bims-metlip 2024-09-29 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–09–29
thirty-two papers selected by
Sofia Costa, Matterworks

A general and rapid LC-MS/MS method for simultaneous determination of voriconazole, posaconazole, fluconazole, itraconazole and hydroxyitraconazole in IFI patients.
Navigating common pitfalls in metabolite identification and metabolomics bioinformatics.
Advances and challenges in liquid chromatography-spectrometry (LC-MS) methodology for quantifying androgens and estrogens in human serum and plasma.
A broad-spectrum LC-MS/MS method for screening and quantification of 100 analytes in clinical and autopsy blood samples.
Identification of bile acids in snake bile by hydrogen/deuterium exchange mass spectrometry and quantitative structure-retention relationship analysis.
[Accurate determination of three new antihypertensive drugs illegally added to food using ultra-high performance liquid chromatography-tandem mass spectrometry].
[Simultaneous determination of 102 synthetic cannabinoids in electronic cigarette oil by liquid chromatography-tandem mass spectrometry].
FAMS-A Targeted Fatty Acid Mass Spectrometry Method for Monitoring Free Fatty Acids from Polysorbate Hydrolysis.
Long-term stability of five atypical antipsychotics (risperidone, olanzapine, paliperidone, clozapine, quetiapine) and the antidepressant mirtazapine in human serum assessed by a validated SPE LC-MS/MS method.
An Expedited Qualitative Profiling of Free Amino Acids in Plant Tissues Using Liquid Chromatography-Mass Spectrometry (LC-MS) in Conjunction With MS-DIAL.
High-performance liquid chromatography-tandem mass spectrometry was used to measure 20 antidepressants in human serum.
Magnetic particle spray mass spectrometry for the determination of beta-blockers in plasma samples.
[Application of chromatographic retention time correction in untargeted screening].
Novel Technique for Simultaneous Ethylene Glycol and Its Metabolites Determination in Human Whole Blood and Urine Samples Using GC-QqQ-MS/MS.
Recent development on the extraction, detection, and quantification of vitamin D from various sources - an update.
Derivatization to reduce background interferences for simultaneous quantitation of trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) using liquid chromatography with tandem mass spectrometry.
Statistical analysis of feature-based molecular networking results from non-targeted metabolomics data.
Deep learning prediction of electrospray ionization tandem mass spectra of chemically derived molecules.
Rapid Characterization of Phospholipids from Biological Matrix Enabled by Indium Tin Oxide (ITO) Coated Slide Assisted Enrichment MALDI Mass Spectrometry.
[Advances in the application of ion chromatography-mass spectrometry in the fields of life and health].
Determination of drugs of abuse and metabolites in plasma microsamples by LC-MS/MS after microQuEChERS extraction.
Knowledge-based in silico fragmentation and annotation of mass spectra for natural products with MassKG.
Paraquat Poisoning: Insights from Autopsy, Histology, and Liquid Chromatography with Tandem Mass Spectrometry in Multidisciplinary Forensic Toxicology Practice.
[Determination of 13 kinds of free and bound phenolic acids in fruits by high-performance liquid chromatography-mass spectrometry].
In-Depth Analysis of Molecular Network Based on Liquid Chromatography Coupled with Tandem Mass Spectrometry in Natural Products: Importance of Redundant Nodes Discovery.
Predicting Collision-Induced-Dissociation Tandem Mass Spectra (CID-MS/MS) Using Ab Initio Molecular Dynamics.
A high-throughput liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of p-cresol sulfate, p-cresol glucuronide, indoxyl-sulfate, and indoxyl glucuronide in HepaRG culture medium and the demonstration of mefenamic acid as a potent and selective detoxifying agent.
Storing liquid chromatographic separations on surface energy traps: decoupling the LC and the mass spectrometer.
Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured Trichodesmium morphotypes.
Boric Acid Functionalized Hypercrosslinked Polymers for Selective Extraction of Trace Catecholamines and Their Metabolites in Rat Serum.
Identification of fentanyl analogs and potential biomarkers in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS).
Quantitation of trace polyfunctional thiols in wine by liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry in parallel reaction monitoring.

J Pharmacol Toxicol Methods. 2024 Sep 23. pii: S1056-8719(24)00075-3. [Epub ahead of print] 107565

A general and rapid LC-MS/MS method for simultaneous determination of voriconazole, posaconazole, fluconazole, itraconazole and hydroxyitraconazole in IFI patients.

Wenwen Xia, Shun Chen, Yunlei Yun, Lili Cui, Zhipeng Wang, Juanjuan Hou, Mao Tang, Chen Bu, Shouhong Gao, Rongzi Shao, Xia Tao.

   OBJECTIVE: To establish a rapid and universal quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring the exposure levels of five triazole antifungal drugs in human plasma, including voriconazole, fluconazole, posaconazole, itraconazole, and hydroxyitraconazole.
METHODS: A triple quadrupole mass spectrometer operating in positive ionization mode was used to detect the analyte, and multiple reaction monitoring mode was employed to gather data. The mobile phase included 0.05 % formic acid in water (phase A) and acetonitrile (phase B). The analytes were separated on an Agilent EclipsePlusC18 RRHD column (30 × 50 mm, 1.8 μm) using gradient elution. The flow rate was 0.3 mL/min with the column temperature set at 35 °C. The acetonitrile was used to pretreat the plasma sample, and the itraconazole-D5 and hydroxyitraconazole-D5 were utilized as the internal standards.
RESULTS: The calibration range was from 100 to 10,000 ng/mL for posaconazole, itraconazole, and hydroxyitraconazole, from 200 to 20,000 ng/mL for fluconazole and from 50 to 5000 ng/mL for voriconazole, with linear correlation coefficients more than 0.99 for all regression curves. The intra- and inter-day accuracy and precision of the method were within ±15 %. The mean extraction recovery of all the analytes ranged from 74.32 % to 117.83 %, and the matrix effect was from 72.54 % to 111.2 %. The results of stability fell into the scope of ±15 % deviation.
CONCLUSION: This newly developed method is sensitive, simple, and robust, and successfully applied in determining triazole antifungal drugs in plasma from 66 IFI patients to provide reference for safe and effective drug administration in clinical practice.

Keywords:  LC-MS/MS; Protein precipitation; Therapeutic drug monitoring; Triazole antifungal drugs

DOI:  https://doi.org/10.1016/j.vascn.2024.107565
Metabolomics. 2024 Sep 21. 20(5): 103

Navigating common pitfalls in metabolite identification and metabolomics bioinformatics.

Elva María Novoa-Del-Toro, Michael Witting.

