False-positive MDA findings in HRMS-based screening of putrefied postmortem blood samples-Identification of the interference as N-acetyltyramine.

An unidentified compound in putrefied postmortem blood samples showed identical accurate mass and chromatographic behavior as 3,4-methylenedioxyamphetamine (MDA) and led to false-positive preliminary screening results. The aim of the study was to identify this unknown interference. Postmortem blood samples were analyzed after protein precipitation on a QExactive Focus high-resolution mass spectrometer (Thermo Fisher, Germany) coupled to a RP C18 column (Macherey-Nagel, Germany). Based on the analysis of mass spectrometry (MS) adducts and isotope ratios using fullscan (m/z 134-330) information, the empiric formula of the protonated molecule [M + H]+ of the unknown compound was found to be C10H14O2N (+ 0.6 ppm). Product ion spectra recorded using normalized collision energy 22% showed a base peak of C8H9O1 (+ 1.5 ppm) and a low-abundant water loss to C7H9 (+ 1.9 ppm), neutral losses of C2H2O and NH3 were found. Based on fullscan and MS-MS information and under consideration of the observed order of neutral losses, the compound was presumptively identified as N-acetyltyramine. This assumption was supported by SIRIUS software showing a SIRIUS score of 99.43% for N-acetyltyramine. Finally, the putative structure annotation was confirmed by a reference compound. The described false-positive MDA findings could be attributed to the presence of N-acetyltyramine in putrefied blood samples. Being an isomer of MDA, N-acetyltyramine could not be distinguished by high-resolution data of the protonated molecules. The presented results once again highlight that false-positive findings may occur even in hyphenatedhigh-resolution mass spectrometry (HRMS) when using full-scan information only.

A liquid chromatographic-mass spectrometric procedure for analysis of pentaerythrityl tetranitrate metabolites - Development, validation and application to ovine serum and human plasma samples.

Pentaerythrityl tetranitrate (PETN) is an established drug in the treatment of coronary heart disease and heart failure. It is assumed, that the vasodilative and vasoprotective effects of PETN also have a positive impact on pregnant patients with impaired placental perfusion and studies evaluating the effect of PETN in risk pregnancies have been carried out. In the context of these clinical trials, measuring of serum levels of PETN and its metabolites pentaerythrityl trinitrate (PETriN), pentaerythrityl dinitrate (PEDN), pentaerythrityl mononitrate (PEMN) and pentaerythritol (PE) were required. To evaluate the transfer of PETN and its metabolites (PEXN) from the mother to the fetus using samples from a human clinical trial and animal study, the present work aimed to develop a rapid and simple method to simultaneously analyze PEXN in human and ovine samples. A method employing a rapid and simple liquid-liquid extraction followed by reversed-phase (C18) liquid chromatography coupled to high-resolution mass spectrometry with negative electrospray ionization was developed and validated for the detection of PETN and PEXN in human and ovine samples. PE could only be qualitatively detected at higher concenrations. Method validation requirements, including accuracy, repeatability and intermediate precision were fulfilled in ovine and human samples for all other PEXN with exception PETriN in human samples. The recovery (RE) in ovine samples was 76.7 % ± 12 % for PEMN, 98 % ± 23 % for PEDN, 94 % ± 22 % for PETriN, in human samples RE was 59 % ± 16 % for PEMN, 67 % ± 19 % for PEDN, 71 % ± 17 %. The matrix effects (ME) in ovine samples were 90 % ± 11 % for PEMN, 70 % ± 30 % for PEDN, 107 % ± 17 % for PETriN, in human samples the ME were 93 % ± 13 % for PEMN, 84 % ± 17 % for PEDN, 98 % ± 16 % for PETriN. The limits of quantification (LOQ) in ovine samples were 1.0 ng/mL for PETriN and 0.1 ng/mL for PEMN and PEDN. The LOQs in human samples were 5.0 ng/mL for PETriN and 0.3 ng/mL for PEMN und PEDN. The newly developed method was used to analyze 184 ovine serum samples and 18 human plasma samples. In ovine maternal samples, the highest observed PEDN concentration was 3.5 ng/mL and the highest PEMN concentration was 10 ng/mL, the respective concentrations in fetal serum samples were 4.9 ng/mL for PEDN and 5.4 ng/mL for PEMN. PETriN was only detected in traces in maternal and fetal samples, whereas PETN could not be detected at all. In human maternal samples, the highest concentration for PEDN was 27 ng/mL and for PEMN 150 ng/mL. In umbilical cord plasma, concentrations of 2.3 ng/mL for PEDN and 73 ng/mL for PEMN were detected. Although the PEMN and PEDN concentrations in the human samples were several times higher than in ovine samples, neither PETN nor PETriN signals could be detected. These results demonstrated that the metabolites were transferred from mother to fetus with a slight time delay.

