bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒10‒23
twelve papers selected by
Sofia Costa

  1. J Chromatogr A. 2022 Oct 08. pii: S0021-9673(22)00747-6. [Epub ahead of print]1684 463556
      In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involving the calculation of individual metabolite recoveries (pre/post extraction spiking U-13C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI- mode. One hundred sixty-one (161) metabolites were successfully detected in ESI+ mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three different biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U-13C-labeled Pichia pastoris (Komagataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter- and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process efficiencies were evaluated following the Matuszewski protocol with U-13C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts.
    Keywords:  13C-cell extract; Hydrophilic interaction liquid chromatography; Stable isotope-labelled internal standards; Targeted metabolomics; UHPLC
  2. J Lipid Res. 2022 Oct 17. pii: S0022-2275(22)00135-3. [Epub ahead of print] 100302
      Oxylipins are important biological regulators that have received extensive research attention. Due to the extremely low concentrations, large concentration variations, and high structural similarity of many oxylipins, the quantitative analysis of oxylipins in biological samples is always a great challenge. Here, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method with high sensitivity, wide linearity, and acceptable resolution for quantitative profiling of oxylipins in multiple biological samples. A total of 104 oxylipins, some with a high risk of detection crosstalk, were well separated on a 150 mm column over 20 min. The method showed high sensitivity with lower limits of quantitation for 87 oxylipins, reaching 0.05-0.5 pg. Unexpectedly, we found that the linear range for 16, 18, and 17 oxylipins reached 10,000, 20,000, and 40,000 folds, respectively. Due to the high sensitivity, while reducing sample consumption to below half the volume of previous methods, 74, 78, and 59 low-abundance oxylipins, among which some were difficult to detect like lipoxins and resolvins, were well quantified in the tested mouse plasma, mouse liver, and human plasma samples, respectively. Additionally, we determined that analytes with multifarious concentrations of over a thousand-fold difference could be well quantified simultaneously due to the wide linearity. In conclusion, most likely due to the instrumental advancement, this method effectively improves the quantitative sensitivity and linear range over existing methods, which will facilitate and advance the study of the physiological and pathophysiological functions of oxylipins.
    Keywords:  Biological regulators; Linear range; Lipoxins; Liquid chromatography-tandem mass spectrometry; Lower limits of quantitation; Plasma samples; Quantitative profiling; Quantitative sensitivity; Reduced sample consumption; Resolvins
  3. J Proteome Res. 2022 Oct 20.
      Lipids play a key role in many biological processes, and their accurate measurement is critical to unraveling the biology of diseases and human health. A high throughput HILIC-based (LC-MS) method for the semiquantitative screening of over 2000 lipids, based on over 4000 MRM transitions, was devised to produce an accessible and robust lipidomic screen for phospholipids in human plasma/serum. This methodology integrates many of the advantages of global lipid analysis with those of targeted approaches. Having used the method as an initial "wide class" screen, it can then be easily adapted for a more targeted analysis and quantification of key, dysregulated lipids. Robustness was assessed using 1550 continuous injections of plasma extracts onto a single column and via the evaluation of columns from 5 different batches of stationary phase. Initial screens in positive (239 lipids, 431 MRM transitions) and negative electrospray ionization (ESI) mode (232 lipids, 446 MRM transitions) were assessed for reproducibility, sensitivity, and dynamic range using analysis times of 8 min. The total number of lipids monitored using these screening methods was 433 with an overlap of 38 lipids in both modes. A polarity switching method for accurate quantification, using the same LC conditions, was assessed for intra- and interday reproducibility, accuracy, dynamic range, stability, carryover, dilution integrity, and matrix interferences and found to be acceptable. This polarity switching method was then applied to lipids important in the stratification of human prostate cancer samples.