   BACKGROUND: Metabolomics, the systematic analysis of small molecules in a given biological system, emerged as a powerful tool for different research questions. Newer, better, and faster methods have increased the coverage of metabolites that can be detected and identified in a shorter amount of time, generating highly dense datasets. While technology for metabolomics is still advancing, another rapidly growing field is metabolomics data analysis including metabolite identification. Within the next years, there will be a high demand for bioinformaticians and data scientists capable of analyzing metabolomics data as well as chemists capable of using in-silico tools for metabolite identification. However, metabolomics is often not included in bioinformatics curricula, nor does analytical chemistry address the challenges associated with advanced in-silico tools.
AIM OF REVIEW: In this educational review, we briefly summarize some key concepts and pitfalls we have encountered in a collaboration between a bioinformatician (originally not trained for metabolomics) and an analytical chemist. We identified that many misunderstandings arise from differences in knowledge about metabolite annotation and identification, and the proper use of bioinformatics approaches for these tasks. We hope that this article helps other bioinformaticians (as well as other scientists) entering the field of metabolomics bioinformatics, especially for metabolite identification, to quickly learn the necessary concepts for a successful collaboration with analytical chemists.
KEY SCIENTIFIC CONCEPTS OF REVIEW: We summarize important concepts related to LC-MS/MS based non-targeted metabolomics and compare them with other data types bioinformaticians are potentially familiar with. Drawing these parallels will help foster the learning of key aspects of metabolomics.

Keywords:  Bioinformatics; Data Analysis; LC-MS/MS; Mass Spectrometry; Metabolite Identification; Metabolite databases; Metabolomics

DOI:  https://doi.org/10.1007/s11306-024-02167-2
J Steroid Biochem Mol Biol. 2024 Sep 21. pii: S0960-0760(24)00166-3. [Epub ahead of print]245 106618

Advances and challenges in liquid chromatography-spectrometry (LC-MS) methodology for quantifying androgens and estrogens in human serum and plasma.

Qingqing Wang, Clementina Mesaros.

  Accurate quantification of androgens and estrogens is critical for elucidating their roles in endocrine disorders and advancing research on their functions in human biology and pathophysiology. This review highlights recent advances and ongoing challenges in liquid chromatography- mass spectrometry (LC- MS) methodology for quantifying androgens and estrogens in human serum and plasma. We summarized current approaches for analyzing the different forms of androgens and estrogens, along with their reported levels in publications from 2010 to the present. These published levels pointed out the inconsistencies in reference intervals across studies. To address these issues, advances in derivatization methods and chromatographic separation techniques are reviewed. Future perspectives for improving the accuracy and consistency of hormone quantification in clinical and research settings were also proposed.

Keywords:  Androgens; Chromatographic separation; Derivatization; Estrogens; Liquid chromatography; Mass spectrometry; Steroid hormones bioanalysis

DOI:  https://doi.org/10.1016/j.jsbmb.2024.106618
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 18. pii: S1570-0232(24)00332-5. [Epub ahead of print]1247 124323

A broad-spectrum LC-MS/MS method for screening and quantification of 100 analytes in clinical and autopsy blood samples.

Dimitra Florou, Mathew Di Rago, Amvrosios Orfanidis, Dimitri Gerostamoulos, Vassiliki A Boumba.

  Liquid chromatography coupled with mass spectrometry (LC-MS) has been tremendously used for screening purposes in forensic toxicology, because of their great adaptability and reasonable time/resource consumption. Herein, a fully validated method based on liquid-liquid extraction (LLE) in human whole blood, by a multiple reaction monitoring (MRM) analysis through LC-MS/MS, is described. The proposed method simultaneously detects 100 analytes (plus three deuterated internal standard compounds) belonging to many different classes, including drugs of abuse, prescription and over-the-counter drugs commonly involved in poisoning and medical malpractice cases in our territory, as well as certain new psychoactive substances (NPS) and toxic substances potentially associated with adverse effects. The optimised LLE employs one extraction step of 200 μL blood using 0.1 M HCl methyl-tert-butyl-ether (MTBE) (acidified with concentrated HCl) proved to be suitable for the extraction of basic and neutral substances; as a reconstitution solvent a mixture of 88:12v/v, 0.1 % formic acid in 10 mM aqueous ammonium acetate, pH 3.5: 0.1 % formic acid in acetonitrile was used, yielding satisfactory recoveries for all analytes. The method was sensitive, showing low LOD/ LOQ for all substances ranging from 0.01 to 5/ 0.05-20 ng/mL, respectively. Linearity ranged between 0.05-500 ng/mL (R2 = 0.9811-0.9995), and the inter- and intra-day precisions ranged between 3-15 % and 7-18 %, respectively. Accuracy was evaluated in terms of percentage recovery, lying within acceptable range. The matrix effect expressed as ion suppression/enhancement of each analyte was in the range ±25 % for all analytes. Post-preparative stability of analytes was higher than 85 %, while no carryover between runs was observed. The developed method has been successfully applied in routine toxicological analyses for the analysis of biological samples from clinical and autopsy cases.

Keywords:  Blood; Drugs; Forensic toxicology; LC-MS/MS; NPS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124323
J Chromatogr A. 2024 Sep 17. pii: S0021-9673(24)00758-1. [Epub ahead of print]1736 465384

Identification of bile acids in snake bile by hydrogen/deuterium exchange mass spectrometry and quantitative structure-retention relationship analysis.

Yinyu Wei, Chenyue Zhu, Xinyuan Ye, Chenxi Wang, Meiling Qi, Yi Chen, Zhifeng Du, Sen Li, Hongliang Jiang.

  Natural bile acids, a class of steroids with a valeric acid side chain at the C-17 position, present significant challenges in separation and analysis due to structural similarities, isomerism, and large polarity differences. Therefore, advanced analytical methods are essential for the accurate identification and quantification of bile acids. This study conducted a comprehensive qualitative analysis of bile acids by integrating liquid chromatography-tandem mass spectrometry (LC-MS/MS), hydrogen-deuterium exchange tandem mass spectrometry (HDX-MS/MS), and quantitative structure-retention relationship (QSRR) methods. Firstly, LC-MS/MS conditions were optimized to enhance chromatographic separation and improve the reliability of characteristic fragment ions. MS/MS fragmentation rules for bile acids were derived from the mass spectral data of bile acid standards and validated through HDX-MS/MS experiments. Secondly, potential bile acids in snake bile were identified based on these validated fragmentation rules, and a QSRR model was established to predict the retention times of the proposed structures. Thirdly, HDX-MS/MS was applied to assist in identifying bile acid isomers. Finally, a total of 150 bile acids, including 11 free bile acids (free BA), 5 glyco-bile acids (GBA) and 134 tauro-bile acids (TBA), were detected in snake bile. Thirteen bile acids were accurately characterized by comparing their retention time and MS/MS spectra with standards. Forty-nine bile acids were reasonably annotated using the QSRR model and HDX-MS/MS. This study is notable for being the first to utilize the QSRR and HDX-MS/MS techniques for the annotation of bile acids in snake bile, providing a robust framework for the structural elucidation of these compounds.

