bims-metlip 2022-01-02 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022‒01‒02
fourteen papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

High Throughput UHPLC-MS-Based Lipidomics Using Vacuum Jacketed Columns.
INCA 2.0: A tool for integrated, dynamic modeling of NMR- and MS-based isotopomer measurements and rigorous metabolic flux analysis.
A clinically validated method to separate and quantify underivatized acylcarnitines and carnitine metabolic intermediates using mixed-mode chromatography with tandem mass spectrometry.
Inorganic Matrices Assisted Laser Desorption/Ionization Mass Spectrometry for Metabolic Analysis in Bio-fluids.
Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the "gold standard" method and integration into clinical practice.
The Foundations and Development of Lipidomics.
Internal calibration as an emerging approach for endogenous analyte quantification: Application to steroids.
Extraction of Metabolites from Cancer Cells.
Evaluation of different internal standardization approaches for the quantification of melatonin in cell culture samples by multiple heart-cutting two dimensional liquid chromatography tandem mass spectrometry.
Quantitative analysis of thiamylal and its metabolite secobarbital using liquid chromatography-tandem mass spectrometry in adipose tissue, serum, and liver.
Developing LC-MS/MS methods to quantify rivaroxabanin human plasma and urine: application to therapeuticdrug monitoring.
Simultaneous Quantification of Aromatase Inhibitors and Estrogens in Postmenopausal Breast Cancer Patients.
The Imprinted PARAFILM as a New Carrier Material for Dried Plasma Spots (DPSs) Utilizing Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) in Phospholipidomics.
Protonation-Induced Chirality Drives Separation by Differential Ion Mobility Spectrometry.

J Proteome Res. 2021 Dec 30.

High Throughput UHPLC-MS-Based Lipidomics Using Vacuum Jacketed Columns.

Robert S Plumb, Giorgis Isaac, Paul D Rainville, Jason Hill, Lee A Gethings, Kelly A Johnson, Joshua Lauterbach, Ian D Wilson.

  Reversed-phase UHPLC-MS is extensively employed for both the profiling of biological fluids and tissues to characterize lipid dysregulation in disease and toxicological studies. With conventional LC-MS systems the chromatographic performance and throughput are limited due to dispersion from the fluidic connections as well as radial and longitudinal thermal gradients in the LC column. In this study vacuum jacketed columns (VJC), positioned at the source of the mass spectrometer, were applied to the lipidomic analysis of plasma extracts. Compared to conventional UHPLC, the VJC-based methods offered greater resolution, faster analysis, and improved peak intensity. For a 5 min VJC analysis, the peak capacity increased by 66%, peak tailing reduced by up to 34%, and the number of lipids detected increased by 30% compared to conventional UHPLC. The narrower peaks, and thus increased resolution, compared to the conventional system resulted in a 2-fold increase in peak intensity as well a significant improvement in MS and MS/MS spectral quality resulting in a 22% increase in the number of lipids identified. When applied to mouse plasma samples, reproducibility of the lipid intensities in the pooled QC ranged from 1.8-12%, with no related drift in tR observed.

Keywords:  NIST 1950 plasma; gefitinib; high-throughput; lipidomics; mouse plasma

DOI:  https://doi.org/10.1021/acs.jproteome.1c00836
Metab Eng. 2021 Dec 26. pii: S1096-7176(21)00195-6. [Epub ahead of print]

INCA 2.0: A tool for integrated, dynamic modeling of NMR- and MS-based isotopomer measurements and rigorous metabolic flux analysis.

Mohsin Rahim, Mukundan Ragavan, Stanislaw Deja, Matthew E Merritt, Shawn C Burgess, Jamey D Young.

