bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒09‒18
29 papers selected by
Sofia Costa

  1. J Cheminform. 2022 Sep 15. 14(1): 62
      MOTIVATION: Compound structure identification is using increasingly more sophisticated computational tools, among which machine learning tools are a recent addition that quickly gains in importance. These tools, of which the method titled Compound Structure Identification:Input Output Kernel Regression (CSI:IOKR) is an excellent example, have been used to elucidate compound structure from mass spectral (MS) data with significant accuracy, confidence and speed. They have, however, largely focused on data coming from liquid chromatography coupled to tandem mass spectrometry (LC-MS). Gas chromatography coupled to mass spectrometry (GC-MS) is an alternative which offers several advantages as compared to LC-MS, including higher data reproducibility. Of special importance is the substantial compound coverage offered by GC-MS, further expanded by derivatization procedures, such as silylation, which can improve the volatility, thermal stability and chromatographic peak shape of semi-volatile analytes. Despite these advantages and the increasing size of compound databases and MS libraries, GC-MS data have not yet been used by machine learning approaches to compound structure identification.RESULTS: This study presents a successful application of the CSI:IOKR machine learning method for the identification of environmental contaminants from GC-MS spectra. We use CSI:IOKR as an alternative to exhaustive search of MS libraries, independent of instrumental platform and data processing software. We use a comprehensive dataset of GC-MS spectra of trimethylsilyl derivatives and their molecular structures, derived from a large commercially available MS library, to train a model that maps between spectra and molecular structures. We test the learned model on a different dataset of GC-MS spectra of trimethylsilyl derivatives of environmental contaminants, generated in-house and made publicly available. The results show that 37% (resp. 50%) of the tested compounds are correctly ranked among the top 10 (resp. 20) candidate compounds suggested by the model. Even though spectral comparisons with reference standards or de novo structural elucidations are neccessary to validate the predictions, machine learning provides efficient candidate prioritization and reduction of the time spent for compound annotation.
    Keywords:  Derivative; Identification; Machine learning; Mass spectrometry; Molecular fingerprint; Prediction; Silylation
  2. J Pharm Anal. 2022 Aug;12(4): 601-609
      A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of total and unbound concentrations of LY3214996, an extracellular signal-regulated kinase inhibitor; abemaciclib, a cyclin-dependent kinase 4/6 inhibitor; and abemaciclib active metabolites, M2 and M20, in human plasma, brain tumor, and cerebrospinal fluid samples. The method was validated over a concentration range of 0.2-500 nM within a total run time of 3.8 min using isocratic elution on a Kinetex™ F5 column. Detection was performed on a Sciex QTRAP 6500+ mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. The intra- and inter-batch accuracy as well as the precision of the method for all matrices was within ±20% and ≤20% at the lower limit of quantification, and within ±15% and ≤15% for other quality control levels for all analytes. The unbound fractions of drugs and metabolites in spiked and patient samples were determined using an optimized equilibrium dialysis. The validated method was successfully applied in a phase 0/2 clinical trial to assess the central nervous system penetration of LY3214996 and abemaciclib.
    Keywords:  Abemaciclib; Brain tumor penetration; Equilibrium dialysis; LC-MS/MS; LY3214996
  3. J Agric Food Chem. 2022 Sep 13.
      Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics has become an important tool to increase our understanding of how diet affects human health. However, public and commercial mass spectral libraries of dietary metabolites are limited, resulting in the greatest challenge in converting mass spectrometry data into biological insights. In this study, we constructed an LC-MS/MS ginger library as an example to demonstrate the importance of dietary libraries for discovering food biomarkers. The functional and exposure biomarkers of ginger were investigated using plasma samples from mice treated with control and ginger extract diets. Our results showed clear discrimination between the metabolome of mice on normal and ginger extract diets. Using the in-house ginger library, we identified 20 ginger metabolites that can be used as exposure biomarkers of ginger. However, without the LC-MS/MS ginger library, none of the ginger metabolites could be accurately identified based on online mass databases. In addition, ginger treatment significantly impacts the endogenous metabolome, especially the purine metabolism and phenylalanine, tyrosine, and tryptophan biosynthesis. Overall, we demonstrated that the construction of LC-MS/MS spectra dietary libraries would enhance the ability to identify potential dietary biomarkers and correlate potential health benefits associated with food consumption.
    Keywords:  biomarkers; dietary metabolites; ginger; gingerols; mass spectral library; shogaols; untargeted metabolomics
  4. Anal Chim Acta. 2022 Sep 22. pii: S0003-2670(22)00841-8. [Epub ahead of print]1227 340270
      In this work, a porous capillary monolithic column was simply prepared by in situ thiol-alkyne click polymerization of dipentaerythritol hexakis (3-mercaptopropionate) and dimethyl dipropargylmalonate in fused-silica capillary. The capillary monolithic column shows excellent permeability, high porosity, and thoiether-rich groups, thereby, a high-efficient capacity for trace estrogens from complex samples are obtained via electron-donor-acceptor π-π interaction and hydrophobic interaction. The highest adsorption efficiency for estrogens is achieved at pH = 7.0 with a flow rate of 0.200 mL min-1. The superior adsorption capacities of the as-prepared capillary column for eight estrogens range from 0.092 mg m-1 to 0.31 mg m-1. A simple, reliable, and sensitive method for the determination of eight estrogens in biological and environmental samples is developed using the monolithic polymer as in-tube solid-phase microextraction coupled with ultrahigh performance liquid chromatography-tandem mass spectrometry (SPME-UPLC-MS/MS), and the total instrumental analysis time for the SPME-UPLC-MS/MS procedures was about 60 min per sample. The developed method shows a wide linear range (0.0500-5.00 μg L-1), and low limits of detection (5.34-9.63 ng L-1) for estrogens. The concentrations of estrogens in serum, urine, and pond water samples are found to be no more than 3.69, 0.741, and 1.04 μg L-1, respectively, and the satisfying recoveries for the eight estrogens range from 80.3% to 113% with relative standard deviations (n = 5) of 1.5-9.4%. The established method is highly potential for extraction and analysis of ultratrace target estrogens in complex matrices, such as biological and environmental samples.