Evaluation of biochemical assays and optimization of LC-MS-MS analysis for the detection of synthetic urine.

Ensuring specimen validity is an essential aspect of toxicological laboratories. In recent years, substituting authentic urine specimens for synthetic urine (SU) has become increasingly popular. Such SU products consist of components expected in normal urine and show physiological values for specific gravity and pH. Thus, standard specimen validity testing may fail in revealing adulteration by SU. The present study investigated three methods to distinguish authentic and SU specimens: enzymatic detection of uric acid, the commercially available Axiom Test True SU and liquid chromatography coupled with (tandem) mass spectrometry (LC-MS-MS) analysis of 10 endogenous biomolecules. Additionally, novel direct markers of SU were investigated. Two specimen sets were analyzed by each method. Specimen set A consisted of eight SU products purchased from the Austrian/German market and 43 urine specimens from volunteers of known authenticity, which underwent double-blind analysis. Specimen set B consisted of 137 real urine specimens submitted for drug testing, which were selected due to initial suspicious test results in adulteration testing and reanalyzed by all three methods. Uric acid and LC-MS-MS-based endogenous biomolecule testing showed 100% sensitivity and specificity for set A. The commercial test had 87.5% sensitivity and 97.7% specificity for set A. For set B, uric acid and LC-MS-MS analysis showed almost similar results, even if uric acid was missing one presumptive authentic urine specimen according to LC-MS-MS findings. Nearly half of the SU assignments for the commercial test were presumptive false positives. New SU markers were observed for SU products from the Austrian/German market. One specimen in set B had both an endogenous biomolecule pattern and SU markers suggesting urine dilution with SU. In conclusion, several analytes or methods should be used rather than one, and the most reliable results are achieved if both indirect and direct markers of urine substitution are analyzed.

Advances in testing for sample manipulation in clinical and forensic toxicology - Part A: urine samples.

In many countries, adherence testing is used to monitor consumption behavior or to prove abstinence. Urine and hair are most commonly used, although other biological fluids are available. Positive test results are usually associated with serious legal or economic consequences. Therefore, various sample manipulation and adulteration strategies are used to circumvent such a positive result. In these critical review articles on sample adulteration of urine (part A) and hair samples (part B) in the context of clinical and forensic toxicology, recent trends and strategies to improve sample adulteration and manipulation testing published in the past 10 years are described and discussed. Typical manipulation and adulteration strategies include undercutting the limits of detection/cut-off by dilution, substitution, and adulteration. New or alternative strategies for detecting sample manipulation attempts can be generally divided into improved detection of established urine validity markers and direct and indirect techniques or approaches to screening for new adulteration markers. In this part A of the review article, we focused on urine samples, where the focus in recent years has been on new (in)direct substitution markers, particularly for synthetic (fake) urine. Despite various and promising advances in detecting manipulation, it remains a challenge in clinical and forensic toxicology, and simple, reliable, specific, and objective markers/techniques are still lacking, for example, for synthetic urine.

Advances in testing for sample manipulation in clinical and forensic toxicology-part B: hair samples.

As a continuation of part A, focusing on advances in testing for sample manipulation of urine samples in clinical and forensic toxicology, part B of the review article relates to hair, another commonly used matrix for abstinence control testing. Similar to urine manipulation, relevant strategies to manipulate a hair test are lowering drug concentrations in hair to undercut the limits of detection/cut-offs, for instance, by forced washout effects or adulteration. However, distinguishing between usual, common cosmetic hair treatment and deliberate manipulation to circumvent a positive drug test is often impossible. Nevertheless, the identification of cosmetic hair treatment is very relevant in the context of hair testing and interpretation of hair analysis results. Newly evaluated techniques or elucidation of specific biomarkers to unravel adulteration or cosmetic treatment often focused on specific structures of the hair matrix with promising strategies recently proposed for daily routine work. Identification of other approaches, e.g., forced hair-washing procedures, still remains a challenge in clinical and forensic toxicology.

