bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒05‒15
nineteen papers selected by
Sofia Costa

  1. Bioinformatics. 2022 May 13. 38(10): 2872-2879
      MOTIVATION: Mass spectrometry-based untargeted lipidomics aims to globally characterize the lipids and lipid-like molecules in biological systems. Ion mobility increases coverage and confidence by offering an additional dimension of separation and a highly reproducible metric for feature annotation, the collision cross-section (CCS).RESULTS: We present a data processing workflow to increase confidence in molecular class annotations based on CCS values. This approach uses class-specific regression models built from a standardized CCS repository (the Unified CCS Compendium) in a parallel scheme that combines a new annotation filtering approach with a machine learning class prediction strategy. In a proof-of-concept study using murine brain lipid extracts, 883 lipids were assigned higher confidence identifications using the filtering approach, which reduced the tentative candidate lists by over 50% on average. An additional 192 unannotated compounds were assigned a predicted chemical class.
    AVAILABILITY AND IMPLEMENTATION: All relevant source code is available at
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
  2. Anal Chem. 2022 May 11.
      Rapid and accurate metabolite annotation in mass spectrometry imaging (MSI) can improve the efficiency of spatially resolved metabolomics studies and accelerate the discovery of reliable in situ disease biomarkers. To date, metabolite annotation tools in MSI generally utilize isotopic patterns, but high-throughput fragmentation-based identification and biological and technical factors that influence structure elucidation are active challenges. Here, we proposed an organ-specific, metabolite-database-driven approach to facilitate efficient and accurate MSI metabolite annotation. Using data-dependent acquisition (DDA) in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to generate high-coverage product ions, we identified 1620 unique metabolites from eight mouse organs (brain, liver, kidney, heart, spleen, lung, muscle, and pancreas) and serum. Following the evaluation of the adduct form difference of metabolite ions between LC-MS and airflow-assisted desorption electrospray ionization (AFADESI)-MSI and deciphering organ-specific metabolites, we constructed a metabolite database for MSI consisting of 27,407 adduct ions. An automated annotation tool, MSIannotator, was then created to conduct metabolite annotation in the MSI dataset with high efficiency and confidence. We applied this approach to profile the spatially resolved landscape of the whole mouse body and discovered that metabolites were distributed across the body in an organ-specific manner, which even spanned different mouse strains. Furthermore, the spatial metabolic alteration in diabetic mice was delineated across different organs, exhibiting that differentially expressed metabolites were mainly located in the liver, brain, and kidney, and the alanine, aspartate, and glutamate metabolism pathway was simultaneously altered in these three organs. This approach not only enables robust metabolite annotation and visualization on a body-wide level but also provides a valuable database resource for underlying organ-specific metabolic mechanisms.
  3. J Exp Bot. 2022 May 13. 73(9): 2938-2952
      Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography-mass spectrometry method for analysing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The liquid chromatography method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared with a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion enhancement or low ion suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyse intermediates more comprehensively in central metabolism of plant tissues.
    Keywords:  Central metabolism; ion suppression and enhancement; isomer separation; isotopic labeling; liquid chromatography–mass spectrometry; metabolite quantification; mixed-mode column chromatography; oilseeds
  4. J Agric Food Chem. 2022 May 12.
      Short-chain fatty acids (SCFAs) are major gut microbiota-derived metabolites, which can reshape the intestine and regulate gut immunity. The application of conventional GC methods has been hampered for quantifying low-concentrated SCFAs, such as in serum, saliva, and digesta of germ-free animals. Herein, we established a LC-MS method to quantify SCFAs after 5-(dimethylamino)-1-carbohydrazide-isoquinoline (DMAQ) derivatization. The DMAQ derivatization significantly enhanced the detection sensitivity and improved separation of SCFAs. 2-methylbutyric acid and 3-methylbutyric acid were separately quantitated. Moreover, the matrix effect was diminished using DMAQ-13C/15N-tagged SCFAs as internal standards. The established quantitation method was successfully applied in the analysis of plasma and cecum digesta collected from neonatal piglets, revealing that significant increases in biological SCFA contents in cecum digesta were closely related to the variation of gut microbial diversity. The established quantitation method is capable of sensitively and comprehensively quantifying SCFAs that may provide insights into underlying gut-microbiota functions.