    Keywords:  high throughput; lipidomics; plasma; prostate cancer; quantification; serum
  4. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Oct 06. pii: S1570-0232(22)00399-3. [Epub ahead of print]1211 123495
      Cannabis remains one of the most commonly used psychotropes. Cannabis use is frequently evaluated via the testing of suspected patient samples. Thus, there is a high demand for simple, accurate and fast assays to support the increasing needs for testing. This report highlights a reliable, simple and fast liquid chromatography - tandem mass spectrometry assay that quantifies the cannabis metabolites THC-COOH and THC-COO(Gluc) in human urine. The assay employs a direct dilute-and-shoot approach, whereby urine samples are diluted 10X before being directly injected on the liquid chromatography and mass spectrometer. The assay quantification is based on an internal calibration approach that used deuterated analogues for THC-COOH and THC-COO(Gluc) as internal standards. The assay's analysis time was 5 min. The quantification was valid over a wide linear range (25 - 8,000 ng/mL) for both analytes and was free of matrix interferences. The within-day and between-day precision was determined to be ≤ 15 % CV for both analytes. The assay was validated based on the College of American Pathologists (CAP) and Clinical Laboratory Standards Institute (CLSI) guidelines.
    Keywords:  Dilute and shoot; LC-MS/MS; Quantitation; THC; Toxicology; Urine
  5. Biomed Chromatogr. 2022 Oct 21. e5531
      Targeted mass spectrometry is extensively used for the quantitative measurement of various molecules present in complex matrices. It is certainly one of the most important analytical duties in a mass spectrometry laboratory. Systematic development of selected-reaction monitoring (SRM), multiple-reaction monitoring (MRM) and parallel-reaction monitoring (PRM) methods for targeted mass spectrometry-based analysis were performed without considering future opportunities. The advancement of hardware and software technologies have resulted in more resolution, more accuracy, more speed and more depth. For sure, SRM, MRM or PRM acquisitions can quantify molecules very accurately at trace levels. However, it does not provide datasets allowing future data mining. Obviously, we cannot truly quantify something that we don't know is there. However, using non-targeted data acquisition for target analysis (nDATA), we can generate a MS1 and MS2 digital libraries of each sample providing future proof datasets. This is instrumental for data mining following new questions potentially arising in time permitting new and deeper processing and interpretation. This perspective article provides thoughts on why we believe it is time to question the status quo in targeted mass spectrometry.
    Keywords:  Bioanalysis; Data-dependent acquisition; Data-independent acquisition; Mass spectrometry; Metabolomics; Proteomics
  6. Adv Sci (Weinh). 2022 Oct 17. e2203339
      Mass spectrometry imaging (MSI) is a powerful analytical technique that generates maps of hundreds of molecules in biological samples with high sensitivity and molecular specificity. Advanced MSI platforms with capability of high-spatial resolution and high-throughput acquisition generate vast amount of data, which necessitates the development of computational tools for MSI data analysis. In addition, computation-driven MSI experiments have recently emerged as enabling technologies for further improving the MSI capabilities with little or no hardware modification. This review provides a critical summary of computational methods and resources developed for MSI data analysis and interpretation along with computational approaches for improving throughput and molecular coverage in MSI experiments. This review is focused on the recently developed artificial intelligence methods and provides an outlook for a future paradigm shift in MSI with transformative computational methods.
    Keywords:  artificial intelligence; computational methods; data mining; data-driven experiments; mass spectrometry imaging
  7. Handb Exp Pharmacol. 2022 Oct 22.
      While NMR-based metabolomics is only about 20 years old, NMR has been a key part of metabolic and metabolism studies for >40 years. Historically, metabolic researchers used NMR because of its high level of reproducibility, superb instrument stability, facile sample preparation protocols, inherently quantitative character, non-destructive nature, and amenability to automation. In this chapter, we provide a short history of NMR-based metabolomics. We then provide a detailed description of some of the practical aspects of performing NMR-based metabolomics studies including sample preparation, pulse sequence selection, and spectral acquisition and processing. The two different approaches to metabolomics data analysis, targeted vs. untargeted, are briefly outlined. We also describe several software packages to help users process NMR spectra obtained via these two different approaches. We then give several examples of useful or interesting applications of NMR-based metabolomics, ranging from applications to drug toxicology, to identifying inborn errors of metabolism to analyzing the contents of biofluids from dairy cattle. Throughout this chapter, we will highlight the strengths and limitations of NMR-based metabolomics. Additionally, we will conclude with descriptions of recent advances in NMR hardware, methodology, and software and speculate about where NMR-based metabolomics is going in the next 5-10 years.