    Keywords:  Estrogens; In-tube solid-phase microextraction; Liquid chromatography-tandem mass spectrometry; Thiol-alkyne click polymerization
  5. Anal Chem. 2022 Sep 13.
      In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.
  6. STAR Protoc. 2022 Sep 13. pii: S2666-1667(22)00548-2. [Epub ahead of print]3(4): 101668
      Peritoneal macrophages (PMs) have been shown to have higher stability compared to other macrophage subtypes. However, obtaining enough PMs from a single mouse is often a limitation for metabolomics analysis. Here, we describe a protocol to isolate metabolites from a small number of mouse primary PMs for 13C-stable glucose tracing and metabolomics. Our protocol uses X for metabolite extraction instead of methanol. Our protocol can consistently extract metabolites from low cell number samples with fewer steps than methanol-based approaches. For complete details on the use and execution of this protocol, please refer to De Jesus et al., (2022).
    Keywords:  Cell isolation; Immunology; Mass spectrometry; Metabolism; Metabolomics
  7. Biosci Rep. 2022 09 15. pii: BSR20221326. [Epub ahead of print]
      The free hormone hypothesis has triggered controversies regarding the measurement of free vitamin D metabolites, such as free 25-hydroxyvitamin D (25(OH)D), as a suitable indicator for total vitamin D for clinical use. This issue can be addressed by developing a precise and accurate method for free 25(OH)D measurement. In this study, a novel assay method for free 25(OH)D3 based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Sample preparation first involved ultrafiltration to remove vitamin D-binding protein-bound and albumin-bound 25(OH)D, followed by extraction with a column, derivatization, evaporation, dissolution, and injection into the LC-MS/MS system. The coefficient of variation of repeatability and reproducibility obtained were 3.8-4.5% and 4.8-5.9%, respectively. Satisfactory linearity (r = 0.999) was obtained up to 80 pg/mL. The lower quantification limit was 0.97 pg/mL and the S/N ratio on the peak of 1.0 pg/mL sample was 24.8 (which is more than the acceptable value of 10). The recovery rate was between 84.5-92.4% with a negligible matrix effect (94.5-104.9%). Levels of free 25(OH)D3, but not total 25(OH)D3, in the serum of the patients with chronic kidney disease (CKD) and hepatic cirrhosis (HC) were substantially lower than those in healthy subjects. The correlation coefficient between total and free 25(OH)D3 was 0.738 in all samples, while the linear regression equations were different between the patients with CKD and HC. In conclusion, LC-MS/MS assay for free 25(OH)D3 might be useful to evaluate high throughput methods, including ELISA.
    Keywords:  free 25(OH)D3; liquid chromatography-tandem mass spectrometry (LC-MS/MS); ultrafiltration; vitamin D metabolites; vitamin D-binding protein (DBP)
  8. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Sep 13. 1-12
      Liquid chromatography-high resolution mass spectrometry (LC-HRMS) is considered an unavoidable extension of low-resolution LC-MS/MS that stretches the capabilities of multi-residue analysis of chemical contaminants in food. However, LC-HRMS acquisitions generate a massive amount of information available for data processing with supplier software that still miss critical calculation features and adapted reporting tools. Consequently, routine laboratories are still reluctant to switch from LC-MS/MS to LC-HRMS, the latter is still perceived as a cumbersome and demanding technology. In that context, we propose a four-step LC-HRMS workflow to speed-up data processing in situations of multi-residue multi-matrix analysis with the goal to maximize the time spent on data interpretation rather than on data formatting. The first three steps of the workflow imply specific settings on the Orbitrap HRMS associated software (TraceFinderTM) while the fourth step is the novelty i.e. a newly coded R-script capable to translate a raw export file into a comprehensive .xlsx report file in a few seconds. As recommended by various international guidelines and in some official methods, standard addition-based applications are fully embedded in this reporting tool whilst still being the main bottleneck of supplier's software. The reporting tool also allows appropriate data formatting, filtering, and color-coding options to provide a clear picture of compounds being detected or not, and those requiring specific attention due to unmet quality control criteria as required by European legislation (European Commission SANTE 11312/2021). It is hoped that additional functionalities compatible with R scripts will be soon fully embedded in the supplier's software for easier data interpretation and reporting.