    Keywords:  chemical derivatization; gut microbiota-derivatized metabolites; isotope-coded derivatization reagents; short-chain fatty acid
  5. Anal Chim Acta. 2022 May 22. pii: S0003-2670(22)00385-3. [Epub ahead of print]1208 339814
      Metabolism studies are one of the important steps in pharmaceutical research. LC-MS combined with metabolomics data-processing approaches have been developed for rapid screening of drug metabolites. Mass defect filter (MDF) is one of the LC/MS-based metabolomics data processing approaches and has been applied to screen drug metabolites. Although MDF can remove most interference ions from an incubation sample, the true positive rate of the retaining ions is relatively low (approximately 10%). To improve the efficacy of MDF, we developed a two-stage data-processing approach by combining MDF and stable isotope tracing (SIT) for metabolite identification. Pioglitazone (PIO), which is an antidiabetic drug used to treat type 2 diabetes mellitus, was taken as an example drug. Our results demonstrated that this new approach could substantially increase the validated rate from about 10% to 74%. Most of these validated metabolite signals (13/14) could be verified as PIO structure-related metabolites. In addition, we applied this approach to identify uncommon metabolite signals (a mass change beyond the window of 50 Da around its parent drug, MDF1). SIT could remove most interference ions (approximately 98%) identified by MDF1, and four out of five validated metabolite signals could be verified as PIO structure-related metabolites. Interestingly, a lot of the verified metabolites (10/17) were novel PIO metabolites. Among these novel metabolites, nine were thiazolidinedione ring-opening signals that might be related to the toxicity of PIO. Our developed approach could significantly improve the efficacy in drug metabolite identification compared with that of MDF.
    Keywords:  High-resolution mass spectrometry; Mass defect filter; Pioglitazone; Stable isotope tracing
  6. FASEB J. 2022 May;36 Suppl 1
      A sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of mycophenolic mofetil (MMF) and its major metabolites, mycophenolic acid (MPA), mycophenolic acyl glucuronide (AcMPAG), and mycophenolic glucuronide (MPAG), in rat plasma. Upon administration, mycophenolic mofetil (MMF), a prodrug, undergoes extensive metabolism in the liver. Following hydrolyzation, the active metabolite MPA is produced. MPA can then be further metabolized to produce its glucuronide conjugated metabolites, AcMPAG and MPAG. This method can simultaneously determine both the parent compound and its major metabolites within biological samples. In addition, liver S9 fraction hydrolysis studies of MMF results in the production of MPA, demonstrating that the parent drug is first hydrolyzed within the liver. A novel UPLC method was developed to simultaneously quantify both compounds following S9 liver enzyme hydrolysis. A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Separation was achieved using an Ultra Biphenyl 5µm column (100 × 2.1mm) with acetonitrile and 0.1% formic acid as the mobile phases. Analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. Additionally, to determine the hydrolysis rate, different concentrations of MMF were incubated with liver S9 fraction (0.5µg/ml final concentration) for 30 mins, after which the reaction was terminated using 6% formic acid in acetonitrile. Samples were prepared and MPA concentrations were determined in the reaction system using UPLC for detection and quantification. The method was linear in the range of 9.77nM - 5000nM with correlation coefficient values > 0.99 for all components. The method has been shown to be reproducible, with intra- and inter-day accuracy and precision ±12.3% of nominal values, for all analytes. The average extraction recovery rates for MMF, MPA, AcMPAG, and MPAG were 91.3%, 97.9%, 98.4%, and 90.5%, respectively. Matrix effect was in the acceptable range (<15%). The analytes in plasma were found to be stable under bench -top, freeze-thaw, and storage (-4°C) conditions. The metabolic studies showed that mycophenolic mofetil can be rapidly hydrolyzed by rat liver S9 fraction with Vmax and Km values of 0.66±0.78 nmol/min/µg and 80.06±123.1µM respectively. This novel UPLC-MS/MS method can comprehensively evaluate and quantify the concentration of both mycophenolic mofetil and its major metabolites, mycophenolic acid, mycophenolic acyl glucuronide, and mycophenolic glucuronide in biological samples. This method can be used as a tool to further investigate and understand the extensive metabolism of mycophenolic mofetil in clinical practice.
