bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒05‒29
39 papers selected by
Sofia Costa

  1. Methods Mol Biol. 2022 ;2482 311-327
      A diverse array of 24-h oscillating hormones and metabolites direct and reflect circadian clock function. Circadian metabolomics uses advanced high-throughput analytical chemistry techniques to comprehensively profile these small molecules (<1.5 kDa) across 24 h in cells, media, body fluids, breath, tissues, and subcellular compartments. The goals of circadian metabolomics experiments are often multifaceted. These include identifying and tracking rhythmic metabolic inputs and outputs of central and peripheral circadian clocks, quantifying endogenous free-running period, monitoring relative phase alignment between clocks, and mapping pathophysiological consequences of clock disruption or misalignment. Depending on the particular experimental question, samples are collected under free-running or entrained conditions. Here we describe both untargeted and targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) and flow injection-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS) based assays we have used for circadian metabolomics studies. We discuss tissue homogenization, chemical derivatization, measurement, and tips for data processing, normalization, scaling, how to handle outliers, and imputation of missing values.
    Keywords:  Circadian; Circadian metabolomics; Flow injection-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS/MS); Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Metabolites; Targeted metabolomics; Untargeted metabolomics
  2. J Mass Spectrom Adv Clin Lab. 2022 Apr;24 107-117
      Introduction: Quantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used.Objectives: To develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma.
    Methods: Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays.
    Results: The method is linear over a wide range of concentrations, 2.0-1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4-10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89-95% relative to the external group mean.
    Conclusion: We have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.
    Keywords:  3NPH, 3-nitrophenylhydrazine; ACN, Acetonitrile; AMR, Analytical measurable range; BCAA, Branched-chain amino acids; BCKD, Branched-chain ketoacid dehydrogenase complex; Branched-chain amino acid; CAP, The College of American Pathologists; CLIA, The Clinical Laboratory Improvement Amendments; CLSI, The Clinical & Laboratory Standards Institute; CN, Cyano; CRR, Clinical Reportable Range; Clinical assay; ESI, Electrospray ionization; FA, Formic Acid; GC–MS, Gas chromatography-mass spectrometry; HILIC, Hydrophilic interaction liquid chromatography; HMDB, Human metabolome database; IEX, Ion exchange; LC, Liquid chromatography; LC-MS/MS, Liquid chromatography-tandem mass spectrometry; LC-UV, Liquid chromatography-ultra violet; LDT, Laboratory-developed tests; LLE, Liquid-liquid extraction; LOD, Limit of detection; LOQ, Limit of quantitation; MSUD, Maple syrup urine disease; Maple syrup urine disease; Mass spectrometry; MeOH, Methanol; NMR, Nuclear magnetic resonance; PBS-BSA, Phosphate buffered saline with bovine serum albumin; PITC, Phenylisothiocyanate; PTFE, Polytetrafluoroethylene; QC, Quality control; Quantitation; RP, Reverse phase; RPLC, Reverse phase liquid chromatography; S/N, Signal-to-noise ratio; SCX, Strong cation exchange; SPE, Solid phase extraction; SRM, Selected reaction monitoring; UHPLC, Ultra-high-performance liquid chromatography; WAX, Weak anion exchange
  3. Molecules. 2022 May 18. pii: 3225. [Epub ahead of print]27(10):
      Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC-MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01-2 ng mL-1 for PREGS, DHEAS, and CRTS and of 0.05-10 ng mL-1 for E2S. The limits of detection (S/N = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL-1, respectively. Moreover, intraday and interday variations were lower than 11.1% (n = 5). The recoveries of these compounds from saliva samples were in the range of 86.6-112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.
    Keywords:  in-tube solid-phase microextraction (IT-SPME); liquid chromatography–tandem mass spectrometry (LC–MS/MS); online automated analysis; saliva; stable isotope dilution; sulfated steroid metabolites
  4. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 May 20. pii: S1570-0232(22)00198-2. [Epub ahead of print]1201-1202 123294
      A development of robust and rapid method with simple sample preparation for the analysis of steroids of C18-, C19-, C21- families is of interest of many research groups. Here we present a novel LC-MS/MS method for the simultaneous quantification of 32 steroid hormones in human plasma. Twenty-two of them were analyzed directly without the need for derivatization, while ten were derivatized with 2-fluoro-1-methylpyridinium p-toluenesulfonate. The steroids were separated on a C18 column with a gradient elution consisting of methanol and water with the addition of 0.1% formic acid. The mass spectrometer was operated in positive ESI mode. Validation demonstrated that the method was applicable for the quantitative analysis of two C18- steroids (estrone, estradiol), nineteen C19- steroids (testosterone, epitestosterone, dihydrotestosterone, 11-ketodihydrotestosterone, 11β-hydroxyandrostenedione, 11β-hydroxytestosterone, 11-ketotestosterone, dehydroepiandrosterone, 7α-hydroxydehydroepiandrosterone, 7β-hydroxydehydroepiandrosterone, 7-ketodehydroepiandrosterone, androsterone, epiandrosterone, androstenedione, androstenediol, 5α-androstane-3α,17β-diol, 5α-androstane-3β,17β-diol, 5β-androstane-3α,17β-diol, 5β-androstane-3β,17β-diol), and eleven C21- steroids (cortisol, 21-deoxycortisol, 11-deoxycortisol, cortisone, corticosterone, 11-deoxycorticosterone, pregnenolone, 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone). The lower limits of quantification are appropriate for analyses in both physiological and various pathophysiological conditions. The accuracy, intra- and inter-day precision values as well as stability tests were in accordance with FDA Guidelines. The method will be a useful tool in investigating the mechanisms of steroid-related diseases and will serve as a steppingstone for the development of other methods for steroid analyses in various biological matrices such as prostate tissue, cerebrospinal fluid, urine, seminal fluid, and saliva.
    Keywords:  Human plasma; LC-MS/MS; Liquid chromatography; Mass spectrometry; Steroids
  5. Curr Protoc. 2022 May;2(5): e454
      The filamentous fungus Neurospora crassa has historically been a model for understanding the relationship between genes and metabolism-auxotrophic mutants of N. crassa were used by Beadle and Tatum to develop the one-gene-one-enzyme hypothesis for which they earned the Nobel Prize in 1958. In the ensuing decades, several techniques have been developed for the systematic analysis of metabolites in N. crassa and other fungi. Untargeted and targeted approaches have been used, with a focus on secondary metabolites over primary metabolism. Here, we describe a pipeline for sample preparation, metabolite extraction, Liquid Chromatography-Mass Spectrometry (LC-MS), and data analysis that can be used for targeted metabolomics of primary metabolites in N. crassa. Liquid cultures are grown with shaking in a defined minimal medium and then collected using filtration. Samples are lyophilized for 2 days at -80°C, pulverized, and mixed with a solution to extract polar metabolites. The metabolites are separated and identified using LC-MS, with downstream analysis using Skyline interpretive software. Relative levels of hundreds of metabolites can be detected and compared across strains. © 2022 Wiley Periodicals LLC. Basic Protocol: Metabolite extraction and detection from Neurospora crassa cell cultures using Liquid Chromatography-Mass Spectrometry.