  Metabolic flux analysis (MFA) combines experimental measurements and computational modeling to determine biochemical reaction rates in live biological systems. Advancements in analytical instrumentation, such as nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), have facilitated chemical separation and quantification of isotopically enriched metabolites. However, no software packages currently exist that can integrate isotopomer measurements from both MS and NMR analytical platforms and have the flexibility to estimate metabolic fluxes from either isotopic steady-state or dynamic labeling experiments. By applying physiologically relevant cardiac and hepatic metabolic models to assess NMR isotopomer measurements, we herein test and validate new modeling capabilities of our enhanced flux analysis software tool, INCA 2.0. We demonstrate that INCA 2.0 can simulate and regress steady-state 13C NMR datasets from perfused hearts with an accuracy comparable to other established flux assessment tools. Furthermore, by simulating the infusion of three different 13C acetate tracers, we show that MFA based on dynamic 13C NMR measurements can more precisely resolve cardiac fluxes compared to isotopically steady-state flux analysis. Finally, we show that estimation of hepatic fluxes using combined 13C NMR and MS datasets improves the precision of estimated fluxes by up to 50%. Overall, our results illustrate how the recently added NMR data modeling capabilities of INCA 2.0 can enable entirely new experimental designs that lead to improved flux resolution and can be applied to a wide range of biological systems and measurement time courses.

Keywords:  INST-MFA; Mass spectrometry; Metabolic flux analysis; Metabolic modeling software; Metabolomics; Nuclear magnetic resonance

DOI:  https://doi.org/10.1016/j.ymben.2021.12.009
J Chromatogr A. 2021 Dec 16. pii: S0021-9673(21)00871-2. [Epub ahead of print]1663 462749

A clinically validated method to separate and quantify underivatized acylcarnitines and carnitine metabolic intermediates using mixed-mode chromatography with tandem mass spectrometry.

Carolina Luna, Chandler Griffin, Marcus J Miller.

  Acylcarnitines are intermediate metabolites of the mitochondria that serve as biomarkers for inherited disorders of fatty acid oxidation and amino acid metabolism. The prevailing clinical method used to quantify acylcarnitines involves flow-injection tandem mass spectrometry, an approach with a number of limitations; foremost the inability to separate and therefore distinguish key isobaric acylcarnitine species. To address these issues, we report a clinically validated liquid chromatography tandem mass spectrometry method to quantify acylcarnitines, free carnitine, and carnitine metabolic intermediates in human plasma. Importantly, this method resolves clinically relevant isobaric and isomeric acylcarnitine species in a single 22 min analysis without the use of ion pairing or derivatization reagents. This unique combination of features is not achievable by existing acylcarnitine methods and is made possible by the use of a novel mixed-mode chromatographic separation. Further clinical validation studies demonstrate excellent limits of quantification, linearity, accuracy, and inter-assay precision for analyses of 38 different calibrated analytes. An additional 28 analytes are semi-quantitatively analyzed using surrogate calibrators. The study of residual patient specimens confirms the clinical utility of this method and suggests expanded applicability to the diagnosis of peroxisomal disorders. In summary, we report a clinically validated acylcarnitine method that utilizes a novel mixed-mode chromatographic separation to provide a number of advantages in terms of specificity, accuracy, sample preparation time, and clinical utility.

Keywords:  Acylcarnitine profile analysis; Carnitine metabolism; Isomeric separation of acylcarnitines; Mixed-mode chromatography; Peroxisomal dicarboxylic acylcarnitine biomarker

DOI:  https://doi.org/10.1016/j.chroma.2021.462749
Chem Asian J. 2021 Dec 28.

Inorganic Matrices Assisted Laser Desorption/Ionization Mass Spectrometry for Metabolic Analysis in Bio-fluids.

Yajie Ding, Congcong Pei, Weikang Shu, Jingjing Wan.

  Metabolic analysis in bio-fluids interprets the end products in the bio-process, emerging as an irreplaceable disease diagnosis and monitoring platform. Laser desorption/ionization mass spectrometry (LDI MS) based metabolic analysis exhibits great potential for clinical applications in terms of high throughput, rapid signal readout, and minimal sample preparation. There are two essential elements to construct the LDI MS-based metabolic analysis: 1) well-designed nanomaterials as matrices; 2) machine learning algorithms for data analysis. This review highlights the development of various inorganic matrices to comprehend the advantages of LDI MS in metabolite detection and the recent diagnostic applications based on target metabolite detection and untargeted metabolic fingerprints in biological fluids.

Keywords:  biological fluids; laser desorption ionization; mass spectrometry; metabolic diagnosis; metabolomics

DOI:  https://doi.org/10.1002/asia.202101310
Mass Spectrom Rev. 2021 Dec 29.

Analysis of vitamin D metabolic markers by mass spectrometry: Recent progress regarding the "gold standard" method and integration into clinical practice.

Anastasia Alexandridou, Pascal Schorr, Caroline S Stokes, Dietrich A Volmer.