  7. J Proteome Res. 2022 May 10.
      Liquid chromatography coupled to mass spectrometry is a key metabolomics/metabonomics technology. Reversed-phase liquid chromatography (RPLC) is very widely used as a separation step, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alternative methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. The combined method was assessed by analyzing a set of liver extracts from aged male and female mice that had been treated with the polyphenol compound ampelopsin. Not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the direction of metabolite change was opposite for males and females.
    Keywords:  NMR spectroscopy; UPLC-MS; ampelopsin; healthy aging; ion-pairing; metabolomics; metabonomics; statistical heterospectroscopy
  8. Int J Environ Res Public Health. 2022 Apr 19. pii: 4966. [Epub ahead of print]19(9):
      The extensive use of herbicides, such as glyphosate and glufosinate, in crop production during recent decades has raised concerns about human exposure. Nevertheless, analysis of trace levels of these herbicides in human biospecimens has been challenging. Here, we describe a method for the determination of urinary glyphosate, its degradation product aminomethylphosphonic acid (AMPA), and glufosinate using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was optimized using isotopically labelled internal standards (13C2, 15N-glyphosate, 13C, 15N, D2-AMPA, and D3-glufosinate) and solid-phase extraction (SPE) with cation-exchange and anion-exchange cartridges. The method provides excellent chromatographic retention, resolution and peak shape of target analytes without the need for strong acidic mobile phases and derivatization steps. The instrument linearity was in the range of 0.1-100 ng/mL, with R &gt; 0.99 in the matrix for all analytes. The method detection limits (MDLs) and the method quantification limits (MQLs) were in the ranges of 0.12 (AMPA and glufosinate)-0.14 (glyphosate) ng/mL and 0.40 (AMPA)-0.48 (glyphosate) ng/mL, respectively. The recoveries of analytes spiked into urine matrix ranged from 79.1% to 119%, with coefficients of variation (CVs) of 4-10%. Repeated analysis of samples for over 2 weeks showed intra-day and inter-day analytical variations of 3.13-10.8% and 5.93-12.9%, respectively. The matrix effects for glyphosate, AMPA, and glufosinate spiked into urine matrix averaged -14.4%, 13.2%, and 22.2%, respectively. The method was further validated through the analysis of external quality assurance proficiency test (PT) urine samples. The method offers optimal sensitivity, accuracy, and precision for the urine-based assessment of human exposure to glyphosate, AMPA, and glufosinate.
    Keywords:  LC–MS/MS; aminomethylphosphonic acid; glufosinate; glyphosate; urine
  9. Molecules. 2022 Apr 30. pii: 2864. [Epub ahead of print]27(9):
      Periodontology is a newer field relative to other areas of dentistry. Remarkable progress has been made in recent years in periodontology in terms of both research and clinical applications, with researchers worldwide now focusing on periodontology. With recent advances in mass spectrometry technology, metabolomics research is now widely conducted in various research fields. Metabolomics, which is also termed metabolomic analysis, is a technology that enables the comprehensive analysis of small-molecule metabolites in living organisms. With the development of metabolite analysis, methods using gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, capillary electrophoresis-mass spectrometry, etc. have progressed, making it possible to analyze a wider range of metabolites and to detect metabolites at lower concentrations. Metabolomics is widely used for research in the food, plant, microbial, and medical fields. This paper provides an introduction to metabolomic analysis and a review of the increasing applications of metabolomic analysis in periodontal disease research using mass spectrometry technology.
    Keywords:  GC-MS; LC-MS/MS; metabolomics research; periodontal disease; periodontitis; proteomic analysis
  10. Lipids Health Dis. 2022 May 10. 21(1): 42
      BACKGROUND: Monoacetyldiglycerides (MAcDG), are acetylated triglycerides (TG) and an emerging class of bioactive or functional lipid with promising nutritional, medical, and industrial applications. A major challenge exists when analyzing MAcDG from other subclasses of TG in biological matrices, limiting knowledge on their applications and metabolism.METHODS: Herein a multimodal analytical method for resolution, identification, and quantitation of MAcDG in biological samples was demonstrated based on thin layer chromatography-flame ionization detection complimentary with C30-reversed phase liquid chromatography-high resolution accurate mass tandem mass spectrometry. This method was then applied to determine the MAcDG molecular species composition and quantity in E. solidaginis larvae. The statistical method for analysis of TG subclass composition and molecular species composition of E. solidaginis larvae was one-way analysis of variance (ANOVA).