    Keywords:  LC-MS; Metabolomics; Neurospora crassa
  6. Nucleic Acids Res. 2022 May 24. pii: gkac383. [Epub ahead of print]
      The CFM-ID 4.0 web server ( is an online tool for predicting, annotating and interpreting tandem mass (MS/MS) spectra of small molecules. It is specifically designed to assist researchers pursuing studies in metabolomics, exposomics and analytical chemistry. More specifically, CFM-ID 4.0 supports the: 1) prediction of electrospray ionization quadrupole time-of-flight tandem mass spectra (ESI-QTOF-MS/MS) for small molecules over multiple collision energies (10 eV, 20 eV, and 40 eV); 2) annotation of ESI-QTOF-MS/MS spectra given the structure of the compound; and 3) identification of a small molecule that generated a given ESI-QTOF-MS/MS spectrum at one or more collision energies. The CFM-ID 4.0 web server makes use of a substantially improved MS fragmentation algorithm, a much larger database of experimental and in silico predicted MS/MS spectra and improved scoring methods to offer more accurate MS/MS spectral prediction and MS/MS-based compound identification. Compared to earlier versions of CFM-ID, this new version has an MS/MS spectral prediction performance that is ∼22% better and a compound identification accuracy that is ∼35% better on a standard (CASMI 2016) testing dataset. CFM-ID 4.0 also features a neutral loss function that allows users to identify similar or substituent compounds where no match can be found using CFM-ID's regular MS/MS-to-compound identification utility. Finally, the CFM-ID 4.0 web server now offers a much more refined user interface that is easier to use, supports molecular formula identification (from MS/MS data), provides more interactively viewable data (including proposed fragment ion structures) and displays MS mirror plots for comparing predicted with observed MS/MS spectra. These improvements should make CFM-ID 4.0 much more useful to the community and should make small molecule identification much easier, faster, and more accurate.
  7. Metabolites. 2022 May 17. pii: 450. [Epub ahead of print]12(5):
      The main concerns in targeted "sphingolipidomics" are the extraction and proper handling of biological samples to avoid interferences and achieve a quantitative yield well representing all the sphingolipids in the matrix. Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture (2:1, v/v) for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences.
    Keywords:  lipidomics; mass spectrometry; sphingoid bases; sphingolipidomics; sphingolipids
  8. Front Med (Lausanne). 2022 ;9 841281
      The gut microbiome and microbial metabolomic influences on liver diseases and their diagnosis, prognosis, and treatment are still controversial. Research studies have provocatively claimed that the gut microbiome, metabolomics understanding, and microbial metabolite screening are key approaches to understanding liver cancer and liver diseases. An advance of logical innovations in metabolomics profiling, the metabolome inclusion, challenges, and the reproducibility of the investigations at every stage are devoted to this domain to link the common molecules across multiple liver diseases, such as fatty liver, hepatitis, and cirrhosis. These molecules are not immediately recognizable because of the huge underlying and synthetic variety present inside the liver cellular metabolome. This review focuses on microenvironmental metabolic stimuli in the gut-liver axis. Microbial small-molecule profiling (i.e., semiquantitative monitoring, metabolic discrimination, target profiling, and untargeted profiling) in biological fluids has been incompletely addressed. Here, we have reviewed the differential expression of the metabolome of short-chain fatty acids (SCFAs), tryptophan, one-carbon metabolism and bile acid, and the gut microbiota effects are summarized and discussed. We further present proof-of-evidence for gut microbiota-based metabolomics that manipulates the host's gut or liver microbes, mechanosensitive metabolite reactions and potential metabolic pathways. We conclude with a forward-looking perspective on future attention to the "dark matter" of the gut microbiota and microbial metabolomics.
    Keywords:  liver diseases; liver therapies; metabolic discrimination; metabolites alteration; microbial metabolomics; short-chain fatty acids; tryptophan metabolism
  9. Metabolites. 2022 May 11. pii: 429. [Epub ahead of print]12(5):
      Gas chromatography-coupled mass spectrometry (GC-MS) has been used in biomedical research to analyze volatile, non-polar, and polar metabolites in a wide array of sample types. Despite advances in technology, missing values are still common in metabolomics datasets and must be properly handled. We evaluated the performance of ten commonly used missing value imputation methods with metabolites analyzed on an HR GC-MS instrument. By introducing missing values into the complete (i.e., data without any missing values) National Institute of Standards and Technology (NIST) plasma dataset, we demonstrate that random forest (RF), glmnet ridge regression (GRR), and Bayesian principal component analysis (BPCA) shared the lowest root mean squared error (RMSE) in technical replicate data. Further examination of these three methods in data from baboon plasma and liver samples demonstrated they all maintained high accuracy. Overall, our analysis suggests that any of the three imputation methods can be applied effectively to untargeted metabolomics datasets with high accuracy. However, it is important to note that imputation will alter the correlation structure of the dataset and bias downstream regression coefficients and p-values.
    Keywords:  HR GC–MS; imputation missing values; metabolomics
  10. J Mass Spectrom Adv Clin Lab. 2022 Apr;24 100-106
      Introduction: Clobazam is a benzodiazepine drug, used to treat Lennox-Gastaut syndrome in patients aged 2 years and older.Objective: To support patient care, our laboratory developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of clobazam (CLB) and its major active metabolite N-desmethylclobazam (N-CLB) in human plasma or serum samples.
    Methods: The chromatographic separation was achieved with an Agilent Zorbax Eclipse Plus C-18 RRHD column with mobile phase consisting of 0.05% formic acid in 5 mM ammonium formate, pH 3.0 and 0.1% formic acid in acetonitrile at a flow rate of 600 µL/minute and an injection volume of 5 µL. The detection was performed on a triple quadrupole mass spectrometer in multiple reaction monitoring mode to monitor precursor-to-product ion transitions in positive electrospray ionization mode.
    Results: The method was validated over a concentration range of 20-2000 ng/mL for CLB and 200-10,000 ng/mL for N-CLB. The lower limit of quantification was 20 ng/mL for CLB and 200 ng/mL for N-CLB with good accuracy and precision. The method performance was successfully evaluated by comparison with two different external laboratories. Retrospective data analysis was performed to evaluate the positivity rate and metabolic patterns for clobazam from our patient population, as a reference laboratory. Among the positive samples, both parent and metabolite were detected in 96.4% of the samples.
    Conclusion: The method was developed to support therapeutic drug monitoring and the data generated from retrospective analysis could be useful for result interpretation in conjunction with clinical patient information.
    Keywords:  CLB, Clobazam; CLIA, Clinical Laboratory Improvement Amendment; CLRW, Clinical Laboratory Reagent Water; Clobazam; DAD, Diode Array Detector; ESI, Electrospray ionization; IRB, Institutional Review Board; LC-MS/MS; LC-MS/MS, liquid chromatography tandem mass spectrometry; LLOQ, lower limit of quantification; LOD, limit of detection; MRM, multiple reaction monitoring; N-CLB, N-desmethylclobazam; N-Desmethylclobazam; Plasma; Retrospective data analysis; TDM, Therapeutic drug monitoring; ULOQ, upper limit of quantification; UV, Ultraviolet
  11. Metabolites. 2022 Apr 29. pii: 408. [Epub ahead of print]12(5):
      The investigation of metabolic fluxes and metabolite distributions within cells by means of tracer molecules is a valuable tool to unravel the complexity of biological systems. Technological advances in mass spectrometry (MS) technology such as atmospheric pressure chemical ionization (APCI) coupled with high resolution (HR), not only allows for highly sensitive analyses but also broadens the usefulness of tracer-based experiments, as interesting signals can be annotated de novo when not yet present in a compound library. However, several effects in the APCI ion source, i.e., fragmentation and rearrangement, lead to superimposed mass isotopologue distributions (MID) within the mass spectra, which need to be corrected during data evaluation as they will impair enrichment calculation otherwise. Here, we present and evaluate a novel software tool to automatically perform such corrections. We discuss the different effects, explain the implemented algorithm, and show its application on several experimental datasets. This adjustable tool is available as an R package from CRAN.