Long time stability of 35 small endogenous biomolecules in dried urine spotted on various surfaces and environmental conditions.

Analysis of endogenous biomolecules is an important aspect of many forensic investigations especially with focus on DNA analysis for perpetrator/victim identification and protein analysis for body fluid identification. Recently, small endogenous biomolecules have been used for differentiation of synthetic "fake" urine from authentic urine and might be also useful for biofluid identification. Therefore, the aim of this study was to adapt and optimize a method for analysis of small EBs and to investigate long time stability of 35 small endogenous biomolecules (including acylcarnitines with their isomers and metabolites as well as amino acids with their metabolites) in spotted urine samples. Urine samples were spotted on seven different surfaces (Whatman 903 Protein Saver Cards, cotton swabs, cotton glove, denim, underwear, and smooth and rough flagstone) and stored under six environmental conditions (reference condition, sunlight, LED light, 4 °C, 37 °C, humidity of 95%). At certain time points (d0, d7, d28 and d56) samples were analyzed in triplicates by an optimized extraction and LC-HRMS approach. In addition, the urine marker Tamm-Horsfall-Protein was determined on cotton swabs at the same time points using a commercial lateral flow test. Twenty-one of 35 small endogenous biomolecules were stable on most materials/surfaces and under most storage conditions. Significant lower endogenous biomolecule peak areas were found for rough flagstone and underwear as well as for high humidity storage. Kynurenic acid proved to be photo labile. While high long time stabilities were found for 19 of 28 acylcarnitines, nine acylcarnitines showed aberrant stability patterns without evident structural reason. For Tamm-Horsfall-Protein degradation within 28 days was observed even under reference conditions. The presented study demonstrated the value of sensitive LC-HRMS analysis for small endogenous biomolecules / pattern. However, further studies will be indispensable for unambiguous body fluid identification by small endogenous biomolecules.

Liquid Chromatography High-Resolution Mass Spectrometry in Forensic Toxicology: What are the Specifics of Method Development, Validation and Quality Assurance for Comprehensive Screening Approaches?

The use of high-resolution mass spectrometry (HRMS) has increased over the past decade in clinical and forensic toxicology, especially for comprehensive screening approaches. Despite this, few guidelines in this field have specifically addressed HRMS issues concerning compound identification, validation, measurement uncertainty and quality assurance. To fully implement this technique, certainly in an era in which the quality demands for laboratories are ever-increasing due to various norms (e.g. the International Organization for Standardization's ISO 17025), these specific issues need to be addressed. This manuscript reviews 26 HRMSbased methods for qualitative systematic toxicological analysis (STA) published between 2011 and 2021. Key analytical data such as samples matrices, analytical platforms, numbers of analytes and employed mass spectral reference databases/libraries as well as the studied validation parameters are summarized and discussed. The article further includes a critical review of targeted and untargeted data acquisition approaches, available HRMS reference databases and libraries as well as current guidelines for HRMS data interpretation with a particular focus on identification criteria. Moreover, it provides an overview on current recommendations for the validation and determination of measurement uncertainty of qualitative methods. Finally, the article aims to put forward suggestions for method development, compound identification, validation experiments to be performed, and adequate determination of measurement uncertainty for this type of wide-range qualitative HRMSbased methods.

HPLC-MS identification of acid degradation products of dolutegravir.

Dolutegravir is an integrase strand transfer inhibitor used for the treatment of human immuno-deficiency virus infections. The present study was conducted in order to identify degradation products formed in acidic solution upon heating. The structures were assigned based on low resolution collision-induced dissociation tandem mass spectra as well as high resolution higher-energy collisional dissociation tandem mass spectra. The major degradation products resulted from hydrolytic opening of the oxepine ring leading to bis-hydroxy diastereomers (DP2 and DP3) as well as a mono-hydroxy derivative (DP1) as the result of dehydration of the diastereomers. Furthermore, two carboxylic acid derivatives (DP4 and DP5) could be identified, which can be explained as the result of the hydrolysis of the exocyclic amide bond of dolutegravir and DP1, respectively. During the fragmentation process of dolutegravir and its degradation products DP1 to DP3 a formal addition of oxygen resulting in the respective carboxylic acid fragments was detected. This could be evidenced based on high resolution masses of the fragments as well as the comparison of the MS/MS spectra of the fragments with the spectra of the carboxylic acids DP4 and DP5.

