bims-metlip 2024-12-15 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–12–15
23 papers selected by
Sofia Costa, Matterworks

Matrix effect evaluation using multi-component post-column infusion in untargeted hydrophilic interaction liquid chromatography-mass spectrometry plasma metabolomics.
Bioinert UHPLC system improves sensitivity and peak shapes for ionic metabolites.
Chiral supercritical fluid chromatography of monoacylglycerol and diacylglycerol enantiomers in biological samples: Adjusting selectivity via column coupling.
Enhancing the practicality along with greenness sustainability of a high throughput HPLC-MS/MS bioanalytical method for the simultaneous determination of amlodipine, perindopril, and its active metabolite perindoprilat in human plasma: Application to a pharmacokinetic study.
Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry.
Quantification of 11 kratom alkaloids including mitragynine and its main metabolites in human plasma using LC-MS/MS.
Development and validation of a liquid chromatography-tandem mass spectrometry assay for the simultaneous analysis of isoniazid and pyrazinamide in cerebrospinal fluid.
Validation of a Dried Blood Spot Assay for Testosterone Measurement Using Liquid Chromatography-Tandem Mass Spectrometry.
Mass-Guided Single-Cell MALDI Imaging of Low-Mass Metabolites Reveals Cellular Activation Markers.
Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry.
Rounded Turn SLIM Design for High-Resolution Ion Mobility Mass Spectrometry Analysis of Small Molecules.
Fragmentation Considerations Using Amidoamine Oxide Homologs.
Development and clinical utility of an ultra performance liquid chromatography - tandem mass spectrometry assay for monitoring omadacycline and tigecycline in severe bacterial infections.
Development of an LC-MS/MS method for the simultaneous quantification of 11 perfluoroalkyl compounds in mouse plasma for toxicokinetic applications.
Holistic Comparison of the Lipidomes Simultaneously From 12 Panax Herbal Medicines By Ultra-High-Performance Supercritical Fluid Chromatography Coupled With Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry.
Quantitative NMR analysis of sugars in natural sweeteners: Profiling in honey, jaggery, date syrup, and coconut sugar.
Sensitive and accurate determination of 32 PFAS in human serum using online SPE-UHPLC-HRMS.
Comprehensive Investigation of Sweroside Metabolism in Rats Using a De Novo Strategy Based on Ultra-High-Performance Liquid Chromatography/Quadrupole-Exactive Mass Spectrometry.
Determination of microcystins and nodularins in ambient freshwater and seawater by liquid chromatography-mass spectrometry including toxin screening and identification.
Cost-efficient analysis of cannabinoids in therapeutic oils using HPLC with UV and mass spectrometry detection.
Forced Degradation Study of an Anti-Diabetic Drug Imeglimin: Impurity Profiling and Structure Elucidation Using LC-Q-ToF-MS/MS and NMR.
Microchip capillary electrophoresis-mass spectrometry for high-throughput simultaneous analysis of B-complex vitamins.
Metabolomic characterization of a new strain of microalgae by GC-MS method with the introduction of a deuterium label.

J Chromatogr A. 2024 Dec 03. pii: S0021-9673(24)00953-1. [Epub ahead of print]1740 465580

Matrix effect evaluation using multi-component post-column infusion in untargeted hydrophilic interaction liquid chromatography-mass spectrometry plasma metabolomics.

Mengle Zhu, Lieke Lamont, Pascal Maas, Amy C Harms, Marian Beekman, P Eline Slagboom, Anne-Charlotte Dubbelman, Thomas Hankemeier.

  Metabolomics based on hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS) is a powerful tool for polar metabolite identification and quantification to further contribute to biomarker discovery and disease mechanism elucidation. However, matrix effect (ME), which may lead to altered ionization efficiency due to co-eluting compounds, is a significant challenge during biological analysis. Therefore, ME evaluation plays a crucial role during method development. Two approaches to evaluate ME are using stable isotope labelled-internal standards (SIL-IS) and post-column infusion (PCI) of standards. In this study, we developed an untargeted HILIC-MS method by applying four PCI standards for ME evaluation. We found PCI is a compelling approach for ME assessment compared to SIL-IS method due to its advantage in untargeted analysis. Through the ME evaluation and chromatographic performance comparison of 18 SIL standards across three columns and three different mobile phase pH conditions, our findings revealed that the BEH-Z-HILIC column operated at pH 4 with 10 mM ammonium formate exhibited minimal ME and superior performance. The method showed exceptional linearity (R² > 0.98), reliable repeatability (RSD < 15 %), good inter-day precision (RSD < 30 %), and acceptable recovery (>75 %) for all SIL standards. Absolute matrix effect (AME) and relative matrix effect (RME) assessment in three plasma donors revealed a high consistency between PCI and SIL-IS approaches. Finally, this method coupled with the PCI approach was applied to 40 plasma samples. Fifty endogenous compounds were detected and their AME and RME were evaluated. Results showed that many compounds experienced severe ion suppression, though their ME variation between 40 samples is low. In conclusion, PCI method is a robust alternative for monitoring ME and evaluating ME of endogenous compounds during untargeted method optimization and biological analysis.

Keywords:  HILIC; Matrix effect; Plasma; Post-column infusion; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.chroma.2024.465580
J Chromatogr A. 2024 Dec 06. pii: S0021-9673(24)00960-9. [Epub ahead of print]1740 465588

Bioinert UHPLC system improves sensitivity and peak shapes for ionic metabolites.

Ondřej Peterka, Alena Langová, Robert Jirásko, Michal Holčapek.