    Keywords:  CorMID; R package; atmospheric pressure chemical ionization; enrichment calculation; flux experiments; mass isotopologue distribution
  12. STAR Protoc. 2022 Jun 17. 3(2): 101408
      Metabolism is important for the regulation of hematopoietic stem cells (HSCs) and drives cellular fate. Due to the scarcity of HSCs, it has been technically challenging to perform metabolome analyses gaining insight into HSC metabolic regulatory networks. Here, we present two targeted liquid chromatography-mass spectrometry approaches that enable the detection of metabolites after fluorescence-activated cell sorting when sample amounts are limited. One protocol covers signaling lipids and retinoids, while the second detects tricarboxylic acid cycle metabolites and amino acids. For complete details on the use and execution of this protocol, please refer to Schönberger et al. (2022).
    Keywords:  Mass Spectrometry; Metabolomics; Stem Cells
  13. Se Pu. 2022 Jun;40(6): 509-519
      Most drugs used to treat diseases are chiral compounds. Drug enantiomers possess similar physical and chemical properties but may feature distinct pharmacological activities. Drug enantiomers may also exhibit different or even opposite functionalities for metabolism, in terms of the metabolic rate and toxicity in the body. Therefore, it is imperative to analyze, separate, and purify the enantiomers of drugs. The separation of chiral compounds is essential for drug research and development. It is also of significance in various fields including biological environments, food, and medicine. Various highly selective and sensitive methods have been developed for the quantitative and qualitative analyses of chiral compounds. A typically employed technique is high performance liquid chromatography-mass spectrometry (HPLC-MS). While HPLC-MS offers high sensitivity and reproducibility, it requires expensive chiral columns and MS-compatible mobile phases for the chromatographic column. Further, the column efficiency and resolution capacity in chiral chromatography packing require improvement. Recent progress has shown that capillary electrophoresis-mass spectrometry (CE-MS) has broad applications in chiral analysis. As a well-established analytical technique, CE-MS combines the highly efficient separation technique of CE with the highly sensitive detection technique of MS. Thus, it offers many essential advantages for analysis. For example, CE-MS has a high separation efficiency and requires very low amounts of samples and reagents. It can also achieve sensitive and selective determination, and the obtained diversified separation modes can be used for different samples. Therefore, CE-MS has proved to be important in analytical chemistry, especially in proteomics and metabolomics. CE can also exhibit excellent performance in chiral separation. Hence, combined with the sensitive detection technique of MS, CE-MS would be ideal for chiral analysis. Chiral CE-MS can provide a wide range of qualitative information on samples simultaneously in a single run, including the migration time, relative molecular mass, and ionic fragments. It addresses the challenges associated with identifying unknown chiral compounds in actual samples (including chiral compounds without UV absorption groups or fluorescence groups). The high-throughput analysis of multiple groups of chiral enantiomers can be achieved while mitigating the matrix effect of biological samples. In the last ten years, high performance chiral analysis strategies based on different CE-MS modes have been developed. These include electrokinetic chromatography-mass spectrometry (EKC-MS), micellar electrokinetic chromatography-mass spectrometry (MEKC-MS), and capillary electrochromatography-mass spectrometry (CEC-MS). CE-MS has been successfully applied in chiral analysis in various fields such as medicine, biology, food, and environmental science. CE-MS is promising in the chiral analysis of drugs, especially for drug development and drug quality control, as well as pharmacokinetics and pharmacodynamics research. Recent studies have focused on the development of MS-friendly and highly selective chiral analytical methods, which will broaden the application of CE-MS. In CEC-MS chiral analysis, more attention has been paid to developing novel capillary chiral stationary phases for monolithic or packed columns. Because of the diversity of chiral selectors for EKC-MS and MEKC-MS, the chiral analysis of drugs using these techniques has attracted intense research interest. Moreover, functional nanoparticles have been employed to increase the surface area of the CEC columns for enhancing the efficiency of chiral analysis. The chiral separation and analysis of miniaturized microchip equipment via CE-MS has also been explored, but remains to be widely used in practical applications. The purpose of this review is to provide insights that would aid in broadening the applications of CE-MS to chiral analysis. In this review, we primarily summarize research progress on the application of CE-MS to chiral analysis, based on the literature published during the years 2011-2021. Chiral selectors (e. g., modified cyclodextrin and polymer surfactants) and their reported applications in CE-MS are presented. The determination results for drug enantiomers using different CE-MS modes are compared. The application of CE-MS in other research fields is also presented, along with the advantages and limitations of different CE-MS methods.
    Keywords:  capillary electrophoresis (CE); chiral compounds; mass spectrometry (MS); review
  14. Methods Mol Biol. 2022 ;2456 349-365
      This chapter describes protocols for the development of consensus chemical phenotypes or "metabolomes" of fungal populations using ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry (UPLC-HRMS). Isolates are cultured using multiple media conditions to elicit the expression of diverse secondary metabolite biosynthetic gene clusters. The mycelium and spent culture media are extracted using organic solvents and profiled by ultra-high pressure chromatography coupled with a high resolution Thermo Orbitrap XL mass spectrometer with the ability to trap and fragment ions to general MS2 spectra. MS data preprocessing is explained and illustrated using the freely available software MZMine 2. Through data processing, binary matrices of mass features can be generated and then combined into a consensus secondary metabolite phenotype of all isolates grown in all media conditions. The production of consensus chemical phenotypes is useful for screening large fungal populations (both inter and intra-species populations) for isolates potentially expressing novel secondary metabolites or analogs of known secondary metabolites.
    Keywords:  Consensus chemical phenotypes; Fungal natural products; Fungi; LCMS; MZMine; Mass spectrometry; Metabolomics; Secondary metabolites; Thermo Orbitrap XL; UPLC-HRMS
  15. Metabolites. 2022 May 18. pii: 455. [Epub ahead of print]12(5):
      Optical microscopy has long been the gold standard to analyse tissue samples for the diagnostics of various diseases, such as cancer. The current diagnostic workflow is time-consuming and labour-intensive, and manual annotation by a qualified pathologist is needed. With the ever-increasing number of tissue blocks and the complexity of molecular diagnostics, new approaches have been developed as complimentary or alternative solutions for the current workflow, such as digital pathology and mass spectrometry imaging (MSI). This study compares the performance of a digital pathology workflow using deep learning for tissue recognition and an MSI approach utilising shallow learning to annotate formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue microarrays (TMAs). Results show that both deep learning algorithms based on conventional optical images and MSI-based shallow learning can provide automated diagnostics with F1-scores higher than 90%, with the latter intrinsically built on biochemical information that can be used for further analysis.
