bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒12‒25
29 papers selected by
Sofia Costa

  1. Metabolites. 2022 Nov 25. pii: 1179. [Epub ahead of print]12(12):
      Glycation products produced by the non-enzymatic reaction between reducing carbohydrates and amino compounds have received increasing attention in both food- and health-related research. Although liquid chromatography mass spectrometry (LC-MS) methods for analyzing glycation products already exist, only a few common advanced glycation end products (AGEs) are usually covered by quantitative methods. Untargeted methods for comprehensively analyzing glycation products are still lacking. The aim of this study was to establish a method for simultaneously characterizing a wide range of free glycation products using the untargeted metabolomics approach. In this study, Maillard model systems consisting of a multitude of heterogeneous free glycation products were chosen for systematic method optimization, rather than using a limited number of standard compounds. Three types of hydrophilic interaction liquid chromatography (HILIC) columns (zwitterionic, bare silica, and amide) were tested due to their good retention for polar compounds. The zwitterionic columns showed better performance than the other two types of columns in terms of the detected feature numbers and detected free glycation products. Two zwitterionic columns were selected for further mobile phase optimization. For both columns, the neutral mobile phase provided better peak separation, whereas the acidic condition provided a higher quality of chromatographic peak shapes. The ZIC-cHILIC column operating under acidic conditions offered the best potential to discover glycation products in terms of providing good peak shapes and maintaining comparable compound coverage. Finally, the optimized HILIC-MS method can detect 70% of free glycation product features despite interference from the complex endogenous metabolites from biological matrices, which showed great application potential for glycation research and can help discover new biologically important glycation products.
    Keywords:  HILIC-MS; Maillard reaction products; advanced glycation end products; non-enzymatic glycation; untargeted analysis
  2. Metabolites. 2022 Nov 24. pii: 1168. [Epub ahead of print]12(12):
      As a comprehensive analysis of all metabolites in a biological system, metabolomics is being widely applied in various clinical/health areas for disease prediction, diagnosis, and prognosis. However, challenges remain in dealing with the metabolomic complexity, massive data, metabolite identification, intra- and inter-individual variation, and reproducibility, which largely limit its widespread implementation. This study provided a comprehensive workflow for clinical metabolomics, including sample collection and preparation, mass spectrometry (MS) data acquisition, and data processing and analysis. Sample collection from multiple clinical sites was strictly carried out with standardized operation procedures (SOP). During data acquisition, three types of quality control (QC) samples were set for respective MS platforms (GC-MS, LC-MS polar, and LC-MS lipid) to assess the MS performance, facilitate metabolite identification, and eliminate contamination. Compounds annotation and identification were implemented with commercial software and in-house-developed PAppLineTM and UlibMS library. The batch effects were removed using a deep learning model method (NormAE). Potential biomarkers identification was performed with tree-based modeling algorithms including random forest, AdaBoost, and XGBoost. The modeling performance was evaluated using the F1 score based on a 10-times repeated trial for each. Finally, a sub-cohort case study validated the reliability of the entire workflow.
    Keywords:  GC-MS; LC-MS; clinical cohort; data modeling; data normalization; metabolomics; quality control
  3. Anal Chem. 2022 Dec 20. 94(50): 17370-17378
      The success of precision medicine relies upon collecting data from many individuals at the population level. Although advancing technologies have made such large-scale studies increasingly feasible in some disciplines such as genomics, the standard workflows currently implemented in untargeted metabolomics were developed for small sample numbers and are limited by the processing of liquid chromatography/mass spectrometry data. Here we present an untargeted metabolomics workflow that is designed to support large-scale projects with thousands of biospecimens. Our strategy is to first evaluate a reference sample created by pooling aliquots of biospecimens from the cohort. The reference sample captures the chemical complexity of the biological matrix in a small number of analytical runs, which can subsequently be processed with conventional software such as XCMS. Although this generates thousands of so-called features, most do not correspond to unique compounds from the samples and can be filtered with established informatics tools. The features remaining represent a comprehensive set of biologically relevant reference chemicals that can then be extracted from the entire cohort's raw data on the basis of m/z values and retention times by using Skyline. To demonstrate applicability to large cohorts, we evaluated >2000 human plasma samples with our workflow. We focused our analysis on 360 identified compounds, but we also profiled >3000 unknowns from the plasma samples. As part of our workflow, we tested 14 different computational approaches for batch correction and found that a random forest-based approach outperformed the others. The corrected data revealed distinct profiles that were associated with the geographic location of participants.
  4. Data Brief. 2023 Feb;46 108802
      Circulating polyunsaturated fatty acids (PUFAs) and lipid mediators were extracted from human red blood cells and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method encompassed 13 different PUFAs and lipid mediators, however, due to instrument capability only five were confidently quantified (EPA, ALA, AA, DHA, and LA). The extraction focused on free polyunsaturated fatty acids since they have a strong correlation with health in humans. The study design was a secondary analysis of the OPPERA-2 study of chronic overlapping pain conditions in adults. The data included are: a) raw LC-MS/MS data (.raw); b) processed data (.xlsx) including chromatographic peak area for each compound and a concentration (ng/mL) based on external calibration with internal standardization using pure analytical grade standards and heavy-isotope labeled internal standards; c) study participant demographics and phenotypes (.xlsx). This dataset consisting of circulating PUFA quantities measured in 605 humans has been made publicly available for analysis and interpretation.