    Keywords:  Applications; Experimental methods; NMR spectroscopy; Targeted metabolomics; Untargeted metabolomics
  8. Anal Bioanal Chem. 2022 Oct 18.
      A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of ginsenosides in three Panax ginseng reference materials (RMs). Extraction procedures were optimized to recover neutral and malonyl-ginsenosides using a methanol-water extraction under basic conditions. Optimized mass fragmentation transitions were obtained for the development of a multiple reaction monitoring (MRM) detection method with electrospray ionization in negative and positive ion mode. Mass fraction values were determined for ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 in the three ginseng materials (rhizomes, extract, and an oral dosage form). Quantitation of these seven compounds was accomplished with 4-methylestradiol and SRM 3389 Ginsenoside Calibration Solution serving as an internal standard (IS) and calibration standards, respectively. Mass fraction values for the seven ginsenosides ranged from 1.27 mg/g to 21.42 mg/g, 3.25 mg/g to 35.81 mg/g, and 0.56 mg/g to 2.51 mg/g for SRM 3384, SRM 3385, and RM 8664, respectively.
    Keywords:  Dietary supplementation; Ginseng; Liquid chromatography-tandem mass spectrometry; Method development; Natural products; Reference materials
  9. Anal Chem. 2022 Oct 18.
      In mass spectrometry (MS), nonvolatile salts contaminate the transmission system and cause ion suppression, hampering MS analysis. When MS is combined with liquid chromatography (LC) that uses a salty mobile phase, the problems become more intractable due to long analysis time. Here, a novel heat-assisted dual neutral spray ionization (HADSI) method was developed, which projected sample solution spray and solvent spray onto a heated plate to achieve online desalting and high ionization. The experimental parameters of HADSI were optimized, which indicated that the plate temperature was crucial for ionization and desalination. Eight drug compounds dissolved in various commonly used buffers were directly analyzed using HADSI-MS, even though the concentration of PBS buffer reached 500 mmol/L. The established method showed considerable sensitivity in the positive ion mode with the limits of detection at the level of nmol/L, and good linearity (R2 > 0.99) was achieved for all the analyzed compounds. The repeatability and intra- and interday precisions of the method were evaluated, demonstrating the feasibility and reliability of the analysis of salty samples by HADSI-MS. Further, the method was demonstrated to tolerate the long-time analysis of high-salt LC eluates and the device was easy to maintain. Finally, a crude roxithromycin product was separated by LC and then analyzed by HADSI-MS, and seven unknown impurities and nine known impurities were successfully detected. Our results indicated that HADSI-MS may have potential applications in academic and industrial fields.
  10. Anal Chim Acta. 2022 Nov 01. pii: S0003-2670(22)01022-4. [Epub ahead of print]1232 340451
      Alternaria toxins are naturally occurring contaminants found in natural products. Given the prevalence of Alternaria toxins and the complexity of oil-rich matrices, achieving ultra-trace analysis has become a daunting task. A new sample pretreatment technique, i.e., cold-induced liquid-liquid microextraction combined with serially-coupled-columns for SIDA-UHPLC-MS/MS, was developed and reported for the first time. Theoretical and experimental investigations on the mechanism and key parameters revealed that the proposed method achieved simultaneous purification and enrichment in one-step sample extraction with a superior limit of quantitation (0.15-1.5 μg kg-1), without further sample manipulation, such as fat removal or solvent exchange procedures prior to LC-MS. The method was validated taking into consideration EU guidelines and showed acceptable linearity (r ≥ 0.9991), accuracy with recoveries between 75 and 114% and precision with RSD≤9.7% for all of the analytes studied. It was successfully applied to the analysis of twenty samples sourced from the Mediterranean region in order to gain first insights into Alternaria toxins contaminations in olive oils. This technical approach is well suited for large-scale studies in a high-throughput and cost-effective quality assurance laboratory environments, and it has the potential to detect ultra-trace levels of toxins in complex samples, which may lead to the development of new and sustainable sample preparation procedures.