    Keywords:  High resolution mass spectrometry; chemical contaminants; data treatment; multi-residue analysis; quality control
  9. Front Pharmacol. 2022 ;13 885386
      Background: Metabolomics can be applied to the clinical diagnosis and treatment evaluation of acquired immune deficiency syndrome (AIDS). AIDS biomarkers have become a new direction of AIDS research providing clinical guidance for diagnosis. Objective: We sought to apply both untargeted and targeted metabolomic profiling to identify potential biomarkers for AIDS patients. Methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based untargeted metabolomic profiling was performed on plasma samples of patients before and after highly active antiretroviral therapy (HAART) treatment as well as healthy volunteers to identify potential AIDS biomarkers. Targeted quantitative analysis was performed on the potential biomarkers screened from untargeted metabolic profiling for verification. Results: Using the Mass Profiler Professional and the MassHunter, several potential biomarkers have been found by LC-MS/MS in the untargeted metabolomic study. High-resolution MS and MS/MS were used to analyze fragmentation rules of the metabolites, with comparisons of related standards. Several potential biomarkers have been identified, including PS(O-18:0/0:0), sphingosine, PE (21:0/0:0), and 1-Linoleoyl Glycerol. Targeted quantitative analysis showed that sphingosine and 1-Linoleoyl Glycerol might be closely related to HIV/AIDS, which may be the potential biomarkers to the diagnosis. Conclusion: We conducted untargeted metabolomic profiling, which indicates that several metabolites should be considered potential biomarkers for AIDS patients. Further targeted metabolomic research verified that d-Sphingosine and 1-Linoleoyl glycerol as the diagnostic biomarker of AIDS.
    Keywords:  AIDS; HIV; LC-MS/MS; biomarker; metabolomics
  10. Talanta. 2022 Aug 31. pii: S0039-9140(22)00697-X. [Epub ahead of print]253 123901
      Non-targeted metabolomic approaches based on direct introduction (DI) through a soft ionization source are nowadays used for large-scale analysis and wide cover-up of metabolites in complex matrices. When coupled with ultra-high-resolution Fourier-Transform ion cyclotron resonance (FTICR MS), DI is generally performed through electrospray (ESI), which, despite the great analytical throughput, can suffer of matrix effects due to residual salts or charge competitors. In alternative, matrix assisted laser desorption ionization (MALDI) coupled with FTICR MS offers relatively high salt tolerance but it is mainly used for imaging of small molecule within biological tissues. In this study, we report a systematic evaluation on the performance of direct introduction ESI and MALDI coupled with FTICR MS applied to the analysis of root exudates (RE), a complex mixture of metabolites released from plant root tips and containing a relatively high salt concentration. Classic dried droplet deposition followed by screening of best matrices and ratio allowed the selection of high ranked conditions for non-targeted metabolomics on RE. Optimization of MALDI parameters led to improved reproducibility and precision. A RE desalted sample was used for comparison on ionization efficiency of the two sources and ion enhancement at high salinity was highlighted in MALDI by spiking desalted solution with inorganic salts. Application of a true lyophilized RE sample exhibited the complementarity of the two sources and the ability of MALDI in the detection of undisclosed metabolites suffering of matrix effects in ESI mode.
    Keywords:  Fourier transform ion cyclotron resonance mass spectrometry; MALDI; Non-targeted metabolomics; Pisum sativum; Root exudates
  11. Front Mol Biosci. 2022 ;9 932261
      Analyses of metabolic compounds inside cells or tissues provide high information content since they represent the endpoint of biological information flow and are a snapshot of the integration of many regulatory processes. However, quantification of the abundance of metabolites requires their careful extraction. We present a comprehensive study comparing ten extraction protocols in four human sample types (liver tissue, bone marrow, HL60, and HEK cells) aiming to detect and quantify up to 630 metabolites of different chemical classes. We show that the extraction efficiency and repeatability are highly variable across protocols, tissues, and chemical classes of metabolites. We used different quality metrics including the limit of detection and variability between replicates as well as the sum of concentrations as a global estimate of analytical repeatability of the extraction. The coverage of extracted metabolites depends on the used solvents, which has implications for the design of measurements of different sample types and metabolic compounds of interest. The benchmark dataset can be explored in an easy-to-use, interactive, and flexible online resource (R/shiny app MetaboExtract: for context-specific selection of the optimal extraction method. Furthermore, data processing and conversion functionality underlying the shiny app are accessible as an R package:
    Keywords:  absolute quantification; extraction protocol; intra-cellular; metabolism; metabolomics
  12. J Chromatogr Sci. 2022 Sep 11. pii: bmac077. [Epub ahead of print]
      This study uses a liquid chromatography-electrospray ionization-tandem mass spectrometry method to determine β-Sitosterol and Ferulic acid in Pygeum africanum extract. Chromatographic separation of the two analytes was performed on an ACQUITY UPLC H-Class system coupled with Xevo TQD mass spectrometer and HSS T3 C18 column (2.1 X 50 mm, 1.8 μm). Mobile phase A consisted of an aqueous solution of 0.1% formic acid (v/v), and mobile phase B was 0.1% formic acid (v/v) in methanol pumped through a gradient elution mode. Mass spectrometer parameters were optimized using an electrospray ionization source in the positive and negative ionization modes. The quantification of the two analytes was performed using multiple reaction monitoring transitions. The method was fully validated per (FDA) guidelines regarding linearity, accuracy, precision, carryover and selectivity. The proposed method was applied successfully to determine the two investigated compounds in commercially available pharmaceutical products.