    Keywords:  Mass spectrometry; Pain; Polyunsaturated fatty acids; Quantitative analysis
  5. Clin Lab. 2022 Dec 01. 68(12):
      BACKGROUND: This study aimed to develop and validate a U-HPLC-MS/MS method for simultaneous determination of four immunosuppressants in human whole blood.METHODS: The method was based on the injection of 20 µL of calibrators and controls pretreated with the liquid phase extraction method for chromatography separation and mass spectrometry determination. LPE offline was performed by adding 0.1 mol/L ZnSO4 and acetonitrile, while separation of target compounds was achieved within 2.5 minutes by a Zorbax Eclipse XDB-C8 column using ammonium acetate and ACN mixed with formic acid as solvents.
    RESULTS: The assay offers ng/mL detection limits (from 1.1 to 12.4 ng/mL), accuracy (% deviation from -4.4% to 5.6%), precision (CV less than 15% at all QC levels), and linearity (from 23.4 to 948 ng/mL for CsA, from 2.11 to 45.5 ng/mL for TAC, SIR and EVR). The recovery and matrix results were acceptable, and the carryover was less than 1%. The results of method comparison show that IA-based methods overestimated the concentration of drugs compared with the MS-based method. Comparing our MS-based method with external LC-MS/MS showed that the results were within 2 SDs.
    CONCLUSIONS: We have developed a reliable assay for the analysis of CsA, TAC, SIR and EVR in whole blood using U-HPLC-MS/MS.
  6. Nat Methods. 2022 Dec 21.
      Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.
  7. Metabolites. 2022 Nov 30. pii: 1198. [Epub ahead of print]12(12):
      Eicosanoids are lipid mediators generated from arachidonic acid with pro- and anti-inflammatory properties. Despite these lipid mediators being known for decades, quantitative determination in biological samples is still challenging due to low abundance, instability, the existence of regio- and stereoisomers, and a wide polarity range that hampers chromatographic separation. In this study, we developed a supercritical fluid chromatography mass spectrometry (SFC-MS) platform for the quantification of relevant eicosanoids. Application of a chiral amylose-based column and modifier combination of 2-propanol/acetonitrile offered separation and sufficient resolution of 11 eicosanoids (5-, 12-, 15-HETE, PGB1, LTB4, t-LTB4, 20-OH-LTB4, PGE2, PGD2, PGF2α, TxB2) with baseline separation of isobaric analytes within 12 min. The method was validated in terms of range (78-2500 ng/mL), linearity, accuracy, precision, and recovery according to EMA guidelines. Finally, we confirmed the method's applicability by quantifying eicosanoid levels in human primary blood cells. In conclusion, we present a validated SFC-MS method for the determination of relevant eicosanoids in biological samples with a wide range of polarity while maintaining baseline separation of isobars, which allows coupling to a single quadrupole mass detector.
    Keywords:  lipid mediators; monocytes; neutrophils; oxylipins; platelets; supercritical fluid chromatography; validation
  8. Prostaglandins Other Lipid Mediat. 2022 Dec 15. pii: S1098-8823(22)00091-0. [Epub ahead of print]165 106701
      Arachidonic acid (AA) is a polyunsaturated fatty acid with a structure of 20:4(ω-6). Cytochrome P450s (CYPs) metabolize AA to several regioisomers and enantiomers of hydroxyeicosatetraenoic acids (HETEs). The hydroxy-metabolites (HETEs) exist as enantiomers in the biological system. The chiral assays developed for HETEs are so far limited to a few assays reported for midchain HETEs. The developed method is capable of quantitative analysis for midchain, subterminal HETE enantiomers, and terminal HETEs in microsomes. The peak area or height ratios were linear over concentrations ranging (0.01 -0.6 µg/ml) with r2 > 0.99. The intra-run percent error and coefficient of variation (CV) were ≤ ± 12 %. The inter-run percent error and coefficient of variation (CV)were ≤ ± 13 %, and ≤ 15 %, respectively. The matrix effect for the assay was also within the acceptable limit (≤ ± 15 %). The recovery of HETE metabolites ranged from 70 % to 115 %. The method showed a reliable and robust performance for chiral analysis of cytochrome P450-mediated HETE metabolites.
    Keywords:  Arachidonic acid; Assay; Enantiomer; Hydroxyeicosatetraenoic acid; LC-MS/MS; Microsome matrix
  9. Rapid Commun Mass Spectrom. 2022 Dec 24. e9463
      RATIONALE: Homocysteine (hcy) is a metabolite in the human body and an important determinant of cardiovascular health. To assist in the assessment of human cardiovascular safety, a rapid and accurate quantitative analysis method must be developed. Laser desorption/ionization mass spectrometry (LDI-MS) has been widely used in biological, chemical and medical analysis due to its excellent characteristics of high throughput, low sample consumption and high speed. Herein, a LDI-MS method based on covalent organic framework (COF) film was developed for rapid and sensitive determination of hcy in human serum using an isotope-labeled internal standard.METHODS: We tried to cultivate COFs on indium-tin oxide (ITO) substrate at room temperature to form thin film, which was used in LDI-MS. Compared with the traditional organic matrices, the COF film showed clean background and high signal response for the detection of hcy. In addition, using COF film as the substrate that can analyze a series of small molecules such as amino acids, bisphenols (Bps), estrogens, drugs, etc., with high signal intensity and clean background. We evaluated the limit of detection (LOD) and the reproducibility of this method. Finally, the calibration curve was made for the quantification of serum hcy using isotope labeled internal standard, and was applied to the rapid determination of hcy in clinical human serum.