    Keywords:  DESI-MSI; FFPE; deep learning; diagnostics; mass spectrometry imaging; shallow learning
  16. Metabolites. 2022 May 10. pii: 426. [Epub ahead of print]12(5):
      As metabolomics increasingly finds its way from basic science into applied and regulatory environments, analytical demands on nontargeted mass spectrometric detection methods continue to rise. In addition to improved chemical comprehensiveness, current developments aim at enhanced robustness and repeatability to allow long-term, inter-study, and meta-analyses. Comprehensive metabolomics relies on electrospray ionization (ESI) as the most versatile ionization technique, and recent liquid chromatography-high resolution mass spectrometry (LC-HRMS) instrumentation continues to overcome technical limitations that have hindered the adoption of ESI for applications in the past. Still, developing and standardizing nontargeted ESI methods and instrumental setups remains costly in terms of time and required chemicals, as large panels of metabolite standards are needed to reflect biochemical diversity. In this paper, we investigated in how far a nontargeted pilot experiment, consisting only of a few measurements of a test sample dilution series and comprehensive statistical analysis, can replace conventional targeted evaluation procedures. To examine this potential, two instrumental ESI ion source setups were compared, reflecting a common scenario in practical method development. Two types of feature evaluations were performed, (a) summary statistics solely involving feature intensity values, and (b) analyses additionally including chemical interpretation. Results were compared in detail to a targeted evaluation of a large metabolite standard panel. We reflect on the advantages and shortcomings of both strategies in the context of current harmonization initiatives in the metabolomics field.
    Keywords:  chemical classification; electrospray ionization; feature statistics; liquid chromatography-high resolution mass spectrometry; method development; method harmonization; nontargeted analysis; quality control
  17. Metabolites. 2022 May 12. pii: 435. [Epub ahead of print]12(5):
      In biological research domains, liquid chromatography-mass spectroscopy (LC-MS) has prevailed as the preferred technique for generating high quality metabolomic data. However, even with advanced instrumentation and established data acquisition protocols, technical errors are still routinely encountered and can pose a significant challenge to unveiling biologically relevant information. In large-scale studies, signal drift and batch effects are how technical errors are most commonly manifested. We developed pseudoDrift, an R package with capabilities for data simulation and outlier detection, and a new training and testing approach that is implemented to capture and to optionally correct for technical errors in LC-MS metabolomic data. Using data simulation, we demonstrate here that our approach performs equally as well as existing methods and offers increased flexibility to the researcher. As part of our study, we generated a targeted LC-MS dataset that profiled 33 phenolic compounds from seedling stem tissue in 602 genetically diverse non-transgenic maize inbred lines. This dataset provides a unique opportunity to investigate the dynamics of specialized metabolism in plants.
    Keywords:  LC–MS; data normalization; maize; metabolomics; signal drift
  18. Mar Drugs. 2022 May 12. pii: 320. [Epub ahead of print]20(5):
      Today, marine natural products are considered one of the main sources of compounds for drug development. Starfish and sea cucumbers are potential sources of natural products of pharmaceutical interest. Among their metabolites, polar steroids, triterpene glycosides, and polar lipids have attracted a great deal of attention; however, studying these compounds by conventional methods is challenging. The application of modern MS-based approaches can help to obtain valuable information about such compounds. This review provides an up-to-date overview of MS-based applications for starfish and sea cucumber bioactive compounds analysis. While describing most characteristic features of MS-based approaches in the context of starfish and sea cucumber metabolites, including sample preparation and MS analysis steps, the present paper mainly focuses on the application of MS-based metabolic profiling of polar steroid compounds, triterpene glycosides, and lipids. The application of MS in metabolomics studies is also outlined.
    Keywords:  lipids; mass spectrometry; metabolomic profiling; metabolomics; polyhydroxysteroids; sea cucumber; starfish; steroid glycosides; triterpene glycosides
  19. Metabolites. 2022 May 18. pii: 453. [Epub ahead of print]12(5):
      The identification of endogenous metabolites has great potential for understanding the underlying tissue processes occurring in either a homeostatic or a diseased state. The application of gas chromatography-mass spectrometry (GC-MS)-based metabolomics on musculoskeletal tissue samples has gained traction. However, limited comparison studies exist evaluating the sensitivity, reproducibility, and robustness of the various existing extraction protocols for musculoskeletal tissues. Here, we evaluated polar metabolite extraction from bone and muscle of mouse origin. The extraction methods compared were (1) modified Bligh-Dyer (mBD), (2) low chloroform (CHCl3)-modified Bligh-Dyer (mBD-low), and (3) modified Matyash (mMat). In particular, the central carbon metabolites (CCM) appear to be relevant for musculoskeletal regeneration, given their role in energy metabolism. However, the sensitivity, reproducibility, and robustness of these methods for detecting targeted polar CCM remains unknown. Overall, the extraction of metabolites using the mBD, mBD-low, and mMat methods appears sufficiently robust and reproducible for bone, with the mBD method slightly bettering the mBD-low and mMat methods. Furthermore, mBD, mBD-low, and mMat were sufficiently sensitive in detecting polar metabolites extracted from mouse muscle; however, they lacked repeatability. This study highlights the need for a re-thinking, towards a tissue-specific optimization of methods for metabolite extractions, ensuring sufficient sensitivity, repeatability, and robustness.
    Keywords:  GC-MS; bone; central carbon metabolism; metabolites; metabolomics; muscle
  20. Methods Mol Biol. 2022 ;2399 151-170
      Data-driven research led by computational systems biology methods, encompassing bioinformatics of multiomics datasets and mathematical modeling, are critical for discovery. Herein, we describe a multiomics (metabolomics-fluxomics) approach as applied to heart function in diabetes. The methodology presented has general applicability and enables the quantification of the fluxome or set of metabolic fluxes from cytoplasmic and mitochondrial compartments in central catabolic pathways of glucose and fatty acids. Additionally, we present, for the first time, a general method to reduce the dimension of detailed kinetic, and in general stoichiometric models of metabolic networks at the steady state, to facilitate their optimization and avoid numerical problems. Representative results illustrate the powerful mechanistic insights that can be gained from this integrative and quantitative methodology.
    Keywords:  Diabetes; Fluxomics; Glucose and fatty acids catabolism; Heart; Kinetic modeling; Metabolomics
  21. Bioinformatics. 2022 May 23. pii: btac344. [Epub ahead of print]
      MOTIVATION: Chromatographic peak picking is among the first steps in data processing workflows of raw LC-HRMS datasets in untargeted metabolomics applications. Its performance is crucial for the holistic detection of all metabolic features as well as their relative quantification for statistical analysis and metabolite identification. Random noise, non-baseline separated compounds and unspecific background signals complicate this task.RESULTS: A machine-learning based approach entitled PeakBot was developed for detecting chromatographic peaks in LC-HRMS profile-mode data. It first detects all local signal maxima in a chromatogram, which are then extracted as super-sampled standardized areas (retention-time vs. m/z). These are subsequently inspected by a custom-trained convolutional neural network that forms the basis of PeakBot's architecture. The model reports if the respective local maximum is the apex of a chromatographic peak or not as well as its peak center and bounding box.In training and independent validation datasets used for development, PeakBot achieved a high performance with respect to discriminating between chromatographic peaks and background signals (accuracy of 0.99). For training the machine-learning model a minimum of 100 reference features are needed to learn their characteristics to achieve high-quality peak-picking results for detecting such chromatographic peaks in an untargeted fashion.PeakBot is implemented in python (3.8) and uses the TensorFlow (2.5.0) package for machine-learning related tasks. It has been tested on Linux and Windows OSs.
