MS2 spectra were recorded using the above-mentioned PRM method to allow identification of significant features. Individual spectra were exported after subtracting the baseline left and right of the peak. After conversion to mzXML format using Proteo Wizard, spectra were imported to NIST MSSEARCH version 2.3. A library search for identification was conducted using the following settings: spectrum search type, identity (MS/MS); precursor ion m/z, in spectrum; spectrum search options, none; presearch, off; other options, none. MS/MS search was conducted using the following settings: precursor tolerance, ± 5 ppm; product ion tolerance, ± 10 ppm; ignoring peaks around precursor, ± m/z 1. The search was conducted by using the following libraries: NIST 14 (nist_msms and nist_msms2 sublibraries) and Wiley METLIN Mass Spectral Database. Metabolites of the investigated NPS were identified by interpreting their spectra in comparison to those of the parent compounds.
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography Tandem MS (LC-MS/MS)"],"chromatography_protocol":["The analysis of UM samples was performed as previously described[1] using a high-performance liquid chromatography system coupled to a high-resolution mass spectrometer (HPLC-HRMS/MS) consisting of a Thermo Fisher Scientific (TF, Dreieich, Germany) Dionex UltiMate 3000 RS pump coupled to a TF Q-Exactive Plus mass spectrometer. The UltiMate 3000 RS system consisted of a degasser, a quaternary pump, an autosampler, and an analytical column heater (column temperature 40 °C). Mass calibration was done prior to analysis according to the manufacturer’s recommendations using external mass calibration. Additionally, before each experiment, the spray shield and capillary were cleaned. The performance of the column and the mass spectrometer was tested using a mixture previously described[2] prior to every experiment. Gradient reversed phase elution was performed on a TF Accucore Phenyl-Hexyl column (100 x 2.1 mm, 2.6 µm). The mobile phases consisted of 2 mM aqueous ammonium formate containing formic acid (0.1%, v/v) and acetonitrile (1%, v/v, pH 3, eluent A), as well as 2 mM ammonium formate in acetonitrile/methanol (1:1, v/v) containing formic acid (0.1%, v/v) and water (1%, v/v, eluent B). The flow rate was set from 1–10 min to 500 µl/min and from 10–13.5 min to 800 µl/min using the following gradient: 0–1.0 min 99% A, 1–10 min to 1% A, 10–11.5 min hold 1% A, 11.5–13.5 min hold 99% A. For normal phase elution, a Macherey-Nagel (Düren, Germany) HILIC Nucleodur column (125 x 3 mm, 3 µm) was used. The mobile phases consisted of 200 mM aqueous ammonium acetate (eluent C) and acetonitrile containing formic acid (0.1%, v/v, eluent D). The flow rate was set to 500 µl/min using the following gradient: 0–1 min 2% C, 1–5 min 20% C, 5–8.5 min 60% C, 8.5–10 min hold 60% C, 10–12 min hold 2% C. The injection volume for every analysis was 1 µl.
Refs:
[1] Manier SK, Keller A, Schäper J, Meyer MR. 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. Sci Rep. 2019 Feb 26;9(1):2741. doi:10.1038/s41598-019-39235-w. PMID:30808896.
[2] Maurer HH, Pfleger K, Weber AA. Mass Spectral Data of Drugs, Poisons, Pesticides, Pollutants and Their Metabolites. Wiley-VCH; Weinheim, Germany: 2016.
"],"publication":["Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes. 10.3390/metabo11010003. PMID:33374857"],"submitter_name":["Sascha K. Manier"],"submitter_affiliation":["Saarland University"],"organism_part":["liver"],"technology_type":["mass spectrometry"],"disease":[""],"extraction_protocol":["Incubations were stopped by addition of 50 µl ice cold acetonitrile and centrifugation for 2 min at 14,000 U/min. The supernatant was transferred to an MS vial and a pooled QC sample was prepared using 10 µl of each prepared sample. All samples were analyzed using HPLC-HRMS/MS as described below.