  13. BMC Bioinformatics. 2022 Sep 16. 23(1): 379
      Metabolomic time course analyses of biofluids are highly relevant for clinical diagnostics. However, many sampling methods suffer from unknown sample sizes, commonly known as size effects. This prevents absolute quantification of biomarkers. Recently, several mathematical post acquisition normalization methods have been developed to overcome these problems either by exploiting already known pharmacokinetic information or by statistical means. Here we present an improved normalization method, MIX, that combines the advantages of both approaches. It couples two normalization terms, one based on a pharmacokinetic model (PKM) and the other representing a popular statistical approach, probabilistic quotient normalization (PQN), in a single model. To test the performance of MIX, we generated synthetic data closely resembling real finger sweat metabolome measurements. We show that MIX normalization successfully tackles key weaknesses of the individual strategies: it (i) reduces the risk of overfitting with PKM, and (ii), contrary to PQN, it allows to compute sample volumes. Finally, we validate MIX by using real finger sweat as well as blood plasma metabolome data and demonstrate that MIX allows to better and more robustly correct for size effects. In conclusion, the MIX method improves the reliability and robustness of quantitative biomarker detection in finger sweat and other biofluids, paving the way for biomarker discovery and hypothesis generation from metabolomic time course data.
    Keywords:  Blood Plasma; Finger Sweat; Metabolomics; PKM; PQN
  14. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Aug 28. pii: S1570-0232(22)00342-7. [Epub ahead of print]1210 123438
      The widespread use of opioid drugs has contributed to escalating rates of addiction, overdoses, and drug-related deaths. Targeted urine drug screening plays an important role in supporting the care of patients with chronic pain or addiction. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can provide excellent sensitivity and specificity, and, as a result, remains the definitive choice for confirmatory urine drug testing. However, the complexities of LC-MS/MS operation present major challenges to the clinical laboratory. In this study, we leveraged upgraded instrumentation to develop and validate a simplified "dilute-and-shoot" LC-MS/MS opioid assay. By modifying the chromatographic gradient, isobaric interferences were well-resolved and eliminated. Analytical ranges were expanded by utilizing alternative mass transitions, and updated quality assurance parameters were established. Results from 204 clinical samples correlated well between the new method and a previous version. The upgraded systems provided better sensitivity, greater dynamic ranges, and the new method reduced carryover, which enabled us to eliminate extra injections and chromatogram reviews. The new method also reduced turnaround time and doubled testing capacity. These improvements could serve as a model for other laboratories approaching a similar transition in mass spectrometric instrumentation.
    Keywords:  Dilute-and-shoot; LC-MS/MS; Opioid; Workflow and capacity
  15. Biomed Chromatogr. 2022 Sep 12. e5504
      OBJECTIVE: A reliable and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of zanubrutinib in the plasma of beagle dogs.METHODS: The column used was an Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm), maintained at 40 °C with an injection volume of 2 μL. The gradient elution program was as follows: 0-1 min (10-10% A), 1-1.1 min (10-90% A), 1.1-2.1 min (90-90% A), 2.1-2.2 min (90-10 % A), 2.2-3.0 min (10-10% A), mobile phase A was 0.1% formic acid, B was acetonitrile, and the total analysis time was 3 min. The mass spectrometry was performed in positive ion mode, and the scanning mode was Multi-reaction monitoring (MRM) mode with ESI as the ion source. m/z 472.2→455.01 for zanubrutinib and m/z 441.03→137.99 for ibrutinib (IS). The plasma samples were processed by protein precipitation.
    RESULTS: The standard curve showed good linearity (r2 =0.999 8) in the range of 1.0-1000 ng/mL (zanubrutinib) with a low limit of quantification of 1 ng/mL. Also, the intra-day and inter-day precision (RSD %) was less than 5.88% and the accuracy (RE%) ranged from -1.56% to 1.08%, the recoveries of zanubrutinib in beagle plasma ranged from 90.12% to 93.53% (RSD: 1.67%~6.42%) and the ME values of zanubrutinib were 98.70%~101.06% (RSD=5.37%~8.49%, n=6). All values meet FDA requirements.
    CONCLUSION: A rapid, highly selective and sensitive method for the determination of zanubrutinib concentration in plasma by UPLC-MS/MS was successfully developed. This method is suitable for pharmacokinetic studies in beagle dogs by following oral administration of zanubrutinib.
    Keywords:  Beagle dogs; Pharmacokinetics; UPLC-MS/MS; Zanubrutinib
  16. Rapid Commun Mass Spectrom. 2022 Sep 13. e9397
      RATIONALE: The development of appropriate analytical screening techniques for pharmaceuticals and personal care products (PPCPs) is the basis for studying the distribution and environmental impact of emerging contaminants (ECs). Mass spectrometry-based screening methods vary with the complexity of the target compounds. It is challenging to compromise both positive and negative ion quantification together with a low detection limit. To establish a set of experimental methods including extraction, chromatography-separation and mass spectrometry screening is one of the most important topics on PPCPs research. This paper describes a universal and efficient qualification and quantification protocol for the simultaneous detection of thirty-four PPCPs in different environmental samples in a single analytical data acquisition run.METHODS: Thirty-four representative PPCPs, which are widely distributed in the environment with high ecological toxicity and complex chemical structures, were selected as representative targets ECs. The extraction of the target PPCPs was achieved only using one SPE cartridge without the need to adjust the pH of samples. The enriched samples were detected by LC-MS/MS in both positive and negative ion modes simultaneously. The protocol was evaluated based on the accuracy, precision, detection limits and matrix effects.
