provisions for Nontargeted Measurement of Urinary Pesticide Biomarkers in the HBM4EU Multisite SPECIMEn Study.

A set of quality assurance/quality control (QA/QC) criteria for nontargeted measurement of pesticide exposure markers in a large-scale study of human urine has been proposed and applied across five laboratories within the HBM4EU project. Quality control material, including reference standards and fortified pooled urine samples (QC urine) were prepared in a centralized way and distributed across participants to monitor analytical performance and consistency of the liquid chromatography coupled to high-resolution mass spectrometry data generated with a harmonized workflow. Signal intensities, mass accuracy, and retention times of selected QA/QC markers covering a broad range of physicochemical properties were monitored across QC solvent standards, QC urine samples, study urine samples, and procedural blanks, setting acceptance thresholds for repeatability and accuracy. Overall, results showed high repeatability of the collected data. The RSDs of the signal intensities were typically below 20-30% in QC and study samples, with good stability of the chromatographic separation (retention time drift within 2-4 s intrabatch and 5 s interbatch) and excellent mass accuracy (average error < 2 ppm). The use of the proposed criteria allowed for the identification of handling errors, instrumental issues, and potential batch effects. This is the first elaboration of harmonized QA/QC criteria applied across multiple laboratories to assess the quality of data generated by nontargeted analysis of human samples.

Ultra-fast retroactive processing by MetAlign of liquid chromatography/high-resolution full-scan Orbitrap mass spectrometry data in WADA Human Urine Sample Monitoring Program.

RATIONALE: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances which are not prohibited but whose potential misuse must be known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on the prevalence of their use. Retroactive processing of old sample datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in previous years. MetAlign is a freeware software with functionality to reduce the size of liquid chromatography (LC)/high-resolution (HR) full-scan (FS) mass spectrometry (MS) datafiles and to perform a fast search for the presence of substances in thousands of reduced datafiles. METHODS: Validation was performed to the search procedure of MetAlign applied to Anti-Doping Lab Qatar (ADLQ)-screened LC/HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and the ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RTs). RESULTS: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FPs) and false negatives (FNs). The search for TRA in 7410 ADLQ routine LC/HR-FS-MS datafiles from the years 2017 to 2020 revealed no false identification (FPs and FNs) compared with the ADLQ WADA reports. ECDY and DESECDY were detected by MetAlign search in approximately 5% of the same cohort of antidoping samples. CONCLUSIONS: MetAlign is a powerful tool for the fast retroactive processing of old reduced datafiles collected in screening by LC/HR-FS-MS to reveal the prevalence of use of antidoping substances. The current study proposed the validation scheme of the MetAlign search procedure, to be implemented per individual substance in the WADA Monitoring program, for the elimination of FNs and FPs.

Ultra-fast retroactive processing of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data in anti-doping screening of human urine.

RATIONALE: Retroactive analysis of previously tested urine samples has become an important sports anti-doping tool. Retroactive reprocessing of old data files acquired from a generic screening procedure can reveal detection of initially unknown substances, like illegal drugs and newly identified metabolites. METHODS: To be able to efficiently search through hundreds to thousands of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data files of anti-doping samples, a combination of MetAlign and HR_MS_Search software has been developed. MetAlign reduced the data size ca 100-fold making possible local storage of a massive volume of data. RESULTS: The newly developed HR_MS_Search module can search through the reduced data files for new compounds (mass or isotope pattern) defined by mass windows and retention time windows. A search for 33 analytes in 940 reduced data files lasted 10 s. The output of the automatic search was compared to the standard manual routine evaluation. The results of searching were evaluated in terms of false negatives and false positives. The newly banned b2-agonist higenamine and its metabolite coclaurine were successfully searched in reduced data files originating from a testing period for which these substances were not banned, as an example of retroactive analysis. CONCLUSIONS: The freeware MetAlign software and its automatic searching module HR_MS_Search facilitated the retroactive reprocessing of reduced full-scan high-resolution liquid chromatography/mass spectrometry screening data files and created a new tool in anti-doping laboratories' network.

Omics analyses of potato plant materials using an improved one-class classification tool to identify aberrant compositional profiles in risk assessment procedures.