Keywords:  Bile acids; Hydrogen-deuterium exchange; LC-MS/MS; Qualitative analysis; Quantitative structure-retention relationship

DOI:  https://doi.org/10.1016/j.chroma.2024.465384
Se Pu. 2024 Oct;42(10): 954-962

[Accurate determination of three new antihypertensive drugs illegally added to food using ultra-high performance liquid chromatography-tandem mass spectrometry].

Ju-Zhou Zhang, Jing Li, Ping-Ping Zhang, Mei Yang, Di Zhou.

  Effective strategies are required to address food safety issues related to the illegal addition of antihypertensive drugs to food and claims of antihypertensive function. In this study, a novel ultra-high performance liquid chromatography-triple-quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of three antihypertensive drugs (azilsartan, candesartan cilexetil, and lacidipine) in 12 food matrices (pressed candies, solid beverages, alternative teas, tea drinks, biscuits, jellies, mixed liquors, oral liquids, medicinal teas, tablets, hard capsules, and soft capsules). Initially, mass spectrometry parameters, such as the collision energies of the three antihypertensive drugs, were optimized. Subsequently, the response intensities and chromatographic separation conditions of the three drugs in different mobile phases were compared. In addition, to enhance the recoveries, various extraction solvents and purification methods, including solid-phase extraction (SPE) columns and the QuEChERS technique, were investigated. In the developed method, sample determination involved three steps. First, the sample was extracted using 0.2% (v/v) formic acid in acetonitrile and then filtered using high-speed centrifugation, in addition, the extracted solution of alternative tea and medicinal tea was purified using the QuEChERS technique. Second, the supernatant was diluted with water, and filtered through a 0.22 μm polytetrafluoroethylene (PTFE) membrane. Finally, the analytes were separated on an Agilent Eclipse Plus RRHD C18 column (50 mm×2.1 mm, 1.8 μm) using a 5 mmol/L ammonium formate aqueous solution and acetonitrile as the mobile phases under gradient elution conditions and then detected using UHPLC-MS/MS with positive electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Quantitative analysis was performed using a matrix-matched external standard method. Methodological validation showed good linear relationships for all three antihypertensive drugs in the investigated concentration ranges, with correlation coefficients (r2) greater than 0.996. The limit of detection (LOD) and limit of quantification (LOQ) of lacidipine were 0.02 mg/kg and 0.04 mg/kg, respectively, whereas those of the other two drugs were 0.01 mg/kg and 0.02 mg/kg, respectively. In the 12 food matrices, the average recoveries of the drugs ranged from 86.6% to 107.5% with relative standard deviations (RSDs) of 1.1%-10.9% (n=6) at low, medium, and high spiked levels. Furthermore, this method was successfully applied to the analysis of real food samples. Overall, the newly developed method is simple, rapid, sensitive, accurate, and suitable for the qualitative and quantitative determination of antihypertensive drugs in different food matrices. This work could provide technical support for food safety agencies in implementing measures against the illegal addition of antihypertensive drugs to food.

Keywords:  antihypertensive drugs; azilsartan; candesartan cilexetil; foods; lacidipine; ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.02025
Se Pu. 2024 Oct;42(10): 943-953

[Simultaneous determination of 102 synthetic cannabinoids in electronic cigarette oil by liquid chromatography-tandem mass spectrometry].

Zhe Yang, Li-Wei Jiang, Si-Yao Yang, Yi-di Wu, Jian-Xia Lyu.

  Synthetic cannabinoids (SCs), which are among the most widely abused new psychoactive substances, are much more potent and have greater efficacy than natural cannabis. SCs can be disguised in various ways and are commonly sold in the form of electronic cigarette oil. SCs belong to a large family with structures consisting of a core with substituents, linker, ring with substituents, and tail. New SCs can be developed by adding substituents, such as halogen, alkyl, and alkoxy groups, to the aromatic ring system or by changing the alkyl chain length. Since the emergence of so-called first-generation SCs, subsequent developments have led to eighth-generation indole/indazole amide-based SCs. As of July 1, 2021, the entire category of SCs was added to the list of controlled substances, but implementation requires urgent improvements in detection technologies. Typically, each method is limited to a few SCs. Owing to the vast number of chemically diverse SCs and their fast update speed, the determination and identification of various types of SCs using a single method is challenging. Therefore, rapid, sensitive, and accurate quantitative methods that includes various types of SCs must be developed to meet the demand for the qualitative and quantitative analysis of new SCs in seized electronic cigarette oil. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of 102 SCs in electronic cigarette oil. The mass spectrometry and liquid-phase conditions influencing SC separation and determination were optimized. Using the external standard method, 102 SCs were successfully identified in electronic cigarette oil. The samples were extracted using methanol. Target analytes were separated on a Shimadzu Shim-pack GIST-HP C18 AQ column (100 mm×2.1 mm, 1.9 μm) at a column temperature of 40 ℃. The mobile phases consisted of (A) 0.1% formic acid aqueous solution and (B) methanol-acetonitrile (1∶1, v/v). The gradient elution conditions were as follows: 0-8 min, 55%A-15%A; 8-15 min, 15%A; 15-16 min, 15%A-55%A; 16-18 min, 55%A. The flow rate was 0.4 mL/min and the injection volume was 1 μL. Operating in the multiple reaction monitoring mode, the 102 SCs were identified within 18 min. Each SC exhibited a good linear relationship in the range of 1-100.0 μg/L with a correlation coefficient (r)≥0.9915. The limits of detection were 0.01-0.30 μg/L and the limits of quantification were 0.04-0.99 μg/L, which meet the requirements for analyzing SCs in actual samples. Precision was determined using standard solutions with 2, 10, and 50 μg/L of the SCs. The precisions (n=6) were 0.3%-6.0%. The recoveries of the 102 SCs, as evaluated by spiking electronic cigarette oil at low (2 μg/mL), medium (10 μg/mL), and high (50 μg/mL) levels, were 80.1%-119.8%. Good performance was observed for the analysis of real samples. The developed method is accurate, rapid, sensitive, and effective for the determination of the 102 SCs in electronic cigarette oil, satisfying the requirements for practical qualitative and quantitative analysis.

Keywords:  electronic cigarette oil; liquid chromatography-tandem mass spectrometry (LC-MS/MS); synthetic cannabinoids (SCs)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.03017
Methods Protoc. 2024 Sep 07. pii: 71. [Epub ahead of print]7(5):

FAMS-A Targeted Fatty Acid Mass Spectrometry Method for Monitoring Free Fatty Acids from Polysorbate Hydrolysis.

Anja Bathke, Sina Hoelterhoff, Jan Wendler, Inn H Yuk, Christian H Bell.

  Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation. We previously developed a user-friendly mass spectrometric method called Fatty Acids by Mass Spectrometry (FAMS) to quantify the free fatty acids. The FAMS method was validated according to ICH Q2 (R1) guidelines and is suitable for a wide range of products, buffers and protein concentrations. The end-to-end workflow can be automated from sample preparation to data analysis. To broaden method accessibility, the QDa detector selected for fatty acid measurement does not require specific mass spectrometry experience. We provide here a detailed procedure for both manual and automated sample preparation for high-throughput analysis. In addition, we highlight in this protocol the critical operational details, procedural watchouts and troubleshooting tips to support the successful execution of this method in another laboratory.