  The analysis of ionic compounds by liquid chromatography is challenging due to the interaction of analytes with the metal surface of the instrument and the column, leading to poor peak shape and decreased sensitivity. The use of bioinert materials in the chromatographic system minimizes these unrequired interactions. In this work, the ultrahigh-performance liquid chromatography (UHPLC) with bioinert components was connected to a high-resolution mass spectrometer to develop a method for untargeted metabolomic analysis. 81 standards of metabolites were used for the development and optimization of the method. In comparison to the conventional chromatographic system, the application of bioinert technology resulted in significantly improved peak shapes and increased sensitivity, especially for metabolites containing phosphate groups. The calibration curves were constructed for the evaluation of the method performance, showing a wide dynamic range, low limit of detection, and linear regression coefficients higher than 0.99 for all standards. The optimized method was applied to the analysis of NIST SRM 1950 human plasma, which allowed the detection of 156 metabolites and polar lipids based on the combination of mass accuracy in the full-scan mass spectra in both polarity modes, characteristic fragment ions in MS/MS, and logical chromatographic behavior leading to the high confidence level of annotation/identification. We have demonstrated an improvement in the peak shapes and sensitivity of ionic metabolites using bioinert technology, which indicates the potential for the analysis of other ionic compounds, e.g., molecules containing phosphate groups.

Keywords:  Bioinert system; Human plasma; Mass spectrometry; Metabolomics; Ultrahigh-performance liquid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2024.465588
J Chromatogr A. 2024 Dec 09. pii: S0021-9673(24)00963-4. [Epub ahead of print]1740 465591

Chiral supercritical fluid chromatography of monoacylglycerol and diacylglycerol enantiomers in biological samples: Adjusting selectivity via column coupling.

Oleksandr Kozlov, Nela Štěrbová, Miroslav Lísa.

  The distinction of lipid isomers is gaining more attention in lipidomics due to their different biochemical properties in the organism. Herein, we aimed to develop a method for the analysis of monoacylglycerol (MG) and diacylglycerol (DG) enantiomers in biological samples using chiral supercritical fluid chromatography and mass spectrometry (SFC-MS). Amylose-based chiral columns showed a certain degree of separation of MG and DG isomers, but low selectivity for the acylglycerol classes in total lipid extracts, which could not be improved by modifier composition or other chromatographic conditions. The coelution of MG and DG enantiomers with highly concentrated triacylglycerols (TGs) negatively affected their MS determination based on the peak area ratio, therefore the interclass selectivity of chiral SFC was adjusted by coupling with an achiral column. The connection of the amylose tris-(3,5-dimethylphenylcarbamate) chiral column with octadecyl achiral column using a methanol as a modifier provided an excellent interclass separation of acylglycerols with the resolution of 5.53 and 15.17 for oleic acid-based MG/DG and DG/TG classes, respectively. The developed method enabled the determination of MG and DG enantiomers in complex total lipid extracts of biological samples in a 15 min gradient without time-consuming sample prefractionation. Chiral SFC-MS analysis of egg yolk, human plasma, and porcine brain samples showed different ratios of enantiomers, suggesting their unique roles within each sample type.

Keywords:  Chiral separation; Column coupling; Diacylglycerol; Monoacylglycerol; Selectivity; Supercritical fluid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2024.465591
J Chromatogr A. 2024 Nov 29. pii: S0021-9673(24)00932-4. [Epub ahead of print]1740 465559

Enhancing the practicality along with greenness sustainability of a high throughput HPLC-MS/MS bioanalytical method for the simultaneous determination of amlodipine, perindopril, and its active metabolite perindoprilat in human plasma: Application to a pharmacokinetic study.

Ehab F Elkady, Marwa A Moffid, Eman A Mostafa, Rawda M Sayed.

  Numerous studies have demonstrated the benefits of amlodipine and perindopril combination therapy in decreasing blood pressure and improving outcomes for high-risk patients. In order to assess the pharmacokinetics of the 2 drugs along with perindoprilat; the active metabolite of perindopril, a simultaneous LC-MS/MS quantification method of amlodipine (AML), perindopril (PER) and perindoprilat (LAT) in human plasma has been developed and validated using amlodipine D4, perindopril D4 and perindoprilat D4 as internal standards (ISs), respectively. A simple and fast protein precipitation method was used to analyze the three analytes from K3EDTA human plasma. The chromatographic separation included the use of a mixture of methanol and acetonitrile (80:20, v/v) and 0.2 % aqueous formic acid (60:40, v/v) as a mobile phase pumped onto a Zorbax® SB-AQ C18 column. Detection was carried out using a tandem mass spectrometer (MS/MS) in multiple reactions monitoring (MRM) mode. This method exhibited great sensitivity (LLOQ of 0.2 ng/mL for AML and PER and 0.4 ng/mL for LAT), linearity, accuracy (ranging from 97.64 % to 110.28 %), precision (ranging from 2.54 % to 7.60 %), and stability. The method showed good linearity over the range of (0.2-10 ng/mL) for AML, (0.2-160 ng/mL) for PER and (0.4-80 ng/mL) for LAT. The average extraction recoveries of AML, PER and LAT samples were between 81.92 % and 85.07 %. Total elution time was as low as 3 min only. In addition, to ensure the practicality of the developed method, BAGI tool was applied, and the current method has achieved the highest score among the compared methods. Moreover, the sustainability of the proposed method was evaluated using AGREE tool and RGB 12 paradigm showing remarkable sustainability. The developed method is fast, accurate, sensitive, reproducible, ecofriendly, sustainable and suitable for the determination of the concentration of the cited analytes in human plasma. Moreover, it was applied for the bioequivalence study of a generic product to the innovator.

Keywords:  AGREE; Amlodipine; BAGI; Bioequivalence; LC-MS/MS; Perindopril; RGB 12

DOI:  https://doi.org/10.1016/j.chroma.2024.465559
JDS Commun. 2024 Nov;5(6): 745-750

Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry.

August Hoppmann, Sebastian I Arriola Apelo.