    RESULTS: COF film-assisted LDI-MS had higher response signal and cleaner background compared to four conventional organic matrices. The LOD of the method for hcy was 0.5 μmol/L, equivalent to 500 fmol. This method exhibited excellent performance for small molecule compounds including amino acids, Bps, drugs, and estrogens. The reproducibilities of this method for shot-to-shot and dot-to-dot were proved to be good. This method was applied to the rapid analysis of hcy in clinical human serum, showing good correlation with those obtained by hospital testing.
    CONCLUSION: The COF film-based LDI-MS method showed simple sample preparation, short analysis time, clean background and good reproducibility for hcy analysis. As an important indicator of human health, the detection of serum hcy is of great significance to human health.
  10. Int J Mol Sci. 2022 Dec 07. pii: 15474. [Epub ahead of print]23(24):
      Oligonucleotides (OGNs) are relatively new modalities that offer unique opportunities to expand the therapeutic targets. Reliable and high-throughput bioanalytical methods are pivotal for preclinical and clinical investigations of therapeutic OGNs. Liquid chromatography-mass spectrometry (LC-MS) is now evolving into being the method of choice for the bioanalysis of OGNs. Ion paring reversed-phase liquid chromatography (IP-RPLC) has been widely used in sample preparation and LC-MS analysis of OGNs; however, there are technical issues associated with these methods. IP-free methods, such as hydrophilic interaction liquid chromatography (HILIC) and anion-exchange techniques, have emerged as promising approaches for the bioanalysis of OGNs. In this review, the state-of-the-art IP-RPLC-MS bioanalytical methods of OGNs and their metabolites published in the past 10 years (2012-2022) are critically reviewed. Recent advances in IP-reagent-free LC-MS bioanalysis methods are discussed. Finally, we describe future opportunities for developing new methods that can be used for the comprehensive bioanalysis of OGNs.
    Keywords:  ASO; HILIC; LC–MS; RNA drugs; bioanalysis; ion-paring; oligonucleotide; siRNA
  11. Proteomics Clin Appl. 2022 Dec 19. e2200056
      PURPOSE: Apolipoprotein monitoring is useful for diagnosing cardiovascular diseases, as they are risk factors of arteriosclerosis and other neutral fat-related diseases. LC-MS/MS is advantageous for simultaneous apolipoprotein quantification, differentiation, and standardization including their isoforms. However, fast and straightforward sample preparation that retains quantification accuracy remains challenging in clinical MS.EXPERIMENTAL DESIGN: We developed a simultaneous assay for serum apolipoprotein A-I, apolipoprotein B100 family, and apolipoprotein C-III using a high-throughput LC-MS/MS platform coupled with a BRAVO system. The assay was simplified by using sodium deoxycholate and trypsin/lys-C without reduction and alkylation steps.
    RESULTS: Simple sample preparation reduced turnaround time by 1.5 h and neat goat serum was chosen as an optimal calibration matrix for accurate protein quantification. Assay precision, linearity, correlation, accuracy, LOD, LLOQ, and carryover were validated according to CLSI guidelines over 41 days using more than 100 human serum samples. Good correlation compared with TIA was observed by Deming regression for all analytes.
    CONCLUSIONS AND CLINICAL RELEVANCE: A high-throughput LC-MS/MS and BRAVO assay for simultaneous apolipoprotein analysis was validated using a simple preparation method with a human serum calibrator in goat serum matrix. The assay is readily expandable to include other target serum proteins and/or their isoforms. This article is protected by copyright. All rights reserved.
    Keywords:  CLSI guidelines; apolipoproteins; high-throughput clinical mass spectrometry; quantification; validation
  12. J Mass Spectrom Adv Clin Lab. 2022 Nov;26 48-59
      Background: Optimizing antimicrobial therapy to attain drug exposure that limits the emergence of resistance, effectively treats the infection, and reduces the risk of side effects is of a particular importance in critically ill patients, in whom normal functions are augmented or/and are infected with pathogens less sensitive to treatment. Achievement of these goals can be enhanced by therapeutic drug monitoring (TDM) for many antibiotics. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is presented here for simultaneous quantification of ten antimicrobials: cefazolin (CZO), cefepime (CEP), cefotaxime (CTA), ceftazidime (CTZ), ciprofloxacin (CIP), flucloxacillin (FLU), linezolid (LIN), meropenem (MER), piperacillin (PIP) and tazobactam (TAZ) in human plasma.Methods: Plasma samples were precipitated with acetonitrile and injected into the LC-MS/MS. Chromatographic separation was on a Waters Acquity BEH C18 column. Compounds were eluted with water and acetonitrile containing 0.1 % formic acid, using a gradient (0.5-65 % B), in 3.8 min. The flow rate was 0.4 mL/min, and the run time was 5.8 min.