Gastric bypass surgery in a rat model alters the community structure and functional composition of the intestinal microbiota independently of weight loss.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) surgery is a last-resort treatment to induce substantial and sustained weight loss in cases of severe obesity. This anatomical rearrangement affects the intestinal microbiota, but so far, little information is available on how it interferes with microbial functionality and microbial-host interactions independently of weight loss. METHODS: A rat model was employed where the RYGB-surgery cohort is compared to sham-operated controls which were kept at a matched body weight by food restriction. We investigated the microbial taxonomy and functional activity using 16S rRNA amplicon gene sequencing, metaproteomics, and metabolomics on samples collected from theileum, the cecum, and the colon, and separately analysed the lumen and mucus-associated microbiota. RESULTS: Altered gut architecture in RYGB increased the relative occurrence of Actinobacteria, especially Bifidobacteriaceae and Proteobacteria, while in general, Firmicutes were decreased although Streptococcaceae and Clostridium perfringens were observed at relative higher abundances independent of weight loss. A decrease of conjugated and secondary bile acids was observed in the RYGB-gut lumen. The arginine biosynthesis pathway in the microbiota was altered, as indicated by the changes in the abundance of upstream metabolites and enzymes, resulting in lower levels of arginine and higher levels of aspartate in the colon after RYGB. CONCLUSION: The anatomical rearrangement in RYGB affects microbiota composition and functionality as well as changes in amino acid and bile acid metabolism independently of weight loss. The shift in the taxonomic structure of the microbiota after RYGB may be mediated by the resulting change in the composition of the bile acid pool in the gut and by changes in the composition of nutrients in the gut. Video abstract.

Ethnomedicinal survey and in vitro confirmation of anti-inflammatory and antispasmodic properties of the termite strain Macrotermes bellicosus used in traditional medicine in the Republic of Benin.

ETHNOPHARMACOLOGICAL RELEVANCE: Insects and insect-derived products play a vital role in traditional medicine in many parts of the world since ancient times. Among these insects, fungus-growing termites like Macrotermes bellicosus (M. bellicosus) are widely used in nutrition and traditional medicine in various societies of sub-Saharan Africa. AIM OF THE STUDY: Aim of the present study was to explore the traditional applications of M. bellicosus and subsequently investigate the anti-inflammatory and spasmolytic activity of samples collected in Benin. MATERIAL AND METHODS: An ethnomedicinal survey with thirty active healers in Benin was conducted and the anti-inflammatory activity of an ethanolic extract of M. bellicosus was investigated. Thus, LPS-induced TNFα release from differentiated human macrophages (THP-1) and IL-8 release from cytokine (IL-1ß/TNFα/IFNγ)-challenged human intestinal epithelial cells (Caco-2) was measured by enzyme-linked immunosorbent assay. Furthermore, the influence of M. bellicosus extract on basal tone and induced contractions in isolated rat small intestinal preparations was determined to examine the influence on intestinal motility. RESULTS: The survey of 30 active healers demonstrated that M. bellicosus and its products (termites' mound and fungus comb) are used in Benin for therapeutic purposes mainly to treat infectious and inflammatory diseases including digestive disorders, snake bites and diarrhea. It was found that M. bellicosus extract inhibited both LPS-induced TNFα release from human macrophages and cytokine-induced IL-8 release from intestinal epithelial cells comparable to budesonide. In addition, isometric contraction measurement with isolated rat small intestinal preparations demonstrated a mild spasmolytic effect of the termite extract in higher concentrations with a suppression of induced contractions and relaxation of basal tone. CONCLUSION: M. bellicosus which is used in traditional medicine in Benin to treat infectious and inflammatory diseases showed anti-inflammatory activity by inhibiting pro-inflammatory cytokine release and a moderate influence on intestinal motility.

Liquid chromatography-mass spectrometry-based determination of ergocristine, ergocryptine, ergotamine, ergovaline, hypoglycin A, lolitrem B, methylene cyclopropyl acetic acid carnitine, N-acetylloline, N-formylloline, paxilline, and peramine in equine hair.

