bims-metlip 2024-07-14 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024‒07‒14
thirty papers selected by
Sofia Costa, Matterworks

Simultaneous measurement of 17 endogenous steroid hormones in human serum by liquid chromatography-tandem mass spectrometry without derivatization.
Metabolomics 2023 workshop report: moving toward consensus on best QA/QC practices in LC-MS-based untargeted metabolomics.
A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues.
Quantification of amino acids secreted by yeast cells by hydrophilic interaction liquid chromatography-tandem mass spectrometry.
Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies.
An FDA-Validated, Self-Cleaning Liquid Chromatography-Mass Spectrometry System for Determining Small-Molecule Drugs and Metabolites in Organoid/Organ-on-Chip Medium.
Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method.
Oversampling for Enhanced Spatial Resolution of Zebrafish by Top-Hat IR-MALDESI-MSI.
Development and validation of an ultrafast method of quantification of rivaroxaban in human serum using laser diode thermal desorption coupled to triple quadrupole mass spectrometry.
Gas chromatography-mass spectrometry determination of nicotine and cotinine in urine: A study of the effect of passive smoking.
Development, validation and long-term evaluation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of amiodarone, desethylamiodarone and mexiletine in human plasma and serum.
Recent Advancement in NMR Based Plant Metabolomics: Techniques, Tools, and Analytical Approaches.
Analysis of Sphingosine and Sphinganine from the Aqueous Humor for Signaling Studies Using Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry.
Detailed Protocol for Solid-Phase Extraction for Lipidomic Analysis.
Development and validation of a sensitive and simultaneous liquid chromatography tandem mass spectrometry method for the determination of eight phytocannabinoids in various CBD products.
Therapeutic drug monitoring of mycophenolic acid and azole antifungals on two distinct LC-MS/MS instruments.
Selective Profiling of Carboxylic Acid in Crude Oil by Halogen Labeling Combined with Liquid Chromatography and High-Resolution Mass Spectrometry.
UPLC-MS/MS determination of 71 neuropsychotropic drugs in human serum.
Accurate Collisional Cross Section Measurement by Multipass Cyclic Ion Mobility Spectrometry.
Improved Characterization of Soil Organic Matter by Integrating FT-ICR MS, Liquid Chromatography Tandem Mass Spectrometry, and Molecular Networking: A Case Study of Root Litter Decay under Drought Conditions.
Effects of a Laparotomy on Targeted Lipidomics Profiles in a Mouse Model of Surgical Stress During Aging.
An analytical method using salting-out assisted liquid-liquid extraction to quantify ceftriaxone from micro volumes of human serum.
Determination of mitomycin C in rat plasma by liquid chromatography-tandem mass spectrometry and its application for determining pharmacokinetics in rat.
Lipidomics Profiling of the Linoleic Acid Metabolites After Whole-Body Vibration in Humans.
Improved high-performance liquid chromatography tandem mass spectrometry method for quantification of infliximab in pediatric plasma and its application in therapeutic drug monitoring.
Accurate Quantification of Ten Methylated Purine Nucleosides by Highly Sensitive and Stable Isotope-Diluted UHPLC-MS/MS.
Optimal conditions for determination of bacitracin, bacitracin zinc and bacitracin methylene disalicylate in animal feed by ultra-performance liquid tandem mass spectrometry.
Resolution Enhancement of Metabolomic J-Res NMR Spectra Using Deep Learning.
Discovery and Identification of Three Homocysteine Metabolites by Chemical Derivatization and Mass Spectrometry Fragmentation.
Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

J Steroid Biochem Mol Biol. 2024 Jul 04. pii: S0960-0760(24)00126-2. [Epub ahead of print]243 106578

Simultaneous measurement of 17 endogenous steroid hormones in human serum by liquid chromatography-tandem mass spectrometry without derivatization.

Marija Gjorgoska, Tea Lanišnik Rižner.

  Mass spectrometric-based steroidomics is a valuable analytical approach that gives a comprehensive understanding of the interlinked steroid biosynthetic pathways. Here, we describe a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed to accurately quantify endogenous steroids in human serum. Sample preparation involved liquid-liquid extraction with methyl tert-butyl ether (MTBE) from 180 µL serum. The targeted steroids for quantification included androgens: dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), 11-oxyandrogens: 11β-hydroxy-androstenedione (11OHA4), 11-keto-androstenedione (11KA4), 11β-hydroxy-testosterone (11OHT), 11-keto-testosterone (11KT), progestogens: 17α-hydroxy-progesterone (17OHP4), progesterone (P4), 11β-hydroxy-progesterone (11OHP4), 11-keto-progesterone (11KP4), mineralocorticoids: aldosterone, corticosterone, and glucocorticoids: 11-deoxycortisol, cortisol, and cortisone. The lower limits of quantification (LLOQ) were 0.05 ng/mL for A4, T, 11KA4, P4, and cortisone, 0.1 ng/mL for DHT, 11OHA4, 11OHT, 11KT, 17OHP4, 11OHP4, 11KP4, corticosterone, aldosterone, 11-deoxycortisol, and cortisol, and 0.5 ng/mL for DHEA. Accuracy, precision, reproducibility, and recovery fell within acceptable limits for bioanalytical method validation. Using serum samples from 29 premenopausal women in different menstrual phases, we demonstrated the clinical utility of our method, which showed sufficient sensitivity to reliably quantify all targeted steroids at levels typically found in circulation, except for 11OHP4 and 11KP4.

Keywords:  11-oxygenenated steroids; Androgens; Glucocorticoids; Liquid chromatography-tandem mass spectrometry; Mineralocorticoids; Progestogens

DOI:  https://doi.org/10.1016/j.jsbmb.2024.106578
Metabolomics. 2024 Jul 09. 20(4): 73

Metabolomics 2023 workshop report: moving toward consensus on best QA/QC practices in LC-MS-based untargeted metabolomics.

Metabolomics Quality Assurance, Quality Control Consortium (mQACC)

  INTRODUCTION: During the Metabolomics 2023 conference, the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) presented a QA/QC workshop for LC-MS-based untargeted metabolomics.OBJECTIVES: The Best Practices Working Group disseminated recent findings from community forums and discussed aspects to include in a living guidance document.
METHODS: Presentations focused on reference materials, data quality review, metabolite identification/annotation and quality assurance.
RESULTS: Live polling results and follow-up discussions offered a broad international perspective on QA/QC practices.
CONCLUSIONS: Community input gathered from this workshop series is being used to shape the living guidance document, a continually evolving QA/QC best practices resource for metabolomics researchers.