    RESULTS: The findings suggest that the proposed analytical method could simultaneously provide a fast, accurate, sensitive, high throughput analysis of MAcDG from other TG subclasses, including the fatty acids, isomers, and molecular species composition.
    CONCLUSION: This method would allow for MAcDG to be included during routine lipidomics analysis of biological samples and will have broad interests and applications in the scientific community in areas such as nutrition, climate change, medicine and biofuel innovations.
    Keywords:  Acylated triglyceride; Eurosta solidaginis; Functional lipids; Liquid chromatography; Mass spectrometry; Thin layer chromatography; Triglyceride analysis
  11. Crit Rev Anal Chem. 2022 May 09. 1-17
      Metabolomics aims to profile the extensive array of metabolites that exists in different types of matrices using modern analytical techniques. These techniques help to separate, identify, and quantify the plethora of chemical compounds at various analytical platforms. Hence, ion mobility spectrometry (IMS) has emerged as an advanced analytical approach, exclusively owing to the 3D separation of metabolites and their isomers. Furthermore, separated metabolites are identified based on their mass fragmentation pattern and CCS (collision cross-section) values. The IMS provides an advanced alternative dimension to separate the isomeric metabolites with enhanced throughput with lesser chemical noise. Thus, the present review highlights the types, factors affecting the resolution, and applications of IMMS (Ion mobility mass spectrometry) for isomeric separations, and ionic contaminants in the plant samples. Furthermore, an overview of IMS-based applications for the identification of plant metabolites (volatile and non-volatile) over the last few decades has been discussed, followed by future assumptions for creating IM-based databases. Such approaches could be significant to accelerate and improve our knowledge of the vast chemical diversity found in plants.
    Keywords:  Collision cross-section; drift tubes; ion mobility; metabolites; metabolomics; plants
  12. FASEB J. 2022 May;36 Suppl 1
      BACKGROUND: Bile is an important liver secretion which aids the digestion of fats in the intestine. Bile acids are the major organic solute component of bile and are synthesized from cholesterol in the liver and stored in the gallbladder. After secretion in the intestine, bile acids are re-absorbed in the distal small intestine (ileum). Imbalances in the reabsorption of bile acids can lead to bile acid diarrhea when bile acid malabsorption occurs. Conversely, when there is an increase in bile acid absorption hepatic disorders such as nonalcoholic fatty liver disease can arise. A wide array of distinct bile acid structures have been identified in vertebrates. Developing and refining experimental tools to identify the role of bile acids in health and disease will enhance our ability to treat complex disorders impacting multiple organ systems.METHODS & RESULTS: We have expanded our existing reverse-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics method to accommodate a simple-to-use standards kit that includes reference standards for 16 bile acids/salts and their corresponding deuterated isotopologs as internal standards. LC-MS/MS method enhancements include scheduled Selected-Reaction Monitoring (sSRM) to reduce instrument duty cycle, and carefully tailored chromatographic conditions to resolve the following groups of structurally similar or isobaric bile acids/salts: i) Glycocholic Acid (GCA) and Taurocholic Acid (TCA); ii) Lithocholic Acid (LCA), Glycolithocholic Acid (GLCA), and Taurolithocholic Acid (TLCA); iii) isobaric Cholic Acid (CA) and beta-Muricholic Acid (b-MCA); iv) isobaric Ursodeoxycholic Acid (UDCA), Chenodeoxycholic Acid (CDCA), and Deoxycholic Acid (DCA); v) isobaric Glycoursodeoxycholic Acid (GUDCA), Glycochenodeoxycholic Acid (GCDCA), and Glycodeoxycholic Acid (GDCA); and vi) isobaric Tauroursodeoxycholic Acid (TUDCA), Taurochenodeoxycholic Acid (TCDCA), and Taurodeoxycholic Acid (TDCA). Our tissue processing and metabolite extraction procedures have been optimized for hepatic and fecal tissues. Individuals with inactivating mutations in the molecular motor, Myosin Vb, have severe diarrhea and often present with liver cholestasis. We used an animal model of loss of Myosin Vb to determine alterations in bile acid composition in the distal small intestine and liver. Whole segments of the ileum and liver were processed in methanol and analyzed by LC-MS/MS.
