bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒10‒09
sixteen papers selected by
Sofia Costa

  1. Anticancer Res. 2022 Oct;42(10): 5077-5081
      BACKGROUND/AIM: Simultaneous assessment of various vitamin D metabolites in human biofluids by liquid chromatography tandem mass spectrometry (LC-MS/MS) represents a new promising tool for the differential diagnosis of vitamin D-related diseases. Particularly, besides 25(OH)VD2/3, low-abundant medicinally relevant vitamin D metabolites, such as 24,25(OH)2VD2/3, 1,25(OH)2VD2/3, and 1,24,25(OH)3VD2/3, along with their 3-epi-derivatives have to be considered.MATERIALS AND METHODS: The assessment of these metabolites by LC-MS/MS requires the development of calibration and reference standards, that is, their labeling with multiple deuterium-, or even better, 13C- atoms.
    RESULTS: Overall, 10 13C-labeled vitamin D metabolites have been chemically synthesized and obtained in good yield and high purity.
    CONCLUSION: Access to a wide variety of 13C-labeled highly pure vitamin D metabolites enables the advancement of LC-MS/MS applications towards a better understanding of differential diagnosis of vitamin D-related diseases.
    Keywords:  Cancer prevention; LC-MS/MS assay development; vitamin D metabolites; vitamin D synthesis
  2. Lipids. 2022 Oct 04.
      Oxysterols play a key role in many (patho)physiological processes and they are potential biomarkers for oxidative stress in several diseases. Here we developed a rapid gas chromatographic-mass spectrometry-based method for the separation and quantification of 11 biologically relevant oxysterols bearing hydroxy, epoxy, and dihydroxy groups. Efficient chromatographic separation (resolution ≥ 1.9) was achieved using a medium polarity 35%-diphenyl/65%-dimethyl polysiloxane stationary phase material (30 m × 0.25 mm inner diameter and 0.25 μm film thickness). Based on thorough analysis of the fragmentation during electron ionization we developed a strategy to deduce structural information of the oxysterols. Optimized sample preparation includes (i) extraction with a mixture of n-hexane/iso-propanol, (ii) removal of cholesterol by solid phase extraction with unmodified silica, and (iii) trimethylsilylation. The method was successfully applied on the analysis of brain samples, showing consistent results with previous studies and a good intra- and interday precision of ≤20%. Finally, we used the method for the investigation of oxysterol formation during oxidative stress in HepG2 cells. Incubation with tert-butyl hydroperoxide led to a massive increase in free radical formed oxysterols (7-keto-chol > 7β-OH-chol >> 7α-OH-chol), while 24 h incubation with the glutathione peroxidase 4 inhibitor RSL3 showed no increase in oxidative stress based on the oxysterol pattern. Overall, the new method described here enables the robust analysis of a biologically meaningful pattern of oxysterols with high sensitivity and precision allowing us to gain new insights in the biological formation and role of oxysterols.
    Keywords:  chromatographic separation; mass spectrometry; oxidative stress; quantification; solid phase extraction
  3. Comput Struct Biotechnol J. 2022 ;20 5085-5097
      LC-MS/MS is a major analytical platform for metabolomics, which has become a recent hotspot in the research fields of life and environmental sciences. By contrast, structure elucidation of small molecules based on LC-MS/MS data remains a major challenge in the chemical and biological interpretation of untargeted metabolomics datasets. In recent years, several strategies for structure elucidation using LC-MS/MS data from complex biological samples have been proposed, these strategies can be simply categorized into two types, one based on structure annotation of mass spectra and for the other on retention time prediction. These strategies have helped many scientists conduct research in metabolite-related fields and are indispensable for the development of future tools. Here, we summarized the characteristics of the current tools and strategies for structure elucidation of small molecules based on LC-MS/MS data, and further discussed the directions and perspectives to improve the power of the tools or strategies for structure elucidation.
    Keywords:  Complex biological samples; LC–MS/MS; Structure elucidation
  4. Anal Chim Acta. 2022 Oct 16. pii: S0003-2670(22)00818-2. [Epub ahead of print]1230 340247
      Mass spectra are an important signature by which compounds can be identified. We recently formulated a mathematical approach for incorporating measurement variability when comparing sets of high-resolution mass spectra. Leveraging replicate mass spectra, we construct high-dimensional consensus mass spectra-representing each of the compared analytes-and compute the similarity between these data structures. In this paper, we present this approach and discuss its applications and limitations when trying to discriminate methamphetamine and phentermine using in-source collision induced dissociation mass spectra collected with direct analysis in real time mass spectrometry.