  Liquid chromatography/tandem mass spectrometry is firmly established today as the gold standard technique for analysis of vitamin D, both for vitamin D status assessments as well as for measuring complex and intricate vitamin D metabolic fingerprints. While the actual mass spectrometry technology has seen only incremental performance increases in recent years, there have been major, very impactful changes in the front- and back-end of MS-based vitamin D assays; for example, the extension to new types of biological sample matrices analyzed for an increasing number of different vitamin D metabolites, novel sample preparation techniques, new powerful chemical derivatization reagents, as well the continued integration of high resolution mass spectrometers into clinical laboratories, replacing established triple-quadrupole instruments. At the same time, the sustainability of mass spectrometry operation in the vitamin D field is now firmly established through proven analytical harmonization and standardization programs. The present review summarizes the most important of these recent developments.

Keywords:  25-hydroxyvitamin D3; HRMS; LC-MS/MS; chemical derivatization; epimers; free vitamin D; sample preparation; vitamin D metabolites; vitamin D standardization

DOI:  https://doi.org/10.1002/mas.21768
J Lipid Res. 2021 Dec 21. pii: S0022-2275(21)00147-4. [Epub ahead of print] 100164

The Foundations and Development of Lipidomics.

Xianlin Han, Richard W Gross.

  For over a century, the importance of lipid metabolism in biology was recognized, but difficult to mechanistically understand due to the lack of sensitive and robust technologies for identification and quantification of lipid molecular species. The enabling technological breakthroughs emerged in the 1980s with the development of soft ionization methods (Electrospray Ionization and Matrix Assisted Laser Desorption/Ionization) that could identify and quantify intact individual lipid molecular species. These soft ionization technologies laid the foundations for what was to be later named the field of lipidomics. Further innovative advances in multistage fragmentation, dramatic improvements in resolution and mass accuracy, and multiplexed sample analysis fueled the early growth of lipidomics through the early 1990s. The field exponentially grew through the use of a variety of strategic approaches, which included direct infusion, chromatographic separation, and charge-switch derivatization, which facilitated access to the low abundance species of the lipidome. In this Thematic Review, we provide a broad perspective of the foundations, enabling advances, and predicted future directions of growth of the lipidomics field.

Keywords:  Lipids; charge-switch derivatization; chromatographic separation; electrospray ionization; lipid metabolism; lipidomics; mass spectrometry; matrix-assisted laser desorption/ionization; shotgun lipidomics; soft ionization

DOI:  https://doi.org/10.1016/j.jlr.2021.100164
Talanta. 2021 Dec 15. pii: S0039-9140(21)01071-7. [Epub ahead of print]240 123149

Internal calibration as an emerging approach for endogenous analyte quantification: Application to steroids.

Gioele Visconti, Eulalia Olesti, Víctor González-Ruiz, Gaëtan Glauser, David Tonoli, Pierre Lescuyer, Nicolas Vuilleumier, Serge Rudaz.

  The use of mass spectrometry methods with triple quadrupole instruments is well established for quantification. However, the preparation of calibration curves can be time-consuming and prone to analytical errors. In this study, an innovative internal calibration (IC) approach using a one-standard calibration with a stable isotope-labeled (SIL) standard version of the endogenous compound was developed. To ensure optimal quantitative performance, the following parameters were evaluated: the stability of the analyte-to-SIL response factor (RF), the chemical and isotopic purities of the SIL, and the instrumental reproducibility. Using six clinically important endogenous steroids and their respective SIL standards, we demonstrated that RFs obtained on different LC-MS platforms were consistent. The quantitative performance of the proposed approach was determined using quality control samples prepared in depleted serum, and showed both satisfactory precision (1.3%-12.4%) and trueness (77.5%-107.0%, with only 3 values outside ±30%). The developed method was then applied to human serum samples, and the results were similar to those obtained with the conventional quantification approach based on external calibration: the Passing-Bablok regression showed a proportional bias of 6.8% and a mean difference of -5.9% between the two methodologies. Finally, we showed that the naturally occurring isotopes of the SIL can be used to provide additional calibration points and increase the accuracy for analytes with low concentrations.

Keywords:  Internal calibration; Multiple isotopologue reaction monitoring; Quantification; Response factor; Steroids

DOI:  https://doi.org/10.1016/j.talanta.2021.123149
Methods Mol Biol. 2022 ;2445 329-335

Extraction of Metabolites from Cancer Cells.