The pool of free, genetically encoded AA in plasma plays an essential role not only as substrate for every protein synthesized in the body but also as signaling molecules that regulate a wide range of physiological processes. Here we present a method for the analysis of 19 of the 20 encoded AA (except Cys) in dairy cow plasma. Isolated plasma or standards for the 19 AA were gravimetrically mixed with an internal standard mix consisting of 13C isotopes of each AA. Plasma proteins were precipitated on acetonitrile and supernatants transferred to glass vials. For precolumn derivatization, plasma supernatants were buffered with sodium borate (pH 9.5-10), and AA were derivatized with 9-fluorenylmethoxycarbonyl (Fmoc) chloride. Analytes were isolated by solid-phase extraction using a strong-anionic ion exchange column and dry eluates were reconstituted in mobile phase consisting of 70% water solution of ammonium formate and 30% acetonitrile. Amino acid derivatives were separated by reverse-phase liquid chromatography over 17.5 min with a C18 column in which acetonitrile increased to 80% over the first 11 min of the method, before returning to initial levels. Electrospray ionization on negative mode was used for most AA, except Arg and Pro, for which positive mode yielded superior results. Single or double (only Lys) derivatives were measured by single quadrupole-mass spectrometry. We hypothesized that precolumn Fmoc derivatization would yield optimal resolution for quantitative analysis of the 19 targeted AA and their respective 13C internal standards, with limits of quantitation beyond physiological ranges. All 19 AA were detected with minimal background noise. An 11-point standard curve was developed for each AA. Limits of quantitation were beyond concentrations observed in plasma samples of lactating dairy cows, except for Gly where upper curve points had to be removed to maintain linearity, limiting quantitation to the upper range of physiological concentration. After removing the 4 highest concentrations from the Gly standard curve, coefficients of determination were greater than 0.999 for all of the AA. Recovery of spiked AA from plasma samples ranged from 89.9% for Phe to 100.3% for Trp. Instrument repeatability averaged 0.91 and ranged from 0.33 for Val to 2.29 for Arg. Meanwhile, sample preparation method repeatability averaged 2.02 and ranged from 1.14 for Tyr to 3.34 for Arg. Although robust methods have been developed, they depend on either availability of sophisticated instruments, mostly limited to core facilities (i.e., tandem MS methods), long and expensive chromatography without specific internal standards for each AA (i.e., HPLC-ultraviolet and HPLC-fluorescence detector), or unstable derivatization (GC-MS). Here we describe a method with high throughput, stable derivatization, high precision and recovery, and potentially more affordable than most existing methods. This method could help dairy nutritionists to consider plasma AA information for diet formulation strategies, potentially reducing feeding costs and N emissions.

DOI: https://doi.org/10.3168/jdsc.2024-0546
Anal Bioanal Chem. 2024 Dec 07.

Quantification of 11 kratom alkaloids including mitragynine and its main metabolites in human plasma using LC-MS/MS.

Cristina Sempio, Jorge Campos-Palomino, Jelena Klawitter, Wanzhu Zhao, Marilyn A Huestis, Uwe Christians, Jost Klawitter.

  Recently in the USA, kratom consumers increasingly report use of the plant for self-treatment of mood ailments, the lack of energy, chronic pain, and opioid withdrawal and dependence. Several alkaloids are present in kratom leaves, but limited data are available on their pharmacokinetics/pharmacodynamics, except for mitragynine. To support clinical studies, a high-performance liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of 11 kratom alkaloids in human plasma was developed and validated. For calibration standards and quality control samples, human plasma was fortified with alkaloids at varying concentrations, and 200 µL were extracted employing a simple one-step protein precipitation procedure. The extracts were analyzed using LC-MS/MS including electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode. The lower limit of quantification was 0.5 ng/mL, and the upper limit of quantification was 400 ng/mL for all analytes. Inter-day analytical accuracy and imprecision ranged from 98.4 to 113% of nominal and from 3.9 to 14.7% (coefficient of variance), respectively. The analysis of plasma samples collected during a clinical trial administering capsules containing kratom leaf extract showed that most samples had quantifiable concentrations of mitragynine, 7-OH-mitragynine, speciogynine, speciociliatine, and paynantheine and that mitragynine, speciogynine, and speciociliatine accumulated in human plasma after daily administration over 15 days. An LC-MS/MS assay for the specific quantification of kratom alkaloids including mitragynine and its main metabolites was developed and successfully validated in human plasma. Human plasma samples collected following multiple oral administrations of a controlled Kratom extract documented accumulation of kratom alkaloids over 15 days.

Keywords:  Kratom; LC-MS/MS; Mitragynine; Pharmacokinetics

DOI:  https://doi.org/10.1007/s00216-024-05689-9
J Pharm Biomed Anal. 2024 Dec 04. pii: S0731-7085(24)00655-1. [Epub ahead of print]255 116613

Development and validation of a liquid chromatography-tandem mass spectrometry assay for the simultaneous analysis of isoniazid and pyrazinamide in cerebrospinal fluid.

Sydwell Poulo Maputla, Anton Joubert, Sandra Castel, Marthinus van der Merwe, Edda Zangenberg, Sean Wasserman, Kelly E Dooley, Lubbe Wiesner.

  For the effective treatment of tuberculosis with first-line anti-tubercular drugs, drug concentrations need to be measured at the site of infection to determine drug exposure. To enable the measurement of the anti-tuberculosis drugs isoniazid and pyrazinamide in the nervous system of patients with tuberculous meningitis, an analytical method was developed and validated for the quantification of these drugs in human cerebrospinal fluid. Samples were prepared by solid phase extraction using Strata-X polymeric extraction plates. The analytes were separated by high-performance liquid chromatography on an Atlantis T3, 100 A, 3 µm, 2.1 mm × 100 mm analytical column with gradient elution, employing a mobile phase that consisted of acetonitrile-methanol-formic acid (50:50:0.1, v/v/v), at a flowrate of 0.25 mL/min. The total run time was 4.5 minutes, and the average retention times of isoniazid and pyrazinamide were 1.1 and 1.3 min, respectively. The analytes and their respective deuterated internal standards were detected on a Sciex API4000 triple quadrupole mass spectrometer applying positive electrospray ionization with multiple reaction monitoring as the detection mode. The method was validated according to the FDA and EMA guidelines. The method was demonstrated to be accurate, reproducible, and robust, showing the necessary sensitivity and specificity for the quantification of isoniazid and pyrazinamide in cerebrospinal fluid. The method was successfully applied to analyze clinical samples from the LASER-TBM and TBM-KIDS clinical studies.