    Keywords:  High-dimensional consensus (HDC) mass spectra; High-resolution mass spectrometry; Mass spectral similarity; Mass spectrometry; Probability distributions; Sample discrimination
  5. Clin Chim Acta. 2022 Sep 30. pii: S0009-8981(22)01316-X. [Epub ahead of print]
      BACKGROUND: Trimethylamine N-oxide (TMAO) and phenylacetylglutamine (PAGln) are associated with acute myocardial infarction (AMI) and type 2 diabetes mellitus (T2DM). This study developed and validated a simple method firstly for simultaneously quantifying serum TMAO and PAGln using liquid chromatography-tandem mass spectrometry (LC-MS/MS).METHODS: Serum samples from patients with T2DM, AMI, and healthy subjects were analyzed using a new LC-MS/MS method to evaluate TMAO and PAGln levels. Statistical analyses were performed to evaluate TMAO and PAGln distributions among different groups.
    RESULTS: Retention and separation of the two metabolites were achieved within 5 min. For both analytes, the assay was linear in a 0.02-10 µg/mL range, with >0.99 average linear correlation coefficients, and quantification limit values of approximately 0.010 µg/mL. The average recoveries of TMAO and PAGln were 96.3% and 96.4%, respectively. The intra-run and total coefficient variations were 3.5-4.8% and 3.9-5.7% respectively for TMAO; and 4.0-5.1% and 4.6-6.3% respectively, for PAGln. TMAO and PAGln showed a moderate correlation (P<0.001) and their levels in patients with T2DM were significantly higher than those in healthy individuals (P<0.001). TMAO levels were higher in patients with T2DM than in patients with AMI (P<0.01). Patients with AMI had higher PAGln levels than healthy individuals (P<0.05). After adjusting for sex and age, the top tertile of PAGln was positively correlated with T2DM and AMI while that of TMAO was positively correlated with T2DM.
    CONCLUSIONS: Overall, a robust isotope dilution LC-MS/MS method was established, which may be beneficial for assessing the association between two metabolites with AMI and T2DM.
    Keywords:  diabetes mellitus; liquid chromatography-tandem mass spectrometry; phenylacetyl-glutamine; trimethylamine N-oxide
  6. RSC Adv. 2022 Sep 05. 12(39): 25528-25548
      Metabolic flux analysis (MFA) quantitatively describes cellular fluxes to understand metabolic phenotypes and functional behaviour after environmental and/or genetic perturbations. In the last decade, the application of stable isotopes became extremely important to determine and integrate in vivo measurements of metabolic reactions in systems biology. 13C-MFA is one of the most informative methods used to study central metabolism of biological systems. This review aims to outline the current experimental procedure adopted in 13C-MFA, starting from the preparation of cell cultures and labelled tracers to the quenching and extraction of metabolites and their subsequent analysis performed with very powerful software. Here, the limitations and advantages of nuclear magnetic resonance spectroscopy and mass spectrometry techniques used in carbon labelled experiments are elucidated by reviewing the most recent published papers. Furthermore, we summarise the most successful approaches used for computational modelling in flux analysis and the main application areas with a particular focus in metabolic engineering.
  7. Int J Anal Chem. 2022 ;2022 4372295
      A new method was developed for the identification and determination of L-ergothioneine in cosmetics based on ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The pretreatment method, chromatographic column, chromatographic conditions, and mass spectrometric conditions of cosmetic samples were optimized. Methanol was chosen as the extraction solvent, 85% acetonitrile with 0.1% FA was selected as the mobile phase, and the Waters CORTECS UPLC hydrophilic interaction liquid chromatography (HILIC) column was chosen for the separation. The sample was extracted with methanol and filtered, then separated by HILIC and detected by triple-quadrupole mass spectrometry. The quantitation was done under the matrix calibration curve using the external standard method. The results showed good linear relationships in the range of 5-200 ng/mL, and the correlation coefficient was greater than 0.999 in cosmetic samples. The limit of detection was in the range of 25-50 μg/kg and the limit of quantitation was in the range of 50-100 μg/kg. The recoveries of the method spiked ranged from 85.3% to 96.2% and the relative standard deviation (RSD) was in the range of 0.84%-2.08% (n = 6). The method is simple, quick, and accurate for the determination of L-ergothioneine in cosmetics, and has great practical value.