Ingestion of hypoglycin A (HGA) in maple seeds or alkaloids produced by symbiotic fungi in pasture grasses is thought to be associated with various syndromes in grazing animals. This article describes analytical methods for monitoring long-term exposure to HGA, its metabolite MCPA-carnitine, as well as ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, N-acetylloline, N-formylloline, peramine, and paxilline in equine hair. After extraction of hair samples separation was achieved using two ultra high performance liquid chromatographic systems (HILIC or RP-C18, ammonium formate:acetonitrile). A benchtop orbitrap instrument was used for high resolution tandem mass spectrometric detection. All analytes were sensitively detected with limits of detection between 1 pg/mg and 25 pg/mg. Irreproducible extraction or ubiquitous presence in horse hair precluded quantitative validation of lolitrem B/paxilline and N-acetylloline/N-formylloline, respectively. For the other analytes validation showed no interferences in blank hair. Other validation parameters were as follows: limits of quantification (LOQ), 10 to 100 pg/mg; recoveries, 18.3 to 91.0%; matrix effects, -48.2 - 24.4%; linearity, LOQ - 1000 pg/mg; accuracy, -14.9 - 6.4%, precision RSDs ≤10.7%. The method allows sensitive detection of all analytes and quantification of ergocristine, ergocryptine, ergotamine, ergovaline, HGA, MCPA-carnitine, and peramine in horse hair. Applicability was proven for N-acetylloline and N-formylloline by analyzing hair of 13 horses.

Comparative study on the metabolism of the ergot alkaloids ergocristine, ergocryptine, ergotamine, and ergovaline in equine and human S9 fractions and equine liver preparations.

1. Ergopeptine alkaloids like ergovaline and ergotamine are suspected to be associated with fescue toxicosis and ergotism in horses. Information on the metabolism of ergot alkaloids is scarce, especially in horses, but needed for toxicological analysis of these drugs in urine/feces of affected horses. The aim of this study was to investigate the metabolism of ergovaline, ergotamine, ergocristine, and ergocryptine in horses and comparison to humans. 2. Supernatants of alkaloid incubations with equine and human liver S9 fractions were analyzed by reversed-phase liquid-chromatography coupled to high-resolution tandem mass spectrometry with full scan and MS2 acquisition. Metabolite structures were postulated based on their MS2 spectra in comparison to those of the parent alkaloids. All compounds were extensively metabolized yielding nor-, N-oxide, hydroxy and dihydro-diole metabolites with largely overlapping patterns in equine and human liver S9 fractions. However, some metabolic steps e.g. the formation of 8'-hydroxy metabolites were unique for human metabolism, while formation of the 13/14-hydroxy and 13,14-dihydro-diol metabolites were unique for equine metabolism. Incubations with equine whole liver preparations yielded less metabolites than the S9 fractions. 3. The acquired data can be used to develop metabolite-based screenings for these alkaloids, which will likely extend their detection windows in urine/feces from affected horses.

Systematic investigations of novel validity parameters in urine drug testing and prevalence of urine adulteration in a two-year cohort.

Urinalysis is well established for drug screening. Various methods of urine adulteration such as dilution, addition of oxidative/reductive chemicals or detergents, and handing over urine-like fluids are used to circumvent a positive screen. Validity parameters such as determination of pH, gravidity, urine temperature, or testing for oxidative/reductive chemicals are therefore used to uncover adulterated urine specimens. However, synthetic urine ("fake urine") has nowadays been used for manipulations, leading to inconspicuous results with common validity test systems. Therefore, the aims of the study were (a) to evaluate additional validity parameters, (b) to evaluate the prevalence of urine adulteration, (c) to identify adulteration markers in purchased fake urine samples. Urine samples (n = 550) submitted for drug abstinence testing were analyzed by a standard urine liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening approach using library-assisted identification of 10 different endogenous biomolecules. The detection rates of biomolecules in authentic samples were phenylalanine (93.4%), tryptophan (97.1%), propionyl-carnitine (67.1%), butyryl-carnitine (99.6%), isovaleryl-carnitine (92.8%), hexanoyl-carnitine (91.0%), heptanoyl-carnitine (97.1%), octanoyl-carnitine (98.9%), and indoleacetylglutamine (98,2%). Phenylacetylglutamine was detected in each authentic sample. Based on the detection rates and measured creatinine levels, six manipulated samples were identified in this study. In two cases, fake urine was handed over, one time fake urine was most likely used for dilution. Once dilution with other fluids was used as adulteration method, while in another sample a detergent solution was handed over. Additionaly, one sample contained reactive chemicals. All fake urine samples were additionally identified by the detection of unique polyglycole patterns, which were observed in purchased fake urine samples.