Elena Kochetkova, Tina Becirovic, Erik Norberg.

  Cancer cells possess an elevated demand for nutrients and metabolites due to their uncontrolled proliferation and need to survive in unfavorable conditions. Autophagy is a conservative degradation pathway that counters lack of nutrients and provides organelle and protein quality control, beyond maintenance of cellular metabolism.Mass spectrometry-based metabolomics is a powerful tool to study the metabolome of a cell. Such analysis requires proper sample preparation including the extraction of metabolites. Here, we provide a protocol for the extraction of metabolites from adherent cancer cells suitable for global metabolome profiling by mass spectrometry.

Keywords:  Autophagy; CE-MS; Cancer metabolism; Chaperone-mediated autophagy; GC-MS; LC-MS; Mass spectrometry; Metabolism; Methanol extraction

DOI:  https://doi.org/10.1007/978-1-0716-2071-7_20
J Chromatogr A. 2021 Dec 17. pii: S0021-9673(21)00874-8. [Epub ahead of print]1663 462752

Evaluation of different internal standardization approaches for the quantification of melatonin in cell culture samples by multiple heart-cutting two dimensional liquid chromatography tandem mass spectrometry.

Amanda Suárez Fernández, Adriana González Gago, Francisco Artime Naveda, Javier García Calleja, Anna Zawadzka, Zbigniew Czarnocki, Juan Carlos Mayo Barrallo, Rosa M Sainz Menéndez, Pablo Rodríguez-González, J Ignacio García Alonso.

  We evaluate here different analytical strategies for the chromatographic separation and determination of N-acetyl-5-methoxytryptamine (MEL) and its oxidative metabolites N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N1-acetyl-5-methoxykynuramine (AMK) and cyclic 3-hydroxymelatonin (c3OHM) in cell culture samples. Two dimensional liquid chromatography (2D-LC) in the multiple heart-cutting mode was compared with regular 1D chromatographic separations of MEL and its oxidative metabolites. Our results showed that the use of trifluoroacetic acid (TFA) as mobile phase modifier was required to obtain a satisfactory resolution and peak shapes particularly for c3OHM. As TFA is not compatible with ESI ionization the application of the MHC mode was mandatory for a proper chromatographic separation. We evaluate also different internal standardization approaches based on the combined use of a surrogate standard (5-methoxytryptophol) and an internal standard (6-methoxytryptamine) for MEL quantification in cell culture samples obtaining unsatisfactory results both by 1D- and 2D-LC-ESI-MS/MS (from 9 ± 2 to 186 ± 38%). We demonstrate that only the application of isotope dilution Mass Spectrometry through the use of an in house synthesized 13C isotopically labelled analogue provided quantitative MEL recoveries both by using 1D- and 2D-LC-ESI-MS/MS (99±1 and 98±1. Respectively) in androgen-insensitive human prostate carcinoma PC3 cells.

Keywords:  Electrospray; Internal standardization; Isotope Dilution; Melatonin; Multiple heart cutting

DOI:  https://doi.org/10.1016/j.chroma.2021.462752
J Forensic Sci. 2021 Dec 30.

Quantitative analysis of thiamylal and its metabolite secobarbital using liquid chromatography-tandem mass spectrometry in adipose tissue, serum, and liver.

Naoki Yoshioka, Migiwa Asano, Azumi Kuse, Takeshi Kondo, Motonori Takahashi, Mai Morichika, Kanako Nakagawa, Makoto Sakurada, Yasuhiro Ueno.