    AVAILABILITY: The package is available free of charge for non-commercial use (CC BY-NC-SA). It is available at
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  22. Anal Bioanal Chem. 2022 May 26.
      LC-MS is one of the most important tools for the comprehensive characterization of N-glycans. Despite many efforts to speed up glycan analysis via optimized sample preparation (e.g., faster enzyme digestion in combination with instant or rapid labeling dyes), a major bottleneck remains the rather long measurement times of HILIC chromatography. Further complication arises from the necessity to concomitantly calibrate with an external standard to allow for accurate retention times and the conversion into more robust GU values. Here we demonstrate the use of an internal calibration strategy for HILIC chromatography to speed up glycan analysis. By reducing the number of utilized dextran oligosaccharides, the calibrant can be spiked directly into the sample such that external calibration runs are no longer required. The minimized dextran ladder shows accurate GU calibration with a minor deviation of well below 1% and can be applied without modifications in sample preparation or data processing. We further demonstrate the simultaneous use of the minimized dextran ladder as calibrant for the estimation of CCS values in traveling wave ion mobility spectrometry. In both cases, the minimized dextran ladder enables the measurement of calibrant and sample in a single HPLC run without losing information or accuracy.
    Keywords:  Calibration; Collision cross sections; Glucose units; HILIC; Ion mobility spectrometry; N-Glycan analysis
  23. Se Pu. 2022 Jun;40(6): 531-540
      A novel method based on ultra-high performance liquid chromatography-orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap HRMS) was developed for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods. In addition, the key points of the database establishment and application are summarized. This research focused on the derivatives of illegally added drugs. An HRMS database was established by comparing the response intensity of each compound in the positive and negative modes. The experimental conditions such as the type of extraction solvent and chromatographic column temperature were explored in detail. The analytes were separated on a Hypersil gold vanquish column (100 mm×2.1 mm, 1.9 μm) by gradient elution with acetonitrile/water (containing 0.1%(v/v) formic acid) as the mobile phase at a flow rate of 0.3 mL/min. Positive and negative ion full scanning/data-dependent secondary scanning mode was used to collect the 32 target compounds within 17 min, and TraceFinder software was used to screen the fragment ions. All the 32 compounds could be well separated within 17 min. The measured and theoretical values of the exact mass of the 32 compounds in the two matrix-spiked solutions were within an error of 5×10-6, and the MS2 fragment ions were within an error of 1×10-5. All the compounds showed an excellent linear relationship, with correlation coefficients (r2) above 0.99. Except dapoxetine, hydroxythiohomo sildenafil, thiohomo sildenafil, thiosildenafil, desmethyl thiosildenafil, the recoveries ranged from 50.5% to 84.5% in the solid matrix, with the relative standard deviations (RSDs) ranging from 1.2% to 13%. The recoveries were 60.4% to 109.3% in the liquid matrix, with the RSDs ranging from 0.77% to 8.2%. The matrix effect (ME) values of the 32 compounds ranged from 0.61 to 0.95 in the solid matrix and from 0.73 to 1.09 in the liquid matrix. Thiohomo sildenafil, desmethyl thiosildenafil, and chlorpretadalafil exhibited strong matrix inhibitory effects in the solid matrix. Therefore, solid and liquid negative matrix extracts were used to prepare a series of mixed standard solutions in order to reduce the ME values. The limits of detection (LODs) were 0.02 mg/kg for the 32 drugs in the liquid sample and 0.02 mg/kg for 29 compounds in the solid sample; the LODs for chlorothalidone, udenafil, and desmethyl thiosildenafil in the solid sample were 0.04 mg/kg. When the retention time in the self-built database matches the sample collection method, it should be used as one of the screening conditions. As for the selection of the matching mode, if the identify mode is selected, the retention time is a necessary condition for compound confirmation. When the retention time does not meet the requirements, subsequent screening of the fragment ions and isotope abundance ratios will not be performed. If the confirm mode is selected, the retention time is the optional condition for compound confirmation. When the retention time does not meet these requirements, subsequent matching of other conditions such as fragment ions and isotope information is required. Isotope information is very important in HRMS and is an effective supplement to the first-order extracted mass. Therefore, its use is recommended, but the isotope abundance ratio will be even lower when the target content is very low in the complex matrix, which may affect isotope matching. In addition, if the fragment ions are not detected in the screening results of the TraceFinder software but can be extracted in the data browser, their intensity threshold in the screening conditions can be further reduced to find the corresponding fragment ions. One positive sample was detected among 48 healthy food samples, with a detection rate of 2.08%. This method has the advantages of simple operation and high accuracy. It can be used for the rapid screening and confirmation of 32 illegally added drugs in slimming and anti-impotence health foods.
    Keywords:  health foods; high-resolution mass spectrometry (HRMS); illegally added drugs; rapid screening; ultra-high performance liquid chromatography (UHPLC)
  24. Biomed Chromatogr. 2022 May 27. e5416
      A reliable and robust bioanalytical method is developed to quantify neratinib, a tyrosine kinase inhibitor in human plasma using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The extraction of neratinib and its deuterated internal standard, neratinib-d6 was successfully performed on hybrid solid phase extraction (SPE) ultra-cartridges to remove the interference of phospholipids and proteins. Chromatographic analysis was done on UPLC BEH C18 (50 × 2.1 mm, 1.7 μm) column using 0.1% formic acid and acetonitrile under gradient conditions. The total analysis time was 1.5 min. The quantification of neratinib was achieved using electrospray ionization source operated in the positive ion multiple reaction monitoring mode. The mass transitions of neratinib and neratinib were m/z 557.3/112.1 and m/z 563.1/118.2, respectively. The linear concentration range for neratinib was 0.5-500 ng/mL, which adequately covers concentration levels expected in real subject samples. The assay was extensively validated for various validation parameters following standard guidelines for a bioanalytical assay. The intra- and inter-batch precision was ≤ 4.6 % and neratinib was found to be stable under various stability conditions. The mean IS-normalized matrix factor and recovery was 0.997 and 95.4 %, respectively. The validated method was successfully applied to a pharmacokinetic study in healthy subjects with different doses.
    Keywords:  Human plasma; HybridSPE; Neratinib; Pharmacokinetic study; Phospholipids; UPLC-MS/MS
  25. Biomolecules. 2022 May 16. pii: 709. [Epub ahead of print]12(5):
      Lipid compositions of cells, tissues, and bio-fluids are complex, with varying concentrations and structural diversity making their identification challenging. Newer methods for comprehensive analysis of lipids are thus necessary. Herein, we propose a targeted-mass spectrometry based lipidomics screening method using a combination of variable retention time window and relative dwell time weightage. Using this method, we identified more than 1000 lipid species within 24-min. The limit of detection varied from the femtomolar to the nanomolar range. About 883 lipid species were detected with a coefficient of variance &lt;30%. We used this method to identify plasma lipids altered due to vitamin B12 deficiency and found a total of 18 lipid species to be altered. Some of the lipid species with ω-6 fatty acid chains were found to be significantly increased while ω-3 decreased in vitamin B12 deficient samples. This method enables rapid screening of a large number of lipid species in a single experiment and would substantially advance our understanding of the role of lipids in biological processes.