Keywords:  Data quality; Liquid chromatography–mass spectrometry (LC–MS); Metabolite annotation; Metabolite identification; Metabolomics; Quality assurance; Quality control (QC) samples; Reference materials

DOI:  https://doi.org/10.1007/s11306-024-02135-w
Biochemistry. 2024 Jul 10.

A Facile LC-MS Method for Profiling Cholesterol and Cholesteryl Esters in Mammalian Cells and Tissues.

Aakash Chandramouli, Siddhesh S Kamat.

Cholesterol is central to mammalian lipid metabolism and serves many critical functions in the regulation of diverse physiological processes. Dysregulation in cholesterol metabolism is causally linked to numerous human diseases, and therefore, in vivo, the concentrations and flux of cholesterol and cholesteryl esters (fatty acid esters of cholesterol) are tightly regulated. While mass spectrometry has been an analytical method of choice for detecting cholesterol and cholesteryl esters in biological samples, the hydrophobicity, chemically inert nature, and poor ionization of these neutral lipids have often proved a challenge in developing lipidomics compatible liquid chromatography-mass spectrometry (LC-MS) methods to study them. To overcome this problem, here, we report a reverse-phase LC-MS method that is compatible with existing high-throughput lipidomics strategies and capable of identifying and quantifying cholesterol and cholesteryl esters from mammalian cells and tissues. Using this sensitive yet robust LC-MS method, we profiled different mammalian cell lines and tissues and provide a comprehensive picture of cholesterol and cholesteryl esters content in them. Specifically, among cholesteryl esters, we find that mammalian cells and tissues largely possess monounsaturated and polyunsaturated variants. Taken together, our lipidomics compatible LC-MS method to study this lipid class opens new avenues in understanding systemic and tissue-level cholesterol metabolism under various physiological conditions.

DOI: https://doi.org/10.1021/acs.biochem.4c00160
J Sep Sci. 2024 Jul;47(13): e2400318

Quantification of amino acids secreted by yeast cells by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Xiongwei Yin, Luís Santos Sousa, Bruno André, Erwin Adams, Ann Van Schepdael.

  Monitoring the levels of amino acids (AAs) in biological cell cultures provides key information to understand the regulation of cell growth and metabolism. Saccharomyces cerevisiae can naturally excrete AAs, making accurate detection and determination of amino acid levels within the cultivation medium pivotal for gaining insights into this still poorly known process. Given that most AAs lack ultraviolet (UV) chromophores or fluorophores necessary for UV and fluorescence detection, derivatization is commonly utilized to enhance amino acid detectability via UV absorption. Unfortunately, this can lead to drawbacks such as derivative instability, labor intensiveness, and poor reproducibility. Hence, this study aimed to develop an accurate and stable hydrophilic interaction liquid chromatography-tandem mass spectrometry analytical method for the separation of all 20 AAs within a short 17-min run time. The method provides satisfactory linearity and sensitivity for all analytes. The method has been validated for intra- and inter-day precision, accuracy, recovery, matrix effect, and stability. It has been successfully applied to quantify 20 AAs in samples of yeast cultivation medium. This endeavor seeks to enhance our comprehension of amino acid profiles in the context of cell growth and metabolism within yeast cultivation media.

Keywords:  HILIC‐MS; amino acids; yeast culture media

DOI:  https://doi.org/10.1002/jssc.202400318
Methods Mol Biol. 2024 ;2816 25-33

Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies.

Alexa Jauregui, Emily J Neag, Amr Almobayed, Alexandra Lens, Sanjoy K Bhattacharya.

  Interconvertible sphingolipid metabolites represent germane constituents of eukaryotic membranes and are vital in the regulation of cellular homeostasis, proliferation, survival, and induction of autophagy. This protocol describes a step-by-step method for extractions of sphingosine and sphinganine from mammalian tissue samples, particularly from the murine optic nerve. These lipids are partitioned into a binary mixture of chloroform and methanol in a modified Bligh and Dyer method. This is followed with reverse phase ultrahigh-performance liquid chromatography fractionation with a C18+ column and subsequent tandem mass spectrometry (UHPLC-MS-MS) analysis of the biological abundance. These free sphingoid bases dissociate to form structurally distinctive carbocation product ions that can be confirmed with annotations of lipidomic databases or in-house fragmentation software.

Keywords:  Bligh and Dyer; Imaging mass spectrometry; Lipidomics; Mass spectrometry; Optic nerve; Regeneration

DOI:  https://doi.org/10.1007/978-1-0716-3902-3_3
Anal Chem. 2024 Jul 10.

An FDA-Validated, Self-Cleaning Liquid Chromatography-Mass Spectrometry System for Determining Small-Molecule Drugs and Metabolites in Organoid/Organ-on-Chip Medium.

Stian Kogler, Gustav Mathingsdal Pedersen, Felipe Martínez-Ramírez, Aleksandra Aizenshtadt, Mathias Busek, Stefan J K Krauss, Steven Ray Wilson, Hanne Røberg-Larsen.

As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography-mass spectrometry (LC-MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC-MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.

DOI: https://doi.org/10.1021/acs.analchem.4c02246
J Chromatogr A. 2024 Jul 04. pii: S0021-9673(24)00497-7. [Epub ahead of print]1730 465123

Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method.

Amir Khajavinia, Deborah Michel, Udoka C Ezeaka, Randy W Purves, Robert B Laprairie, Anas El-Aneed.

  Psilocybin is a psychedelic compound found in some hallucinogenic "magic mushrooms". Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.

Keywords:  HILIC; LC-MS; Psilocin; Quantfication

DOI:  https://doi.org/10.1016/j.chroma.2024.465123
J Am Soc Mass Spectrom. 2024 Jul 10.

Oversampling for Enhanced Spatial Resolution of Zebrafish by Top-Hat IR-MALDESI-MSI.

Alexandria L Sohn, Andrew P Bowman, Morgan M Barnes, Seth W Kullman, David C Muddiman.