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry assay for nine toxic alkaloids from endophyte-infected pasture grasses in horse serum.

Endophyte fungi (e.g. Epichloë ssp. and Neotyphodium ssp.) in symbiosis with pasture grasses (e.g. Festuca arundinacaea and Lolium perenne) can produce toxic alkaloids, which are suspected to be involved in equine diseases such as fescue toxicosis, ryegrass staggers, and equine fescue oedema. The aim of this study was, therefore, to develop and validate a quantification method for these and related alkaloids: ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum. Horse serum samples (1.5mL) were worked up by solid-phase extraction (OASIS HLB). The extracts were analyzed by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS). Chromatographic separation was achieved by gradient elution with ammonium formate buffer and acetonitrile on a RP18 column (100×2.1mm; 1.7µm). HRMS/MS detection was performed on a QExactive Focus instrument with heated positive electrospray ionization and operated in the parallel reaction monitoring (PRM) mode. Method validation included evaluation of selectivity, matrix effect, recovery, linearity, limit of quantification (LOQ), limit of detection (LOD), accuracy, and stability. With exception of lolitrem B solid phase extraction yielded high recoveries (73.6-104.6%) for all analytes. Chromatographic separation of all analytes was achieved with a run time of 25min. HRMS/MS allowed sensitive detection with LODs ranging from 0.05 to 0.5ng/mL and LOQs from 0.1 to 1.0ng/mL. Selectivity experiments showed no interferences from matrix or IS, but N-acetylloline and N-formylloline were found to be ubiquitous in horse serum. Newborn calf serum was therefore used as surrogate matrix for the validation study. Calibration ranges were analyte-dependent and in total covered concentrations from 0.1 to 50ng/mL. Lolitrem B and paxilline could be sensitively detected, but did not meet quantification requirements. For the other analytes, accuracy and precision were shown for 3 different concentrations (QC low, medium, high) with acceptable bias (-10, 5%-7.9%) and precision (CV 2.6%-12.5%). Matrix effects varied from 55.0% to 121% (RSD 7.8-18.5%) and were adequately compensated by IS. Matrix effects of N-acetylloline and N-formylloline could only be estimated in newborn calf serum (n=1) and ranged from 52.5-88.3%. All analytes were stable under autosampler conditions and over 3 freeze and thaw cycles. Applicability was proven by analyzing authentic horse serum samples (n=24). In conclusion, the presented method allows a sensitive detection of ergocrisitine, ergocryptine, ergotamine, ergovaline, lolitrem B, lysergic acid, N-acetylloline, N-formylloline, peramine, and paxilline in horse serum and reliable quantification of all but lolitrem B and paxilline.

MEST mediates the impact of prenatal bisphenol A exposure on long-term body weight development.

Background: Exposure to endocrine-disrupting chemicals can alter normal physiology and increase susceptibility to non-communicable diseases like obesity. Especially the prenatal and early postnatal period is highly vulnerable to adverse effects by environmental exposure, promoting developmental reprogramming by epigenetic alterations. To obtain a deeper insight into the role of prenatal bisphenol A (BPA) exposure in children's overweight development, we combine epidemiological data with experimental models and BPA-dependent DNA methylation changes. Methods: BPA concentrations were measured in maternal urine samples of the LINA mother-child-study obtained during pregnancy (n = 552), and BPA-associated changes in cord blood DNA methylation were analyzed by Illumina Infinium HumanMethylation450 BeadChip arrays (n = 472). Methylation changes were verified by targeted MassARRAY analyses, assessed for their functional translation by qPCR and correlated with children's body mass index (BMI) z scores at the age of 1 and 6 years. Further, female BALB/c mice were exposed to BPA from 1 week before mating until delivery, and weight development of their pups was monitored (n ≥ 8/group). Additionally, human adipose-derived mesenchymal stem cells were treated with BPA during the adipocyte differentiation period and assessed for exposure-related epigenetic, transcriptional and morphological changes (n = 4). Results: In prenatally BPA-exposed children two CpG sites with deviating cord blood DNA-methylation profiles were identified, among them a hypo-methylated CpG in the promoter of the obesity-associated mesoderm-specific transcript (MEST). A mediator analysis suggested that prenatal BPA exposure was connected to cord blood MEST promoter methylation and MEST expression as well as BMI z scores in early infancy. This effect could be confirmed in mice in which prenatal BPA exposure altered Mest promoter methylation and transcription with a concomitant increase in the body weight of the juvenile offspring. An experimental model of in vitro differentiated human mesenchymal stem cells also revealed an epigenetically induced MEST expression and enhanced adipogenesis following BPA exposure. Conclusions: Our study provides evidence that MEST mediates the impact of prenatal BPA exposure on long-term body weight development in offspring by triggering adipocyte differentiation.