    RESULTS: This method achieved simultaneous detection of PPCPs in both positive and negative ion modes, with a single analytical cycle of 12 minutes. The observed SPE recoveries were between 40% and 115%. The instrumental detection limits (IDL) varied from 0.01 to 1 picogram; method detection limits (MDL) were between 0.002 and 3.323 ng/L in different matrices. Most of the PPCPs were subjected to matrix suppression below 30%. The method was successfully applied for quantitative analysis of the PPCPs in different environmental samples, including river samples, wastewater treatment plant (WWTP) sample and soil samples.
    CONCLUSIONS: This protocol developed a rapid and efficient detection method to simultaneous qualitative and quantification thirty-four representative PPCPs in the environment. The instrument detection limit (IDL) ranged from 0.01 to 1 picogram and the method detection limit (MDL) ranged from 0.002 to 3.323 ng/L in different matrices. The detection limit was one order of magnitude lower compared to previous studies. The protocol also provided a wide application range to different environmental matrices, which permitted exploring the migration and transformation of PPCPs.
  17. J Chem Inf Model. 2022 Sep 15.
      Here, we provide an algorithm that introduces excited states into the molecular dynamics prediction of the 70 eV electron ionization mass spectra. To decide the contributions of different electronic states, the ionization cross section associated with relevant molecular orbitals was calculated by the binary-encounter-Bethe (BEB) model. We used a fast orthogonalization model/single and double state configuration interaction (OM2/CISD) method to implement excited states calculations and combined this with the GFN1-xTB semiempirical model. Demonstrated by predicting the mass spectrum of urocanic acid, we showed better accuracies to experimental spectra using excited-state molecular dynamics than calculations that only used the ground-state occupation. For several histidine pathway intermediates, we found that excited-state corrections yielded an average of 73% more true positive ions compared to the OM2 method when matching to experimental spectra and 16% more true positive ions compared to the GFN method. Importantly, the exited state models also correctly predict several fragmentation reactions that were missing from both ground-state methods. Overall, for 48 calculated molecules, we found the best average mass spectral similarity scores for the mixed excited-state method compared to the ground-state methods using either cosine, weighted dot score, or entropy similarity calculations. Therefore, we recommend adding excited-state calculations for predicting the electron ionization mass spectra of small molecules in metabolomics.
  18. Food Chem. 2022 Sep 08. pii: S0308-8146(22)02113-6. [Epub ahead of print]401 134151
      In this study, a two-step extraction strategy (TSES) and targeted metabolomics combined with chemometrics was successfully applied for profiling of phenolic compounds in different colored rapeseeds. To this end, organic solvent extraction followed by deep eutectic solvent extraction made up the TSES with improved extraction coverage of free phenolics and enhanced extraction yield of conjugated phenolics, which combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) for further profiling of phenolics. TSES-LC-MS/MS method was established with determination coefficients for phenolic compounds greater than 0.9989. Finally, the relationship between color differences and phenolic compounds in rapeseeds was investigated upon TSES-LC-MS/MS method combined with chemometrics. Syringin, kaempferol, isorhamnetin, and sinapic acid were found to be the differential phenolics for the six different colored rapeseeds and their spatial distribution in rapeseeds were presented. Consequently, our method showed great potential for future studies based on comprehensive extraction and profiling of phenolics from complex matrices.
    Keywords:  Deep eutectic solvents; Phenolic compounds; Rapeseed; Targeted metabolomics; Two-step extraction strategy
  19. Anal Chim Acta. 2022 Sep 22. pii: S0003-2670(22)00869-8. [Epub ahead of print]1227 340298
      The separation of chiral amino acids (AAs) and their derivatives has always been a research difficulty in the field of biochemistry due to the high similarity of enantiomeric structures. In this work, a simple and quick method using natamycin (Nat) as chiral selector has been developed to simultaneously separate chiral AAs and their derivatives of carbobenzoxy/benzyl-AAs (Cbz/Bzl-AAs) by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). Specifically, 12 groups of the Cbz-AAs and Bzl-AAs can get baseline mobility separation by simple mixing with Nat to form binary diastereomeric complex ions [Nat+(Cbz-D/L-AA)+H]+ and [Nat+(Bzl-D/L-AA)+H]+. While for the remained 5 groups of Bzl-D/L-AAs and 16 groups of D/L-AAs with unsatisfying separation, by further adding P-toluenesulfonic acid (PTS), the formed ternary complexes can allow their baseline chiral separation. Specifically, Bzl-D-AAs and Bzl-L-AAs get much improved separation effect by the formed diastereomeric complexes of [Nat+(Bzl-AA)2+PTS2+H]+, which the Rp-p was improved from 0 to 2.40; while the D/L-AAs can get baseline separation by the formed diastereomeric complexes of [Nat + AA + PTS + H]+, [Nat + AA+(PTS)2+H]+, and [Nat+(AA)2+(PTS)2+H]+, with the Rp-p ranged from 0.44 to 3.53. Definitely, PTS is the first time reported as the ligand to improve the separation effect for the enantiomers, and with the higher assembly of chiral analyte, Nat, and PTS might enable better chiral separation for the chiral amino acid and their derivatives. Moreover, method validation of relative quantification and accuracy for the D/L-AA and their derivatives were measured in a mixture, yielding R2 greater than 0.99 and RSD% ≤ 2.68%. Overall, Nat and PTS as chiral selector and ligand can be widely used for chiral AAs and their derivatives mobility separation, and potentially for the separation of other AA-related chiral molecules.