Keywords:  Cerebrospinal fluid; Isoniazid; LC-MS/MS; Pyrazinamide; Tuberculosis meningitis

DOI:  https://doi.org/10.1016/j.jpba.2024.116613
Anal Sci Adv. 2024 Dec;5(11-12): e202400035

Validation of a Dried Blood Spot Assay for Testosterone Measurement Using Liquid Chromatography-Tandem Mass Spectrometry.

Yehudah Gruenstein.

  Testosterone is a critical hormone involved in regulating various physiological processes in both men and women. Accurate testosterone measurement is essential for diagnosing endocrine disorders such as hypogonadism and polycystic ovary syndrome and for routine testing. Traditionally, testosterone levels are measured using serum or plasma samples, which present challenges in sample collection, storage, and transport, particularly in resource-limited settings. Dried blood spot (DBS) sampling has emerged as an effective alternative for hormone analysis, offering significant advantages in terms of sample stability, ease of collection, and simplified logistics. This study aimed to validate a DBS-based testosterone assay using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to ensure accuracy and precision comparable to conventional serum-based methods. Drops of whole blood samples were collected from adult volunteers using a single-use safety lancet for finger pricks, with blood applied onto DBS cards (PerkinElmer 226 Spot Saver RUO card) for further analysis. The testosterone was extracted from DBS using a liquid-liquid method and analyzed with LC-MS/MS. The assay demonstrated excellent linearity across a wide concentration range (0.1-100 ng/mL) with a correlation coefficient (r2) of 0.999 and achieved a lower limit of detection of 0.058 ng/mL and a lower limit of quantification of 0.086 ng/mL. The method showed high precision, with intra- and inter-day coefficients of variation below 10%, and satisfactory recovery rates. Hematocrit correction and matrix effect evaluations confirmed the robustness of the assay for clinical and research applications. Additionally, the assay displayed a strong clinical correlation between testosterone levels in DBS and venous serum samples, supporting its reliability for testosterone monitoring. This validation study supports that the DBS-based LC-MS/MS testosterone assay is a reliable tool for testosterone quantification for routine testing.

Keywords:  assay validation; dried blood spot (DBS); hematocrit effect; liquid chromatography‐mass spectrometry (LC‐MS/MS); testosterone

DOI:  https://doi.org/10.1002/ansa.202400035
Adv Sci (Weinh). 2024 Dec 12. e2410506

Mass-Guided Single-Cell MALDI Imaging of Low-Mass Metabolites Reveals Cellular Activation Markers.

James L Cairns, Johanna Huber, Andrea Lewen, Jessica Jung, Stefan J Maurer, Tobias Bausbacher, Stefan Schmidt, Pavel A Levkin, Daniel Sevin, Kerstin Göpfrich, Philipp Koch, Oliver Kann, Carsten Hopf.

  Single-cell MALDI mass spectrometry imaging (MSI) of lipids and metabolites >200 Da has recently come to the forefront of biomedical research and chemical biology. However, cell-targeting and metabolome-preserving methods for analysis of low mass, hydrophilic metabolites (<200 Da) in large cell populations are lacking. Here, the PRISM-MS (PRescan Imaging for Small Molecule - Mass Spectrometry) mass-guided MSI workflow is presented, which enables space-efficient single cell lipid and metabolite analysis. In conjunction with giant unilamellar vesicles (GUVs) as MSI ground truth for cell-sized objects and Monte Carlo reference-based consensus clustering for data-dependent identification of cell subpopulations, PRISM-MS enables MSI and on-cell MS2-based identification of low-mass metabolites like amino acids or Krebs cycle intermediates involved in stimulus-dependent cell activation. The utility of PRISM-MS is demonstrated through the characterization of complex metabolome changes in lipopolysaccharide (LPS)-stimulated microglial cells and human-induced pluripotent stem cell-derived microglia. Translation of single cell results to endogenous microglia in organotypic hippocampal slice cultures indicates that LPS-activation involves changes of the itaconate-to-taurine ratio and alterations in neuron-to-glia glutamine-glutamate shuttling. The data suggests that PRISM-MS can serve as a standard method in single cell metabolomics, given its capability to characterize larger cell populations and low-mass metabolites.

Keywords:  MALDI mass spectrometry imaging; giant unilamellar vesicles (GUVs); human induced pluripotent stem cells (hiPSC); microglia; neurodegeneration; single cell; spatial metabolomics

DOI:  https://doi.org/10.1002/advs.202410506
J Vis Exp. 2024 Nov 22.

Detection of Regulated Ergot Alkaloids in Food Matrices by Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight Mass Spectrometry.

Alfonso Narváez, Laura Gámiz-Gracia, Ana M García-Campaña, Maykel Hernández-Mesa.

Ion mobility mass spectrometry (IMS) acts as an additional separation dimension when integrated into liquid chromatography-mass spectrometry (LC-MS) workflows. LC-IMS-MS methods provide higher peak resolution, enhanced separation of isobaric and isomeric compounds, and improved signal-to-noise ratio (S/N) compared to traditional LC-MS methods. IMS provides another molecular characteristic for the identification of analytes, namely the collision cross section (CCS) parameter, reducing false positive results. Therefore, LC-IMS-MS methods address important analytical challenges in the field of food safety (i.e., detection of compounds at trace levels in complex food matrices and unambiguous identification of isobaric and isomeric molecules). Ergot alkaloids (EAs) are a family of mycotoxins produced by fungi that attack a wide variety of grass species, including small grains such as rye, triticale, wheat, barley, millet, and oats. Maximum levels (MLs) of these mycotoxins have been established in several foodstuffs, as detailed in the Commission Regulation EC/2023/915. This new legislation includes six main EAs and their corresponding epimers, so an efficient methodology is required to properly distinguish these isomeric molecules considering their co-occurrence. Therefore, the goal of this protocol is to show how the integration of IMS in LC-MS workflows contributes to the separation of isomeric EAs, enhancing the selectivity of the analytical method. Additionally, it illustrates how the generation of CCS libraries through the characterization of analytical standards provides higher confidence for the identification of mycotoxins. This protocol is designed to clearly explain the benefits of implementing IMS in food safety, taking as an example the determination of EAs in cereals. A QuEChERS-based extraction followed by an LC-trapped ion mobility spectrometry (TIMS)-MS analysis provided limits of quantification ranging from 0.65 to 2.6 ng/g with acceptable accuracy (although low recovery for ergotaminine) at 1.5x, 1x, and 0.5x the ML and exhibited a negligible matrix effect.

