14 research outputs found
Impact of the used solvent on the reconstitution efficiency of evaporated biosamples for untargeted metabolomics studies
Introduction
Untargeted metabolomics intends to objectively analyze a wide variety of compounds. Their diverse physicochemical properties make it difficult to choose an appropriate reconstitution solvent after sample evaporation without influencing the chromatography or hamper column sorbent integrity.
Objectives
The study aimed to identify the most appropriate reconstitution solvent for blood plasma samples in terms of feature recovery, four endogenous compounds, and one selected internal standard.
Methods
We investigated several reconstitution solvent mixtures containing acetonitrile and methanol to resolve human plasma extract and evaluated them concerning the peak areas of tryptophan-d5, glucose, creatinine, palmitic acid, and the phophatidylcholine PC(P-16:0/P-16:0), as well as the total feature count
Results
Results indicated that acetonitrile containing 30% methanol was best suited to match all tested criteria at least for human blood plasma samples.
Conclusion
Despite identifying the mixture of acetonitrile and methanol being suitable as solvent for human blood plasma extracts, we recommend to systematically test for an appropriate reconstitution solvent for each analyzed biomatrix
Untargeted metabolomics by high resolution mass spectrometry coupled to normal and reversed phase liquid chromatography as a tool to study the in vitro biotransformation of new psychoactive substances
In 2016, several synthetic cathinones were seized by the State Bureau of Criminal Investigation Bavaria in Germany. Due to their previous appearances in other countries their metabolism was already investigated in human urine as well as different in vitro models. These investigations were conducted using ordinary metabolism studies for drugs of abuse by using general knowledge about drug metabolism and visual comparison of mass spectra. The present study aimed to use untargeted metabolomics to support and improve those methods that highly depend on the investigators experience. Incubations were conducted using pooled human liver microsomes (pHLM) and the two cathinones 1-phenyl-2-(1-pyrrolidinyl)-1-butanone and 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone. Samples were analyzed by LC-HRMS/MS using a metabolomics workflow consisting of a reversed phase or normal phase separation followed by electrospray ionization and full scan in positive or negative mode. LC-MS data was afterwards statistically evaluated using principal component analysis, t-distributed stochastic neighborhood embedding, and hierarchical clustering. Significant features were then identified using MS/MS. The workflow revealed 24 significant features after 1-phenyl-2-(1-pyrrolidinyl)-1-butanone and 39 after 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone incubation, consisting of adducts, artifacts, isomers, and metabolites. The applied untargeted metabolomics strategy was able to find almost all of the metabolites that were previously described for 1-phenyl-2-(1-pyrrolidinyl)-1-butanone in literature as well as three additional metabolites. Concerning 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone biotransformation in pHLM, merely four metabolites described in primary human hepatocytes and human urine were not found. This study revealed that untargeted metabolomics workflows are well suited to support biotransformation studies at least of the investigated compounds in pHLM
The metabolic fate of two new psychoactive substances - 2-aminoindane and N-methyl-2-aminoindane - studied in vitro and in vivo to support drug testing
The aim of this study was to characterize the in vitro and in vivo metabolism of 2-aminoindane (2,3-dihydro-1H-inden-2-amine, 2-AI), and N-methyl-2-aminoindane (N-methyl-2,3-dihydro-1H-inden-2-amine, NM-2-AI) after incubations using pooled human liver microsomes (pHLMs), pooled human liver S9 fraction (pS9), and rat urine after oral administration. After analysis using liquid chromatography coupled to high-resolution mass spectrometry, pHLM incubations revealed that 2-AI was left unmetabolized, while NM-2-AI formed a hydroxylamine and diastereomers of a metabolite formed after hydroxylation in beta position. Incubations using pS9 led to the formation of an acetyl conjugation in the case of 2-AI and merely a hydroxylamine for NM-2-AI. Investigations on rat urine showed that 2-AI was hydroxylated also forming diasteromers as described for NM-2-AI or acetylated similar to incubations using pS9. All hydroxylated metabolites of NM-2-AI except the hydroxylamine were found in rat urine as additional sulfates. Assuming similar patterns in humans, urine screening procedures might be focused on the parent compounds but should also include their metabolites. An activity screening using human recombinant N-acetyl transferase (NAT) isoforms 1 and 2 revealed that 2-AI was acetylated exclusively by NAT2, which is polymorphically expressed
Toxicometabolomics of the new psychoactive substances α-PBP and α-PEP studied in HepaRG cell incubates by means of untargeted metabolomics revealed unexpected amino acid adducts