    Keywords:  dwell time; isomers; lipidomics; mass spectrometry; plasma lipidome; scheduled MRM; variable RT window; vitamin B12
  26. Int J Mol Sci. 2022 May 20. pii: 5743. [Epub ahead of print]23(10):
      Sulfation is an important reaction in nature, and sulfated phenolic compounds are of interest as standards of mammalian phase II metabolites or pro-drugs. Such standards can be prepared using chemoenzymatic methods with aryl sulfotransferases. The aim of the present work was to obtain a large library of sulfated phenols, phenolic acids, flavonoids, and flavonolignans and optimize their HPLC (high performance liquid chromatography) analysis. Four new sulfates of 2,3,4-trihydroxybenzoic acid, catechol, 4-methylcatechol, and phloroglucinol were prepared and fully characterized using MS (mass spectrometry), 1H, and 13C NMR. The separation was investigated using HPLC with PDA (photodiode-array) detection and a total of 38 standards of phenolics and their sulfates. Different stationary (monolithic C18, C18 Polar, pentafluorophenyl, ZICpHILIC) and mobile phases with or without ammonium acetate buffer were compared. The separation results were strongly dependent on the pH and buffer capacity of the mobile phase. The developed robust HPLC method is suitable for the separation of enzymatic sulfation reaction mixtures of flavonoids, flavonolignans, 2,3-dehydroflavonolignans, phenolic acids, and phenols with PDA detection. Moreover, the method is directly applicable in conjunction with mass detection due to the low flow rate and the absence of phosphate buffer and/or ion-pairing reagents in the mobile phase.
    Keywords:  Desulfitobacterium hafniense; HPLC analysis; aryl sulfotransferase; flavonoids; phenolic acid; polyphenols; sulfates
  27. Biomedicines. 2022 May 20. pii: 1189. [Epub ahead of print]10(5):
      Targeted analytical methods for the determination of free fatty acids (FFAs) in human plasma are of high interest because they may help in identifying biomarkers for diseases and in monitoring the progress of a disease. The determination of FFAs is of particular importance in the case of metabolic disorders because FFAs have been associated with diabetes. We present a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which allows the simultaneous determination of 74 FFAs in human plasma. The method is fast (10-min run) and straightforward, avoiding any derivatization step and tedious sample preparation. A total of 35 standard saturated and unsaturated FFAs, as well as 39 oxygenated (either hydroxy or oxo) saturated FFAs, were simultaneously detected and quantified in plasma samples from 29 subjects with type 2 diabetes mellitus (T2D), 14 with type 1 diabetes mellitus (T1D), and 28 healthy subjects. Alterations in the levels of medium-chain FFAs (C6:0 to C10:0) were observed between the control group and T2D and T1D patients.
    Keywords:  LC-HRMS; diabetes; free fatty acids; hydroxy fatty acids; oxo fatty acids
  28. Genes (Basel). 2022 May 13. pii: 878. [Epub ahead of print]13(5):
      Triple quadrupole mass spectrometry coupled to liquid chromatography (LC-TQ-MS) can detect and quantify modified nucleosides present in various types of RNA, and is being used increasingly in epitranscriptomics. However, due to the low resolution of TQ-MS and the structural complexity of the many naturally modified nucleosides identified to date (&gt;160), the discrimination of isomers and mass-analogs can be problematic and is often overlooked. This study analyzes 17 nucleoside standards by LC-TQ-MS with separation on three different analytical columns and discusses, with examples, three major causes of analyte misidentification: structural isomers, mass-analogs, and isotopic crosstalk. It is hoped that this overview and practical examples will help to strengthen the accuracy of the identification of modified nucleosides by LC-TQ-MS.
    Keywords:  HILIC; LC-MS/MS; RNA modification; epitranscriptomics; nucleoside misidentification; tRNA
  29. Pharmaceuticals (Basel). 2022 May 20. pii: 629. [Epub ahead of print]15(5):
      Pentacyclic triterpenoids (PCTs) are a widely distributed class of plant secondary metabolites. These compounds have high bioactive properties, primarily antitumor and antioxidant activity. In this study, a method was developed for the quantitative analysis of pentacyclic triterpenoids in plants using supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). Separation of ten major PCTs (friedelin, lupeol, β-amyrin, α-amyrin, betulin, erythrodiol, uvaol, betulinic, oleanolic and ursolic acids) was studied on six silica-based reversed stationary phases. The best results (7 min analysis time in isocratic elution mode) were achieved on an HSS C18 SB stationary phase using carbon dioxide-isopropanol (8%) mobile phase providing decisive contribution of polar interactions to the retention of analytes. It was shown that the use of atmospheric pressure chemical ionization (APCI) is preferred over atmospheric pressure photoionization (APPI). The combination of SFC with APCI-MS/MS mass spectrometry made it possible to achieve the limits of quantification in plant extracts in the range of 2.3-20 μg·L-1. The developed method was validated and tested in the analyses of birch outer layer (Betula pendula) bark, and licorice (Glycyrrhiza glabra) root, as well as lingonberry (Vaccinium vitis-idaea), cranberry (Vaccinium oxycoccos), apple (Malus domestica "Golden Delicious" and Malus domestica "Red Delicious") peels.
    Keywords:  pentacyclic triterpenoids; plant feedstock; supercritical fluid chromatography; tandem mass spectrometry
  30. Molecules. 2022 May 23. pii: 3358. [Epub ahead of print]27(10):
      The growing demand in natural matrices that represent a source of dietary and nutraceutical molecules has led to an increasing interest in Cannabis sativa, considered to be a multipurpose, sustainable crop. Particularly, the considerable content in essential fatty acids (FAs) makes its derived-products useful food ingredients in the formulation of dietary supplements. In this research, the FA and triacylglycerol (TAG) composition of hempseed oils and flours were investigated using gas chromatography coupled to mass spectrometry and flame ionization detection as well as liquid chromatography coupled to mass spectrometry (LC-MS), respectively. Furthermore, a recently introduced linear retention index (LRI) approach in LC was successfully employed as a useful tool for the reliable identification of TAG species. A total of 30 FAs and 62 glycerolipids were positively identified in the investigated samples. Relative quantitative analyses confirmed linoleic acid as the most abundant component (50-55%). A favorable omega6/omega3 ratio was also measured in hemp-derived products, with the α-linolenic acid around 12-14%. Whereas, γ-linolenic acid was found to be higher than 1.70%. These results confirm the great value of Cannabis sativa as a source of valuable lipids, and the further improvement of the LRI system paves the way for the automatization of the identification process in LC.