The objective of this study was to quantitatively assess potato omics profiles of new varieties for meaningful differences from analogous profiles of commercial varieties through the SIMCA one-class classification model. Analytical profiles of nine commercial potato varieties, eleven experimental potato varieties, one GM potato variety that had acquired Phytophtora resistance based on a single insert with potato-derived DNA sequences, and its non-GM commercial counterpart were generated. The ten conventional varieties were used to construct the one-class model. Omics profiles from experimental non-GM and GM varieties were assessed using the one-class SIMCA models. No potential unintended effects were identified in the case of the GM variety. The model showed that varieties that were genetically more distant from the commercial varieties were recognized as aberrant, highlighting its potential in determining whether additional evaluation is required for the risk assessment of materials produced from any breeding technique, including genetic modification.

Use of omics analytical methods in the study of genetically modified maize varieties tested in 90 days feeding trials.

Genetically modified (GM) maize and their non-modified counterparts were compared using MON810 varieties, the only GMO event cultivated in Europe. The differences in grain samples were analysed by omics profiles, including transcriptomics, proteomics and metabolomics. Other cultivated maize varieties were analysed as a reference for the variability that will exist between cultivated varieties. The observed differences between modified and non-modified maize varieties do not exceed typical differences between non-modified varieties. The use of these advanced analytical approaches to analyse novel plant materials as compared to the results from animal feeding trials with whole foods is assessed. No indications were observed for changes in the GM varieties that warrant further investigations. Furthermore, it was shown that such indications will be obtained if maize samples of inferior quality are analysed similarly. Omics data provide detailed analytical information of the plant material, which facilitates a risk assessment procedure of new (GM) plant varieties.

Comparison of gas chromatography/quadrupole time-of-flight and quadrupole Orbitrap mass spectrometry in anti-doping analysis: I. Detection of anabolic-androgenic steroids.

RATIONALE: The World Anti-Doping Agency (WADA) encourages drug-testing laboratories to develop screening methods that can detect as many doping substances as possible in urine. The use of full-scan high-resolution acquisition (FS/HR) with gas chromatography/mass spectrometry (GC/MS) for the detection of known and unknown trimethylsilyl (TMS) derivatives of anabolic-androgenic steroids (AAS) provides anti-doping testing bodies with a new analytical tool. METHODS: The AAS were extracted from urine samples by generic liquid-liquid extraction, after enzymatic hydrolysis, and TMS derivatization. The extracted urine was analyzed by GC/Q-TOF and GC/Q-Orbitrap to compare the performance of the two instrument types for the detection of 46 AAS in human urine. The quantitation of endogenous anabolic steroids and the ability of the two analytical platforms to comply with the requirements for testing as part of the WADA Athlete Biological Passport (ABP) were also assessed. RESULTS: The data presented show that the analytical performance for both instruments complies with the WADA specifications. The limits of detection (LODs) for both instruments are well below the WADA 50% Minimum Required Performance Levels. The mass errors in the current study for the GC/Q-Orbitrap platform are lower than those obtained for the GC/Q-TOF instrument. CONCLUSIONS: The data presented herein proved that both molecular profiling platforms can be used for antidoping screening. The mass accuracies are excellent in both instruments; however, the GC/Q-Orbitrap performs better as it provides higher resolution than the GC/Q-TOF platform.

High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis.

The aim of this paper is to present the development and validation of a high-resolution full scan (HR-FS) electrospray ionization (ESI) liquid chromatography coupled to quadrupole Orbitrap mass spectrometer (LC/Q/Orbitrap MS) platform for the screening of prohibited substances in human urine according to World Antidoping Agency (WADA) requirements. The method was also validated for quantitative analysis of six endogenous steroids (epitestosterone, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, androsterone and etiocholanolone) in their intact sulfates form. The sample preparation comprised a combination of a hydrolyzed urine liquid-liquid extraction and the dilute & shoot addition of original urine in the extracted aliquot. The HR-FS MS acquisition mode with Polarity Switching was applied in combination of the Quadrupole-Orbitrap mass filter. The HR-FS acquisition of analytical signal, for known and unknown small molecules, allows the inclusion of all analytes detectable with LC-MS for antidoping investigations to identify the use of known or novel prohibited substances and metabolites after electronic data files' reprocessing. The method has been validated to be fit-for-purpose for the antidoping analysis.

Gas chromatographic quadrupole time-of-flight full scan high resolution mass spectrometric screening of human urine in antidoping analysis.