Keywords:  automation; biopharmaceuticals; free fatty acid; high-throughput; liquid chromatography; mass spectrometry; method validation; polysorbate degradation; single quad

DOI:  https://doi.org/10.3390/mps7050071
Basic Clin Pharmacol Toxicol. 2024 Sep 23.

Long-term stability of five atypical antipsychotics (risperidone, olanzapine, paliperidone, clozapine, quetiapine) and the antidepressant mirtazapine in human serum assessed by a validated SPE LC-MS/MS method.

Palle Bach Nielsen Fruekilde, M Flemming Nielsen.

  Long-term sample stability of five atypical antipsychoticdrugs risperidone, paliperidone, clozapine, quetiapine and olanzapine and the antidepressant drug mirtazapine in serum was studied by use of a newly developed and validated analytical method based on solid-phase extraction and liquid chromatography-tandem mass spectrometry. Ascorbic acid was used as an antioxidative agent to stabilize olanzapine during storage and sample preparation. We assessed analyte stability on long-term storage in serum samples at 25°C, 5°C, -20°C and -80°C, and during five freeze-thaw cycles. Analytes were stable for 23 days at room temperature except for olanzapine and mirtazapine (17 days). All analytes were stable for at least 30 days at 5°C. All analytes were stable for 270 days at -20°C, except for paliperidone and mirtazapine with 60 days and 180 days, respectively. All analytes were stable for 270 days at -80°C. Furthermore, all analytes were stable for five freeze-thaw cycles. We recommend storage at -80°C when samples drawn for analysis of antipsychotic drugs are stored for more than 60 days, whereas a temperature of -20°C is sufficient for storage less than 60 days.

Keywords:  LC–MS/MS; SPE; antidepressant; antipsychotics; clozapine; mirtazapine; olanzapine; paliperidone; quetiapine; risperidone; serum; stability

DOI:  https://doi.org/10.1111/bcpt.14080
J Mass Spectrom. 2024 Oct;59(10): e5094

An Expedited Qualitative Profiling of Free Amino Acids in Plant Tissues Using Liquid Chromatography-Mass Spectrometry (LC-MS) in Conjunction With MS-DIAL.

Anish Kaachra, Anish Tamang, Vipin Hallan.

  The estimation of relative levels of amino acids is crucial for understanding various biological processes in plants, including photosynthesis, stress tolerance, and the uptake and translocation of nutrients. A wide range of liquid chromatography (LC; HPLC/UHPLC)-based methods is available for measuring the quantity of amino acids in plants. Additionally, the coupling of LC with mass spectrometry (MS) significantly enhanced the robustness of existing chromatographic methods used for amino acid quantification. However, accurate annotation and integration of mass peaks can be challenging for plant biologists with limited experience in analyzing MS data, especially in studies involving large datasets with multiple treatments and/or replicates. Further, there are instances when the experiment demands an overall view of the amino acids profile rather than focusing on absolute quantification. The present protocol provides a detailed LC-MS method for obtaining a qualitative amino acids profile using MS-DIAL, a versatile and user-friendly program for processing MS data. Free amino acids were extracted from the leaves of control and Tomato leaf curl Palampur virus (ToLCPalV)-infected Nicotiana benthamiana plants. Extracted amino acids were derivatized and separated using UHPLC-QTOF, with each amino acid subsequently identified by aligning mass data with a custom text library created in MS-DIAL. Further, MS-DIAL was employed for internal standard-based normalization to obtain a qualitative profile of 15 amino acids in control and virus-infected plants. The outlined method aims to simplify the processing of MS data to quickly assess any modulation in amino acid levels in plants with a higher degree of confidence.

Keywords:   Nicotiana benthamiana ; MS‐DIAL; UHPLC‐QTOF; amino acids

DOI:  https://doi.org/10.1002/jms.5094
Bioanalysis. 2024 Sep 24. 1-12

High-performance liquid chromatography-tandem mass spectrometry was used to measure 20 antidepressants in human serum.

Huanchang Luo, Jianwei Zhou, Guanfeng Lin, Xiaojing Huang, Yuhang Yang, Yingsong Wu.

  Aim: This study used high performance liquid chromatography-tandem mass spectrometry to quantify the blood concentrations of 20 antidepressants, such as bupropion and fluoxetine, in human serum samples.Methods: After direct precipitation with a 1:9 protein precipitant of methanol and acetonitrile, serum samples were examined using high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS). The material was separated using a Poroshell 120 EC-C18 column (3.0 mm × 50 mm, 2.7 μm) and gradient elution. The mobile phases were phase A 0.01% formic acid aqueous solution (containing 2 mmol/ml ammonium acetate) and phase B methanol solution. A 0.45 ml/min flow rate was used to divide the sample and inject 5 μl. Electrospray ionization source positive ion mode and multiple reaction monitoring modes were used for analysis. Measurement was quantified using an internal standard technique.Results: Accuracy ranged from 90.3 to 114.3%, intra-day precision from 100.1 to 112.3%, inter-day precision from 100.4 to 112.6%, extraction recoveries from 85.5 to 114.5% and matrix effect from 85.6 to 98.7%.Conclusion: This approach is fast, accurate, sensitive and repeatable. It can identify 20 antidepressants in blood simultaneously. This can be used to monitor blood drug levels and medication metabolism.

Keywords:  LC-MS/MS; antidepressants; high performance liquid chromatography; mass spectrometry; therapeutic drug monitoring

DOI:  https://doi.org/10.1080/17576180.2024.2401283
Mikrochim Acta. 2024 09 25. 191(10): 620

Magnetic particle spray mass spectrometry for the determination of beta-blockers in plasma samples.

Elizângela Maria de Castilho Alves Morais, Tássia Venga Mendes, Estela Domingos Arnoni, Luiz Fernando Gorup, Mariana Azevedo Rosa, Ailton José Moreira, Eduardo Costa Figueiredo.