  Mass spectrometry imaging (MSI) has become a significant tool for measuring chemical species in biological tissues, where much of the impact of these platforms lies in their capability to report the spatial distribution of analytes for correlation to sample morphology. As a result, enhancement of spatial resolution has become a frontier of innovation in the field, and necessary developments are dependent on the ionization source. More particularly, laser-based imaging sources may require modifications to the optical train or alternative sampling techniques. These challenges are heightened for systems with infrared (IR) lasers, as their operating wavelength generates spot sizes that are inherently larger than their ultraviolet counterparts. Recently, the infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) source has shown the utility of a diffractive optical element (DOE) to produce square ablation patterns, termed top-hat IR-MALDESI. If the DOE optic is combined with oversampling methods, smaller ablation volumes can be sampled to render higher spatial resolution imaging experiments. Further, this approach enables reproducible spot sizes and ablation volumes for better comparison between scans. Herein, we investigate the utility of oversampling with top-hat IR-MALDESI to enhance the spatial resolution of measured lipids localized within the head of sectioned zebrafish tissue. Four different spatial resolutions were evaluated for data quality (e.g., mass measurement accuracy, spectral accuracy) and quantity of annotations. Other experimental parameters to consider for high spatial resolution imaging are also discussed. Ultimately, 20 μm spatial resolution was achieved in this work and supports feasibility for use in future IR-MALDESI studies.

Keywords:  high spatial resolution; mass spectrometry imaging; oversampling; top-hat IR-MALDESI

DOI:  https://doi.org/10.1021/jasms.4c00219
Rapid Commun Mass Spectrom. 2024 Sep 15. 38(17): e9855

Development and validation of an ultrafast method of quantification of rivaroxaban in human serum using laser diode thermal desorption coupled to triple quadrupole mass spectrometry.

Mégane Moreau, Serge Auger, Pierre Picard, Jean Lacoursière, Pedro A Segura.

RATIONALE: Rivaroxaban is an anticoagulant prescribed to patients who are at risk of medical conditions such as deep-vein thrombosis, pulmonary embolisms, and strokes caused by blood clots. The administration of this drug is monitored to adjust the dosage and evaluate patients' blood concentration. Rapid quantification of this drug in plasma could make it possible to ensure that the dose present in the blood of patients does not represent a danger for the medical intervention to be carried out.METHODS: Liquid chromatography-tandem mass spectrometry is usually employed to quantify rivaroxaban in blood, plasma, and serum. Here, an alternative method of analysis based on laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS) was developed and comprehensively validated. This new method allows the quantification of rivaroxaban in less than 13 s from sample to sample. The extraction of rivaroxaban in human serum was done by a salting-out liquid-liquid extraction with acetonitrile and a saturated sodium chloride solution.
RESULTS: The proposed method allows the quantification of rivaroxaban in less than 13 s from sample to sample. During validation, all criteria were respected. The accuracy was <15% of the nominal value, the precision was <15%CV, and the recovery was ≥89.9%. There were no observed carryover or matrix effects. Analysis of the extracted samples established the stability of dry (24 h) and wet samples (1 week) when samples cannot be analyzed immediately, a considerable advantage in a clinical setting.
CONCLUSIONS: This method improves sample throughput by more than 1200% compared to liquid chromatography-tandem mass spectrometry methods of analysis of rivaroxaban and decreases analysis costs by reducing solvent consumption and instrument time.

DOI: https://doi.org/10.1002/rcm.9855
Rapid Commun Mass Spectrom. 2024 Sep 30. 38(18): e9864

Gas chromatography-mass spectrometry determination of nicotine and cotinine in urine: A study of the effect of passive smoking.

Adamantios Krokos, Amvrosios Orfanidis, Orthodoxia Mastrogianni, Foteini Mitsa, Maria Avgeri, Maria Eboriadou, Georgios Theodoridis, Nikolaos Raikos.

RATIONALE: Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine's metabolite cotinine (COT) in urine samples, using gas chromatography-mass spectrometry (GC-MS).METHODS: Analysis was performed using a gas chromatograph Agilent Technologies 7890A with an MS 5975C inert XL, EI/CI MSD with Triple-Axis detector. For sample preparation, liquid-liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. Injection of 2 μL was performed using GC-MS, and selected ion monitoring (SIM) analysis was performed with the following ions (m/z): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and 9.743 min for COT.
RESULTS: The method was validated following international principles, assessing characteristics such as absolute recovery, carryover, linearity, specificity, selectivity, accuracy, precision, and stability. The method showed a linear dynamic range from 0.5 to 50 ng/mL, and the limits of detection and quantification were for both NCOT and COT 0.2 and 0.5 ng/mL, respectively. Validation results were found satisfactory. Finally, the method was applied to the analysis of 60 clinical pediatric samples obtained from Aristotle University's pediatric clinic to check for possible exposure to smoke. Concentration levels ranged between 0.5 and 16.2 ng/mL for NCOT and between 1.0 and 25.1 ng/mL for COT.
CONCLUSIONS: A rapid, sensitive, accurate, and simple method was developed and used as a tool for the confirmation of passive smoking in children. It is the first method applied to the analysis of such samples belonging to nonsmokers of young age. The total runtime of the GC-MS analysis was short (20 min), and the pretreatment protocol was simple, giving the ability for analysis of a large number of samples on a daily routine basis.

DOI: https://doi.org/10.1002/rcm.9864
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jul 08. pii: S1570-0232(24)00242-3. [Epub ahead of print]1243 124233

Development, validation and long-term evaluation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of amiodarone, desethylamiodarone and mexiletine in human plasma and serum.

Martinus W A Braakhuis, Marcel C M Pistorius, Pieter G Postema, Carolina E M Hollak, Eleonora L Swart.

  Amiodarone and mexiletine are used for ventricular arrhythmias, for which a combination therapy of both anti-arrhythmic drugs (AADs) is not uncommon. Therapeutic drug monitoring (TDM) can be beneficial for clinical guidance of therapy, especially to correctly identify adverse events. Desethylamiodarone, the active metabolite of amiodarone, accumulates over time and is associated with serious adverse events. Therefore, simultaneous TDM for amiodarone, desethylamiodarone and mexiletine is advantageous in clinical practice. The presented LC-MS/MS method was validated for selectivity, matrix effect, linearity, accuracy, precision, carry-over and stability. The method was continuously evaluated during eight months of clinical use. The method was shown to be linear within the measured range of 0.1 to 10 mg/L for each component. The matrix effect was considered negligible. No interfering responses were found for amiodarone, desethylamiodarone and the isotopic-labeled internal standards. A constant and reproducible within-run contribution of 45.3 %, originating from the system, was identified for mexiletine. The systemic contribution to the peak area of the lowest quantifiable concentration of mexiletine affected the selectivity and carry-over effect measurements. Multiple measurements showed that regression adjusted concentrations were accurate and reproducible, indicating calibration correction was applicable. Sample stability was found to be within limits for all storage conditions and freeze-thaw cycles. Furthermore, long-term method evaluation with external controls resulted in stable measurements with a percentage coefficient of variance between 1.3 % and 6.3 %. The presented practical and reliable method is applicable for clinical TDM and will allow clinical practitioners to guide drug therapy of amiodarone and mexiletine.