    Results: The calibration curves were linear across the tested concentration ranges (0.5-250, CZO, CEP, CTA, CTZ and FLU; 0.2-100, MER and TAZ; 0.1-50, CIP and LIN and 1-500 mg/L, PIP). The intra and inter-day imprecision was < 11 %. Accuracy ranged from 95 to 114 %. CTZ and MER showed ionization suppression while CIP showed ionization enhancement, which was normalized with the use of the internal standard.
    Conclusion: An LC-MS/MS method for simultaneous quantification of ten antimicrobials in human plasma was developed for routine TDM.
    Keywords:  Antimicrobials; CEP, cefepime; CIP, ciprofloxacin; CTA, cefotaxime; CTZ, ceftazidime; CV, coefficient of variation; CZO, cefazolin; ESI, electrospray ionization; FLU, flucloxacillin; HPLC, high performance liquid chromatography; ICU, intensive care unit; LC–MS/MS; LC–MS/MS, liquid chromatography tandem mass spectrometry; LIN, linezolid; LLOQ, lower limit of quantification; MER, meropenem; MIC, minimum inhibitory concentration; MRM, multiple reaction monitoring; NOR, norfloxacin; PIP, piperacillin; PK, pharmacokinetic; QC, quality control; R, resistant organism; RT, room temperature; Routine therapeutic drug monitoring; Rt, retention time; S, susceptible, wild type organism; SIL-IS, stable isotope labelled internal standard; TAZ, tazobactam; TDM, therapeutic drug monitoring; ULOQ, upper limit of quantitation; r2, coefficient of determination; β-Lactams
  13. Chem Sci. 2022 Dec 07. 13(47): 14114-14123
      The importance of chiral amino acids (AAs) in living organisms has been widely recognized since the discovery of endogenous d-AAs as potential biomarkers in several metabolic disorders. Chiral analysis by ion mobility spectrometry-mass spectrometry (IMS-MS) has the advantages of high speed and sensitivity but is still in its infancy. Here, an N α-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (FDAA) derivatization is combined with trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) for chiral AA analysis. For the first time, we demonstrate the simultaneous separation of 19 pairs of chiral proteinogenic AAs in a single fixed condition TIMS-MS run. The utility of this approach is presented for mouse brain extracts by direct-infusion TIMS-MS. The robust separation ability in complex biological samples was proven in matrix-assisted laser desorption/ionization (MALDI) TIMS mass spectrometry imaging (MSI) as well by directly depositing 19 pairs of chiral AAs on a tissue slide following on-tissue derivatization. In addition, endogenous chiral amino acids were also detected and distinguished. The developed methods show compelling application prospects in biomarker discovery and biological research.
  14. Anal Sci. 2022 Dec 24.
      Recently, biodegradable aminopolycarboxylic acid chelating agents have attracted attention as an alternative to environmentally persistent chelating agents such as ethylenediamine-N,N,N',N'-tetraacetic acid. However, the detection of chelating agents requires complexation with metals or derivatization by esterification reagents, and their direct detection using the currently available analytical methods still represents a challenge. Herein, we describe a direct analytical method for the biodegradable chelating agents ethylenediamine-N,N'-disuccinic acid, 3-hydroxy-2,2'-iminodisuccinic acid, methylglycine-N,N'-diacetic acid, and N,N-bis(carboxymethyl)-L-glutamic acid, via ultra-performance liquid chromatography/electrospray ionization quadrupole/time-of-flight mass spectrometry. Satisfactory retention and separation with a good peak shape were successfully achieved using a metal-free hydrophilic interaction liquid chromatographic column. The calibration curves showed good linearity in the range of 1.0-50 μM with correlation coefficients greater than 0.9988. The detection limits ranged from 0.04 to 0.12 μM. Furthermore, the developed method could be applied to the quantitative analysis of the four chelating agents in biodegradation and photodegradation experiments at the laboratory level. The proposed method, which offers the advantages of quickness, sensitivity, and requiring no complicated pretreatment steps, is expected to contribute significantly to the practical analysis of chelating agents in environmental water samples.
    Keywords:  Biodegradable chelating agent; Biodegradation; Hydrophilic interaction liquid chromatography; Liquid chromatography/electrospray ionization mass spectrometry; Metal-free column; Photodegradation
  15. Wei Sheng Yan Jiu. 2022 Nov;51(6): 1002-1010
      OBJECTIVE: A method for simultaneous determination of 11 kinds of organophosphorus flame retardants in fish was established by using the EMR-Lipid cleaning agent and ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).METHODS: The samples were extracted by ultrasonic with 0.5% formic acid acetonitrile solution. The lipid removal product EMR-lipid was used for lipid purification. The co-extracts were further removed by magnesium sulfate, N-propyl ethylene amine(PSA) and graphitized carbon black(GCB) purification agent. The target compounds were separated on an ACQUITY UPLC® BEH C_(18) column(100 mm×2.1 mm, 1.7 μm), detected by electrospray ionization(ESI) and positive ion multiple reaction monitoring mode, the internal standard method and external standard method are quantitatively evaluated, and external standard method was adopted.
    RESULTS: The 11 kinds of organophosphorus flame retardants had a good linear relationship in the range of 0.5-50 μg/L(tris(2-ethylhexyl) phosphate 0.05-5 μg/L) with r&gt;0.999. The limits of detection were 0.004-1.029 μg/kg and the limits of quantitation were 0.012-3.094 μg/kg. The average recoveries at three spiked levels(low, medium and high) were 80.0%-111.2% with the relative standard deviations all less than 10%(n=6).