  Thiamylal is an ultrashort-acting barbiturate used for intravenous administration or general anesthesia induction. However, some cases of poisoning and suicide with thiamylal administration have been reported. Additionally, there are few reports on its analysis in the organs and adipose tissue, which requires purification by column chromatography and evaporation. A rapid and sensitive method was developed for quantifying thiamylal and its metabolite, secobarbital, in the adipose tissue, serum, and liver using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were prepared using modified QuEChERS extraction. For adipose tissue samples, an acetonitrile-hexane partitioning step was added to the extraction. This method was applied to investigate a suspected self-poisoning autopsy case. The quantitation accuracy for thiamylal added to porcine pericardial fat (0.18 µg/g), human serum (0.015 µg/mL), and porcine liver (0.18 µg/g) was 103%, 113%, and 95.3%, respectively. The quantitation limits calculated for porcine pericardial fat, human serum, and porcine liver at a signal-to-noise ratio of 10 were 0.06 µg/g, 0.005 µg/mL, and 0.06 µg/g, respectively. In addition, the thiamylal and secobarbital levels in the forensic autopsy case were 140 and 1.5 µg/g, respectively, in myocardial fat; 3.5-4.9 and 0.12-0.20 µg/mL, respectively, in serum; and 6.2-42 and 0.58-1.1 µg/g, respectively, in liver tissue. Thiamylal is especially distributed in the adipose tissue. The thiamylal-to-fat ratio may help estimate the time from administration to death. The developed modified QuEChERS extraction method with acetonitrile-hexane partitioning is suitable for analyzing hydrophobic compounds, such as thiamylal, in the adipose tissue.

Keywords:  LC-MS/MS; QuEChERS; adipose tissue; liquid chromatography-tandem mass spectrometry; secobarbital; thiamylal

DOI:  https://doi.org/10.1111/1556-4029.14965
Biomed Chromatogr. 2021 Dec 29. e5306

Developing LC-MS/MS methods to quantify rivaroxabanin human plasma and urine: application to therapeuticdrug monitoring.

Xin Zheng, Chen Chen, Huitao Gao, Xuefeng Sun, Yanbao Zhang, Juhong Shi, Xiaohong Han.

  Rivaroxaban is an oral anticoagulant directly inhibiting Factor Xa activity, which is widely used for prophylaxis of thromboembolic disorders and is required for therapeutic drug monitoring (TDM) during therapyfor individual dosage adjustment. Thisstudyaimed at developing a liquidchromatography/tandem mass spectrometry method that was suitable forrivaroxaban TDM in human plasma and urine and exploring the feasibility of urine drug monitoring in medical care. The 3-min run of LC-MS/MS methodswereestablishedby employingan Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) column using gradient elution of 10 mmol/L ammonium acetate containing 0.1% formic acid - 0.1% formic acid acetonitrile as mobile phase at a flow rate of 0.4 mL/min with the calibration range of 0.5 ng/mL-400 ng/mL and 10 ng/mL-10000 ng/mLfor human plasma and urine, respectively. Rivaroxaban wasdetected on a triple quadrupole tandem mass spectrometer with electrospray ionization source in positive ion mode. Themethods showed good linearity within the calibration range. Theprecision and accuracy, matrix effect, extraction recovery and stability inboth human matrices were all validatedand meet the international guideline requirements. These validated methods had been successfullyapplied to support TDMofan aged patient receiving rivaroxaban for therapy.

Keywords:  Human plasma; Rivaroxaban; TDM; UPLC-MS/MS; human urine

DOI:  https://doi.org/10.1002/bmc.5306
J Clin Endocrinol Metab. 2021 Dec 27. pii: dgab923. [Epub ahead of print]

Simultaneous Quantification of Aromatase Inhibitors and Estrogens in Postmenopausal Breast Cancer Patients.

Bjørn-Erik Bertelsen, Kristin Viste, Thomas Helland, Magnus Hagland, Håvard Søiland, Jürgen Geisler, Tone Hoel Lende, Per Eystein Lønning, Jørn V Sagen, Gunnar Mellgren, Bjørg Almås.

  CONTEXT: Currently there are no assays that can simultaneously quantify serum levels of the third generation aromatase inhibitors (AI); letrozole, anastrozole, and exemestane, and the ultralow levels of estrogens in postmenopausal breast cancer patients on AI treatment. Such measurements may be pivotal for the determination of optimal and individualized treatment regimens. We aimed at developing an LC-MS/MS method for simultaneous assessment of letrozole, anastrozole, exemestane and 17-hydroxy-exemestane as well as subpicomolar levels of estradiol and estrone.METHODS: Internal standards, calibrators, serum samples and quality controls were in fully automated steps transferred to a deepwell plate for a two-step liquid-liquid-extraction. The extracts were reconstituted and analytes were separated chromatographically using two serially coupled columns, then subject to MS/MS in ESI mode. The method was thoroughly validated and is traceable to two accredited estrogen-methods.
RESULTS: Measurement range for estrone and estradiol was 0.2 - 12 000 pmol/L and 0.8 - 13 000 pmol/L, and covered the expected therapeutic range for the AIs. All analytes had precision ≤ 13 %, and accuracies within 100 ± 8 %. As proof-of-concept, AI and estrogen levels were determined in sera from postmenopausal breast cancer patients under treatment.
CONCLUSIONS: We present here an assay suitable for the simultaneous measurement of serum levels of all third generation AIs and ultralow levels of estrogens, providing a powerful new tool to study drug efficacy and compliance. The method is highly valuable for postmenopausal patients whose pretreatment estradiol levels are below threshold of detection for most routine assays, but still require suppression.