  8. Crit Rev Anal Chem. 2022 Oct 07. 1-50
      Mass spectrometry (MS) is a formidable analytical tool for the analysis of non-polar to polar compounds individually and/or from mixtures, providing information on the molecular weights and chemical structures of the analytes. During the last more than one-decade, ambient ionization mass spectrometry (AIMS) has developed quickly, producing a wide range of platforms and proving scientific improvements in a variety of domains, from biological imaging to quick quality control. These methods have made it possible to detect target analytes in real time without sample preparation in an open environment, and they can be connected to any MS system with an atmospheric pressure interface. They also have the ability to analyze explosives, illicit drugs, disease diagnostics, drugs in biological samples, adulterants in food and agricultural products, reaction progress, and environmental monitoring. The development of novel ambient ionization techniques, such as probe electrospray ionization, paper spray ionization, and fiber spray ionization, employed even at picolitre to femtolitre solution levels to provide femtogram to attogram levels of the target analytes. The special characteristic of this ambient ion source, which has been extensively used, is the noninvasive property of PESI of examination of biological real samples. The results in the current review supports the idea that AIMS has emerged as a pioneer in MS-based approaches and that methods will continue to be developed along with improvements to existing ones in the near future.
    Keywords:  Ambient mass spectrometry; high throughput analysis
  9. J Pharm Biomed Anal. 2022 Sep 29. pii: S0731-7085(22)00506-4. [Epub ahead of print]222 115085
      An ultra-rapid and eco-friendly method for the determination of loganic acid and gentiopicroside in Gentianae Macrophyllae Radix (GMR) was developed by vortex-assisted matrix solid-phase dispersion extraction (VAMSPD) and liquid chromatography with mass spectrometry (LC-MS). The optimized VAMSPD parameters are as follow: sample-dispersant (diatomaceous earth) ratio is 1:5, grinding for 0.5 min and whirling with 0.5 mL 15 % ethanol for 0.5 min. The LC separation is performed on a Poroshell 120 EC-C18 column (30 ×2.1 mm, 2.7 µm) and eluted by an eco-friendly mobile phase (14 % ethanol containing 0.1 % formic acid) at a flow rate of 0.5 mL min-1 in isocratic mode, and detected by mass spectrometry (MS). The developed method exhibits a good linearity for the analytes (r > 0.9990). The RSDs of precision and repeatability are less than 4.0 %, the recoveries for loganic acid and gentiopicroside are 106.5 % (RSD=3.6 %) and 95.7 % (RSD=8.0 %), respectively. The developed method was successfully applied in the analysis of loganic acid and gentiopicroside in GMR samples. The total analysis time is 2 min, including 1 min for sample extraction and 1 min for LC-MS analysis. In addition, the method only requires 0.3 mL of ethanol.
    Keywords:  Eco-friendly; Gentianae Macrophyllae Radix; LC-MS; Ultra-rapid; Vortex-assisted matrix solid-phase dispersion
  10. J Pharm Biomed Anal. 2022 Sep 16. pii: S0731-7085(22)00480-0. [Epub ahead of print]221 115059
      Acquiring accurate and reliable results are crucial in pharmaceutical and biomedical analyses. There is a demand for the ongoing development and validation of advanced analytical and bioanalytical methods. Mass spectrometry (MS) has become an extremely powerful tool for the identification, quantification, and characterization of small and macro-(bio)molecules. Capillary electrophoresis (CE) presents fast and high-resolution separation and in combination with MS allows sensitive and selective identification and detailed characterization. To date, CE-MS has been used to analyze a wide range of molecules, including pharmaceuticals, biopharmaceuticals, metabolites, peptides, and proteins. This review provides an update on recent applications and approaches of CE-MS relevant to biomedical and (bio)pharmaceuticals between January 2018 and May 2022. Furthermore, the latest developments on the hyphenation of CE with MS, as well as different CE modes including capillary isotachophoresis, capillary zone electrophoresis, and micellar electrokinetic capillary chromatography along with on-capillary on-line analyte stacking methods such as field-amplified sample injection, transient isotachophoresis, dynamic pH junction, and solid-phase extraction are discussed.