    CONCLUSION: The method could be used for the determination of trace organophosphorus flame retardants in freshwater fish with accurate and reliable result.
    Keywords:  EMR-Lipid; fish; organophosphorus flame retardants; ultra-high performance liquid chromatography-tandem mass spectrometry
  16. Talanta. 2022 Dec 09. pii: S0039-9140(22)00978-X. [Epub ahead of print]254 124182
      Isoprenoids give rise to many functional products used today such as flavours, fragrances and even pharmaceutical compounds. Mevalonate pathway metabolites are the key intermediates that affect the production yield of isoprenoids. With increasing demand and benefit of isoprenoids, the present study adopts Analytical Quality-by-Design (AQbD) approach to establish an efficacious extraction protocol prior to the determination of mevalonate pathway metabolites in an engineered Escherichia coli model. The statistical experimental design approach, described in this work, has successfully validated an optimised sample preparation method i.e., using acetonitrile: 50 mM ammonium formate (pH 9.5) (7:3) (ACN73) at -20 °C for 10 min without solvent evaporation to retain the targeted mevalonate metabolites in engineered E. coli strain. The study also demonstrates the use of liquid chromatography paired with a Time-of-Flight Mass Spectrometer (LC-ToF-MS) for the quantitative analysis of the mevalonate pathway metabolites in E. coli. The analytical method was validated in accordance with guidelines in Metabolomics Standards Initiative and ICH Q2 (R1) with analyte spike recoveries at 80% and above. In short, the present study overcomes the one-variable-at-a-time (OVAT) limitations in analytical development, minimises metabolite losses and gives better cost and time efficiencies by eliminating the solvent evaporation and swapping process. This work highlights the importance of analytical methods development in microbial metabolomics studies.
    Keywords:  Design of experiment (DoE); Engineered Escherichia coli; Extraction; Fast filtration sampling; LC-ToF-MS; Mevalonate pathway
  17. Life (Basel). 2022 Dec 15. pii: 2122. [Epub ahead of print]12(12):
      We present a systematic analysis of a large number of mass spectra accumulated as the number of ion fragments recorded in unit mass-to-charge detector channels. The method retrieves the abundances of detected species using an efficient deconvolution algorithm, which relies on fragment pattern recognition, mass calibration, and background correction. The abundance analysis identifies target species, amino acids, and mycotoxins through their characteristic fragmentation patterns in the presence of an increasing number of interfering species. The method offered robust and efficient retrieval of abundances of metabolic molecules in complex mixtures obscured by a wide range of toxic compounds.
    Keywords:  collision induced dissociation; deconvolution; electron impact ionization; mass spectrometry
  18. Drug Test Anal. 2022 Dec 23.
      Gamma-hydroxybutyric acid (GHB) represents an important drug in clinical and forensic toxicology, particularly in the context of drug-facilitated crimes. Analytically, GHB remains a major challenge given its endogenous occurrence and short detection window. Previous studies identified a number of potential interesting novel conjugates of GHB with carnitine, amino acids (AA, glutamate, glycine, and taurine) or fatty acids. As a basis for comprehensive studies on the suitability of these novel biomarkers, we developed and validated a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method in human urine. Additionally, already knowns markers 2,4-dihydroxy butyric acid (2,4-DHB), 3,4-DHB, glycolic acid, succinic acid, succinylcarnitine, and GHB glucuronide were included. The method was fully validated according to (inter)national guidelines. Synthetic urine proved suitable as a surrogate matrix for calibration. Matrix effects were observed for all analytes with suppression effects of about 50% at QC LOW, and approximately 20 to 40% at QC HIGH, but with consistent standard deviation of < 25% at QC LOW and <15% at QC HIGH, respectively. All analytes showed acceptable intra- and inter-day imprecision of below 20%, except for inter-day variation of GHB taurine and FA conjugates at the lowest QC. Preliminary applicability studies proved the usefulness of the method and pointed towards GHB glycine, followed by other AA conjugates as the most promising candidates to improve GHB detection. FA conjugates were not detected in urine samples yet. The method can be used now for comprehensive sample analysis on (controlled) GHB administration to prove the usefulness of the novel GHB biomarkers.