Keywords:  Amiodarone; Desethylamiodarone; LC-MS/MS; Mexiletine; Therapeutic drug monitoring; Validation

DOI:  https://doi.org/10.1016/j.jchromb.2024.124233
Crit Rev Anal Chem. 2024 Jul 11. 1-25

Recent Advancement in NMR Based Plant Metabolomics: Techniques, Tools, and Analytical Approaches.

Nitish Kumar, Vikas Jaitak.

  Plant metabolomics, a rapidly advancing field within plant biology, is dedicated to comprehensively exploring the intricate array of small molecules in plant systems. This entails precisely gathering comprehensive chemical data, detecting numerous metabolites, and ensuring accurate molecular identification. Nuclear magnetic resonance (NMR) spectroscopy, with its detailed chemical insights, is crucial in obtaining metabolite profiles. Its widespread application spans various research disciplines, aiding in comprehending chemical reactions, kinetics, and molecule characterization. Biotechnological advancements have further expanded NMR's utility in metabolomics, particularly in identifying disease biomarkers across diverse fields such as agriculture, medicine, and pharmacology. This review covers the stages of NMR-based metabolomics, including historical aspects and limitations, with sample preparation, data acquisition, spectral processing, analysis, and their application parts.

Keywords:  1D-nmr; 2D-nmr; Metabolomics; NMR-based metabolomics; targeted metabolomics; untargeted metabolomics

DOI:  https://doi.org/10.1080/10408347.2024.2375314
Methods Mol Biol. 2024 ;2816 35-40

Analysis of Sphingosine and Sphinganine from the Aqueous Humor for Signaling Studies Using Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry.

Meher A Saleem, Molly Kennedy, Omar Badla, Emily J Neag, Sanjoy K Bhattacharya.

  Sphingolipids, including sphingosine and sphinganine, are one of the major classes of lipids. They serve as constituents of cell membranes and lipid rafts and aid in the performance of cell-cell communication and adhesion. Abnormal levels of sphingolipids in the aqueous humor can indicate impaired sphingolipid metabolism and associated ocular pathologies. Sphingolipids can be extracted from the aqueous humor by the methyl-tert-butyl ether (MTBE) lipid extraction method and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). This chapter describes a modified protocol for an MTBE lipid extraction from the aqueous humor, followed by analysis with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS).

Keywords:  Aqueous humor; LC; LC-MS; Lipid extraction; Lipidomics; Liquid chromatography; MS; MTBE; Mass spectrometry; Sphinganine; Sphingolipidomics; Sphingolipids; Sphingosine; UHPLC; UHPLC-MS; Ultrahigh-performance liquid chromatography

DOI:  https://doi.org/10.1007/978-1-0716-3902-3_4
Methods Mol Biol. 2024 ;2816 151-159

Detailed Protocol for Solid-Phase Extraction for Lipidomic Analysis.

Marian N Aziz, Leticia Brotto, Ahmed S Yacoub, Kamal Awad, Marco Brotto.

  Developing robust analytical techniques is a vital phase to facilitate understanding the roles and impacts of various omic profilings in cellular functions. The comprehensive analysis of various biological molecules within a biological system requires a precise sample preparation technique. Solid-Phase Extraction (SPE) has proven to be an indispensable method in lipidomic analysis, providing an uncomplicated and user-friendly technique for extraction and purification of lipid components from complex biological matrices. Of all the factors influencing the reliability and success of SPE, column or adsorbent materials, flow rate, and storage conditions are paramount in terms of their significance. In this chapter, we will discuss in detail the SPE steps for lipidomic analysis in biofluid samples (serum and plasma) and muscle tissues.

Keywords:  SPE for lipidomic; SPE for muscle; SPE for plasma; SPE for serum; Sample preparation

DOI:  https://doi.org/10.1007/978-1-0716-3902-3_15
J Pharm Biomed Anal. 2024 Jul 05. pii: S0731-7085(24)00381-9. [Epub ahead of print]249 116341

Development and validation of a sensitive and simultaneous liquid chromatography tandem mass spectrometry method for the determination of eight phytocannabinoids in various CBD products.

Kyosuke Minamoto, Takahiro Takayama, Hidenao Katehashi, Munehiro Katagi, Koichi Inoue.

  In this study, we developed and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of eight phytocannabinoids in various cannabidiol (CBD) products from Japanese market. This method was combined with electrospray ionization in positive mode and sample preparation with QuEChERS. Three types of commercial products such as honey, chocolate, and gummies were used to perform accurate quantification with unified protocol of LC-MS/MS and QuEChERS. The limit of detection and quantification were 5-20 µg g-1 and 10-40 µg g-1, respectively. Reproducibility was ensured using matrices free of target foods, resulting in an accuracy within ±10 % and a precision with a relative standard deviation of less than 5 % for all targets. Finally, this analytical method was applied to 8 series of commercial samples from the Japanese market. This unified protocol will serve as a reference as an official method in Japan.

Keywords:  Chocolate; Gummy; Honey; LC-MS/MS; Phytocannabinoids; QuECHERS

DOI:  https://doi.org/10.1016/j.jpba.2024.116341
J Mass Spectrom Adv Clin Lab. 2024 Aug;33 7-13

Therapeutic drug monitoring of mycophenolic acid and azole antifungals on two distinct LC-MS/MS instruments.

Jill K Wolken, Wenjing Cao, Min Cui, Zhicheng Jin.