  Magnetic particle spray mass spectrometry (MPS-MS), an innovative ambient ionization technique proposed by our research group, was employed to determine beta-blockers in human plasma samples. A dispersive solid phase extraction of atenolol, metoprolol, labetalol, propranolol, nadolol, and pindolol was carried out using magnetic molecularly imprinted polymer (M-MIP) particles that were attached to the tip of a metal probe, which was placed in the mass spectrometer inlet. A solvent (1% formic acid in methanol) was dispensed on the particles, and the Taylor cone was formed around them (in high voltage). The analytes were desorbed/ionized and determined by a triple quadrupole mass spectrometer. M-MIP was synthesized with oxprenolol as a pseudo-template, demonstrating good selectivity to beta-blockers compared with no-analog molecules, with an adsorption process occurring in monolayers, according to isotherm studies. Kinetic experiments indicated chemisorption as the predominant M-MIP/analyte interaction. The analytical curves were linear (R2 > 0.98), and the limit of quantification was 3 µg L-1 for all the analytes. Limits of detection ranged from 0.64 to 2.41 µg L-1. Precisions (relative standard deviation) and accuracies (relative error) ranged from 3.95 to 21.20% and -17.05 to 18.93%, respectively. MPS-MS proved to be a simple, sensitive, and advantageous technique compared with conventional approaches. The analyses were fast, requiring no chromatographic separation and without ionic suppression. The method is aligned with green chemistry principles, requiring minimal sample, solvent, and sorbent amounts. MPS-MS successfully integrates sample preparation and ambient ionization mass spectrometry and holds great potential for application with other sorbents, samples, and analytes.

Keywords:  Ambient ionization; Dispersive solid phase extraction; Magnetic particle spray mass spectrometry; Magnetic sorbent; Molecularly imprinted polymers

DOI:  https://doi.org/10.1007/s00604-024-06698-2
Wei Sheng Yan Jiu. 2024 Sep;53(5): 783-789

[Application of chromatographic retention time correction in untargeted screening].

Fei Shen, Yifei Fang, Zongli Huo, Feng Zhu, Hao Zhang, Yonglin Zhou.

   OBJECTIVE: Chromatographic retention time correction is one of the important steps to effectively improve the accuracy of identification. This article proposed a strategy for untargeted screening of biological samples based on retention time correction.
METHODS: A pre-treatment method for biological samples was established. The conditions of liquid chromatography and mass spectrometry were optimized. Fourteen compounds were selected as calibration agents. The retention time correction of different samples, different injection time, different brands of instruments, changing chromatographic column and changing mobile phase were investigated.
RESULTS: Calibration agents had a wide coverage, good stability and no interference with sample determination. They could be uniformly distributed in the chromatogram in both positive and negative ion modes. The chromatogram was divided into several time intervals. Calibration agents in each time period were used for retention time linear correction, and the correction effects were good.
CONCLUSION: The retention time correction method could eliminate the retention time drift caused by experimental conditions, improve the accuracy of qualitative analysis, and help to solve the problem of high false positive result based on mass spectrum information.

Keywords:  retention time correction; ultra high performance liquid chromatography-high resolution mass spectrometry; untargeted screening

DOI:  https://doi.org/10.19813/j.cnki.weishengyanjiu.2024.05.015
J Xenobiot. 2024 Aug 27. 14(3): 1143-1164

Novel Technique for Simultaneous Ethylene Glycol and Its Metabolites Determination in Human Whole Blood and Urine Samples Using GC-QqQ-MS/MS.

Kaja Tusiewicz, Olga Wachełko, Marcin Zawadzki, Paweł Szpot.

  Toxicological analyses often necessitate the identification of compounds belonging to diverse functional groups. For GC-MS analyses, derivatization of compounds belonging to different functional groups can pose a challenge and requires the development of comprehensive methods of analysis. One example could be ethylene glycol, whose widespread use is related to possible unintentional or suicidal intoxications. This fact clearly indicates the need to develop sensitive methods for the determination of ethylene glycol and its metabolites in biological material, as only such complex analysis allows for proper toxicological expertise. A simultaneous GC-QqQ-MS/MS method for the determination of ethylene glycol together with its metabolites, glyoxal and glycolic acid, as well as the detection of glyoxylic acid and oxalic acid, was developed and fully validated. A novel approach for simultaneous derivatization of substances from different groups (alcohols, aldehydes, and carboxylic acids) was established. Sample preparation included the addition of three internal standards (BHB-d4, ethylene glycol-d4 and methylglyoxal), precipitation with acetonitrile and subsequent derivatization with N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), as well as pentafluorophenylhydrazine (PFPH). Detection was carried out with the use of triple quadrupole mass spectrometer. The ionization method was electron impact, and quantitative analysis was carried out in multiple reaction monitoring mode. The lower limit of quantification was 1 μg/mL, 0.1 μg/mL, and 500 μg/mL for ethylene glycol, glyoxal, and glycolic acid, respectively. The presented method was applied in three authentic postmortem cases of ethylene glycol intoxication.

Keywords:  GC–QqQ–MS/MS; MTBSTFA derivatization; PFPH derivatization; ethylene glycol intoxication; postmortem biological material

DOI:  https://doi.org/10.3390/jox14030065
Anal Methods. 2024 Sep 24.

Recent development on the extraction, detection, and quantification of vitamin D from various sources - an update.

G S Amithabh, M P Gireesh Kumar, K Selvapandian, B Baskar.

Recent advancements in analytical methods for vitamin D and its metabolites have substantially enhanced our capacity to precisely determine and quantify these substances in a wide range of sources, such as biological fluids, fungus, natural and fortified foods. This study focuses on the latest advancements in sample preparation procedures, including solid-phase extraction and environmentally friendly extraction methods. These approaches aim to enhance efficiency and minimize the use of solvents. In addition, we explore the growing popularity of chromatographic methods, specifically LC-MS/MS and developing supercritical fluid chromatography (SFC), which provide improved sensitivity, selectivity, and faster analytical times for comprehensive vitamin D profiling. These developments are crucial for overcoming the challenges presented by low concentrations and complex matrices in the investigation of vitamin D.

DOI: https://doi.org/10.1039/d4ay01114g
J Pharm Biomed Anal. 2024 Sep 20. pii: S0731-7085(24)00522-3. [Epub ahead of print]252 116480

Derivatization to reduce background interferences for simultaneous quantitation of trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) using liquid chromatography with tandem mass spectrometry.

Maria A Valdivia-Garcia, Yang Bi, Kaoutar Abaakil, Jia V Li.

  Trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) play a crucial role in many biochemical processes within diverse organisms including animals, plants, fungi and bacteria. Studies have linked these metabolites with cardiovascular and kidney diseases; however, emerging evidence demonstrates their protective properties. Owing to these controversies and co-existence of these metabolites in biological samples, it is crucial to accurately quantify these metabolites to associate their concentrations with various physiological and pathophysiological conditions to elucidate their potential roles. We reported interferences on TMA quantification without derivatizing the analyte. A combined sample preparation method, including sample derivatization with ethyl bromoacetate and use of ion pairing reagent (sodium heptanesulfonate), minimized these interferences and provided improved accuracy and precision for simultaneous quantification of TMA and TMAO. The linearity for TMAO ranged from 0.01 µM to 300 µM and 0.1 µM - 300 µM for TMA. With the application of this method, we reported that the circulating concentrations of TMA was 4 times higher in male mice (33.1 ± 5.9 µmol/L) compared to females (8.3 ± 1.39 µmol/L), whereas TMAO levels were 6 times lower in male (7.2 ± 0.4 µmol/L) than female mice (42.1 ± 4.5 µmol/L). In contrast, concentrations of TMA and TMAO in the colonic tissue did not differ significantly between males and females. The robust analytical method for simultaneously quantifying TMA and TMAO presents a significant value in facilitating investigations on TMA and TMAO biology.