    Keywords:  GC-FID/MS; UHPLC-MS; fatty acids; hempseed flour; hempseed oil; industrial hemp; linear retention indices; lipids; triacylglycerols
  31. Anal Chem. 2022 May 25.
      Metabolomics and fluxomics are core approaches to directly profile and interrogate cellular metabolism in response to various genetic or environmental perturbations. In order to accurately measure the abundance and isotope enrichment of intracellular metabolites, cell culture samples must be rapidly harvested and cold quenched to preserve the in vivo metabolic state of the cells at the time of sample collection. When dealing with suspension cultures, this process is complicated by the need to separate the liquid culture media from cellular biomass prior to metabolite extraction. Here, we examine the efficacy of several commonly used metabolic quenching methods, using the model cyanobacterium Synechocystis sp. PCC 6803 as an example. Multiple 13C-labeled compounds, including 13C-bicarbonate, 13C-glucose, and 13C-glutamine, were used as tracers during the sample collection and the cold-quenching process to assess the extent of metabolic turnover after cells were harvested from culture flasks. We show that the combination of rapid filtration followed by 100% cold (-80 °C) methanol quenching exhibits the highest quenching efficiency, while mixing cell samples with a partially frozen 30% methanol slurry (-24 °C) followed by centrifugation is slightly less effective at quenching metabolism but enables less laborious sample processing. By contrast, rapidly mixing the cells with a saline ice slurry (∼0 °C) is less effective, as indicated by high isotope-labeling rates after sample harvest, while mixing the cells with 60% cold methanol (-65 °C) prior to centrifugation causes significant metabolite loss. This study demonstrates a rigorous, quantitative, and broadly applicable method for assessing the metabolic quenching efficacy of protocols used for sample collection in metabolomics and fluxomics studies.
  32. J Pharm Biomed Anal. 2022 May 06. pii: S0731-7085(22)00242-4. [Epub ahead of print]217 114821
      The use of small amounts of sample presents advantages in chromatographic analyses that have made this a current trend following the development of increasingly sensitive analytical techniques. Biological sample preparation methods, especially for rigid or semi-rigid matrices, are also under constant development, focusing on a more efficient extraction and in obtaining cleaner residues for analysis. In this context, the aim of this study was to present a validated a liquid chromatography-mass spectrometry (LC-MS) method for the quantification of famprofazone and its metabolites, methamphetamine and amphetamine in liver, using enzymatic cell dispersion promoted by collagenase, followed by protein precipitation and solid phase extraction (SPE) for sample extraction, concentration and clean-up. Potentially relevant variables for enzymatic cell dispersion concerning efficiency, such as enzyme concentration, temperature, buffering, agitation, and mechanical effect of stainless-steel spheres were assessed. Recovery evaluations were performed during the optimization of each step to ensure minimal loss of analytes. Linearity, the limit of detection (LOD) and limit of quantification (LOQ), stability, carryover, matrix effect, precision and bias were evaluated using fortified blank samples. An authentic sample was obtained from a controlled daily oral administration of 200 mg famprofazone to pigs for five days. The procedure was optimized for 500 mg of liver tissue, obtaining 99.9 ± 9.3% of digested collagen and 90.2 ± 1.7% of dispersed cells, without the tissue losses that usually ensue during crushing or grinding processes. Precision (CV%) was ≤ 10% and bias was ≤ 13% for all analytes. The LOQ was 5 ng/g for all analytes. The mean famprofazone concentration was 9.3 ± 0.53 ng/g, and mean metabolite concentrations were 16.7 ± 1.67 and 24.3 ± 1.36 ng/g for amphetamine and methamphetamine, respectively.
    Keywords:  Amphetamines; Collagenase; Enzymatic digestion; Famprofazone; Liver
  33. Toxins (Basel). 2022 May 04. pii: 328. [Epub ahead of print]14(5):
      Natural toxins include a wide range of toxic metabolites also occurring in food and products, thus representing a risk for consumer health. In the last few decades, several robust and sensitive analytical methods able to determine their occurrence in food have been developed. Liquid chromatography mass spectrometry is the most powerful tool for the simultaneous detection of these toxins due to its advantages in terms of sensitivity and selectivity. A comprehensive review on the most relevant papers on methods based on liquid chromatography mass spectrometry for the analysis of mycotoxins, alkaloids, marine toxins, glycoalkaloids, cyanogenic glycosides and furocoumarins in food is reported herein. Specifically, a literature search from 2011 to 2021 was carried out, selecting a total of 96 papers. Different approaches to sample preparation, chromatographic separation and detection mode are discussed. Particular attention is given to the analytical performance characteristics obtained in the validation process and the relevant application to real samples.
    Keywords:  analytical methods; food; liquid chromatography-mass spectrometry; natural toxins; simultaneous detection
  34. Biomed Chromatogr. 2022 May 22. e5415
      The determination of azithromycin in human plasma of pediatric patients was performed with a ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay. A simple sample preparation of protein precipitation was used and the separation was achieved on a C18 column by the gradient mixture of mobile phase A ( 0.1% acetic acid and 3 mM ammonium acetate in water) and the mobile phase B (0.1% acetic acid and 3 mM ammonium acetate in the solution of acetonitrile, methanol and water, 47.5/47.5/5, V/V/V). The multiple reaction monitoring (MRM) mode was adopted to monitor the precursor-to-product ion transitions of m/z 749.6→m/z 591.5 for azithromycin and m/z 753.6→m/z 595.5 for azithromycin-13 C-d3 (IS) at positive ionization mode. The calibration curve ranged between 0.5 ng·mL-1 and 500.0 ng·mL-1 and the correlation coefficient was greater than 0.99. The intra- and inter-batch precision was less than 13.7%. Accuracy determined at four concentrations ranged from 99.5% to 110.8%. The extraction recoveries were more than 95% and the matrix effects were 98% - 100%. The stability under various conditions were acceptable with the accuracy deviation within 9.2%. In conclusion, our method was simple, sensitive and reliable for quantification of azithromycin in plasma among pediatric patients.
    Keywords:  LC-MS/MS; azithromycin; interleukin-10; interleukin-6; polymorphism
  35. Curr Pharm Des. 2022 May 26.
      The use of High Resolution Mass Spectrometry (HRMS) has increased over the past decade in clinical and forensic toxicology, especially for comprehensive screening approaches. Despite this, few guidelines of this field have specifically addressed HRMS issues concerning compound identification, validation, measurement uncertainty and quality assurance. To fully implement this technique, certainly in an era in which the quality demands for laboratories are ever increasing due to various norms (e.g. the International Organization for Standardization's ISO 17025), these specific issues need to be addressed. This manuscript reviews 26 HRMS-based methods for qualitative systematic toxicological analysis (STA) published between 2011 and 2021. Key analytical data such as samples matrices, analytical platforms, numbers of analytes and employed mass spectral reference databases/libraries as well as the studied validation parameters are summarized and discussed. The article further includes a critical review of targeted and untargeted data acquisition approaches, available HRMS reference databases and libraries as well as current guidelines for HRMS data interpretation with a particular focus on identification criteria. Moreover, it provides an overview on current recommendations for the validation and determination measurement uncertainty of qualitative methods. Finally, the article aims to put forward suggestions for method development, compound identification, validation experiments to be performed, and adequate determination of measurement uncertainty for this type of wide-range qualitative HRMS-based methods.