    Keywords:  Capillary electrophoresis; Electrospray ionization; Mass spectrometry; Preconcentration; Sample preparation
  11. Eur J Mass Spectrom (Chichester). 2022 Oct 06. 14690667221130160
      In this study the use of negative electrospray ionisation mode as a confirmation tool for identifying derivatized amino acids using LC-ESI-MS, was evaluated. The derivatization reagent was based on azobenzene N-hydroxysuccinimide carbamate. The results indicate that even though negative ionisation mode produced less intense peaks, the ratio of peak area of quantifier ion (obtained in positive mode) to the qualifier (or identifier) ion measured in negative mode meets the requirements established by two prominent validation guidelines: SANTE/11312/2021 and 2002/657/EC. Therefore, the use of product ions obtained via negative transitions as qualifier ions, while using product ions from positive transitions as quantifier ions is a fruitful approach that widens the choice of transitions to choose from for obtaining suitable qualifier ions. This methodology was applied to the LC-ESI-MS/MS determination of amino acids in different beverages (tomato juice, watermelon juice, kvass).
    Keywords:  ESI; LC-MS; MS/MS; amino acids; derivatization
  12. Food Chem. 2022 Sep 24. pii: S0308-8146(22)02369-X. [Epub ahead of print]403 134407
      Liquid chromatography coupled with quadrupole linear ion trap tandem mass spectrometry (LC-QLIT-MS/MS) technology operated in electrospray ionization (ESI) was developed for tracing anesthetic (AETs) and sedatives (SDTs) in fish. Sampling procedure was achieved by using acetonitrile extraction followed by dispersive solid phase extraction (DSPE) clean-up. Under the optimized laboratory conditions, reliable qualitative confirmation was obtained through the multiple reaction monitoring-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode. Results indicated a favorable linear in the concentration range of 1-100 μg∙kg-1 (0.1-10 μg∙kg-1 for MS-222), with regression coefficient not less than 0.9997. The detection limit ranges from 0.03 to 0.4 μg∙kg-1 (S/N = 3). The validated method was applied to determine AETs and SDTs in fish with satidfied recoveries of 86.3 %-111.7 % and the relative standard deviations (RSD) of 1.9 %-8.9 % (n = 6). Practical samples analysis indicated that the proposed method is simple, rapid, sensitive and accurate for identification of AETs and SDTs.
    Keywords:  Anesthetic; Dispersive solid phase extraction (DSPE); Fish; Liquid chromatography tandem mass spectrometry (LC–MS/MS); Sedatives
  13. J Am Soc Mass Spectrom. 2022 Oct 07.
      Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical and biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue to evolve and bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly evolve in size. Therefore, high-throughput (HT) MS and automated deconvolution methods are key for success. Additionally, newer therapeutics such as bispecific T-cell engagers and nucleic acid-based modalities will also require MS characterization. Herein, we present a modality and target agnostic HT solid-phase extraction (SPE) MS method that affords the analysis of a 96-well plate in 41.4 min, compared to the traditional rpLC-MS method that would typically take 14.4 h. The described method can accurately determine the molecular weights for monodispersed and highly polydispersed biotherapeutic species and membrane proteins; determine levels of glycosylation, glycation, and formylation; detect levels of chain mispairing; and determine accurate drug-to-antibody ratio values.
  14. Anal Bioanal Chem. 2022 Oct 01.
      1,2-Unsaturated pyrrolizidine alkaloids (PA), their corresponding N-oxides (PANO), and tropane alkaloids (TA) are toxic secondary plant metabolites. Their possible transfer into the milk of dairy cows has been studied in feeding trials; however, only few data on the occurrence of these toxins in milk are available. In this study, the development of a sensitive analytical approach for the simultaneous detection and quantification of a broad range of 54 PA/PANO as well as of the TA atropine and scopolamine in milk of dairy cows is presented. The method optimisation focused on sensitivity and separation of PA/PANO isomers. Milk samples were extracted using liquid-liquid extraction with aqueous formic acid and n-hexane, followed by a cation-exchange solid-phase extraction for purification. Reversed phase liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using alkaline solvent conditions. Validation proved low limits of detection and quantification of 0.005 to 0.054 µg/L and of 0.009 to 0.123 µg/L, respectively. For 51 of the 54 tested PA/PANO and both TA, the recovery rates ranged from 64 to 127% with repeatability (RSDr) values below 15% at concentration levels of 0.05 and 0.50 µg/L and below 8% at a concentration level of 3.00 µg/L. Only three PANO did not match the validation criteria and were therefore regarded as semiquantitative. The final method was applied to 15 milk samples obtained from milk vending stations at farms and from local marketers in Bavaria, Germany. In three of the milk samples, traces of PA were detected.