This paper presents the development and validation of a high-resolution full scan (FS) electron impact ionization (EI) gas chromatography coupled to quadrupole Time-of-Flight mass spectrometry (GC/QTOF) platform for screening anabolic androgenic steroids (AAS) in human urine samples. The World Antidoping Agency (WADA) enlists AAS as prohibited doping agents in sports, and our method has been developed to comply with the qualitative specifications of WADA to be applied for the detection of sports antidoping prohibited substances, mainly for AAS. The method also comprises of the quantitative analysis of the WADA's Athlete Biological Passport (ABP) endogenous steroidal parameters. The applied preparation of urine samples includes enzymatic hydrolysis for the cleavage of the Phase II glucuronide conjugates, generic liquid-liquid extraction and trimethylsilyl (TMS) derivatization steps. Tandem mass spectrometry (MS/MS) acquisition was applied on few selected ions to enhance the specificity and sensitivity of GC/TOF signal of few compounds. The full scan high resolution acquisition of analytical signal, for known and unknown TMS derivatives of AAS provides the antidoping system with a new analytical tool for the detection designer drugs and novel metabolites, which prolongs the AAS detection, after electronic data files' reprocessing. The current method is complementary to the respective liquid chromatography coupled to mass spectrometry (LC/MS) methodology widely used to detect prohibited molecules in sport, which cannot be efficiently ionized with atmospheric pressure ionization interface.

Validation of an automated screening method for persistent organic contaminants in fats and oils by GC×GC-ToFMS.

An screening method, comprised of straightforward sample treatment based on silica clean-up, GC×GC-ToFMS detection and automated data processing with the non-proprietary free downloadable software MetAlignID, has been successfully validated with respect to false negatives for the sum PCB 28, 52, 101, 138, 153 and 180), for the sum of BDE 28, 47, 99, 100, 153, 154 and 183, for the four markers of PAHs and for a number of emerging brominated flame retardants. A screening detection limit (SDL) equal to or lower than the maximum regulatory level was always achieved. MetAlignID considerably decreased the time needed for data treatment from 20 to 5min/file. Automated identification of the signature mass spectral patterns was applied to identify chlorinated- and brominated-containing substances with more than two halogen atoms, and PAH derivates. Although the success rate was variable and needs to be further improved, the tool was considered to be of added value.

Improved batch correction in untargeted MS-based metabolomics.

INTRODUCTION: Batch effects in large untargeted metabolomics experiments are almost unavoidable, especially when sensitive detection techniques like mass spectrometry (MS) are employed. In order to obtain peak intensities that are comparable across all batches, corrections need to be performed. Since non-detects, i.e., signals with an intensity too low to be detected with certainty, are common in metabolomics studies, the batch correction methods need to take these into account. OBJECTIVES: This paper aims to compare several batch correction methods, and investigates the effect of different strategies for handling non-detects. METHODS: Batch correction methods usually consist of regression models, possibly also accounting for trends within batches. To fit these models quality control samples (QCs), injected at regular intervals, can be used. Also study samples can be used, provided that the injection order is properly randomized. Normalization methods, not using information on batch labels or injection order, can correct for batch effects as well. Introducing two easy-to-use quality criteria, we assess the merits of these batch correction strategies using three large LC-MS and GC-MS data sets of samples from Arabidopsis thaliana. RESULTS: The three data sets have very different characteristics, leading to clearly distinct behaviour of the batch correction strategies studied. Explicit inclusion of information on batch and injection order in general leads to very good corrections; when enough QCs are available, also general normalization approaches perform well. Several approaches are shown to be able to handle non-detects-replacing them with very small numbers such as zero seems the worst of the approaches considered. CONCLUSION: The use of quality control samples for batch correction leads to good results when enough QCs are available. If an experiment is properly set up, batch correction using the study samples usually leads to a similar high-quality correction, but has the advantage that more metabolites are corrected. The strategy for handling non-detects is important: choosing small values like zero can lead to suboptimal batch corrections.

Interindividual variation in gene expression responses and metabolite formation in acetaminophen-exposed primary human hepatocytes.