    Keywords:  Amino acid-derivatives; Amino acids; Chiral separation; Natamycin; P-toluenesulfonic acid; Trapped ion mobility spectrometry
  20. J Chromatogr A. 2022 Sep 06. pii: S0021-9673(22)00671-9. [Epub ahead of print]1681 463479
      Solid-phase microextraction (SPME) directly coupled to MS is a widespread technique for determining small molecules in different matrices in many application fields. Here we present a modified microfluidic open interface (MOI) connected to a passive-flow-splitter device (PFS) for the direct coupling of SPME to a liquid-electron ionization (LEI) interface in a tandem mass spectrometer for the analysis of complex biological samples. No chromatographic separation is involved. The new MOI-PFS configuration was designed to speed up the sample transfer to MS, improving the signal-to-noise ratio and peak shape and leading to fast and sensitive results. MOI-PFS-LEI-MS/MS experiments were conducted using fentanyl as a model compound in water and blood serum. The method uses a C18 Bio-SPME fiber by direct immersion (3 min) in 300 µL of the sample followed by rapid desorption (1 min) in a flow isolated volume (MOI chamber, 2.5 µL) filled with 100% acetonitrile. The PFS permits the rapid transfer of a fraction of the sample into the MS via the LEI interface. The optimal conditions were obtained at a flow rate of 10 µL·min-1 and a 1:20 split ratio. Altogether, extraction, desorption, and analysis require approximately 5 min. Good interday and intraday precision, excellent linearity and LOQs in the µg·L-1 range were obtained for fentanyl in water and serum. Greenness evaluation demonstrated a limited environmental impact of this technique.
    Keywords:  Direct mass spectrometry; Liquid-EI interface – LEI; Microfluidic open interface – MOI; Passive Flow Splitter (PFS); SPME
  21. Anal Methods. 2022 Sep 14.
      Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and low-solvent-consumption technique. However, almost every mass in the low mass-to-charge-ratio region of the mass spectrum appears as strongly fluctuating matrix background signals. Thus, it is difficult to identify small molecules using this technique. In this study, we used methanol to methylate valsartan, an angiotensin II receptor blocker that is commonly used to treat high blood pressure and heart failure. The methylation derivatization of valsartan enhanced the detection sensitivity and transformed the detection m/z ratio. The liquid-phase microextraction of valsartan in human plasma (20 μL) was achieved by acidifying valsartan with HCl aqueous solution and extracting it with toluene. An acetyl chloride/anhydrous methanol mixture was added for methylation derivatization, which was completed within 30 min at 30 °C. Finally, the residue was re-dissolved in irbesartan methanolic solution, which together with the matrix 2-mercaptobenzothiazole was spotted on an AnchorChip target plate for MALDI-TOF MS analysis. Liquid-phase microextraction was performed and the methylation-derivatization parameters were investigated. The valsartan calibration range was 0.2-10 μg mL-1 with good linearity in human plasma. In the within- and between-run analyses, the relative standard deviation and relative error were both <11.32%. This method was successfully applied to determine the valsartan concentration in the plasma of 10 patients with hypertension.
  22. Rapid Commun Mass Spectrom. 2022 Sep 13. e9398
      Gas chromatography-mass spectrometry (GC-MS) is an analytical technique widely used in materials science, biomedicine, and other fields. The target compound in the experiment is identified by searching for its mass spectrum in a large mass spectrum database using some algorithms. This work introduces the use of deep learning ranking for small molecules identification using low-resolution electron ionization mass spectrometry.RATIONALE: Because different spectra are often very similar, the algorithm produces wrong search results, and the search accuracy needs improvement. Due to the library's large amount of data, the algorithm sometimes requires a large amount of calculation and is very time-consuming.
    METHODS: Given these two problems, this work aims to develop a model for ranking based on mass-to-charge ratio (m/z) pre-retrieval method combined with deep learning to improve search accuracy and reduce the algorithm's computational time. The master spectral library maintained by the National Institute of Standards and Technology is used as the reference library for all the experiments, and the replicate library is used as the query library to evaluate the method's performance.
    RESULTS: Compared with non-machine learning algorithms, the combination of m/z matching pre-retrieval and deep learning significantly improves library retrieval accuracy by about 4%. Moreover, compared with the deep learning sorting algorithm that does not use the pre-retrieval process, it also improves the accuracy of spectral library retrieval by about 0.1% and reduces the computational time of the algorithm by more than 2 hours.
    CONCLUSIONS: This method identifies compounds more efficiently and accurately than non-machine learning and deep learning algorithms without a pre-retrieval process.