DOI: https://doi.org/10.3791/67484
Anal Chem. 2024 Dec 11.

Rounded Turn SLIM Design for High-Resolution Ion Mobility Mass Spectrometry Analysis of Small Molecules.

Liulin Deng, Jody C May, Joshua K McBee, Adam Rosen, Leonard C Rorrer, Ryan Clingman, Miriam Fico, John A McLean, Daniel DeBord.

Various rounded turn designs in Structures for Lossless Ion Manipulation (SLIM) were explored via ion trajectory simulations. The optimized design was integrated into a SLIM ion mobility (IM) system coupled with a time-of-flight (TOF) mass spectrometer (MS) for further experimental investigation. The SLIM-TOF IM-MS system was assessed for IM resolution and ion transmission efficiency across a wide m/z range using various RF frequencies and buffer gas combinations. High ion transmission efficiency and high resolution ion mobility (HRIM) separation were achieved for Agilent tune mix ions through a ∼12.8 m serpentine separation path in both nitrogen and helium. In helium, ion transmission for low m/z ions was enhanced at higher RF trapping frequency, enabling the detection of ions with m/z below 50 and all 17 amino acids from a standard mixture. Lossless ion transmission was observed for glycine (m/z 76) in both passthrough and HRIM modes. HRIM resolution was benchmarked using L-isoleucine, L-leucine, and various other isobaric and isomeric metabolites with m/z values of 60-89. This work demonstrates a rounded turn SLIM design that enables HRIM measurements for small molecule analytes, with a particular focus on metabolomics, where IM offers a means to enhance the speed, robustness, and specificity of analytical workflows.

DOI: https://doi.org/10.1021/acs.analchem.4c03808
Mass Spectrom (Tokyo). 2024 ;13(1): A0158

Fragmentation Considerations Using Amidoamine Oxide Homologs.

Atsushi Yamamoto, Naoji Tokai, Rie Kakehashi, Daisuke Saigusa.

  This study investigates the mass spectrometric analysis of 10 novel amidoamine oxide compounds, which are innovative hydrogelators for polar solvents. This research aims to identify characteristic fragment patterns for these amide compounds using high-resolution mass spectrometry. Methanol solutions of the compounds were analyzed in positive and negative ion modes, and MS1 and MS2 spectra at 6 collision energy levels were obtained via electrospray ionization and hybrid tandem mass spectrometry. The importance of low-intensity peaks in structure elucidation was emphasized because low-intensity fragments could provide crucial structural information, especially for compounds with similar structures. Chain-length-dependent fragmentation patterns were observed, which could aid in predicting the structures of related compounds. This research highlights the challenges of balancing informative low-intensity peaks with accurate spectral matching in databases. Based on our results, combining mass spectrometry with separation techniques, such as liquid chromatography, could enhance structural elucidation for unknown compounds. This study contributes to the broader field of mass spectrometry and structural chemistry, particularly in the analysis of amide compounds, and future directions are proposed for developing robust algorithms for selecting and interpreting low-intensity peaks to improve compound identification in complex mixtures.

Keywords:  amidoamine oxide; chain-length dependence of fragmentation; structural elucidation

DOI:  https://doi.org/10.5702/massspectrometry.A0158
J Mass Spectrom Adv Clin Lab. 2024 Nov;34 46-54

Development and clinical utility of an ultra performance liquid chromatography - tandem mass spectrometry assay for monitoring omadacycline and tigecycline in severe bacterial infections.

Chang Wang, Bingfeng Luo, Wenqing Liu, Chen Jia, Haile Chen, Jingjing Ma, Xia Song, Xingfang Ji, Aijia Cao, Yinliang Bai, Wen Qiu.

   Objective: We aimed to develop a rapid, simple, and precise ultra performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) technique for simultaneous measurement of omadacycline (OMA) and tigecycline (TGC) in the bloodstream of individuals suffering from serious bacterial infections.
Methods: All analytes were extracted using a 0.2 % formic acid-water dilution and acetonitrile plasma protein precipitation. The quantification was performed by electrospray ionization-triple quadrupole mass spectrometry with selected reaction monitoring and positive ion mode detection. Tetracycline was used as an internal standard in this experiment, with the mobile phase composed of water (with 0.1 % formic acid) and acetonitrile (using gradient elution) flowing at a rate of 0.35 ml/min, and the column temperature set at 30 °C. Each individual analysis was completed in under 3.5 min.
Results: The method was validated based on FDA recommendations, including the assessment of extraction recovery (92.65-101.72 %) and matrix effects (86.22-91.12 %). The standard curve ranges for both OMA and TGC are 0.025 µg/mL to 2.5 µg/mL. The plasma samples were found to be consistent after undergoing three rounds of freezing and thawing at room temperature for 24 h, being placed in an automated sample injector for 24 h, and then frozen for 45 days. Clinical cases were used to demonstrate the application of the therapeutic drug monitoring (TDM) assay, showing how an analytical test can quickly provide information on antibiotic levels in patients and impact their treatment.
Conclusion: Multiplex UPLC-MS/MS assays for the simultaneous measurement of plasma OMA and TGC concentrations are the ideal choice for clinically TDM applications.

Keywords:  Omadacycline; Plasma concentration; Severe infection; Tigecycline; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jmsacl.2024.11.001
J Pharm Biomed Anal. 2024 Nov 30. pii: S0731-7085(24)00638-1. [Epub ahead of print]255 116596

Development of an LC-MS/MS method for the simultaneous quantification of 11 perfluoroalkyl compounds in mouse plasma for toxicokinetic applications.