Keywords:  Derivatisation; Ethyl bromoacetate; Sodium heptanesulfonate; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2024.116480
Nat Protoc. 2024 Sep 20.

Statistical analysis of feature-based molecular networking results from non-targeted metabolomics data.

Abzer K Pakkir Shah, Axel Walter, Filip Ottosson, Francesco Russo, Marcelo Navarro-Diaz, Judith Boldt, Jarmo-Charles J Kalinski, Eftychia Eva Kontou, James Elofson, Alexandros Polyzois, Carolina González-Marín, Shane Farrell, Marie R Aggerbeck, Thapanee Pruksatrakul, Nathan Chan, Yunshu Wang, Magdalena Pöchhacker, Corinna Brungs, Beatriz Cámara, Andrés Mauricio Caraballo-Rodríguez, Andres Cumsille, Fernanda de Oliveira, Kai Dührkop, Yasin El Abiead, Christian Geibel, Lana G Graves, Martin Hansen, Steffen Heuckeroth, Simon Knoblauch, Anastasiia Kostenko, Mirte C M Kuijpers, Kevin Mildau, Stilianos Papadopoulos Lambidis, Paulo Wender Portal Gomes, Tilman Schramm, Karoline Steuer-Lodd, Paolo Stincone, Sibgha Tayyab, Giovanni Andrea Vitale, Berenike C Wagner, Shipei Xing, Marquis T Yazzie, Simone Zuffa, Martinus de Kruijff, Christine Beemelmanns, Hannes Link, Christoph Mayer, Justin J J van der Hooft, Tito Damiani, Tomáš Pluskal, Pieter Dorrestein, Jan Stanstrup, Robin Schmid, Mingxun Wang, Allegra Aron, Madeleine Ernst, Daniel Petras.

Feature-based molecular networking (FBMN) is a popular analysis approach for liquid chromatography-tandem mass spectrometry-based non-targeted metabolomics data. While processing liquid chromatography-tandem mass spectrometry data through FBMN is fairly streamlined, downstream data handling and statistical interrogation are often a key bottleneck. Especially users new to statistical analysis struggle to effectively handle and analyze complex data matrices. Here we provide a comprehensive guide for the statistical analysis of FBMN results, focusing on the downstream analysis of the FBMN output table. We explain the data structure and principles of data cleanup and normalization, as well as uni- and multivariate statistical analysis of FBMN results. We provide explanations and code in two scripting languages (R and Python) as well as the QIIME2 framework for all protocol steps, from data clean-up to statistical analysis. All code is shared in the form of Jupyter Notebooks ( https://github.com/Functional-Metabolomics-Lab/FBMN-STATS ). Additionally, the protocol is accompanied by a web application with a graphical user interface ( https://fbmn-statsguide.gnps2.org/ ) to lower the barrier of entry for new users and for educational purposes. Finally, we also show users how to integrate their statistical results into the molecular network using the Cytoscape visualization tool. Throughout the protocol, we use a previously published environmental metabolomics dataset for demonstration purposes. Together, the protocol, code and web application provide a complete guide and toolbox for FBMN data integration, cleanup and advanced statistical analysis, enabling new users to uncover molecular insights from their non-targeted metabolomics data. Our protocol is tailored for the seamless analysis of FBMN results from Global Natural Products Social Molecular Networking and can be easily adapted to other mass spectrometry feature detection, annotation and networking tools.

DOI: https://doi.org/10.1038/s41596-024-01046-3
Nat Commun. 2024 Sep 27. 15(1): 8396

Deep learning prediction of electrospray ionization tandem mass spectra of chemically derived molecules.

Bin Chen, Hailiang Li, Rongfu Huang, Yanan Tang, Feng Li.

Chemical derivatization is a powerful strategy to enhance sensitivity and selectivity of liquid chromatography-mass spectrometry for non-targeted analysis of chemicals in complex mixtures. However, it remains impossible to obtain large sets of reference spectra for chemically derived molecules (CDMs), representing a major barrier in real-world applications. Herein, we describe a deep learning approach that enables accurate prediction of electrospray ionization tandem mass spectra for CDMs (DeepCDM). DeepCDM is established by transfer learning from a generic spectrum predicting model using a small set of experimentally acquired tandem mass spectra of CDMs, which converts a generic model with low predictability for CDMs into a specialized model with high predictability. We demonstrate DeepCDM by predicting electrospray ionization tandem mass spectra of dansylated molecules. The success in establishing Dns-MS further enables the development of DnsBank, a dansylation-specialized in silico spectral library. DnsBank achieves significant increases of accurate annotation rates of dansylated molecules, facilitating discovery of new hazardous pollutants from an environmental study of leather industrial wastewater. DeepCDM is also highly versatile for other classes of CDMs. Therefore, we envision that DeepCDM will pave a way for high-throughput identification of CDMs in non-targeted analysis to dig unknowns with potential health impacts from emerging anthropogenic chemicals.

DOI: https://doi.org/10.1038/s41467-024-52805-5
Anal Sens. 2024 May;pii: e202300097. [Epub ahead of print]4(3):

Rapid Characterization of Phospholipids from Biological Matrix Enabled by Indium Tin Oxide (ITO) Coated Slide Assisted Enrichment MALDI Mass Spectrometry.

Penghsuan Huang, Hua Zhang, Yuan Liu, Lingjun Li.

  Lipidomic analysis of human serum is essential to monitor the individual's health status. Herein, we develop a facile strategy for rapid characterization of phospholipids in human serum via indium tin oxide (ITO) coated glass slide solid phase extraction MALDI mass spectrometry (ITO-SPE-MALDI-MS). Phospholipid species are retained on ITO slide via solid phase extraction owing to the unique property of the ITO material; the measurement of phospholipid species from 1 μl human serum within 2 min is achievable. A comparison of ITO-SPE strategy with conventional extraction methods was further carried out using liquid chromatography-mass spectrometry (LC-MS) and ion-mobility mass spectrometry (IM-MS), resulting in a comparable enrichment performance for the phospholipid analysis. Furthermore, rapid lipidomic profiling of serum samples from human colorectal cancer patients and cell lines was demonstrated. Our results indicate that ITO-SPE-MALDI-MS provides a higher throughput strategy for the analysis of phospholipid species in complex biological mixtures, showcasing its potential for applications in the analysis of clinical biofluids.

Keywords:  Human serum; ITO-enrichment; LC-MS/MS; Lipidomic; MALDI

DOI:  https://doi.org/10.1002/anse.202300097
Se Pu. 2024 Oct;42(10): 923-934

[Advances in the application of ion chromatography-mass spectrometry in the fields of life and health].

Yu-Xin Yi, Mi-Cong Jin.