  23. J Pharm Biomed Anal. 2022 Sep 08. pii: S0731-7085(22)00455-1. [Epub ahead of print]221 115034
      The goal of this study was to develop a method for the simultaneous quantification of 23 commonly used antineoplastic drugs in a hospital pharmacy, using ultra-high pressure liquid chromatography separation coupled to tandem mass spectrometry detection (UHPLC-MS/MS). The following drugs were investigated: 5-fluorouracil, cytarabine, ganciclovir, gemcitabine, dacarbazine, methotrexate, pemetrexed, busulfan, topotecan, rentitrexed, ifosfamide, cyclophosphamide, etoposide, irinotecan, doxorubicin/epirubicin, vincristine, docetaxel, paclitaxel, daunorubicin, idarubicin, vinblastine, oxaliplatin and carboplatin. The chromatographic separation was performed on a phenyl-hexyl column (2.1 ×100 mm, 1.7 µm) with a gradient elution of methanol and water containing 10 mM ammonium formate adjusted to pH 4.9. All compounds were analyzed in less than 13 min and detected with a triple quadrupole mass spectrometer operating in MRM mode. Limits of detection (LODs) and limits of quantification (LOQs) were comprised between 0.01 and 5 ng.mL-1, and between 0.5 and 5 ng.mL-1, respectively. Accuracies ranged between 117% and 83% at the LOQ, intermediate and upper LOQ concentrations, with relative standard deviations (RSD) inferior to 8%, for all the antineoplastic drugs. Finally, the UHPLC-MS/MS method was successfully applied to the analysis of surface samples to evaluate the chemical contamination by these highly toxic compounds in a chemotherapy preparation unit in a hospital pharmacy with the purpose of monitoring the exposure of health care professionals.
    Keywords:  Antineoplastic drug; Mass spectrometry; Reversed phase liquid chromatography; Surface analyses; Trace analysis
  24. Prog Nucl Magn Reson Spectrosc. 2022 Jun-Aug;130-131:pii: S0079-6565(22)00008-5. [Epub ahead of print]130-131 1-46
      2D NMR is extensively used in many different fields, and its potential for the study of complex biochemical or chemical mixtures has been widely demonstrated. 2D NMR gives the ability to resolve peaks that overlap in 1D spectra, while providing both structural and quantitative information. However, complex mixtures are often analysed in situations where the data acquisition time is a crucial limitation, due to an ongoing chemical reaction or a moving sample from a hyphenated technique, or to the high-throughput requirement associated with large sample collections. Among the great diversity of available fast 2D methods, ultrafast (or single-scan) 2D NMR is probably the most general and versatile approach for complex mixture analysis. Indeed, ultrafast NMR has undergone an impressive number of methodological developments that have helped turn it into an efficient analytical tool, and numerous applications to the analysis of mixtures have been reported. This review first summarizes the main concepts, features and practical limitations of ultrafast 2D NMR, as well as the methodological developments that improved its analytical potential. Then, a detailed description of the main applications of ultrafast 2D NMR to mixture analysis is given. The two major application fields of ultrafast 2D NMR are first covered, i.e., reaction/process monitoring and metabolomics. Then, the potential of ultrafast 2D NMR for the analysis of hyperpolarized mixtures is described, as well as recent developments in oriented media. This review focuses on high-resolution liquid-state 2D experiments (including benchtop NMR) that include at least one spectroscopic dimension (i.e., 2D spectroscopy and DOSY) but does not cover in depth applications without spectral resolution and/or in inhomogeneous fields.
    Keywords:  Complex mixtures; Metabolomics; Monitoring; NMR spectroscopy; Single-scan; Ultrafast
  25. J Pharm Biomed Anal. 2022 Aug 22. pii: S0731-7085(22)00424-1. [Epub ahead of print]221 115003
      The probable carcinogenic nitrosamine impurities, such as N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA), have been detected from various pharmaceuticals in recent years. The sensitive chromatographic methods, including liquid chromatography (LC) and gas chromatography (GC), have been applied for analyzing nitrosamines in the pharmaceutical substrates, such as sartans, ranitidine and metformin. In comparison of LC, the efficacy of GC for analyzing multiple nitrosamines in diverse pharmaceuticals will be limited or attenuated owing to the chemical properties of target analytes or matrix hinderance of pharmaceutical substrates. To extend the applicability of GC analysis for multiple nitrosamines in pharmaceuticals, this study presented a gas chromatograph tandem mass (GC-MS/MS) method for monitoring 14 nitrosamines within 44 pharmaceuticals, whereas the headspace-solid phase microextraction (HS-SPME) sampling mode was introduced. Chromatographic separation was achieved on a DB-heavyWax column (30 m × 0.25 mm; i.d., 0.25 µm), whereas the HS-SPME sampling mode with a 50/30 µm DVB/CAR/PDMS extracting fiber was applied for comparison of the direct injection mode. Meanwhile, the HS-SPME conditions were optimized to evaluate the effects of the parameters on analyzing total nitrosamines in pharmaceuticals by GC-MS/MS. The optimal conditions of HS-SPME were as follows: extracting solution of 90% NaCl, HS incubation time 1 min, SPME adsorbing at 80 ℃ for 30 min, and desorbing at 250 ℃ for 5 min. The limit of quantification (LOQ) for 14 nitrosamines in pharmaceutical matrices under the optimal conditions was 0.05 μg/g for the optimal HS-SPME, whereas the value was 0.05-0.25 μg/g for direct injection.