  Background: As an active metabolite of a commonly prescribed immunosuppressant, mycophenolic acid (MPA) levels are often monitored to prevent organ rejection following a transplant. Triazoles are often prescribed for treatment of invasive fungal infections in immunocompromised patients. Due to the variability in individual pharmacokinetics and drug-drug interactions, therapeutic drug monitoring is recommended for triazole antifungals. A multiplex LC-MS/MS assay has been developed that can quantify both MPA and triazole drugs in serum.Methods: A sample preparation procedure was established to spike in internal standard compounds and precipitate proteins. Reversed-phase chromatographic separation was performed on a C18 column with an analysis time of five minutes per sample. The mass spectrometer was operated in multiple reaction monitoring mode. The method was validated on two HPLC systems interfaced with either a Triple Quad 6500 or an API 4000 instrument.
Results: The multiplex assay was linear over a wide dynamic range with analyte measurable ranges of 0.4-48 μg/mL for MPA, 0.1-12 μg/mL for posaconazole, and 0.2-24 μg/mL for voriconazole, itraconazole, hydroxyitraconazole, and isavuconazole. The between-day and intraday imprecisions were less than 10 %. Limits of detection were below 0.04 ug/mL with limits of quantitation below 0.2 μg/mL. Method comparison studies against the current in-house method met acceptance criteria. The instrument comparison study demonstrated a strong correlation between data collected from the two systems.
Conclusion: A robust multiplex LC-MS/MS assay was developed and validated for monitoring MPA and triazoles drug levels in a clinical laboratory. The assay performance on two distinct instruments was acceptable and comparable.

Keywords:  Antifungal; Immunosuppressant; Liquid chromatography; Mass spectrometry; Mycophenolate mofetil; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jmsacl.2024.06.001
J Am Soc Mass Spectrom. 2024 Jul 10.

Selective Profiling of Carboxylic Acid in Crude Oil by Halogen Labeling Combined with Liquid Chromatography and High-Resolution Mass Spectrometry.

Khoa Huynh, Karen Louise Feilberg, Jonas Sundberg.

Carboxylic acids are a small but essential compound class within petroleum chemistry, influencing crude oil behaviors in production and processing and causing environmental impacts. Detailed structural information is fundamental to understanding their influence on petroleum characteristics. However, characterizing acids in crude oil remains challenging due to matrix effects, structural diversity, and low abundance. In this work, we present a new methodology for profiling carboxylic acids by liquid-liquid extraction and selective derivatization using 4-bromo-N-methylbenzylamine (4-BNMA) followed by liquid chromatography and electrospray ionization Orbitrap mass spectrometry (LC-ESI-Orbitrap MS). The fragmentation of 4-BNMA derivatives produces a unique product ion pair, m/z 169/171, enabling the identification of chromatographic fractions containing carboxylic acids. The mass spectra of the corresponding fractions are extracted, and the acids are further computationally isolated based on the isotopic pattern. The method was optimized and validated using acid standards and systematic experimental designs, assuring robustness and sensitivity for nontarget screening purposes. This method detected up to 380 carboxylic acids in six Danish North Sea crude oils, with up to two carboxyl and other heteroatom functionalities (NSO). The results indicated that the most populated species are fatty acids (double bond equivalent (DBE) = 1) and small aromatic acids (DBE = 2-6). The predominance and diversities of compound classes in different samples are consistent with their corresponding bulk properties. Polyfunctional acids (Ox, NxOx, and SxOx) were observed due to exposure to oxidation and biodegradation. Also, the approach's applicability benefits high-resolution MS analysis by simplifying data processing for crude oil and potentially other high-organic and aqueous samples.

DOI: https://doi.org/10.1021/jasms.4c00085
Heliyon. 2024 Jun 30. 10(12): e32274

UPLC-MS/MS determination of 71 neuropsychotropic drugs in human serum.

Weifeng Jin, Jianhua Wang, Shuzi Chen, Qing Chen, Dan Li, Mengyuan Zhu, Xiaomei Fu, Yingyu Huang, Ping Lin.

  In this study, a UPLC-MS/MS method was developed for the rapid detection of 71 neuropsychotropic drugs in human serum for drug concentration monitoring and toxicity screening. The analytes were separated from the biological matrix by protein precipitation using a methanol-acetonitrile solvent mixture. The chromatographic separation was performed on a Kromasil ClassicShell C18 column (2.1*50 mm, 2.5 μ m) with gradient elution using acetonitrile-0.2 % acetic acid and 10 mM ammonium acetate as the mobile phases (flow rate 0.4 mL/min, column temperature 40 °C, injection volume 5 μL). An electrospray ion source in both positive and negative ion modes with multiple ion monitoring was used. The total run time was 6 min. All compounds were quantified using the isotope internal standard method. Totally, 71 drugs were detected within their linear ranges with correlation coefficients greater than 0.990. The intra- and inter-batch precision relative standard deviations (RSDs) for the low, medium, and high concentration points were less than 15 %, with an accuracy of 90%-110 %. All compounds except Moclobemide N-oxindole are stabilised within 7 days. The relative matrix effect results for each analyte were within ±20 % of the requirements. The method is validated according to Clinical and Laboratory Standards Institute guidelines, easy to use, and has a low cost.

Keywords:  Neuropsychotropic drugs; TDM; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.heliyon.2024.e32274
Anal Chem. 2024 Jul 11.

Accurate Collisional Cross Section Measurement by Multipass Cyclic Ion Mobility Spectrometry.

Chaoshuang Xia, Elias Mernie, Joseph Zaia, Catherine E Costello, Cheng Lin.

Ion mobility-mass spectrometry (IM-MS) is a powerful analytical tool for structural characterization. IM measurement provides collision cross section (CCS) values that facilitate analyte identification. While CCS values can be directly calculated from mobility measurements obtained using drift tube ion mobility spectrometry (DT-IMS), this method has limited mobility resolution due to the practical constraints on the length of the ion drift path. Consequently, DT-IMS cannot differentiate analytes with similar mobilities or resolve fine mobility features of individual ions. Cyclic IMS (cIMS) instruments leverage a cyclic path enabled by traveling wave ion mobility (TWIM) technology and offer increased mobility solution to address this challenge. While TWIM devices must first be calibrated to enable CCS measurements, current calibration strategies are primarily tailored for single-pass analyses. This preference is partly attributed to the challenges associated with multipass calibration methods, which require both calibrants and analytes to experience the same number of passes. Achieving this consistency can be complicated due to factors like peak splitting and diffusion, and may not be feasible for online IM-MS analyses. A recent report employed average ion velocities obtained from multiple measurements under different separation pathlengths as a path length-independent metric for CCS calibration. However, the ability to exploit this averaging approach is limited by observed variation in ion drift time/velocity in these measurements. In this study, we introduce a novel calibration strategy designed for multipass cIMS analyses, directly targeting the root cause for the path length- and mobility-dependent variations in ion drift time. With this method, we demonstrate that CCS values derived from multipass measurements closely align with those obtained from single-pass analyses, with an average deviation of 0.1%. We apply this method to characterize four isomeric trisaccharides. Our approach not only results in excellent agreement between our measured cIMSCCS values and the reported DTCCS values, with an average difference of only 0.5%, but also allows us to effectively identify subtle mobility characteristics of each compound and determine their respective CCS values. This level of detail and accuracy was previously unattainable using DT-IMS or single-pass cIMS measurements. We developed an algorithm for reconstructing arrival time distribution in cases where wrap-around has resulted in peak splitting. Collectively, the new calibration strategy and the reconstruction procedure maintain reproducibility and precision in CCS measurements while largely eliminating the need for meticulous selection of separation times. We expect that our method will empower researchers to harness the high mobility resolution offered by multipass cIMS analyses without compromising the accuracy of CCS measurement, making it appropriate for straightforward use across a wide range of applications.