"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS2218"],"author":["Florian Schwermer.","Niels Eckstein.","Sascha Manier. Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Center for Molecular Signaling (PZMS), Homburg, Germany. Kirrberger Str 100 – Building 46, 66421 Homburg, Germany. Sascha.Manier@uks.eu. +49 (0)6841 16 26438.","Markus Meyer. Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Center for Molecular Signaling (PZMS), Homburg, Germany. Kirrberger Str 100 – Building 46, 66421 Homburg, Germany. Markus.Meyer@uks.eu. +49 (0)6841 16 26438.","Lea Wagmann."],"data_transformation_protocol":["The proprietary TF raw data format files were converted to the open data format mzXML using ProteoWizard's MSConvert (version 3.0.1)[1]. Subsequently, QC files were used for peak detection parameter optimization as previously described[2]. Peak picking parameters that were used in this study are summarized in Table S1 of the paper associated with this study. Preprocessing and statistical evaluation was conducted using XCMS Online (version 3.7.1) and MetaboAnalyst (via https://www.metaboanalyst.ca/) (version 4.0). The converted MS data sets of Blank, Low, High, and QC were uploaded, and the parameters were adjusted according to the optimized values generated through the algorithm. Afterwards, features with valid extracted ion chromatograms and p-values lower than or equal to 0.01 after Kruskal-Wallis analysis were kept. Chromatograms were assumed valid when the peak detection integrated gaussian-like chromatographic peaks rather than arbitrary baseline fluctuation. Samples were also checked for within group outliers and removed from the study in case of high deviation. The obtained feature table was subsequently uploaded to MetaboAnalyst for further statistical evaluation. In MetaboAnalyst, missing value estimation was skipped because in no case missing values were present. Data filtering was skipped as well because in every case, less than 5000 features were submitted and therefore a need to compress the data was not given. After log transformation, the data set was submitted to multivariate statistical analysis. Hierarchical clustering was performed using Euclidian distances and complete clustering. Color contrast in the corresponding heatmap was set to 'Heat Color', samples were not reorganized, and normalized data was selected to be displayed. Additionally, features were autoscaled for this analysis. Finally, a principal component analysis (PCA) was performed to inspect the samples clustering in the corresponding score plots and the features’ influence on the clustering in the corresponding loading plots. The data set was not scaled prior to PCA. Names of the features were adopted from XCMS Online using 'M' followed by the rounded mass and “T” followed by the retention time in seconds (e.g., 'M206T264' as given in Table S2 of the paper associated with this study for protonated 4-MPD at m/z 206.1543 and a retention time of 264 s using reversed phase chromatography).
Refs:
[1] Chambers MC, Maclean B, Burke R, Amodei D, Ruderman DL, Neumann S, Gatto L et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol. 2012 Oct;30(10):918-20. doi:10.1038/nbt.2377. PMID:23051804.
[2] Manier SK, Keller A, Meyer MR. Automated optimization of XCMS parameters for improved peak picking of liquid chromatography-mass spectrometry data using the coefficient of variation and parameter sweeping for untargeted metabolomics. Drug Test Anal. 2019 Jun;11(6):752-761. doi:10.1002/dta.2552. PMID:30479047.
Pooled human liver microsomes (pHLM) (pool of 25 donors, 20 mg microsomal protein/ml) were obtained from Corning (Amsterdam, The Netherlands). After delivery, the pHLM were aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.
Microsomal incubations were performed as previously described[1] with some modifications. Stock solutions of the investigated substances were prepared at concentrations of 125 and 62.5 µM in 100 mM phosphate buffer. The incubation mixture of each sample had a total volume of 50 µl and contained 90 mM phosphate buffer, 200 U/ml superoxide dismutase, 5 mM isocitrate, 5 mM MgCl2, 1.2 mM NADP+, 0.5 U/mL isocitrate dehydrogenase, 1 mg protein/ml pHLM, and 25 µM (further referred to as group High), 12.5 µM (further referred to as group Low), or 0 µM (further referred to as group Blank) substrate. Each concentration was prepared in 5 replicates. The substrate was added after preincubation of the incubation mixture in the orbital shaker (10 min, 37 °C, 200 rpm) to incubate for another 60 min (37 °C, 200 rpm).
