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.

Current state-of-the-art approaches for mass spectrometry in clinical toxicology: an overview.

INTRODUCTION: Hyphenated mass spectrometry (MS) has evolved into a very powerful analytical technique of high sensitivity and specificity. It is used to analyze a very wide spectrum of analytes in classical and alternative matrices. The presented paper will provide an overview of the current state-of-the-art of hyphenated MS applications in clinical toxicology primarily based on review articles indexed in PubMed (1990 to April 2023). AREAS COVERED: A general overview of matrices, sample preparation, analytical systems, detection modes, and validation and quality control is given. Moreover, selected applications are discussed. EXPERT OPINION: A more widespread use of hyphenated MS techniques, especially in systematic toxicological analysis and drugs of abuse testing, would help overcome limitations of immunoassay-based screening strategies. This is currently hampered by high instrument cost, qualification requirements for personnel, and less favorable turnaround times, which could be overcome by more user-friendly, ideally fully automated MS instruments. This would help making hyphenated MS-based analysis available in more laboratories and expanding analysis to a large number of organic drugs, poisons, and/or metabolites. Even the most recent novel psychoactive substances (NPS) could be presumptively identified by high-resolution MS methods, their likely presence be communicated to treating physicians, and be confirmed later on.

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.

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.

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.

Corrigendum: Monocyte-induced recovery of inflammation-associated hepatocellular dysfunction in a biochip-based human liver model.

This corrects the article DOI: 10.1038/srep21868.

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.

Preservation of Cell Structure, Metabolism, and Biotransformation Activity of Liver-On-Chip Organ Models by Hypothermic Storage.

The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research.

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.

Update of Standard Practices for New Method Validation in Forensic Toxicology.

International agreement concerning validation guidelines is important to obtain quality forensic bioanalytical research and routine applications as it all starts with the reporting of reliable analytical data. Standards for fundamental validation parameters are provided in guidelines as those from the US Food and Drug Administration (FDA), the European Medicines Agency (EMA), the German speaking Gesellschaft fur Toxikologie und Forensische Chemie (GTFCH) and the Scientific Working Group of Forensic Toxicology (SWGTOX). These validation parameters include selectivity, matrix effects, method limits, calibration, accuracy and stability, as well as other parameters such as carryover, dilution integrity and incurred sample reanalysis. It is, however, not easy for laboratories to implement these guidelines into practice as these international guidelines remain nonbinding protocols, that depend on the applied analytical technique, and that need to be updated according the analyst's method requirements and the application type. In this manuscript, a review of the current guidelines and literature concerning bioanalytical validation parameters in a forensic context is given and discussed. In addition, suggestions for the experimental set-up, the pros and cons of statistical approaches and adequate acceptance criteria for the validation of bioanalytical applications are given.

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.

Recent advances of liquid chromatography-(tandem) mass spectrometry in clinical and forensic toxicology - An update.

Liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) is a well-established and widely used technique in clinical and forensic toxicology as well as doping control especially for quantitative analysis. In recent years, many applications for so-called multi-target screening and/or quantification of drugs, poisons, and or their metabolites in biological matrices have been developed. Such methods have proven particularly useful for analysis of so-called new psychoactive substances that have appeared on recreational drug markets throughout the world. Moreover, the evolvement of high resolution MS techniques and the development of data-independent detection modes have opened new possibilities for applications of LC-(MS/MS) in systematic toxicological screening analysis in the so called general unknown setting. The present paper will provide an overview and discuss these recent developments focusing on the literature published after 2010.

Search for fungi-specific metabolites of four model drugs in postmortem blood as potential indicators of postmortem fungal metabolism.

Fungi colonizing cadavers are capable of drug metabolism and may thus change the metabolite pattern or concentration of drugs in forensic postmortem samples. The purpose of this study was to check for the presence of such changes by searching fungi-specific metabolites of four model drugs (amitriptyline, metoprolol, mirtazapine, and zolpidem) in decomposed postmortem blood samples from 33 cases involving these drugs. After isolation and identification of fungal strains present in the samples, each isolate was incubated in Sabouraud medium at 25°C for up to 120h with each model drug. One part of the supernatants was directly analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), another after liquid-liquid extraction with chlorobutane and concentration. From 21 out of 33 decomposed postmortem blood samples (64%) a total of 30 different strains could be isolated, one from the class of Ascomycete and the rest belonging to 15 species from 8 different genera (number of species): Aspergillus (2), Botrytis (1), Candida (8), Fusarium (1), Mucor (1), Penicillium (1), and Rodothorula (1). In the in vitro studies, these microorganisms were found capable of N-demethylation and N-oxidation of amitriptyline and mirtazapine, O-demethylation followed by side chain oxidation of metoprolol as well as hydroxylation of all four-model drugs. In two of the postmortem blood samples, from which the fungi Aspergillus jensenii, Candida parapsilosis. and Mucor circinelloides had been isolated, a fungi-specific hydroxy zolpidem metabolite was detected. The presence of this metabolite in postmortem samples likely indicates postmortem fungal biodegradation.