  Ion chromatography is a technique commonly used to separate strongly polar and ionizable substances; it can be used to separate, identify, and quantify ionizable compounds in complex samples when coupled with mass spectrometry, and is currently being used in the application of food analysis, drug analysis, metabolomics and clinical poisoning analysis. Herein, we review the development of ion chromatography-mass spectrometry (IC-MS), its progress over the past 20 years, and future trends in the abovementioned areas. The IC-MS research progress and applications for the determination of inorganic anions, organic acids, polar pesticides, biogenic amines, and sugars in the food field are discussed. Drug analysis applications are discussed mainly in relation to the analysis of drug impurities, identifying drug degradation products, and determination of plasma concentration, while the separation and analysis of strongly polar metabolites, such as organic acids, sugar phosphates, and nucleotides in biological matrices are discussed in relation to metabolomics. Advances in the analysis of strongly polar or ionizable toxic compounds, such as alkyl methylphosphonic acid, methylphosphonic acid, glyphosate, 3-nitropropionic acid, and indandione rodenticides, are mainly discussed in clinical poisoning analysis field. This paper is expected to become a useful reference for the further expansion and application of IC-MS in the life and health fields.

Keywords:  clinical poisoning analysis; drug analysis; food analysis; ion chromatography-mass spectrometry (IC-MS); metabolomics; review

DOI:  https://doi.org/10.3724/SP.J.1123.2023.11001
Bioanalysis. 2024 Sep 27. 1-11

Determination of drugs of abuse and metabolites in plasma microsamples by LC-MS/MS after microQuEChERS extraction.

Mariana Smidt, Marcos Frank Bastiani, Roberta Zilles Hahn, Lilian de Lima Feltraco Lizot, Amanda Pacheco Bondan, Giovana Piva Peteffi, Náthaly Cbf Dos Santos, José Luiz da Costa, Rafael Lanaro, Rafael Linden.

  Aim: Identifying drugs of abuse and their metabolites in plasma is vital in both forensic and clinical toxicology. While the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method offers an efficient approach to sample preparation, its application is complex due to the wide-ranging properties of target analytes and the challenges posed by biological matrix interferences. This study aims to develop a microQuEChERS approach for the quantification of 14 drugs of abuse and metabolites utilizing minimal sample and solvent volumes.Methods: The microQuEChERS method involved using 10 μl plasma samples, 25 mg of a salt mixture and 150 μl of acetonitrile. Extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), with a 7.5 min run. The assay was validated according to bioanalytical guidelines.Results: The accuracy was 96.8-112.4%. The within-assay precision was within 2.0-8.9% and the between-assay precision was within 3.2-8.2%. Matrix effects were found to range from -5.7 to 13.5%. The extraction yield was higher than 74.7%.Conclusion: This study described a microQuEChERS sample preparation approach for determining drugs of abuse and metabolites using plasma microsamples and LC-MS/MS. The approach is efficient, environmentally friendly and suitable for scenarios with limited amounts of biological samples.

Keywords:  LC-MS/MS; MicroQuEChERS; drugs of abuse; plasma microsamples

DOI:  https://doi.org/10.1080/17576180.2024.2404383
Comput Struct Biotechnol J. 2024 Dec;23 3327-3341

Knowledge-based in silico fragmentation and annotation of mass spectra for natural products with MassKG.

Bingjie Zhu, Zhenhao Li, Zehua Jin, Yi Zhong, Tianhang Lv, Zhiwei Ge, Haoran Li, Tianhao Wang, Yugang Lin, Huihui Liu, Tianyi Ma, Shufang Wang, Jie Liao, Xiaohui Fan.

  Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a potent analytical technique utilized for identifying natural products from complex sources. However, due to the structural diversity, annotating LC-MS/MS data of natural products efficiently remains challenging, hindering the discovery process of novel active structures. Here, we introduce MassKG, an algorithm that combines a knowledge-based fragmentation strategy and a deep learning-based molecule generation model to aid in rapid dereplication and the discovery of novel NP structures. Specifically, MassKG has compiled 407,720 known NP structures and, based on this, generated 266,353 new structures using chemical language models for the discovery of potential novel compounds. Furthermore, MassKG demonstrates exceptional performance in spectra annotation compared to state-of-the-art algorithms. To enhance usability, MassKG has been implemented as a web server for annotating tandem mass spectral data (MS/MS, MS2) with a user-friendly interface, automatic reporting, and fragment tree visualization. Lastly, the interpretive capability of MassKG is comprehensively validated through composition analysis and MS annotation of Panax notoginseng, Ginkgo biloba, Codonopsis pilosula, and Astragalus membranaceus. MassKG is now accessible at https://xomics.com.cn/masskg.

Keywords:  Deep learning; In silico fragmentation; MS annotation; Mass spectrometry; Natural products

DOI:  https://doi.org/10.1016/j.csbj.2024.09.001
Toxics. 2024 Sep 16. pii: 675. [Epub ahead of print]12(9):

Paraquat Poisoning: Insights from Autopsy, Histology, and Liquid Chromatography with Tandem Mass Spectrometry in Multidisciplinary Forensic Toxicology Practice.

Issarapong Nuwongsa, Tanyarat Markmee, Nareerat Pholpo, Manoch Chockjamsai, Tawachai Monum, Yutti Amornlertwatana, Preechaya Tajai.

  The herbicide paraquat (PQ) is responsible for a significant number of fatalities resulting from self-poisoning. Nevertheless, only a limited number of comprehensive studies focusing on fatal PQ poisoning, which include examination of autopsy findings, histopathology, and quantitative analysis of post-mortem samples, have been published. This study aimed to evaluate autopsy findings, histopathology, and quantitative analysis of PQ in post-mortem human serum samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a simple, sensitive, and specific method. Autopsies were performed on all deaths due to PQ poisoning, and serum samples were sent to the toxicology laboratory for chemical analysis. The method was successfully applied to seven human serum samples, and the results indicate its reliability for detecting PQ. The study reports fatal serum PQ levels ranging from 0.5 to 372.0 µg/mL. The comprehensive data presented in this study can be useful for further research and practical applications.

Keywords:  LC-MS/MS; autopsy; histology; paraquat; poisoning

DOI:  https://doi.org/10.3390/toxics12090675
Wei Sheng Yan Jiu. 2024 Sep;53(5): 790-796

[Determination of 13 kinds of free and bound phenolic acids in fruits by high-performance liquid chromatography-mass spectrometry].

Jun'an Yan, Hongxing Tan, Zengxu Tang, Xiaoqian Liu, Wanna Xu, Zhu Wang, Weisheng Xu, Wenxu Hong.