    Keywords:  Alternariol (5359485); Alternariol monomethyl ether (5360741); Cold-induced phase separation; Liquid chromatography; Mass spectrometry; Serially coupled columns; Stable isotope dilution assay; Tentoxin (5281143); Tenuazonic acid (54678599)
  11. Anal Chim Acta. 2022 Nov 01. pii: S0003-2670(22)01028-5. [Epub ahead of print]1232 340457
      Different chemical forms of sex hormones including free/conjugated metabolites as well as their protein/DNA adducts in human serum are a panel of important indicators of health conditions. It is, however, hard to quantify all species simultaneously due to the lack of general extraction, derivatization, and de-conjugation methods. Here we developed a label-free and de-conjugation-free workflow to quantify 11 free/conjugated estrogen metabolites including depurinating DNA and protein adduct forms of 4-hydroxyestradiol (4OHE2) in human serum. Acetonitrile acts as an excellent solvent to purify adducted and non-adducted human serum albumin (HSA) by precipitation as well as to extract free/conjugated metabolites and depurinating DNA adducts from the supernatant by salting-out effect. The adduction level of 4OHE2 on HSA was determined by proteomics; free/conjugated metabolites were quantified by a newly developed microflow liquid chromatography (microflow LC)-nanoelectrospray ionization (nanoESI)-multiple reaction monitoring (MRM) method with high reproducibility (7-22% RSD, n > 3) and sub-picogram levels (0.6-20 pg/mL) of quantification limits (S/N = 8) by using non-pulled capillary as nano-ESI emitter. This workflow was demonstrated to reveal endogenous adduction level of 4OHE2 on HSA as well as circulation levels of free/conjugated metabolites in clinical samples. 4OHE2 in human serum were solely detected as protein-bound form, indicating the merit of such integrated platform covering unstable or active metabolites. Compared to traditional methods using labeling or de-conjugation reaction, this workflow is much simplier, more sensitive, and more specific. Moreover, it can be widely applied in omics to concurrently access various bio-transformed known and un-known markers or drugs.
    Keywords:  Catechol estrogen; Conjugated metabolites; DNA adduct; Estrogen; Metabolites; Protein adduct; Protein precipitation; Salting-out extraction; nanoESI
  12. J Pharm Biomed Anal. 2022 Oct 05. pii: S0731-7085(22)00511-8. [Epub ahead of print]222 115090
      Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90-3802 pM for 1-AG, 90-724 pM for 2-AG, 12-5226 pM for LEA, 33-13,942 for OEA, 34-23,850 pM for 2-AGE, 72-30,190 for PEA and 10-4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of - 14.7 to + 12.3% at LLOQ and - 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and <15% for the remaining levels) as well. It was finally used to quantify endocannabinoids in human cerebrospinal fluid obtained from 118 donors. Accurate quantification of endogenous compounds in biological samples was achieved by using two different principal approaches (surrogate matrix for AEA, 2-AG, OEA, 2-AGE, LEA and PEA, and surrogate calibrant for AEA only) and they were evaluated by use of the Passing-Bablok regression. Concentrations (median) of CSF samples of patients suffering from CNS infection and controls were found to be around 160 pM for 1- and 2-AG, 86 pM for AEA, 62 for 2-AGE, 58 for LEA, 93 pM for PEA, and 83 pM for OEA.
    Keywords:  2-arachidonoylglycerol; Anandamide; Cerebrospinal fluid; Endocannabinoid; Surrogate matrix; UHPLC-MS/MS