Toxicometabolomics, essentially applying metabolomics to toxicology of endogenous compounds such as drugs of abuse or new psychoactive substances (NPS), can be investigated by using different in vitro models and dedicated metabolomics techniques to enhance the number of relevant findings. The present study aimed to study the toxicometabolomics of the two NPS α-pyrrolidinobutiophenone (1-phenyl-2-(pyrrolidin-1-yl)butan-1-one, α-PBP) and α-pyrrolidinoheptaphenone (1-phenyl-2-(pyrrolidin-1-yl)heptan-1-one, α-PEP, PV8) in HepaRG cell line incubates. Evaluation was performed using reversed-phase and normal-phase liquid chromatography coupled with high-resolution mass spectrometry in positive and negative ionization mode, respectively, to analyze cells and cell media. Statistical evaluation was performed using one-way ANOVA, principal component discriminant function analysis, as well as hierarchical clustering. In general, the analysis of cells did not mainly reveal any features, but the parent compounds of the drugs of abuse. For α-PBP an increase in N-methylnicotinamide was found, which may indicate hepatotoxic potential of the substance. After analysis of cell media, significant features led to the identification of several metabolites of both compounds. Amino acid adducts with glycine and alanine were found, and these have not been described in any study before and are likely to appear in vivo. Additionally, significant changes in the metabolism of cholesterol were revealed after incubation with α-PEP. In summary, the application of metabolomics techniques after HepaRG cells exposure to NPS did not lead to an increased number of identified drug metabolites compared to previously published studies, but gave a wider perspective on the physiological effect of the investigated compounds on human liver cells
Abuse of nutmeg seeds: Detectable by means of liquid chromatography-mass spectrometry techniques?
Numerous case reports of intoxications with nutmeg seeds (Myristica fragrans, Houtt.)
can be found in literature often following their abuse, as psychotropic effects were
described after ingestions of large doses. The successful detection of the main ingredients of the nutmeg seeds essential oil elemicin, myristicin, and safrole, as well as
their metabolites in human urine by gas chromatography coupled to mass spectrometry (GC-MS) was already described. The aim of this study was to investigate the
detectability of the main ingredients of nutmeg seeds and their metabolites in human
blood and urine samples using liquid chromatography coupled to linear ion trap mass
spectrometry (LC-LIT-MSn
) and liquid chromatography coupled to high-resolution
mass spectrometry (LC-HRMS/MS) after nutmeg seed abuse. Sample material of
three individuals was retrospectively investigated after a systematic screening
approach indicated an intoxication with nutmeg seeds as a likely cause of symptoms.
Metabolic patterns in plasma and urine using GC-MS were comparable with those
described in earlier publications. Investigations using hyphenated liquid chromatography techniques lead to the detection of myristicin and safrole, as well as further
metabolites not described using GC-MS and revealed sulfation as an additional Phase
II metabolic pathway. These results might help to detect or confirm future intoxications with nutmeg seeds by using LC-MS techniques
Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes
Synthetic cathinones belong to the most often seized new psychoactive substances on
an international level. This study investigated the toxicometabolomics, particularly the in vitro
metabolism of 2-(methylamino)-1-(4-methylphenyl)-1-pentanone (4-MPD) and 2-(ethylamino)-1-(4-
methylphenyl)-1-pentanone (4-MEAP) in pooled human liver microsomes (pHLM) using untargeted
metabolomics techniques. Incubations were performed with the substrates in concentrations ranging
from 0, 12.5, and 25 µM. Analysis was done by means of high-performance liquid chromatography
coupled to high-resolution mass spectrometry (HPLC-HRMS/MS) in full scan only and the obtained
data was evaluated using XCMS Online and MetaboAnalyst. Significant features were putatively
identified using a separate parallel reaction monitoring method. Statistical analysis was performed
using Kruskal-Wallis test for prefiltering significant features and subsequent hierarchical clustering,
as well as principal component analysis (PCA). Hierarchical clustering or PCA showed a distinct
clustering of all concentrations with most of the features z-scores rising with the concentration of
the investigated substances. Identification of significant features left many of them unidentified
but revealed metabolites of both 4-MPD and 4-MEAP. Both substances formed carboxylic acids,
were hydroxylated at the alkyl chain, and formed metabolites after combined hydroxylation and
reduction of the cathinone oxo group. 4-MPD additionally formed a dihydroxy metabolite and a
hydroxylamine. 4-MEAP was additionally found reduced at the cathinone oxo group, N-dealkylated,
and formed an oxo metabolite. These findings are the first to describe the metabolic pathways
of 4-MPD and to extend our knowledge about the metabolism of 4-MEAP. Findings, particularly
the MS data of the metabolites, are essential for setting up metabolite-based toxicological (urine)
screening procedures
Toxicokinetic studies of the four new psychoactive substances 4-chloroethcathinone, N-ethylnorpentylone, N-ethylhexedrone, and 4-fluoro-alpha-pyrrolidinohexiophenone
Purpose
The presented study aimed to elucidate the toxicokinetics of the four synthetic cathinones 4-chloroethcathinone (4-CEC), N-ethylnorpentylone (N-ethylpentylone, ephylone), N-ethylhexedrone (NEH), and 4-fluoro-alpha-pyrrolidinohexiophenone (4-fluoro-alpha-pyrrolidinohexanophenone, 4-F-α-PHP, 4F-alpha-PHP, 4F-PHP).