  19. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Dec 09. pii: S1570-0232(22)00473-1. [Epub ahead of print]1214 123568
      In this study, an automated online micro-solid-phase extraction (μSPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of metabolites of cannabidiol (CBD), Δ8-tetrahydrocannabinol (Δ8-THC), and Δ9-tetrahydrocannabinol (Δ9-THC), particularly 7-carboxy- cannabidiol (7-COOH-CBD), 11-nor-9-carboxy-Δ8-tetrahydrocannabinol (Δ8-THCCOOH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (Δ9-THCCOOH), and 11-nor-9-carboxy-Δ9- tetrahydrocannabinol-glucuronide (Δ9-THCCOOH-glu) in urine. An instrument top sample preparation (ITSP) cartridge was introduced to increase the sensitivity toward analytes and decrease the matrix effect of the urine. LC-MS/MS analysis was performed in the multiple-reaction monitoring mode, and the analytes were separated using an Acquity UPLC HSS T3 (2.1 × 100 mm, 1.8 µm) column and gradient elution with water containing 0.05 % acetic acid and methanol as the mobile phase. The calibration range was 0.5-200 ng/mL for all the analytes, with a correlation coefficient (r) of ≥0.996 and a weighting factor of 1/x2. The limits of detection for 7-COOH-CBD, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-THCCOOH-glu were 0.06, 0.02, 0.03, and 0.1 ng/mL, respectively. The intra- and inter-day accuracy ranged from -8.0 to 6.2 % and -7.3 to 7.8 % with a precision of ≤7.2 % and ≤6.2 %, respectively. The method was also validated for selectivity, recovery, matrix effect, stability, and dilution integrity. The developed method was successfully applied to the analysis of 78 urine samples, and 7-COOH-CBD, Δ8-THCCOOH, Δ9-THCCOOH, and Δ9-THCCOOH-glu were detected in 54 urine samples at normalized concentrations of 1.1, 0.6-939.1, 0.9-2595.0, and 1.3-527.6 ng/mg creatinine, respectively.
    Keywords:  11-nor-9-carboxy-Δ(8)-tetrahydrocannabinol; 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol; 7-carboxy-cannabidiol; LC–MS/MS; Online μSPE; Urine
  20. J Magn Reson Open. 2022 Dec;pii: 100082. [Epub ahead of print]12-13
      Human blood is the most widely used biospecimen in the clinic and the metabolomics field. While both mass spectrometry and NMR spectroscopy are the two premier analytical platforms in the metabolomics field, NMR exhibits several unsurpassed characteristics for blood metabolite analysis, the most important of which are its ability to identify unknown metabolites and its quantitative nature. However, the relatively small number of metabolites accessible by NMR has restricted the scope of its applications. Enhancing the limit of identified metabolites in blood will therefore greatly impact NMR-based metabolomics. Continuing our efforts to address this major issue, our current study describes the identification of 12 metabolites, which expands the number of quantifiable blood metabolites by ~15%. These results, in combination with our earlier efforts, now provide access to nearly 90 metabolites, which is the highest to date for a simple 1D 1H NMR experiment that is widely used in the metabolomics field. Metabolites were identified based on the comprehensive investigation of human blood and plasma using 1D/2D NMR techniques. The newly identified metabolites were validated based on chemical shift databases, spectra of authentic compounds obtained under conditions identical to blood/plasma, and, finally, spiking experiments using authentic compounds. Considering the high reproducibility of NMR and the sensitivity of chemical shifts to altered sample conditions, experimental protocols and peak annotations are provided for the newly identified metabolites, which serve as a template for identification of blood metabolites for routine applications. Separately, the identified metabolites were evaluated for their sensitivity to preanalytical conditions. The results reveal that among the newly identified metabolites, inosine monophosphate (IMP) and nicotinamide are associated with labile coenzymes and their levels are sensitive to preanalytical conditions. The study demonstrates the expansion of quantifiable blood metabolites using NMR to a new height and is expected to greatly impact blood metabolomics.
    Keywords:  1d nmr, 2d nmr; Blood metabolomics; Energy coenzymes; Labile metabolites; Redox coenzymes; Unknown metabolite identification
  21. Metabolites. 2022 Dec 16. pii: 1275. [Epub ahead of print]12(12):
      Recent developments in molecular networking have expanded our ability to characterize the metabolome of diverse samples that contain a significant proportion of ion features with no mass spectral match to known compounds. Manual and tool-assisted natural annotation propagation is readily used to classify molecular networks; however, currently no annotation propagation tools leverage consensus confidence strategies enabled by hierarchical chemical ontologies or enable the use of new in silico tools without significant modification. Herein we present ConCISE (Consensus Classifications of In Silico Elucidations) which is the first tool to fuse molecular networking, spectral library matching and in silico class predictions to establish accurate putative classifications for entire subnetworks. By limiting annotation propagation to only structural classes which are identical for the majority of ion features within a subnetwork, ConCISE maintains a true positive rate greater than 95% across all levels of the ChemOnt hierarchical ontology used by the ClassyFire annotation software (superclass, class, subclass). The ConCISE framework expanded the proportion of reliable and consistent ion feature annotation up to 76%, allowing for improved assessment of the chemo-diversity of dissolved organic matter pools from three complex marine metabolomics datasets comprising dominant reef primary producers, five species of the diatom genus Pseudo-nitzchia, and stromatolite sediment samples.
    Keywords:  CANOPUS; annotation propagation; dissolved organic matter; metabolomics
  22. Food Chem. 2022 Dec 10. pii: S0308-8146(22)03144-2. [Epub ahead of print]408 135182
      Mycotoxins can produce toxic effects on humans; hence, it is of high importance to determine their presence in food products. This work presents a reliable method for the quantification of 32 mycotoxins in cheese. The analysis procedure was optimized based on a QuEChERS extraction process and the ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) detection. The analysis method was validated for four cheese varieties (emmental, blue, brie and camembert) in terms of linearity, sensitivity, matrix effect, accuracy and precision. Satisfactory precision and accuracy values were achieved, with recoveries above 70% for most mycotoxins. The developed method was applied to the analysis of 38 commercial cheese samples. A high occurrence of beauvericin and enniatins were found, ranging from 31% for enniatin A to 100% for enniatin B. The ochratoxin A was detected in three samples at concentrations that may pose a risk to human health.