    Keywords:  Carcinogens; Gas chromatography; Headspace-solid phase microextraction; Mass spectrometry; Nitrosamines; Optimization
  26. Talanta. 2022 Aug 27. pii: S0039-9140(22)00625-7. [Epub ahead of print]253 123829
      Thermal Extraction-Desorption (TED) using a thermobalance coupled to a gas chromatograph (GC) with mass spectrometer (MS) detector is an extended method for polymers identification in complex matrixes. A new TED-GC/MS method for microplastics identification is developed in this study, where the whole filter with solids collected from water is thermal treated in a furnace, instead of using a small portion in a Thermogravimetric analysis (TGA) device, avoiding sample handling. Pyrolyzing the whole filter in a tubular furnace has advantages with respect to the standard procedure of using a TGA with a small crucible in TED-GC/MS. The main advantage is the easy manipulation of the sample, since the filter does not have to be manipulated to extract the sample or cut some portions, avoiding sample losses during handling and ensuring that inhomogeneity on the filter surface is not a problem. Furthermore, there are no limitations on the weight of the sample beyond the adsorbent's ability to trap decomposition compounds without becoming saturated, so high intensity signals can be obtained in order to avoid confuse signals with noise, false negatives or values so close of the quantification limit.
    Keywords:  Filter; Microplastic; Polymer; TED-GC/MS; Thermodecomposition
  27. STAR Protoc. 2022 Sep 15. pii: S2666-1667(22)00559-7. [Epub ahead of print]3(4): 101679
      This protocol outlines a translational lipidomic approach to discover lipid biomarkers that could predict morphometric body and histological organ measurements (e.g., weight and adiposity gains) during specific stages of life (e.g., early life). We describe procedures ranging from animal experimentation and histological analyses to downstream analytical steps through lipid profiling, both in mice and humans. This protocol represents a reliable and versatile approach to translate and validate candidate lipid biomarkers from animal models to a human cohort. For complete details on the use and execution of this protocol, please refer to Olga et al. (2021).
    Keywords:  Clinical protocol; Health sciences; Mass spectrometry; Metabolism; Metabolomics; Systems biology
  28. J Agric Food Chem. 2022 Sep 11.
      A wide range of secondary metabolites has been described for various Hydrangea species, including the sweet-tasting phenyldihydroisocoumarin phyllodulcin, which is found in the leaves of Hydrangea macrophylla ssp. serrata. This work aims at the development and validation of an analytical workflow for comprehensive semi-polar metabolite profiling using liquid chromatography coupled with electrospray ionization ion mobility quadrupole time-of-flight mass spectrometry (UPLC-ESI-IMS-QToF-MS) to complement existing analytical studies. The unsupervised analysis of this data set demonstrates the capability of this analytical workflow to distinguish different H. macrophylla ssp. serrata cultivars. In combination with supervised analysis, a list of metabolites responsible for the differentiation of the cultivars studied has been obtained. Suspect screening of phenyldihydroisocoumarins provides comprehensive information, which could help in the search for key enzymes related to the biosynthesis of phyllodulcin.
    Keywords:  Hydrangeaceae; dihydroisocoumarins; ion-mobility high resolution mass spectrometry; liquid chromatography; plants
  29. J Pharm Biomed Anal. 2022 Sep 01. pii: S0731-7085(22)00448-4. [Epub ahead of print]221 115027
      Establishing dihydropyrimidine dehydrogenase (DPD) activity is highly important in determining the correct starting dose of fluoropyrimidines such as 5-fluorouracil and capecitabine. The concentration ratio of endogenous uracil with its metabolite dihydrouracil (DHU) is a well-known parameter that is linked to DPD activity. Concentration ratios such as thymine over its DPD-converted metabolite dihydrothymine (DHT) is less described and may serve as an alternative diagnostic biomarker for DPD activity. In this study, we describe the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for the quantification of uracil, DHU, thymine, and DHT in human plasma. In addition, stability experiments were performed. Uracil and thymine were quantified up to 80.0 ng/mL and DHU and DHT up to 800 ng/mL. Intra- and inter-assay precision were maximum 8.0 % and 7.6 %. respectively. Also, recovery was adequate and significant matrix-effects and carry-over were excluded. Stability experiments showed that uracil concentrations increased with 27-52 % when stored for 1 or 2 h at ambient temperatures compared to cold storage. Thymine, DHU, and DHT concentrations remained stable, thymine after 1 h in plasma excluded, showing the DHT:T ratio might be a more robust marker for DPD activity than DHU:U. In conclusion, we present here a novel assay capable of quantifying uracil, thymine, DHU and DHT in a single analytical run. We provide additional data showing increased stability for DHU, thymine and DHT compared to uracil. This assay may be used as a diagnostic test in future studies, establishing the association of these endogenous biomarker concentrations with DPD activity and safety to treatment with fluoropyrimidines. In addition, future research should also be focused on reducing pre-analytical instability. Standardization in this field is essential to set proper reference values and to allow inter-study comparison on clinical outcomes.
    Keywords:  Biomarker; Dihydropyrimidine dehydrogenase; Fluorouracil; Mass spectrometry; Thymine; Uracil