    CONCLUSION: We have developed a targeted metabolomics method to accurately identify and quantify bile acids by LC-MS/MS and this technique can be used to investigate changes in bile acid composition in gastrointestinal disorders.
  13. Plants (Basel). 2022 May 02. pii: 1234. [Epub ahead of print]11(9):
      The detection of chemical species and understanding their respective localisations in tissues have important implications in plant science. The conventional methods for imaging spatial localisation of chemical species are often restricted by the number of species that can be identified and is mostly done in a targeted manner. Mass spectrometry imaging combines the ability of traditional mass spectrometry to detect numerous chemical species in a sample with their spatial localisation information by analysing the specimen in a 2D manner. This article details the popular mass spectrometry imaging methodologies which are widely pursued along with their respective sample preparation and the data analysis methods that are commonly used. We also review the advancements through the years in the usage of the technique for the spatial profiling of endogenous metabolites, detection of xenobiotic agrochemicals and disease detection in plants. As an actively pursued area of research, we also address the hurdles in the analysis of plant tissues, the future scopes and an integrated approach to analyse samples combining different mass spectrometry imaging methods to obtain the most information from a sample of interest.
    Keywords:  chemical imaging; mass spectrometry; plant
  14. J Inherit Metab Dis. 2022 May 11.
      Untargeted metabolomics (UM) allows for the simultaneous measurement of hundreds of metabolites in a single analytical run. The sheer amount of data generated in UM hampers its use in patient diagnostics because manual interpretation of all features is not feasible. Here, we describe the application of a pathway-based metabolite set enrichment analysis method (MSEA) to prioritise relevant biological pathways in UM data. We validate our method on a set of 55 patient samples with a diagnosed inherited metabolic disorder (IMD), and show that it complements feature-based prioritisation of biomarkers by placing the features in a biological context. In addition, we find that by taking enriched pathways shared across different IMDs we can identify common drugs and compounds that could otherwise obscure genuine disease biomarkers in an enrichment method. Finally, we demonstrate the potential of this method to identify novel candidate biomarkers for known IMDs. Our results show the added value of pathway-based interpretation of UM data in IMD diagnostics context.
    Keywords:  Biochemical pathways; Biomarkers; Cystathionine ß-synthase; Inborn errors of metabolism; Inherited metabolic disorders; Mass spectrometry; Metabolite set enrichment analysis; Next generation metabolic screening; Untargeted metabolomics
  15. Sci Rep. 2022 May 13. 12(1): 7933
      The AbsoluteIDQ p400 HR kit is a commercial product for targeted metabolomics. While the kit has been validated for human plasma and serum, adherent cell lysates have not yet been evaluated. We have optimized the detection of polar and lipid metabolites in cell lysates using the kit to enable robust and repeatable analysis of the detected metabolites. Parameters optimized include total cell mass, loading volume and extraction solvent. We present a cell preparation and analytical method and report on the performance of the kit with regard to detectability of the targeted metabolites and their repeatability. The kit can be successfully used for a relative quantification analysis of cell lysates from adherent cells although validated only for human plasma and serum. Most metabolites are below the limit of the Biocrates' set quantification limits and we confirmed that this relative quantification can be used for further statistical analysis. Using this approach, up to 45% of the total metabolites in the kit can be detected with a reasonable analytical performance (lowest median RSD 9% and 13% for LC and FIA, respectively) dependent on the method used. We recommend using ethanol as the extraction solvent for cell lysates of osteosarcoma cell lines for the broadest metabolite coverage and 25 mg of cell mass with a loading volume of 20 µL per sample.
  16. Electrophoresis. 2022 May 12.
      The composition of wine is determined by a complex interaction between environmental factors, genetic factors (i.e., grape varieties) and winemaking practices (including technology and storage). Metabolomics using NMR spectroscopy, GC-MS and/or LC-MS has shown to be a useful approach for assessing the origin, authenticity and quality of various wines. Nonetheless, the use of additional analytical techniques with complementary separation mechanisms may aid in the deeper understanding of wine's metabolic processes. In this study, we demonstrate that capillary electrophoresis-mass spectrometry (CE-MS) is a very suitable approach for the efficient profiling of polar ionogenic metabolites in wines. Without using any sample preparation or derivatization, wine was analyzed using a 10 minute CE-MS workflow with interday RSD values for 31 polar and charged metabolites below 3.8% and 23% for migration times and peak areas, respectively. The utility of this workflow for the global profiling of polar ionogenic metabolites in wine was evaluated by analyzing different cool-climate Polish wine samples. This article is protected by copyright. All rights reserved.