    Keywords:  High resolution mass spectrometry; identification; measurement uncertainty; method development; quality assurance; systematic toxicological analysis.; validation
  36. J Chromatogr A. 2022 May 13. pii: S0021-9673(22)00339-9. [Epub ahead of print]1674 463146
      Lipophilicity can be measured with different methods, such as Shake-Flask or liquid chromatography. HPLC presents the advantage of overcoming solubility issues and therefore extending the range of lipophilicity to high values. A specific HPLC method, called ELogD, had been developed 20 years ago on a C16-amide stationary phase, enhancing hydrophobic and hydrogen bond interactions to mimic octanol-water partition. The emergence of novel stationary phases and the need for a less complex mobile phase have led to the development of a new HPLC assay called alphaLogD, applicable to neutral and basic compounds at pH 7.4, that combines superficially porous particles with a high number of equilibriums between solutes and stationary phase, leading to a lower number of isocratic methods to determine the logk'w at a higher throughput. Statistical studies have been run to successfully evaluate the alphaLogD method compared to the Shake-Flask method and to allow this lipophilicity measurement into the so-called Beyond-Rule-of-5-molecules space.
    Keywords:  Beyond-rule-of-5; High performance liquid chromatography; Lipophilicity; Shake-flask method; Superficially porous particle
  37. Electrophoresis. 2022 May 22.
      Migration time fluctuation strongly affects peak alignment and identification of unknown compounds, making migration time correction an essential step in capillary electrophoresis (CE)-based metabolomics. To obtain more reliable information, metabolites with different apparent mobilities are analyzed by tandem mass spectrometry. Applying a small pressure is a common practice for reducing the analysis time of anions in a positive mode CE, known as the pressure-assisted CE (PACE). However, applying pressure may reduce the separation efficiency and can be undesirable for cation analysis. A simple way to address this issue is to increase the pressure after a certain time, during the separation. We term this practice as dual pressure CE. However, changing the pressure during the CE separation complicates migration time correction. Previous migration time correction methods were established based on a consistent electroosmotic flow and a constant pressure driven bulk-flow velocity. We proposed a new correction method to support the peak alignment when dual pressure CE is used. A Python-based script was developed to implement dual pressure CE migration time correction for semi-targeted metabolomics study performed by a multiple reaction monitoring (MRM)-based method. This script can help select suitable endogenous metabolites as correction markers, perform migration time correction, and conduct peak alignment. A case study showed that migration time precision of 156 metabolites in 32 samples can be improved from 4.8 to 11.4%RSD to less than 1.8%RSD. This article is protected by copyright. All rights reserved.
    Keywords:  capillary electrophoresis mass spectrometry; dual pressure capillary electrophoresis; migration time correction; semi-targeted metabolomics
  38. Se Pu. 2022 Jun;40(6): 541-546
      Colon cancer (CC) is one of the most common malignant tumors worldwide. As there are no effective biomarkers for the early diagnosis and intervention tracking, the incidence of CC is increasing every year. Cholesterol is an important component of cell membrane, and it has been shown to be associated with CC. Oxysterol is an oxidized derivative of cholesterol, which plays an important role in many malignant tumors. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine serum cholesterol and ten oxysterol metabolites related to cholesterol in CC patients and healthy controls, and qualitative and quantitative analyses were carried out. Raw data were processed and analyzed using GraphPad Prism 8.3.0 and the MetaboAnalyst 5.0 platform ( To perform the independent sample t-test, it was necessary to ensure that all the sample data followed a normal distribution; therefore, the normal distribution test was performed in advance. The Mann-Whitney U test, which is a nonparametric test, was adopted for samples without a normal distribution. For the processed data, we used the statistical analysis function module of the MetaboAnalyst 5.0 platform to perform partial least-square discriminant analysis (PLS-DA) and orthogonal partial least-square discriminant analysis (OPLS-DA). Both PLS-DA and OPLS-DA are supervised discriminant analysis methods. The OPLS-DA model is based on the PLS-DA model and eliminates variables that are unrelated to the experiment. In both models, the samples from the two groups were well separated by the score plot. In the PLS-DA model, the horizontal and vertical coordinates of the score plot represent the interpretation rates of the principal components of the model. The horizontal coordinates show the differences between groups, and the vertical coordinates show the differences within groups. In addition to the score plot in the PLS-DA model, another crucial factor is variable importance in the projection (VIP). When VIP>1, the compound makes an important contribution to the model and is also used as a criterion for screening differential metabolites. Based on 10-fold cross-validation (CV) of the PLS-DA model, the performance of the model was the best when the number of components was three. To avoid overfitting of the data, three metabolic markers were selected by using not only the VIP values of metabolites of the PLS-DA model, but also the optimal compositions and K-mean clusters. The three biomarkers were 4β-hydroxycholesterol (4β-OHC), cholestane-3β,5α,6β-triol (Triol), and cholesterol. A receiver operating characteristic (ROC) curve was constructed. The area under the curve (AUC) was generally between 0.5 and 1.0. In the case of AUC>0.5, the closer the AUC is to 1, the better is the performance of the model. In this study, the area under the ROC curve constructed jointly by the three metabolic markers was 0.998, indicating that their combined ability to predict CC was strong and that the diagnostic performance was excellent. In addition, to understand the role of the three metabolic markers in the pathogenesis of CC, the genes associated with the metabolic markers were identified using GeneCards ( Finally, 110 genes were identified. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the biological processes, metabolic pathways, and possible roles in the body. GO enrichment showed that the three markers are mainly distributed in the endoplasmic reticulum lumen and coated vesicles, and they are mainly involved in biological processes such as cholesterol metabolism, transportation, and low-density lipoprotein particle remodeling. Their molecular functions are cholesterol transfer activity and low-density lipoprotein particle receptor binding. KEGG pathway analysis showed that biomarkers are enriched in steroid biosynthesis, PPAR (peroxisome proliferator-activated receptor) signaling pathways, and ABC (ATP-binding cassette) transport pathways. The results of this study are helpful to understand the role of cholesterol and oxysterol in the pathogenesis of CC and to elucidate the pathogenesis of CC.
    Keywords:  colon cancer (CC); liquid chromatography-tandem mass spectrometry (LC-MS/MS); metabolomics; oxysterol
  39. J Am Soc Mass Spectrom. 2022 May 24.
      Solid-phase microextraction (SPME)-direct mass spectrometry (MS) has proven to be an efficient tool for the rapid screening and quantitation of target compounds at trace levels. However, it is challenging to perform screening using both positive and negative modes in one analytical run without compromising scanning speed and detection sensitivity. To take advantage of the special geometry of a coated blade spray (CBS) blade, which consists of two flat sides coated with the same SPME coating, we developed a CBS-MS method that enables desorption and ionization to be performed in positive ionization mode on one side of a coated blade and negative ionization mode on the other side of the same blade. By simply flipping the blade 180°, MS analysis in both ionization modes on different sides can be completed in 40 s. Combining this approach with an automated Concept 96-blade-based SPME system allowed analysis for one sample in positive and negative modes to be completed in less than 1 min. The workflow was optimized by using a biocompatible polyacrylonitrile as an undercoating layer and a binder of polyacrylonitrile/hydrophilic-lipophilic balance (HLB) particles, which enabled the rapid analysis of 20 drugs of abuse in saliva samples in both positive and negative modes. The proposed method provided low limits of quantification (between 0.005 and 10 ng/mL), with calibration linear correlation coefficients ⩾ 0.9925, accuracy between 72% and 126%, and relative precision < 15% for three validation points.