Development and validation of an ultrahigh performance liquid chromatography-high resolution tandem mass spectrometry quantification method for hypoglycin A and methylene cyclopropyl acetic acid carnitine in horse serum in cases of atypical myopathy.

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (HILIC) column (100 x 2.1 mm, 1.7 µm). Serum samples (250 µL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 to 2000 ng/mL for HGA and from 10 to 1000 ng/mL for MCPA-carnitine. HGA and MCPA-carnitine showed acceptable accuracy and precision (bias -3.0% to 1.1%; RSD 9.2% to 12.4%). The limit of quantification (LOQ) was defined as the lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from -79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples, respectively. Applicability was tested in 10 authentic AM cases. In all specimens, relevant amounts of HGA and MCPA-carnitine were found (570-2000 ng/mL; ~8.5-150 ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA-carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM.

Acidotolerant Bacteria and Fungi as a Sink of Methanol-Derived Carbon in a Deciduous Forest Soil.

Methanol is an abundant atmospheric volatile organic compound that is released from both living and decaying plant material. In forest and other aerated soils, methanol can be consumed by methanol-utilizing microorganisms that constitute a known terrestrial sink. However, the environmental factors that drive the biodiversity of such methanol-utilizers have been hardly resolved. Soil-derived isolates of methanol-utilizers can also often assimilate multicarbon compounds as alternative substrates. Here, we conducted a comparative DNA stable isotope probing experiment under methylotrophic (only [13C1]-methanol was supplemented) and combined substrate conditions ([12C1]-methanol and alternative multi-carbon [13Cu]-substrates were simultaneously supplemented) to (i) identify methanol-utilizing microorganisms of a deciduous forest soil (European beech dominated temperate forest in Germany), (ii) assess their substrate range in the soil environment, and (iii) evaluate their trophic links to other soil microorganisms. The applied multi-carbon substrates represented typical intermediates of organic matter degradation, such as acetate, plant-derived sugars (xylose and glucose), and a lignin-derived aromatic compound (vanillic acid). An experimentally induced pH shift was associated with substantial changes of the diversity of active methanol-utilizers suggesting that soil pH was a niche-defining factor of these microorganisms. The main bacterial methanol-utilizers were members of the Beijerinckiaceae (Bacteria) that played a central role in a detected methanol-based food web. A clear preference for methanol or multi-carbon substrates as carbon source of different Beijerinckiaceae-affiliated phylotypes was observed suggesting a restricted substrate range of the methylotrophic representatives. Apart from Bacteria, we also identified the yeasts Cryptococcus and Trichosporon as methanol-derived carbon-utilizing fungi suggesting that further research is needed to exclude or prove methylotrophy of these fungi.

Method Development in Forensic Toxicology.

BACKGROUND: In the field of forensic toxicology, the quality of analytical methods is of great importance to ensure the reliability of results and to avoid unjustified legal consequences. A key to high quality analytical methods is a thorough method development. METHODS: The presented article will provide an overview on the process of developing methods for forensic applications. RESULTS: This includes the definition of the method's purpose (e.g. qualitative vs quantitative) and the analytes to be included, choosing an appropriate sample matrix, setting up separation and detection systems as well as establishing a versatile sample preparation. CONCLUSION: Method development is concluded by an optimization process after which the new method is subject to method validation.