    Keywords:  capillary electrophoresis; mass spectrometry; metabolomics; polish wines
  17. Molecules. 2022 Apr 22. pii: 2702. [Epub ahead of print]27(9):
      Catecholamines (CAs) and their metabolites play significant roles in many physiological processes. Changes in CAs concentration in vivo can serve as potential indicators for the diagnosis of several diseases such as pheochromocytoma and paraganglioma. Thus, the accurate quantification of CAs and their metabolites in biological samples is quite important and has attracted great research interest. However, due to their extremely low concentrations and numerous co-existing biological interferences, direct analysis of these endogenous compounds often suffers from severe difficulties. Employing suitable sample preparation techniques before instrument detection to enrich the target analytes and remove the interferences is a practicable and straightforward approach. To date, many sample preparation techniques such as solid-phase extraction (SPE), and liquid-liquid extraction (LLE) have been utilized to extract CAs and their metabolites from various biological samples. More recently, several modern techniques such as solid-phase microextraction (SPME), liquid-liquid microextraction (LLME), dispersive solid-phase extraction (DSPE), and chemical derivatizations have also been used with certain advanced features of automation and miniaturization. There are no review articles with the emphasis on sample preparations for the determination of catecholamine neurotransmitters in biological samples. Thus, this review aims to summarize recent progress and advances from 2015 to 2021, with emphasis on the sample preparation techniques combined with separation-based detection methods such capillary electrophoresis (CE) or liquid chromatography (LC) with various detectors. The current review manuscript would be helpful for the researchers with their research interests in diagnostic analysis and biological systems to choose suitable sample pretreatment and detection methods.
    Keywords:  catecholamines; chemical derivatization; chromatography; sample preparation
  18. J Am Soc Mass Spectrom. 2022 May 13.
      Vitamin D compounds are a group of secosteroids derived from cholesterol that are vital for maintaining bone health in humans. Recent studies have shown extraskeletal effects of vitamin D, involving vitamin D metabolites such as the dihydroxylated vitamin D3 compounds 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. Differentiation and characterization of these isomers by mass spectrometry can be challenging due to the zero-mass difference and minor structural differences between them. The isomers usually require separation by liquid chromatography (LC) prior to mass spectrometry, which adds extra complexity to the analysis. Herein, we investigated and revisited the use of fragmentation methods such as collisional induced dissociation (CID), infrared multiphoton dissociation (IRMPD), electron induced dissociation (EID), and ultraviolet photodissociation (UVPD), available on a 12T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) to generate characteristic fragments for the dihydroxylated vitamin D3 isomers that can be used to distinguish between them. Isomer-specific fragments were observed for the 1,25-dihydroxyvitamin D3, which were clearly absent in the 24,25-dihydroxyvitamin D3 MS/MS spectra using all fragmentation methods mentioned above. The fragments generated due to cleavage of the C-6/C-7 bond in the 1,25-dihydroxyvitamin D3 compound demonstrate that the fragile OH groups were retained during fragmentation, thus enabling differentiation between the two dihydroxylated vitamin D3 isomers without the need for prior chromatographic separation or derivatization.
  19. FASEB J. 2022 May;36 Suppl 1
      We describe the design and synthesis for neutron encoded (NeuCode) tags for highly multiplexed targeted sample analysis for use in high resolution mass spectrometry (HRMS) based metabolomics. A set of aldehyde and carboxylic acid reactive tags were synthesized with a quaternary ammonium polar head group which limits off-target ionization and promotes coelution of differentially tagged metabolites. A four carbon linker allowed us to synthesize highly multiplexed NeuCode tags by providing many areas for isotope substitution and the obtained amine tags can be easily converted to other functional groups to target a wide array of metabolites. Tags were used to analyze levels of targeted metabolites in differentially treated cell lysates producing tagged samples which were mixed 1:1 and run in a single LC-MS injection.