Chloé Ml Argoul, Yannick Dauwe, Laïla Lakhal, Pierre-Louis Toutain, Nicole Picard-Hagen, Véronique Gayrard, Marlène Z Lacroix.

  Following regulatory pressure, the manufacture of long-chain per- and polyfluoroalkyl substances (PFAS) has been phased out, and alternatives such as short-chain homologs and ether-PFAS have replaced the bioaccumulative long-chain PFAS. However, data are lacking regarding the toxicokinetic (TK) properties of certain PFAS, particularly emergent substitutes for long-chain compounds. Additionally, the existing analytical methods used for TK studies measure a single compound or only a few simultaneously. For this reason, an LC-MS/MS method was developed for the simultaneous quantification in mouse plasma of 11 PFAS representative of some of the most important categories of these compounds, for application in TK studies. The method was successfully validated in the range of 0.5-1000 ng/mL, in accordance with the European Medicines Agency guidelines, and applied to a 24-h pilot TK study conducted in mice. All compounds were monitored over 24 hours in the pilot study. The present method is therefore suitable for the simultaneous quantification of PFAS in plasma samples and can be applied for future TK studies.

Keywords:  Liquid chromatography; Mass spectrometry; Perfluoroalkyl substances; Toxicokinetic

DOI:  https://doi.org/10.1016/j.jpba.2024.116596
J Sep Sci. 2024 Dec;47(23): e70040

Holistic Comparison of the Lipidomes Simultaneously From 12 Panax Herbal Medicines By Ultra-High-Performance Supercritical Fluid Chromatography Coupled With Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry.

Lili Hong, Yu Wang, Simiao Wang, Ying Xiong, Bei Xu, Qinhua Chen, Yang Yang, Mengxiang Ding, Hongda Wang, Wenzhi Yang.

  Researches regarding quality control of ginseng focusing on the lipids are rare. Herein, ultra-high-performance supercritical fluid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPSFC/IM-QTOF-MS) combined with untargeted metabolomic analysis was utilized to holistically characterize and compare the lipidomic difference among 12 Panax-derived herbal medicines. The established UHPSFC/IM-QTOF-MS method, using a Torus 1-AA column with CO2/CH3OH (modifier) as the mobile phase, well resolved the ginseng lipidome within 30 min. The lipid isomers and those easily co-eluted by conventional reversed-phase chromatography got separated, and integrated analyses of the positive-/negative-mode MS data and IM-derived collision cross section (CCS) greatly enhanced lipids identification. By the pattern recognition chemometric analysis of 90 batches of ginseng samples, the root ginseng samples showed significant differences in lipidome composition from those stem/leaf and flower samples. In contrast, red ginseng also contained lipids significantly different from the other root ginseng. Totally 82 potential differential lipids were discovered and identified based on the positive-mode data and 90 ones in the negative mode. Some of these lipid markers might be diagnostic for their authentication. Conclusively, we first report the lipidomic difference among 12 ginseng varieties, and the information obtained can lay foundation for the accurate identification of ginseng from the lipidome level.

Keywords:  ultra‐high‐performance supercritical fluid chromatography, ion mobility‐quadrupole time‐of‐flight mass spectrometry, lipidome, untargeted metabolomic analysis, Panax

DOI:  https://doi.org/10.1002/jssc.70040
Food Res Int. 2025 Jan;pii: S0963-9969(24)01428-5. [Epub ahead of print]199 115358

Quantitative NMR analysis of sugars in natural sweeteners: Profiling in honey, jaggery, date syrup, and coconut sugar.

Anisha Biswas, Babasaheb Bhaskarrao Borse, Sachin R Chaudhari.

  Due to the high demand for natural sweeteners and their perceived health benefits, it is crucial to use analytical techniques for accurately profiling natural sweeteners. The present study describes a simple and fast approach for the analysis of sweeteners using 1D - 1H NMR spectroscopy. This method is based on the direct detection of protons in sugar molecules with an internal standard, without the need for complex derivatization steps. The presented approach offers a faster and more convenient way of quantifying mono-saccharides mainly glucose and fructose and di-saccharides like sucrose in various selected sweeteners. These includes honey, jaggery, coconut sugar, and date syrup. The direct 1D - 1H NMR method with an internal standard yields accurate and precise quantification results with good reproducibility and minute analysis times. This information is of increasing importance to both consumers and the food industry, as it provides a reliable and accurate method for characterizing and verifying natural sweeteners. Overall, 1D - 1H NMR spectroscopy can be a valuable tool for the rapid and easy analysis of sugar content in food products, and it may have potential applications in the food industry.

Keywords:  (1)H NMR spectroscopy; Food chemistry; Multiscale structure; Natural sweeteners; Sugar profiling; UPLC; q-NMR

DOI:  https://doi.org/10.1016/j.foodres.2024.115358
J Hazard Mater. 2024 Dec 04. pii: S0304-3894(24)03361-2. [Epub ahead of print]485 136780

Sensitive and accurate determination of 32 PFAS in human serum using online SPE-UHPLC-HRMS.

Masho Hilawie Belay, Elisa Robotti, Arianna Ghignone, Alessia Fabbris, Jessica Brandi, Daniela Cecconi, Maria Angela Masini, Francesco Dondero, Emilio Marengo.