DOI: https://doi.org/10.1021/acs.analchem.4c01758
Anal Chem. 2024 Jul 11.

Improved Characterization of Soil Organic Matter by Integrating FT-ICR MS, Liquid Chromatography Tandem Mass Spectrometry, and Molecular Networking: A Case Study of Root Litter Decay under Drought Conditions.

Nicole DiDonato, Albert Rivas-Ubach, William Kew, Noah W Sokol, Chaevien S Clendinen, Jennifer E Kyle, Carmen Enid Martínez, Megan M Foley, Nikola Tolić, Jennifer Pett-Ridge, Ljiljana Paša-Tolić.

Understanding of how soil organic matter (SOM) chemistry is altered in a changing climate has advanced considerably; however, most SOM components remain unidentified, impeding the ability to characterize a major fraction of organic matter and predict what types of molecules, and from which sources, will persist in soil. We present a novel approach to better characterize SOM extracts by integrating information from three types of analyses, and we deploy this method to characterize decaying root-detritus soil microcosms subjected to either drought or normal conditions. To observe broad differences in composition, we employed direct infusion Fourier-transform ion cyclotron resonance mass spectrometry (DI-FT-ICR MS). We complemented this with liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify components by library matching. Since libraries contain only a small fraction of SOM components, we also used fragment spectral cosine similarity scores to relate unknowns and library matches through molecular networks. This integrated approach allowed us to corroborate DI-FT-ICR MS molecular formulas using library matches, which included fungal metabolites and related polyphenolic compounds. We also inferred structures of unknowns from molecular networks and improved LC-MS/MS annotation rates from ∼5 to 35% by considering DI-FT-ICR MS molecular formula assignments. Under drought conditions, we found greater relative amounts of lignin-like vs condensed aromatic polyphenol formulas and lower average nominal oxidation state of carbon, suggesting reduced decomposition of SOM and/or microbes under stress. Our integrated approach provides a framework for enhanced annotation of SOM components that is more comprehensive than performing individual data analyses in parallel.

DOI: https://doi.org/10.1021/acs.analchem.4c00184
Methods Mol Biol. 2024 ;2816 87-100

Effects of a Laparotomy on Targeted Lipidomics Profiles in a Mouse Model of Surgical Stress During Aging.

Jian Huang, Kamal Awad, Lauren Harlow, Carlos A Toro, Marco Brotto, Christopher P Cardozo.

  Laparotomy (EL) is one of the most common procedures performed among surgical specialties. Previous research demonstrates that surgery is associated with an increased inflammatory response. Low psoas muscle mass and quality markers are associated with increased mortality rates after emergency laparotomy. Analysis of lipid mediators in serum and muscle by using liquid chromatography-mass spectrometry (LC-MS)-based lipidomics has proven to be a sensitive and precise technique. In this chapter, we describe an LC-MS/MS protocol for the profiling and quantification of signaling lipids formed from Eicosapentaenoic Acid (EPA) and Eicosatetranoic acid (ETA) by 5, 12, or 15 lipoxynases. This protocol has been developed for and validated in serum and muscle samples in a mouse model of surgical stress caused by laparotomy.

Keywords:  Laparotomy; Lipidomics; Muscle; Omics; Serum; Surgical stress

DOI:  https://doi.org/10.1007/978-1-0716-3902-3_9
Biomed Chromatogr. 2024 Jul 08. e5955

An analytical method using salting-out assisted liquid-liquid extraction to quantify ceftriaxone from micro volumes of human serum.

Yuji Mukai, Narushi Sugii, Kosuke Doki, Masato Homma.

  Ceftriaxone (CTRX) is a commonly used cephalosporin antibiotic. It is suggested that monitoring plasma/serum concentrations is helpful for its safe use. This study aimed to develop and validate an analytical method for measuring CTRX concentrations in human serum according to International Conference on Harmonization guideline M10. Ten microliters of serum sample was purified using a salting-out assisted liquid-liquid extraction procedure with magnesium sulfate. The upper layer was then diluted threefold and analyzed using a liquid chromatography-tandem mass spectrometry-based method with a total run time of 12 min. The linear calibration curve was obtained over the concentration range 5-500 μg/ml. The within-run accuracy varied from 0.2 to 6.5%, and the precision was ≤8.0%. The between-run accuracy and precision ranged from 0.7% to 5.6% and ≤6.4%, respectively. Significant carryover was resolved by injecting four blanks after high-concentration CTRX samples. The recovery rates from spiked serum at low and high concentrations were 44.4 and 43.4%, respectively. Other factors, including selectivity, matrix effects, stability, dilution integrity and reinjection reproducibility also met the acceptance criteria. Serum concentrations in 14 samples obtained from two participants receiving 2 g/day of CTRX were successfully determined using this method.

Keywords:  ceftriaxone; liquid chromatography–tandem mass spectrometry; magnesium sulfate; salting‐out assisted liquid–liquid extraction

DOI:  https://doi.org/10.1002/bmc.5955
Heliyon. 2024 Jun 30. 10(12): e32927

Determination of mitomycin C in rat plasma by liquid chromatography-tandem mass spectrometry and its application for determining pharmacokinetics in rat.

Kyung-Sun Moon, Jong-Min Song, JiMin Yi, Quynh Khoa Pham, Sung-Hoon Ahn.