Acetaminophen (APAP) is a readily available over-the-counter drug and is one of the most commonly used analgesics/antipyretics worldwide. Large interindividual variation in susceptibility toward APAP-induced liver failure has been reported. However, the exact underlying factors causing this variability in susceptibility are still largely unknown. The aim of this study was to better understand this variability in response to APAP by evaluating interindividual differences in gene expression changes and APAP metabolite formation in primary human hepatocytes (PHH) from several donors (n = 5) exposed in vitro to a non-toxic to toxic APAP dose range. To evaluate interindividual variation, gene expression data/levels of metabolites were plotted against APAP dose/donor. The correlation in APAP dose response between donors was calculated by comparing data points from one donor to the data points of all other donors using a Pearson-based correlation analysis. From that, a correlation score/donor for each gene/metabolite was defined, representing the similarity of the omics response to APAP in PHH of a particular donor to all other donors. The top 1 % highest variable genes were selected for further evaluation using gene set overrepresentation analysis. The biological processes in which the genes with high interindividual variation in expression were involved include liver regeneration, inflammatory responses, mitochondrial stress responses, hepatocarcinogenesis, cell cycle, and drug efficacy. Additionally, the interindividual variation in the expression of these genes could be associated with the variability in expression levels of hydroxyl/methoxy-APAP and C8H13O5N-APAP-glucuronide. The before-mentioned metabolites or their derivatives have also been reported in blood of humans exposed to therapeutic APAP doses. Possibly these findings can contribute to elucidating the causative factors of interindividual susceptibility toward APAP.

Integrative cross-omics analysis in primary mouse hepatocytes unravels mechanisms of cyclosporin A-induced hepatotoxicity.

The liver is responsible for drug metabolism and drug-induced hepatotoxicity is the most frequent reason for drug withdrawal, indicating that better pre-clinical toxicity tests are needed. In order to bypass animal models for toxicity screening, we exposed primary mouse hepatocytes for exploring the prototypical hepatotoxicant cyclosporin A. To elucidate the mechanisms underlying cyclosporin A-induced hepatotoxicity, we analyzed expression levels of proteins, mRNAs, microRNAs and metabolites. Integrative analysis of transcriptomics and proteomics showed that protein disulfide isomerase family A, member 4 was up-regulated on both the protein level and mRNA level. This protein is involved in protein folding and secretion in the endoplasmic reticulum. Furthermore, the microRNA mmu-miR-182-5p which is predicted to interact with the mRNA of this protein, was also differentially expressed, further emphasizing endoplasmic reticulum stress as important event in drug-induced toxicity. To further investigate the interaction between the significantly expressed proteins, a network was created including genes and microRNAs known to interact with these proteins and this network was used to visualize the experimental data. In total 6 clusters could be distinguished which appeared to be involved in several toxicity related processes, including alteration of protein folding and secretion in the endoplasmic reticulum. Metabonomic analyses resulted in 5 differentially expressed metabolites, indicative of an altered glucose, lipid and cholesterol homeostasis which can be related to cholestasis. Single and integrative analyses of transcriptomics, proteomics and metabonomics reveal mechanisms underlying cyclosporin A-induced cholestasis demonstrating that endoplasmic reticulum stress and the unfolded protein response are important processes in drug-induced liver toxicity.

Ultrafast PubChem searching combined with improved filtering rules for elemental composition analysis.

A new and improved software tool for elemental composition annotation of molecular ions detected in mass spectrometry, based on improved filtering rules followed by ultrafast querying in publicly available compound databases, is provided. Pubchem is used as a general source of 1.3 million unique chemical formulas. A plant metabolomics database containing ca. 100,000 formulas is used as a source of naturally occurring compounds. Four modes with different sets of rules for heuristic filtering of candidate formulas coming from elemental composition analysis are incorporated and tested on both databases. The elemental composition analysis is then coupled to ultrafast PubChem searching based on a mass-indexed intermediate system. The performance of the filters is compared and discussed. When reactive compounds are assumed not to be present, 99.95% of the 1.3 million PubChem formulas is correctly found, while ca. 30% less formulas per mass are given compared to previously published rules. For the ca. 100,000 plant metabolomics based formulas, 100% fit the improved rules.

Identification of cisplatin-regulated metabolic pathways in pluripotent stem cells.

The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR) signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells.

metAlignID: a high-throughput software tool set for automated detection of trace level contaminants in comprehensive LECO two-dimensional gas chromatography time-of-flight mass spectrometry data.