    Keywords:  Cheese; Mass spectrometry; Mycotoxin; QuEChERS; UHPLC
  23. J Chromatogr A. 2022 Dec 08. pii: S0021-9673(22)00889-5. [Epub ahead of print]1688 463698
      We developed a novel chiral mass spectrometry derivatization reagent (S)-(3-(4-carboxythiazolidin-3-yl)-3-oxopropyl) diphenylsulfonium (CTOD) with a positively charged sulfur-containing structure for high-sensitivity detection of the chiral resolution of amino acid enantiomers. CTOD reacted with DL-amino acids at 60oC for 60 min to generate the corresponding diastereomers, fifteen chiral amino acid-derived products were separated. Resolution (Rs) values were of the range 1.54-4.36, except Asn 1.07, achieving good separation. A highly sensitive and selective UHPLC-MS/MS method for the simultaneous determination and chiral separation of five chiral amino acids (Pro, Ala, Glu, Asp, and Phe) based on CTOD derivatization was established and applied to the detection of chiral amino acids in different wines. The diastereomeric resolution of the five amino acids was 1.71-5.42, and an excellent linear relationship was obtained in the range of 0.25-500 pmol (R2 ≥0.9993). The detection limit was 0.05-0.25 pmol. The intra- and inter-day precisions were 0.51-5.76% and 0.78-5.18%, respectively, and the average recovery was 90.03-99.99%. In addition, the metabolic concentration of chiral amino acids was monitored after drinking red wine and white wine, and the fitting curve of metabolic concentration was drawn.
    Keywords:  Chiral amino enantiomer; Chiral separation; Diphenyl sulfide; UHPLC-MS/MS; Wine
  24. Nat Rev Methods Primers. 2022 ;2(1): 96
      Mass spectrometry is a powerful analytical tool used for the analysis of a wide range of substances and matrices; it is increasingly utilized for clinical applications in laboratory medicine. This Primer includes an overview of basic mass spectrometry concepts, focusing primarily on tandem mass spectrometry. We discuss experimental considerations and quality management, and provide an overview of some key applications in the clinic. Lastly, the Primer discusses significant challenges for implementation of mass spectrometry in clinical laboratories and provides an outlook of where there are emerging clinical applications for this technology.
    Keywords:  Mass spectrometry; Medical research
  25. Int J Environ Res Public Health. 2022 Dec 12. pii: 16680. [Epub ahead of print]19(24):
      The ability to effectively detect N-nitrosamine compounds by liquid chromatography-tandem mass spectrometry presents a challenge due to the problems of high detection limits and difficulty in simultaneous N-nitrosamine compound detection. In order to overcome these limitations, this study reduced the detection limit of N-nitrosamine compounds by applying n-hexane pre-treatment to remove non-polar impurities before the conventional process of column extraction. In addition, ammonium acetate was used as the mobile phase to enhance the retention of nitrosamine target substances on the chromatographic column, with formic acid added to the mobile phase to improve the ionization level of N-nitrosodiphenylamine, to achieve the simultaneous detection of multiple N-nitrosamine compounds. Applying these modifications to the established detection method allowed the rapid and accurate detection of N-nitrosamine in water within 12 min. The linear relationship, detection limit, quantification limit and sample spiked recovery rate of nine types of nitrosamine compound were investigated, showing that the correlation coefficient ranged from 0.9985-0.9999, while the detection limits of the instrument and the method were 0.280-0.928 µg·L-1 and 1.12-3.71 ng·L-1, respectively. The spiked sample recovery rate ranged from 64.2-83.0%, with a standard deviation of 2.07-8.52%, meeting the requirements for trace analysis. The method was applied to the detection of N-nitrosamine compounds in nine groundwater samples in Wuhan, China, and showed that the concentrations of N-nitrosodimethylamine and NDEA were relatively high, highlighting the need to monitor water bodies with very low levels of pollutants and identify those requiring treatment.
    Keywords:  groundwater; n-nitrosamine compounds; solid phase extraction (SPE); ultra-high performance liquid chromatography triple quadrupole instrument (UHPLC-MS/MS)
  26. Anal Chem. 2022 Dec 20.
      Large cohorts of samples from multiple batches are usually required for global metabolomic studies to characterize the metabolic state of human disease. As such, it is critical to eliminate systematic variation and truly reveal the biologically associated alterations. In this study, we proposed a reference material-based approach (Ref-M) for data correction by liquid chromatography-mass spectrometry and represented by an analysis of multibatch human serum samples. The reference material was generated by mixing serum from healthy donors and distributed to each extraction batch of subject samples. Pooled quality control samples and isotopic internal standards were then applied in each acquisition batch for data quality control. Finally, each metabolite in subject samples was normalized by its counterpart in the reference serum. We demonstrated that Ref-M significantly enhanced the numbers of efficient features and effectively eliminated the batch variation of 522 serum samples of healthy individuals, benign pulmonary nodules, and lung cancer patients. Twenty differential metabolites were identified to distinguish lung cancer from healthy controls in the training set. The discriminant model was validated in an independent data set with an area under the receiver operating characteristics (ROC) curve (AUC) of 0.853. Another 40 serum samples further tested with Ref-M were achieved an AUC of 0.843 by the established model. Our results showed that the reference material-based approach presents the potential to improve the data comparability and precision for biomarker discovery in large-scale metabolomic studies.