  To develop the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring mitomycin C in rat plasma, samples were processed using solid-phase extraction, with the internal standard being carbamazepine. A reversed phased C18 column was utilized for the LC-MS/MS study, and mobile phases consisting of 0.1 % formic acid in acetonitrile and water were injected into it at a rate of 0.3 mL/min. Multiple reaction monitoring in positive-ion mode with precursor-product ion pairs 335.3 → 242.3 (mitomycin C) and 237.1 → 194.1 (carbamazepine) was employed to quantify the compounds. The linear range in plasma was found to be 10-4000 ng/mL (r2 = 0.992). The inter-batch and intra-batch precision were <14.3 % (LLOQ: 14.7 %) and 13.4 % (LLOQ: 16.1 %), respectively. The recovery and the matrix effect of mitomycin C in plasma were 113 % and 111 %, respectively. Mitomycin C was stable under the conditions of this assay method. In the end, this approach proved effective in a pharmacokinetic investigation with the intravenous and oral administration of mitomycin C to rats.

Keywords:  LC-MS/MS; Mitomycin C; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.heliyon.2024.e32927
Methods Mol Biol. 2024 ;2816 241-252

Lipidomics Profiling of the Linoleic Acid Metabolites After Whole-Body Vibration in Humans.

Kamal Awad, Stephen Leroy Newhart, Leticia Brotto, Marco Brotto.

  Bioactive lipids have been identified as dynamic signaling lipid mediators (LMs). These fats have the ability to activate responses and control bodily functions either directly or indirectly. Linoleic Acid (LA) and Alpha Linoleic Acid (ALA) are types of omega 3 fatty acids that possess inflammatory properties and promote resolution of inflammation either through their own actions or through their metabolites known as oxylipins. In this chapter, we provide an explanation of a method that combines chromatography with tandem mass spectroscopy (LC MS/MS) to identify and measure all the metabolites derived from LA and ALA. Additionally, we employed the described methodology to analyze human serum samples obtained before and after whole-body vibration exercise training. The results indicated an increase in some of the LA and ALA LMs that have beneficial effects in regulating the cardiovascular system.

Keywords:  Alpha linoleic acid; LC-MS/MS; Linoleic acid; Lipidomics; Whole-body vibration

DOI:  https://doi.org/10.1007/978-1-0716-3902-3_21
Rapid Commun Mass Spectrom. 2024 Sep 30. 38(18): e9865

Improved high-performance liquid chromatography tandem mass spectrometry method for quantification of infliximab in pediatric plasma and its application in therapeutic drug monitoring.

Boran Yu, Siyao Jin, Jiaqi Han, Xiaona Li, Jiamin Xu, Ning Sun, Liang Sun, Xiaoling Wang, Libo Zhao.

RATIONALE: The application of infliximab (IFX) to immune-mediated disease is limited by the significant individual variability and associated clinical nonresponse, emphasizing the importance of therapeutic drug monitoring (TDM). Because of the cross-reactivity, limited linear range, and high costs, the clinical application of the previous reported methods was limited. Here, an improved high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed to address the issues.METHODS: This study developed an improved bioanalytical HPLC-MS/MS method coupling nanosurface and molecular-orientation limited proteolysis technology. The commercially available compound P14R was selected as the internal standard. This method was developed with fewer volume of reagents and was thoroughly validated. The validated method was applied to TDM in pediatric inflammatory bowel disease (IBD).
RESULTS: Chromatography was performed using a Shim-pack GISS-HP C18 metal-free column (3 μm, 2.1 × 100 mm) with a gradient elution of 0.1% formic acid in water and acetonitrile at 0.4 mL/min. Detection and quantitation were performed using electrospray ionization (ESI) and multiple reaction monitoring in the positive ion mode. The method was validated to demonstrate its selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability. The method exhibited a linear dynamic range of 0.3-100 μg/mL, with intra- and inter-day precision and relative errors below 15%. The recovery and matrix effect were measured as 87.28%-89.72% and 41.98%-67.17%, respectively, which were effectively compensated by the internal standard. A total of 32 samples collected from 24 pediatric patients with IBD were analyzed using the validated method, and only 46.9% achieved the reported targeted trough level.
CONCLUSION: This study developed an improved HPLC-MS/MS method for the quantitative determination of IFX concentration in human plasma. The accurate, reliable, and cost-effective method was validated and utilized in the analysis of clinical samples. The results confirmed the importance of TDM on IFX and the clinical application prospects of the improved method.

DOI: https://doi.org/10.1002/rcm.9865
Anal Chem. 2024 Jul 06.

Accurate Quantification of Ten Methylated Purine Nucleosides by Highly Sensitive and Stable Isotope-Diluted UHPLC-MS/MS.

Lyuye Zhang, Weibing Zhang, Hailin Wang.

The dynamic landscape of cellular nucleotides/nucleosides associated with RNA metabolism, particularly in diseases like cancer, has spurred intensive interest. Here, we report a robust stable isotope-diluted UHPLC-ESI-MS/MS method for accurate quantification of 12 purine ribonucleosides, including 10 methylated purine nucleosides. By the use of thermally decomposable ammonium bicarbonate (NH4HCO3) as a mobile phase additive for UHPLC-MS/MS detection, the ESI-MS/MS signal responses of these target compounds were enhanced by 1.7-24.5 folds. Noteworthily, three methylated guanosine isomers (m1G, m2G, and m7G) and two methylated adenosine isomers (m1A and m6A) that are indistinguishable directly by mass spectrometry were well resolved with optimal UHPLC separation. Combined with methanol extraction and solid-phase extraction (SPE) pretreatment, the method quantified intracellular concentrations of three modified nucleosides (Gm, m1G, and m2G), which would otherwise be undetectable because of significant suppression of their signals by the interfering cellular matrix. Nine purine nucleosides were simultaneously quantified in 293T cells, and their concentrations ranged by 4 orders of magnitude. Overall, the method presents high recovery rates over 90% for endogenous modified purine nucleosides in cultured cells, along with good precision, linearity, and LOD ranging from 0.30 fmol to 0.37 pmol per 5 × 105 cells. The developed UHPLC-MS/MS method holds potential for screening purine nucleosides as diagnostic and prognostic biomarkers and for quantifying purine epigenetic nucleosides post-cell metabolome analysis, thereby providing a valuable analytical tool for intracellular nucleoside quantification in future clinical research.