Ref:
[1] Welter J, Meyer MR, Wolf EU, Weinmann W, Kavanagh P, Maurer HH. 2-methiopropamine, a thiophene analogue of methamphetamine: studies on its metabolism and detectability in the rat and human using GC-MS and LC-(HR)-MS techniques. Anal Bioanal Chem. 2013 Apr;405(10):3125-35. doi:10.1007/s00216-013-6741-4. PMID:23361230.
"],"omics_type":["Metabolomics"],"study_design":["Synthetic cathinone","Toxicology","tandem mass spectrometry","high-performance liquid chromatography-mass spectrometry","untargeted metabolites"],"curator_keywords":["Synthetic cathinone","Toxicology","tandem mass spectrometry","high-performance liquid chromatography-mass spectrometry","untargeted metabolites"],"mass_spectrometry_protocol":["The Q-Exactive Plus was equipped with a heated electrospray ionization source (HESI-II) and was operated in both, positive and negative ionization mode. The spray voltage was 3.50 kV, capillary temperature, 320 °C; heater temperature, 320 °C; S-lens RF level, 50.0; sheath gas flow rate, 60 AU; auxiliary gas flow rate, 10 AU and sweep gas, 3 AU. Mass spectrometry for UM was performed according to a previously optimized workflow[1] using full scan (FS) only. The resolution was 140,000 full width at half maximum (FWHM) at mass to charge ration (m/z) 200; microscans, 1; automatic gain control target, 5 x 10^5; maximum injection time, 200 ms; scan range, m/z 50-750; polarity negative or positive and spectrum data type centroid. Thermo Fisher Scientific (TF, Dreieich, Germany) Xcalibur version 4.0.27.19 software was used for data acquisition and manipulation. The analysis was performed using a randomized sequence order with 5 injections of pure methanol (Phenyl-Hexyl column) or eluent D (HILIC column) samples at the beginning of the sequence for apparatus equilibration, followed by 5 injections of the pooled quality control (QC) sample. Additionally, 1 QC injection was performed every 5 samples.
Statistically significant features were putatively identified using parallel reaction monitoring (PRM): resolution, 70,000 FWHM; microscans, 1; automatic gain control target, 5 x 10^5; maximum injection time, 200 ms; isolation window, 0.4 m/z; normalized collision energy, 10, 20, and 40 eV; spectrum data type, centroid.
Ref:
[1] Manier SK, Keller A, Meyer MR. Automated optimization of XCMS parameters for improved peak picking of liquid chromatography-mass spectrometry data using the coefficient of variation and parameter sweeping for untargeted metabolomics. Drug Test Anal. 2019 Jun;11(6):752-761. doi:10.1002/dta.2552. PMID:30479047.
"],"metabolite_name":["4-MEAP"],"pubmed_abstract":["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."],"pubmed_title":["Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes."],"pubmed_authors":["Manier Sascha K SK, Schwermer Florian F, Wagmann Lea L, Eckstein Niels N, Meyer Markus R MR"],"additional_accession":[]},"is_claimable":false,"name":"Liquid Chromatography-High-Resolution Mass Spectrometry-Based In Vitro Toxicometabolomics of the Synthetic Cathinones 4-MPD and 4-MEAP in Pooled Human Liver Microsomes","description":"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.","dates":{"publication":"2023-11-28","submission":"2020-11-04"},"accession":"MTBLS2218","cross_references":{"MetaboLights":["MTBLC228252","MTBLC228253"],"pubmed":["33374857"],"ChEBI":["CHEBI:228252","CHEBI:228253"]}}