  27. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Dec 16. pii: S1570-0232(22)00482-2. [Epub ahead of print]1215 123577
      Organoids are laboratory-grown 3D organ models, mimicking human organs for e.g. drug development and personalized therapy. Islet organoids (typically 100-200 µm), which can be grown from the patient́s own cells, are emerging as prototypes for transplantation-based therapy of diabetes. Selective methods for quantifying insulin production from islet organoids are needed, but sensitivity and carry-over have been major bottlenecks in previous efforts. We have developed a reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method for studying the insulin secretion of islet organoids. In contrast to our previous attempts using nano-scale LC columns, conventional 2.1 mm inner diameter LC column (combined with triple quadrupole mass spectrometry) was well suited for sensitive and selective measurements of insulin secreted from islet organoids with low microliter-scale samples. Insulin is highly prone to carry-over, so standard tubings and injector parts were replaced with shielded fused silica connectors. As samples were expected to be very limited, an extended Box-Behnken experimental design for the MS settings was conducted to maximize performance. The finale method has excellent sensitivity, accuracy and precision (limit of detection: ≤0.2 pg/µL, relative error: ≤±10%, relative standard deviation: <10%), and was well suited for measuring 20 µL amounts of Krebs buffer containing insulin secreted from islet organoids.
    Keywords:  Box-Behnken design; Insulin secretion; Islet organoids; Liquid chromatography-mass spectrometry; Stem cell-derived islets
  28. Pharmaceuticals (Basel). 2022 Dec 19. pii: 1583. [Epub ahead of print]15(12):
      The aim of Quantitative mass spectrometry imaging (Q-MSI) is to provide distribution analysis and quantitation from one single mass-spectrometry-based experiment, and several quantitation methods have been devised for Q-MSI. Mimetic tissue models based on spiked tissue homogenates are considered one of the most accurate ways to perform Q-MSI, since the analyte is present in a well-defined concentration in a sample matrix highly similar to the one of the unknown sample to be analyzed. The delivery of drugs in skin is among the most frequent types of pharmaceutical MSI studies. Here, a mimetic tissue model is extended for use on the skin, which, due to its high collagen content, is different from most other tissue as the homogenates become extremely viscous. A protocol is presented which overcomes this by the addition of water and the handling of the homogenate at an elevated temperature where the viscosity is lower. Using a mimetic tissue model, a method was developed for the quantitative imaging of bleomycin in skin. To compensate for the signal drift and the inhomogeneities in the skin, an internal standard was included in the method. The method was tested on skin from a pig which had had an electropneumatic injection of bleomycin into the skin. Quantification was made at several regions in a cross section of the skin at the injection site, and the results were compared to the results of a quantitative LC-MS on a neighboring tissue biopsy from the same animal experiment. The overall tissue concentration determined by the LC-MS was within the range of the different regions quantified by the Q-MSI. As the model provides the results of the same order of magnitude as a LC-MS, it can either be used to replace LC-MS in skin studies where MSI and LC-MS are today carried out in combination, or it can add quantitative information to skin studies which are otherwise carried out by MSI alone.
    Keywords:  MALDI-MS; drug distribution; mass spectrometry imaging; quantitation
  29. Environ Pollut. 2022 Dec 16. pii: S0269-7491(22)02092-9. [Epub ahead of print]318 120877
      Most studies on the biodegradation of textile azo dyes use color as parameter for measuring the efficiency of degradation. Although widely employed, spectrophotometric methods are susceptible to the interference of metabolites or degradation products from the biological treatment. We propose a method for determination of a model sulfonated azo dye (Direct Black 22, DB22) in wastewater using solid-phase extraction (SPE) and liquid chromatography - electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). MS analysis in negative electrospray ionization mode showed DB22 as the most abundant precursor ion, corresponding to [M-3Na + H]2-, which yields two radical anions of m/z 370.1 and m/z 645 after MS/MS fragmentation by collision-induced dissociation (CID). Calibration curve presented adequate linearity and precision in the range of 120-1500 ng mL-1, and recovery and detection limit were appropriate to the typically employed working concentrations. Nevertheless, we observed that standard heating of DB22 under alkaline conditions to simulate the production of wastewater during dye-baths resulted in loss of MS/MS signal, without affecting color. Further analysis showed that DB22 undergoes hydrolysis and does not remain unaltered in solution. Alternative methods of hydrolysis evaluated resulted in no MS/MS signal as well. SPE-LC-ESI-MS/MS analysis evidenced the structural change of DB22 in aqueous solution while the dyeing-capacity was preserved. This technique has also the potential of being tailored to consider the detection of the hydrolyzed fragments of azo dyes in wastewater for appropriate quantification, but it was not the scope of the current step of this research. Color remains as a more reliable parameter for monitoring azo compounds which are unstable in aqueous solution, while a more robust and holistic method needs to be developed for the speciation of the DB22 products of thermal hydrolysis.
    Keywords:  Biological treatment; Liquid chromatography; Mass spectrometry; Reductive decolorization; Textile wastewater