  Per- and polyfluoroalkyl substances' (PFAS) extreme persistence has been linked to many adverse effects on human health including increased risk of certain cancers. This study presents the development and validation of a new, highly sensitive method for the quantification of 32 PFAS in human serum using online solid-phase extraction (SPE) coupled with ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Legacy and emerging PFAS were targeted. Main steps of sample pretreatment include protein precipitation (PP), pellet rinsing, centrifugation, preconcentration through solvent evaporation, and online SPE using a weak anion-exchange polymeric sorbent. The PP and pellet-rinsing procedures were optimized through a comprehensive exploration of solvent combinations. Following this, a pretreatment that offers the best compromise for the targeted PFAS was identified using principal component analysis. The method demonstrated excellent linearity (R² = 0.977-0.997) with limits of quantification ranging from 8.9 to 27 ng/L, 5 to 15 times lower than previous methods. Precision (intraday 2.6-14.0 % and interday 1.3-11.0 % relative standard deviation) and accuracy (recoveries 72.7-106 %) were robust. The method was validated in accordance with ISO/IEC 17025 and successfully applied to five human serum samples, confirming its suitability for high-throughput profiling of PFAS in biomonitoring studies. This method is the first to use online SPE for the simultaneous determination of a broad range of PFAS, including ether congeners such as perfluoro(2-ethoxyethane) sulfonic acid and Nafion byproduct 2. Furthermore, control charts were employed to assess instrument performance during routine analysis and implement necessary actions.

Keywords:  High resolution mass spectrometry; Human biomonitoring; Method development; PFAS; Serum pretreatment

DOI:  https://doi.org/10.1016/j.jhazmat.2024.136780
J Sep Sci. 2024 Dec;47(23): e70048

Comprehensive Investigation of Sweroside Metabolism in Rats Using a De Novo Strategy Based on Ultra-High-Performance Liquid Chromatography/Quadrupole-Exactive Mass Spectrometry.

Xuelin Sun, Zhanpeng Shang, Yatong Zhang, Jiantao Qiu, Xueying Tan.

  Sweroside, a natural secoiridoid glycoside derived from various medicinal plants, is known for its anti-tumor, anti-inflammatory, and hepatoprotective properties. However, its pharmacological significance is not fully supported by its low systemic exposure. In this study, a de novo strategy was proposed to investigate the metabolism of sweroside in rats, including drug administration, sample pretreatment, ultra-high-performance liquid chromatography/Quadrupole-Exactive mass spectrometry data acquisition, data processing, and semi-quantitative analysis. First, following oral administration of sweroside to rats, plasma, urine, and feces were collected, and respectively mixed using the area-under-the-curve pooling method. Secondly, data-dependent, dynamic exclusion, and parallel reaction monitoring scan modes were employed to build a tandem mass spectra database. Using a neutral loss fragment-based method, target metabolites were effectively filtered. As a result, sweroside was demonstrated to be extensively metabolized, while 18 metabolites were identified and nine of them were newly reported. Sweroside predominantly underwent phase II metabolism, including glycosylation, sulfonation, glucuronidation, and deglycosylation, and were primarily excreted via the kidney. Notably, N-heterocyclization (M7 and M10) was likely catalyzed by intestinal bacteria. This study not only elucidates the in vivo drug elimination of sweroside but also offers an efficient approach for profiling the metabolism of specific molecules.

Keywords:  area‐under‐the‐curve pooling; de novo strategy; drug metabolism; neutral loss fragment; sweroside

DOI:  https://doi.org/10.1002/jssc.70048
Anal Chim Acta. 2025 Jan 15. pii: S0003-2670(24)01250-9. [Epub ahead of print]1335 343449

Determination of microcystins and nodularins in ambient freshwater and seawater by liquid chromatography-mass spectrometry including toxin screening and identification.

Zhihong Wang, Christina M Mikulski, Makayla Kent, Tod Leighfield, Gregory J Doucette, John S Ramsdell.

   BACKGROUND: Microcystins (MCs) and nodularins (NODs) produced by cyanobacteria occur in ambient freshwaters and across the freshwater-marine continuum, and pose health threats through drinking and recreational waters, as well as food resources. Approximately 300 MC and NOD toxins have been published, but less than 15 of them are commercially available as toxin standards. Our aim herein was to rapidly identify and quantify all toxin congeners, including those without standards, in water samples even at low abundance by reversed-phase solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) to provide insights into toxin levels and potential toxicity.
RESULTS: Trizma instead of acid was used as an ion pairing reagent to increase retention of MCs without arginine residues on Strata X SPE. Toxin elution from Oasis HLB SPE was complicated by their hydrophilic and lipophilic interactions with HLB sorbent. Three stable isotope-labeled (SIL) toxin standards representing MCs carrying 2, 1, and 0 arginine residues served as internal standards (IS) for toxin determination and evaluation of cyanobacterial cell lysis methods. Average recoveries of toxin standards in lake water and seawater using the HLB sorbent for validation ranged from 90 to 109 % except MC-WR & LW (71-87 %) with detection limits from 1.3 to 23.7 ng L-1. Doubly charged protonated toxin molecular ions were employed as precursors for tandem MS to screen and identify toxin congeners using a hybrid triple quadrupole linear ion trap MS. More than 30 (4 new) MCs were detected in Microcystis aeruginosa strain LE-3 culture.
SIGNIFICANCE: This is the first report to explain the issues with and possible mechanisms for SPE extraction of MCs from water, to use SIL-IS to evaluate methods for lysing cyanobacterial cells for MC release, and to show that doubly rather than singly charged toxin molecular ions as MS/MS precursors enabled efficient screening, identification, and quantification of all toxins at low levels. A MC with homoalanine residue at position 1 was reported for the first time.

Keywords:  Freshwater; Liquid chromatography-mass spectrometry; Microcystin; Nodularin; Seawater; Solid-phase extraction

DOI:  https://doi.org/10.1016/j.aca.2024.343449
Nat Prod Res. 2024 Dec 13. 1-9

Cost-efficient analysis of cannabinoids in therapeutic oils using HPLC with UV and mass spectrometry detection.

Alexander Fries, Carlos Alberto Moldes, Laura S Mazzaferro.

  Cannabis oil, derived from Cannabis sativa plants, is increasingly used for therapeutic purposes across a wide range of diseases. Accurate quantification of cannabinoids is essential, especially for cannabis products sourced from informal markets where supply origins are uncertain. This study aimed to develop a cost-effective, robust analytical methodology using liquid chromatography in combination with UV- and mass detectors for the quantification of key cannabinoids (THC, CBD and CBN) and the identification of THCA and CBDA. Utilising an isocratic flow, the method achieved effective separation within 17 min, ensuring simplicity and reproducibility. The methodology validation was aligned with ICH guidelines' requirements for selectivity, linearity, precision, accuracy, and matrix effects. Successful application of this method to both homemade and commercial cannabis oil samples underscores its relevance for adjusting therapeutic doses and optimising CBD:THC ratios for specific disease treatments.