Keywords:  anastrozole; aromatase inhibitors; breast cancer; estradiol; exemestane; letrozole

DOI:  https://doi.org/10.1210/clinem/dgab923
Front Chem. 2021 ;9 801043

The Imprinted PARAFILM as a New Carrier Material for Dried Plasma Spots (DPSs) Utilizing Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) in Phospholipidomics.

Jiansong Chen, Yue Hu, Congxiang Shao, Haiyun Zhou, Zhiyue Lv.

  The application of desorption electrospray ionization mass spectrometry (DESI-MS) and dried blood spot (DBS) sampling has been successfully implemented several times. However, the difficulty of combining DBS sampling with DESI-MS is still the carrier material used for the blood samples. In this study, a new, easily obtained, and cost-effective carrier substrate for dried plasma spot (DPS) sampling and DESI-MS analysis and its application in phospholipidomics studies was described. First, the effects of several carrier materials, including cellulose-based materials (31 ET paper and filter paper) and non-cellulose-based materials (PARAFILM and its shape-modified material, PTFE-printed glass slide and polyvinylidene fluoride film), were tested. Second, a method combining DPS sampling with DESI-MS for phospholipidomics analysis was established, and parameters affecting compound signal intensities, such as sample volume and sprayer solvent system, were optimized. In conclusion, the total signal intensity obtained from shape-modified PARAFILM was the strongest. The suitable plasma sample volume deposited on PARAFILM carriers was 5 μl, and acetonitrile (ACN) was recommended as the optimal spray solvent for phospholipid (PL) profiling. Repeatability (87.5% of compounds with CV < 30%) and stability for data acquisition (48 h) were confirmed. Finally, the developed method was applied in phospholipidomics analysis of schistosomiasis, and a distinguished classification between control mice and infected mice was observed by using multivariate pattern recognition analysis, confirming the practical application of this new carrier material for DPS sampling and DESI-MS analysis. Compared with a previously reported method, the rapid metabolomics screening approach based on the implementation of DPS sampling coupled with the DESI-MS instrument developed in this study has increased analyte sensitivity, which may promote its further application in clinical studies.

Keywords:  carrier material; desorption electrospray ionization mass spectrometry; dried plasma spots; parafilm; phospholipidomics

DOI:  https://doi.org/10.3389/fchem.2021.801043
Angew Chem Int Ed Engl. 2021 Dec 28.

Protonation-Induced Chirality Drives Separation by Differential Ion Mobility Spectrometry.

Christian Ieritano, J C Yves Le Blanc, Bradley B Schneider, Justine R Bissonnette, Alexander Haack, W Scott Hopkins.

  Upon development of a workflow to analyze (±)-Verapamil and its metabolites using differential mobility spectrometry (DMS), we noticed that the ionogram of protonated Verapamil consisted of two peaks. This was inconsistent with its metabolites, as each exhibited only a single peak in the respective ionograms. The unique behaviour of Verapamil was attributed to protonation at its tertiary amino moiety, which generated a stereogenic quaternary amine. The introduction of additional chirality upon N-protonation of Verapamil renders four possible stereochemical configurations for the protonated ion: ( R,R ), ( S,S ), ( R,S ), or ( S,R ). The ( R,R )/( S,S ) and ( R,S )/( S,R ) enantiomeric pairs are diastereomeric and thus exhibit unique conformations that are resolvable by linear and differential ion mobility techniques. Protonation-induced chirality appears to be a general phenomenon, as N -protonation of 12 additional chiral amines generated diastereomers that were readily resolved by DMS.

Keywords:  mass spectrometry, ion mobility, chiral derivatization, verapamil, diastereomer

DOI:  https://doi.org/10.1002/anie.202116794