Methods
First, their metabolism was studied using human urine and blood samples. Analysis of specimens was performed by liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) and gas chromatography–mass spectrometry (GC–MS). LC-HRMS/MS was also used to analyze in vitro incubations of the new psychoactive substances using pooled human liver S9 fraction (pS9), to identify the monooxygenases involved in the initial metabolic steps, and determination of plasma concentrations after a standard addition method. Metabolic stability was tested in pooled human liver microsomes incubations analyzed by LC-ion trap MS.
Results
Using LC-HRMS/MS, 47 metabolites in total were found in patient samples and pS9 incubations. Using GC–MS, 4-CEC, ephylone, NEH, and five of their metabolites were detectable in urine. The following main phase I reactions were observed: carbonyl group reduction, N-deethylation, hydroxylation, lactam formation (4F-PHP), and demethylenation (ephylone). Mainly glucuronidations were observed as phase II reactions besides conjugates with the dicarboxylic acids malonic, succinic, and glutaric acid (4-CEC), sulfation, methylation (both ephylone), and N-acetylation (NEH). A broad range of monooxygenases was involved in the initial steps with exception of NEH (only CYP1A2 and CYP2C19). 4F-PHP had the shortest in vitro half-life (38 min) and highest intrinsic clearance (15.7 mL/min/kg). Plasma concentrations ranged from 0.8 to 8.5 ng/mL.
Conclusions
Our results are expected to help toxicologists to reliably identify these substances in case of suspected abuse and allow them a thorough risk assessment
Comparison of Three Untargeted Data Processing Workflows for Evaluating LC-HRMS Metabolomics Data
The evaluation of liquid chromatography high-resolution mass spectrometry (LC-HRMS)
raw data is a crucial step in untargeted metabolomics studies to minimize false positive findings.
A variety of commercial or open source software solutions are available for such data processing.
This study aims to compare three different data processing workflows (Compound Discoverer 3.1,
XCMS Online combined with MetaboAnalyst 4.0, and a manually programmed tool using R) to
investigate LC-HRMS data of an untargeted metabolomics study. Simple but highly standardized
datasets for evaluation were prepared by incubating pHLM (pooled human liver microsomes)
with the synthetic cannabinoid A-CHMINACA. LC-HRMS analysis was performed using normaland reversed-phase chromatography followed by full scan MS in positive and negative mode.
MS/MS spectra of significant features were subsequently recorded in a separate run. The outcome of
each workflow was evaluated by its number of significant features, peak shape quality, and the results
of the multivariate statistics. Compound Discoverer as an all-in-one solution is characterized by its
ease of use and seems, therefore, suitable for simple and small metabolomic studies. The two open
source solutions allowed extensive customization but particularly, in the case of R, made advanced
programming skills necessary. Nevertheless, both provided high flexibility and may be suitable for
more complex studies and questions
Addendum: Hemmer, S., et al. Comparison of Three Untargeted Data Processing Workflows for Evaluating LC-HRMS Metabolomics Data. Metabolites 2020, 10, 378
The authors wish to make the following comment to this paper [...
Evaluation of extraction methods for untargeted metabolomic studies for future applications in zebrafish larvae infection models
Sample preparation in untargeted metabolomics should allow reproducible extractions of as many
molecules as possible. Thus, optimizing sample preparation is crucial. This study compared six
diferent extraction procedures to fnd the most suitable for extracting zebrafsh larvae in the context
of an infection model. Two one-phase extractions employing methanol (I) and a single miscible
phase of methanol/acetonitrile/water (II) and two two-phase methods using phase separation
between chloroform and methanol/water combinations (III and IV) were tested. Additional bead
homogenization was used for methods III and IV (III_B and IV_B). Nine internal standards and 59
molecules of interest (MoInt) related to mycobacterial infection were used for method evaluation.
Two-phase methods (III and IV) led to a lower feature count, higher peak areas of MoInt, especially
amino acids, and higher coefcients of variation in comparison to one-phase extractions. Adding bead
homogenization increased feature count, peak areas, and CVs. Extraction I showed higher peak areas
and lower CVs than extraction II, thus being the most suited one-phase method. Extraction III and IV
showed similar results, with III being easier to execute and less prone to imprecisions. Thus, for future
applications in zebrafsh larvae metabolomics and infection models, extractions I and III might be
chosen