    Keywords:  Liquid chromatography tandem mass spectrometry; Method development; Milk; Pyrrolizidine alkaloids; Tropane alkaloids
  15. RSC Adv. 2022 Sep 12. 12(40): 26016-26022
      An analytical method was developed and validated for the simultaneous determination of 12 anti-obesity drugs (methylephedrine (MER), amphetamine (AMP), fenfluramine (FEN), bupropion (BUP), fluoxetine (FLU), sibutramine (SIBU), bisacodyl (BISA), bumetanide (BUM), lovastatin (LOVA), simvastatin (SIM), rimonabant (RIMO), and fenofibrate (FENO)) in human plasma by a 96-well protein precipitation plate combined with high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). The 96-well protein precipitation plate was chosen for simultaneous pretreatment of large sample volumes, making the whole process more efficient and faster. Drugs were separated on an Agilent Poroshell 120 EC-C18 column, and detected by MS/MS under multiple reaction monitoring (MRM) mode. The developed method was validated in terms of linearity, matrix effect, accuracy and precision. A good linearity was obtained in the range of 0.1-20.0 ng mL-1 for fenfluramine, bupropion, fluoxetine, sibutramine, bisacodyl, and rimonabant; and 0.5-20.0 ng mL-1 for methylephedrine, amphetamine, bumetanide, lovastatin, simvastatin, and fenofibrate with a correlation coefficient above 0.995. The method was fully validated with an acceptable accuracy of 75.63-108.21%, matrix effect of 80.41-117.71% except for fenofibrate (76.07% at low concentration levels), and precision of 0.32-13.12%. Owing to the advantages of simple operation, high accuracy and sensitivity, this method is suitable for the rapid and simultaneous detection of 12 anti-obesity drugs in human plasma, providing support for clinically monitoring the development of adverse reactions and guiding the rational and appropriate use of weight-loss drugs for obese people.
  16. Anal Sci. 2022 Oct 05.
      Analytical processes involving sample preparation, separation, and quantifying analytes in complex mixtures are indispensable in modern-day analysis. Each step is crucial to enriching correct and informative results. Therefore, sample preparation is the critical factor that determines both the accuracy and the time consumption of a sample analysis process. Recently, several promising sample preparation approaches have been made available with environmentally friendly technologies with high performance. As a result of its many advantages, solid-phase extraction (SPE) is practiced in many different fields in addition to the traditional methods. The SPE is an alternative method to liquid-liquid extraction (LLE), which eliminates several disadvantages, including many organic solvents, a lengthy operation time and numerous steps, potential sources of error, and high costs. SPE advanced sorbent technology reorients with various functions depending on the structure of extraction sorbents, including reversed-phase, normal-phase, cation exchange, anion exchange, and mixed-mode. In addition, the commercial SPE systems are disposable. Still, with the continual developments, the restricted access materials (RAM) and molecular imprinted polymers (MIP) are fabricated to be active reusable extraction cartridges. This review will discuss all the theoretical and practical principles of the SPE techniques, focusing on packing materials, different forms, and performing factors in recent and future advances. The information about novel methodological and instrumental solutions in relation to different variants of SPE techniques, solid-phase microextraction (SPME), in-tube solid-phase microextraction (IT-SPME), and magnetic solid-phase extraction (MSPE) is presented. The integration of SPE with analytical chromatographic techniques such as LC and GC is also indicated. Furthermore, the applications of these techniques are discussed in detail along with their advantages in analyzing pharmaceuticals, biological samples, natural compounds, pesticides, and environmental pollutants, as well as foods and beverages.
    Keywords:  Chromatographic analysis; Cleanup; SPE; Separation technique