DOI: https://doi.org/10.1021/acs.analchem.4c01446
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jul 06. pii: S1570-0232(24)00243-5. [Epub ahead of print]1243 124234

Optimal conditions for determination of bacitracin, bacitracin zinc and bacitracin methylene disalicylate in animal feed by ultra-performance liquid tandem mass spectrometry.

Feng Xu, Jiayong Yu, Yinliang Wu.

  An integrated method combining solid-phase extraction (SPE) with ultra-performance liquid tandem mass spectrometry (UPLC-MS/MS) has been established for quantifying bacitracin (BTC), bacitracin zinc (BZ), and bacitracin methylene disalicylate (BMD) in animal feed. A pretreatment procedure that can effectively, quickly, and simultaneously extract and purify BTC, BZ, or BMD in feed was developed for the first time through the optimization of extraction and SPE conditions. After extraction with acetonitrile + methanol + 15 % ammonia solution (1:1:1, v:v:v) and dilution with EDTA solution (1.5 mmol/L, pH 7.0), a SPE procedure was carried out with C18 cartridge. Following LC-MS/MS analysis utilized a Waters Peptide BEH C18 column with a gradient elution of 0.1 % formic acid in water/acetonitrile with. This method demonstrated a strong linear correlation (R2 > 0.9980) across a 0.01-1.0 mg/L concentration span, based on a matrix-matched standard curve. Satisfactory recoveries of BTC (bacitracin A, B1, B2, and B3), BZ, and BMD in different feeds were obtained from 80.7 % to 108.4 %, with relative standard deviations below 15.7 %. Low limits of quantification ranging within 7.2-20 μg/kg were achieved for bacitracin A, B1, B2, and B3. This method provided an effective and reliable detection method to prevent the addition of BTC and different BTC formulations in feeds.

Keywords:  Bacitracin; Bacitracin methylene disalicylate; Bacitracin zinc; Feed; LC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124234
Anal Chem. 2024 Jul 11.

Resolution Enhancement of Metabolomic J-Res NMR Spectra Using Deep Learning.

Yan Yan, Michael T Judge, Toby Athersuch, Yuchen Xiang, Zhaolu Liu, Beatriz Jiménez, Timothy M D Ebbels.

J-Resolved (J-Res) nuclear magnetic resonance (NMR) spectroscopy is pivotal in NMR-based metabolomics, but practitioners face a choice between time-consuming high-resolution (HR) experiments or shorter low-resolution (LR) experiments which exhibit significant peak overlap. Deep learning neural networks have been successfully used in many fields to enhance quality of natural images, especially with regard to resolution, and therefore offer the prospect of improving two-dimensional (2D) NMR data. Here, we introduce the J-RESRGAN, an adapted and modified generative adversarial network (GAN) for image super-resolution (SR), which we trained specifically for metabolomic J-Res spectra to enhance peak resolution. A novel symmetric loss function was introduced, exploiting the inherent vertical symmetry of J-Res NMR spectra. Model training used simulated high-resolution J-Res spectra of complex mixtures, with corresponding low-resolution spectra generated via blurring and down-sampling. Evaluation of peak pair resolvability on J-RESRGAN demonstrated remarkable improvement in resolution across a variety of samples. In simulated plasma data, 100% of peak pairs exhibited enhanced resolution in super-resolution spectra compared to their low-resolution counterparts. Similarly, enhanced resolution was observed in 80.8-100% of peak pairs in experimental plasma, 85.0-96.7% in urine, 94.4-98.9% in full fat milk, and 82.6-91.7% in orange juice. J-RESRGAN is not sample type, spectrometer or field strength dependent and improvements on previously acquired data can be seen in seconds on a standard desktop computer. We believe this demonstrates the promise of deep learning methods to enhance NMR metabolomic data, and in particular, the power of J-RESRGAN to elucidate overlapping peaks, advancing precision in a wide variety of NMR-based metabolomics studies. The model, J-RESRGAN, is openly accessible for download on GitHub at https://github.com/yanyan5420/J-RESRGAN.

DOI: https://doi.org/10.1021/acs.analchem.4c00563
Anal Chem. 2024 Jul 08.

Discovery and Identification of Three Homocysteine Metabolites by Chemical Derivatization and Mass Spectrometry Fragmentation.

Julius Agongo, Scott F Grady, Kevin Cho, Gary J Patti, Benjamin J Bythell, Christopher K Arnatt, James L Edwards.

Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.

DOI: https://doi.org/10.1021/acs.analchem.4c01706
J Mass Spectrom. 2024 Aug;59(8): e5070

Dependency of fentanyl analogue protomer ratios on solvent conditions as measured by ion mobility-mass spectrometry.

Copeland R Johnson, Heidi M Sabatini, Ralph Aderorho, Christopher D Chouinard.

  Recently, our group has shown that fentanyl and many of its analogues form prototropic isomers ("protomers") during electrospray ionization. These different protomers can be resolved using ion mobility spectrometry and annotated using mobility-aligned tandem mass spectrometry fragmentation. However, their formation and the extent to which experimental variables contribute to their relative ratio remain poorly understood. In the present study, we systematically investigated the effects of mixtures of common chromatographic solvents (water, methanol, and acetonitrile) and pH on the ratio of previously observed protomers for 23 fentanyl analogues. Interestingly, these ratios (N-piperidine protonation vs. secondary amine/O = protonation) decreased significantly for many analogues (e.g., despropionyl ortho-, meta-, and para-methyl fentanyl), increased significantly for others (e.g., cis-isofentanyl), and remained relatively constant for the others as solvent conditions changed from 100% organic solvent (methanol or acetonitrile) to 100% water. Interestingly, pH also had significant effects on this ratio, causing the change in ratio to switch in many cases. Lastly, increasing conditions to pH ≥ 4.0 also prompted the appearance of new mobility peaks for ortho- and para-methyl acetyl fentanyl, where all previous studies had only showed one single distribution. Because these ratios have promise to be used qualitatively for identification of these (and emerging) fentanyl analogues, understanding how various conditions (i.e., mobile phase selection and/or chromatographic gradient) affect their ratios is critically important to the development of advanced ion mobility and mass spectrometry methodologies to identify fentanyl analogues.

Keywords:  fentanyl; ion mobility‐mass spectrometry; protomers

DOI:  https://doi.org/10.1002/jms.5070