Keywords:  HPLC-MS; Isocratic flow; cannabinoids; cost-effective; validation

DOI:  https://doi.org/10.1080/14786419.2024.2439024
Rapid Commun Mass Spectrom. 2025 Mar;39(5): e9960

Forced Degradation Study of an Anti-Diabetic Drug Imeglimin: Impurity Profiling and Structure Elucidation Using LC-Q-ToF-MS/MS and NMR.

Shiva Kumar Gogikar, Sibu Sen, Shivashankar Pathinti, Gananadhamu Samanthula, Amol G Dikundwar.

   RATIONALE: The present study aims to establish structures of the degradation products of an anti-diabetic drug, Imeglimin (IMG) approved for the treatment of type 2 diabetes mellitus in the year 2021. Degradation pathways are proposed along with in silico toxicity assessments of the observed degradation products (DPs) of the drug.
METHODS: A reversed-phase high-performance liquid chromatography (RP-HPLC), equipped with a photodiode array detector, was used to separate the observed DPs with a Phenomenex Luna PFP (250 × 4.6 mm, 5 μm) column, using 10 mM ammonium formate (pH 4.5) and methanol as mobile phase. Liquid chromatography quadrupole time of flight mass spectrometry (LC-Q-ToF-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed for structural elucidation. Zeneth and Derek suites were used for in silico assessments.
RESULTS: A total of four degradation products were observed, which were successfully separated on an RP-HPLC. The structural characterization of three of the four DPs was achieved using LC-Q-TOF-MS/MS by employing electro spray ionization as well as atmospheric pressure chemical ionization. Additionally, DP-3 was isolated using a preparative HPLC and was characterized by NMR. Computationally predicted structures were compared with the experimental observations.
CONCLUSION: An HPLC method, capable of separating the Imeglimin and its four DPs, was developed and validated as per the ICH Q2(R1) guideline. Structure elucidation reveals a variety of products with metformin as one of the identified DPs along with a metabolite. The toxicity potential of DPs was assessed through docking studies.

Keywords:  Imeglimin; LC‐Q‐ToF‐MS/MS; forced degradation; metformin; toxicity

DOI:  https://doi.org/10.1002/rcm.9960
J Chromatogr A. 2024 Dec 06. pii: S0021-9673(24)00961-0. [Epub ahead of print]1740 465589

Microchip capillary electrophoresis-mass spectrometry for high-throughput simultaneous analysis of B-complex vitamins.

Liu Yang, Shuang Wang, Xiaolong Li, Weimin Wang, Fuxing Xu, Chuan-Fan Ding.

  B-complex vitamins are essential micronutrients that maintaining health, and provide (individually/simultaneously) many important biological actions in organism. Therefore, sensitive, reliable analytical method to determine B-complex vitamins simultaneously in actual samples is significant. Conventional analytical methods for vitamins analysis are usually labor-intensive, time-consuming and mostly do not allow the simultaneous determination. Few papers have been published on capillary electrophoresis-mass spectrometry (CE-MS) analysis of individual vitamin B or B-complex vitamins. Microchip capillary electrophoresis (ME) offers less consumption of background electrolyte and reagents than CE, and fast separation as having several channels for high-throughput. ME based on ZipChip integrates sample injection, electrophoretic separation, and ESI functions into one platform, herein, for the first time, in this paper, the ME-MS based on ZipChip platform was investigated and demonstrated preliminarily a significant improvement over existing techniques. Because: the ME-MS method has a linear response with excellent precision for replicate injections and LODs lower than 4 ppb; The method is simple and fast (<3 min per sample), using a small amount of sample (∼5 nL) with low reagent consumption, making it suitable for high-throughput applications; The inter/intraday reproducibility (RSD %) is less than 3 %, which is important for clinic application.

Keywords:  B-complex vitamins; Microchip capillary electrophoresis-mass spectrometry; Simultaneous analysis; ZipChip

DOI:  https://doi.org/10.1016/j.chroma.2024.465589
Biochimie. 2024 Dec 05. pii: S0300-9084(24)00293-1. [Epub ahead of print]

Metabolomic characterization of a new strain of microalgae by GC-MS method with the introduction of a deuterium label.

Anna Vishnevskaya, Anton Bashilov, Sergey Osipenko, Albert Kireev, Maria Sinetova, Eugene Nikolaev, Yury Kostyukevich.

  Microalgae are active producers of various compounds, including toxic substances. However, their metabolism is very diverse and insufficiently known. We demonstrate an approach that includes growing a new strain of cyanobacterium Leptolyngbya sp. (IPPAS B-1204) on an isotopically labeled medium (D2O) and evaluating the metabolomic composition of these microorganisms after deuterium uptake. Despite the low resolution of the GC-MS method, the interpretation of the obtained spectra allowed us to find out not only the amount of the embedded isotope label but also to assume the position in the structure where the label is embedded. We present the results of reliably detecting more than 30 compounds with isotope labels belonging to various classes of biological compounds produced by this cyanobacterium, revealing the metabolic pathways of entry of this label. We also demonstrate that the synthesis of unsaturated fatty acids is suppressed under the growth on D2O medium. In addition, we found an isotopic effect in the chromatographic separation of isotopically labeled compounds in gas chromatography. These data can be used in the future both for the identification of compounds and the analysis of the biosynthesis pathways of secondary biologically active compounds and in the analysis of the production of isotopically labeled standards of compounds.

Keywords:  Cyanobacterium; Gas chromatography; H/D exchange; Heavy water administration; Mass spectrometry; Microalgae; Plant metabolomics; Stable isotope labeling

DOI:  https://doi.org/10.1016/j.biochi.2024.11.015