A new alternative data processing tool set, metAlignID, is developed for automated pre-processing and library-based identification and concentration estimation of target compounds after analysis by comprehensive two-dimensional gas chromatography with mass spectrometric detection. The tool set has been developed for and tested on LECO data. The software is developed to run multi-threaded (one thread per processor core) on a standard PC (personal computer) under different operating systems and is as such capable of processing multiple data sets simultaneously. Raw data files are converted into netCDF (network Common Data Form) format using a fast conversion tool. They are then preprocessed using previously developed algorithms originating from metAlign software. Next, the resulting reduced data files are searched against a user-composed library (derived from user or commercial NIST-compatible libraries) (NIST=National Institute of Standards and Technology) and the identified compounds, including an indicative concentration, are reported in Excel format. Data can be processed batch wise. The overall time needed for conversion together with processing and searching of 30 raw data sets for 560 compounds is routinely within an hour. The screening performance is evaluated for detection of pesticides and contaminants in raw data obtained after analysis of soil and plant samples. Results are compared to the existing data-handling routine based on proprietary software (LECO, ChromaTOF). The developed software tool set, which is freely downloadable at www.metalign.nl, greatly accelerates data-analysis and offers more options for fine-tuning automated identification toward specific application needs. The quality of the results obtained is slightly better than the standard processing and also adds a quantitative estimate. The software tool set in combination with two-dimensional gas chromatography coupled to time-of-flight mass spectrometry shows great potential as a highly-automated and fast multi-residue instrumental screening method.

MetAlign 3.0: performance enhancement by efficient use of advances in computer hardware.

A new, multi-threaded version of the GC-MS and LC-MS data processing software, metAlign, has been developed which is able to utilize multiple cores on one PC. This new version was tested using three different multi-core PCs with different operating systems. The performance of noise reduction, baseline correction and peak-picking was 8-19 fold faster compared to the previous version on a single core machine from 2008. The alignment was 5-10 fold faster. Factors influencing the performance enhancement are discussed. Our observations show that performance scales with the increase in processor core numbers we currently see in consumer PC hardware development.

Screening for modulatory effects on steroidogenesis using the human H295R adrenocortical cell line: a metabolomics approach.

The recently OECD validated H295R steroidogenesis assay provides an in vitro alternative to evaluate the potential interference of exogenous compounds with endogenous steroid hormone synthesis. Currently, this assay is used for a simple negative-positive screening of compounds using testosterone and estradiol levels as end points, measured with specific enzyme immunoassays (EIAs) or targeted liquid chromatography (LC) and gas chromatography (GC)-mass spectrometry (MS) methods. However, recent developments in LC-MS and bioinformatics allow for more comprehensive approaches to evaluate changes in steroid profiles. In the current work, the H295R cell model was combined with a metabolomics approach to monitor changes in metabolite profiles in both a targeted and untargeted way. H295R cells were exposed for 48 h to model compounds, i.e., forskolin, abiraterone, prochloraz, ketoconazole, trilostane, formestane, aminoglutethimide, fadrozole, etomidate, and metyrapone, known to affect steroidogenesis. After exposure, the levels of 9 natural steroids were determined by a quantitative targeted GC-MS/MS method and compared to a metabolomics method using Ultra Performance Liquid Chromatography-Time-of-Flight-Mass Spectrometry (UPLC-ToF-MS). Like the EIAs, both methods were suited for negative-positive screening, but the MS methods also generated specific fingerprints, allowing chemical class prediction of the compound under investigation. Although the targeted GC-MS/MS was more sensitive, which was an advantage regarding analysis of the estrogens 17ß-estradiol and estrone, the untargeted UPLC-ToF-MS was able to evaluate effects on the synthesis of the corticosteroids. Moreover, untargeted comparison of the aligned chemical profiles allowed identification of all m/z-values that are differential between exposed and nonexposed H295R cells. In conclusion, application of a comprehensive metabolite profiling methodology not only provides a tool to screen compounds for steroidogenic modulating properties, but also allows chemical class prediction. As such, steroid profiling methodologies in conjunction with the H295R assay can contribute to the prioritization of chemicals for additional safety testing.

Data (pre-)processing of nominal and accurate mass LC-MS or GC-MS data using MetAlign.

This paper gives a step-by-step account of how to install, set up, and run MetAlign software, which can be downloaded freely ( http://www.metalign.wur.nl/UK/Download+and+publications ). The software is used for accurate mass and nominal mass data coming from different kinds of GC-MS and LC-MS platforms. The algorithms are beyond the scope of this paper and were published separately.