   OBJECTIVE: To establish a high-performance liquid chromatography-mass spectrometry(HPLC-MS/MS) method for detecting 13 kind of free and bound phenolic acids(chlorogenic acid, protocatechuic acid, ferulic acid, p-coumaric acid, gallic acid, gentisic acid, vanillic acid, caffeic acid, syringic acid, sinapic acid, rosmarinic acid, salicylic acid, p-hydroxybenzoic acid) in fruits, and optimize the pre-treatment conditions to meet the detection requirements for phenolic acid content in various types of fruits.
METHODS: Free phenolic acids in fruits were extracted using methanol through ultrasonic extraction. Conjugated phenolic acids in the centrifuged residue were released by alkaline hydrolysis and extracted with ethyl acetate. The two extracts were combined, concentrated, and analyzed using HPLC-MS/MS. Separation was achieved using an Agilent ZORBAX SB-C_(18) chromatography column(3.0 mm×100 mm, 3.5 μm), and detection was performed in multiple reaction monitoring(MRM) mode.
RESULTS: All 13 standard phenolic acids achieved complete separation within 10 minutes, with linear correlation coefficients greater than 0.998 and detection limits ranging from 0.172 to 3.471 ng/mL. After optimization of the pre-treatment method, the recovery rates of the method for four types of fruits-apples, strawberries, oranges, and peaches-ranged from 80.0% to 119.4%, and the precision were lower than 7.00%(n=6). The result of testing on four categories of twelve types of fruits demonstrated significant variations in the content of phenolic acids among different fruits, and within the same category, the composition of phenolic acids did not exhibit consistency.
CONCLUSION: The HPLC-MS/MS method exhibits high sensitivity, precision, and accuracy. It is suitable for the detection of both free and bound phenolic acids in various types of fruits.

Keywords:  HPLC-MS/MS; fruits; phenolic acids

DOI:  https://doi.org/10.19813/j.cnki.weishengyanjiu.2024.05.016
Anal Chem. 2024 Sep 23.

In-Depth Analysis of Molecular Network Based on Liquid Chromatography Coupled with Tandem Mass Spectrometry in Natural Products: Importance of Redundant Nodes Discovery.

Yuhao Zhang, Jingyu Liao, Wanqi Le, Weidong Zhang, Gaosong Wu.

The identification of molecules within complex mixtures is a major bottleneck in natural products (NPs) research. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as the main tool for the high-throughput characterization of NPs. The large amount of data sets by LC-MS/MS presents a challenge for data processing and interpretation, and the LC-MS/MS molecular network (MN) is one of the most prominent tools for analyzing large MS/MS data sets, widely used for rapid classification, identification, and structural speculation of unknown compounds. However, the existence of a large number of redundant nodes leads to false-positive results. To solve this problem, we proposed the in-depth analysis of MN. In this study, in-depth analysis of MN of five NPs representing the common structures of saponin, steroid, flavonoid, alkaloid, and phenolic acid revealed the presence of redundant nodes (including other adducts, isotope, and in-source fragmentation) in addition to the normal nodes, which can lead to false-positive identification results. Additionally, the reasons for different redundant nodes are discussed and experimentally verified, and it was found that the impact of redundant nodes can be mitigated by optimizing the experimental conditions and employing Feature-Based Molecular Networking. Furthermore, Ion Identity Molecular Networking can rapidly discover and screen redundant nodes, simplifying the in-depth analysis of MN and improving the network connectivity of structurally related molecules. Finally, a combination formulation of 7 NPs is used as an example to provide a guide for in-depth analysis of MN for comprehensive characterization of complex systems. This study highlights the importance of an in-depth analysis of MN for better understanding and utilization of MS/MS data in complex systems to reduce the false-positive rate of identification by screening and filtering redundant nodes.

DOI: https://doi.org/10.1021/acs.analchem.4c02230
J Chem Inf Model. 2024 Sep 27.

Predicting Collision-Induced-Dissociation Tandem Mass Spectra (CID-MS/MS) Using Ab Initio Molecular Dynamics.

Jesi Lee, Dean Joseph Tantillo, Lee-Ping Wang, Oliver Fiehn.

Compound identification is at the center of metabolomics, usually by comparing experimental mass spectra against library spectra. However, most compounds are not commercially available to generate library spectra. Hence, for such compounds, MS/MS spectra need to be predicted. Machine learning and heuristic models have largely failed except for lipids. Here, quantum chemistry software can be used to predict mass spectra. However, quantum chemistry predictions for collision induced dissociation (CID) mass spectra in LC-MS/MS are rare. We present the CIDMD (Collision-Induced Dissociation via Molecular Dynamics) framework to model CID-based MS/MS spectra. It uses first-principles molecular dynamics (MD) to simulate the physical process of molecular collisions in CID tandem mass spectrometry. First, molecular ions are constructed at specific protonation sites. Using density functional theory, these protonated ions are targeted by argon collider gas atoms at user-specified velocities. Subsequent bond breakages are simulated over time for at least 1,000 fs. Each simulation is repeated multiple times from various collisional directions. Fragmentations are accumulated over those repeated collisions to generate CIDMD in silico mass spectra. Twelve small metabolites (<205 Da) were selected to test the accuracy of this framework in comparison to experimental MS/MS spectra. When testing different protomers, collider velocities, number of simulations, simulation time and impact factor b cutoffs, we yielded 261 predicted mass spectra. These in silico spectra resulted in entropy similarity scores of an average 624 ± 189 for all 261 spectra compared to their corresponding experimental spectra, which improved to 828 ± 77 when using optimal parameters of the most probable protomers for 12 molecules. With increasing molecular mass, higher velocities achieved better results. Similarly, different protomers showed large differences in fragmentation; hence, with increasing numbers of protomers and tautomers, the average CIDMD prediction accuracy decreased. Mechanistic details showed that specific fragment ions can be produced from different protomers via multiple fragmentation pathways. We propose that CIDMD is a suitable tool to predict mass spectra of small metabolites like produced by the gut microbiome.

DOI: https://doi.org/10.1021/acs.jcim.4c00760
Expert Opin Drug Metab Toxicol. 2024 Sep 26.

A high-throughput liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of p-cresol sulfate, p-cresol glucuronide, indoxyl-sulfate, and indoxyl glucuronide in HepaRG culture medium and the demonstration of mefenamic acid as a potent and selective detoxifying agent.

Ala'a R Al-Dajani, Tony K L Kiang.

   BACKGROUND: p-cresol and indole are diet-derived uremic compounds which undergo sulfonation to generate the highly toxic p-cresol sulfate (pCS) and indoxyl sulfate (IxS). They are also subjected to glucuronidation to produce the less toxic p-cresol glucuronide (pCG) and indoxyl glucuronide (IG). We developed and validated a liquid chromatography-mass spectrometry assay to quantify these metabolites in the HepaRG model. We also tested the effects of mefenamic acid, a noncompetitive sulfotransferase inhibitor, on the in-situ formations of these metabolites in relation to the development of cellular necrosis.
RESEARCH DESIGN AND METHODS: the assay, developed on Shimadzu LCMS-8050, was used to measure metabolites generated in HepaRG cells exposed to p-cresol or indole (0-1 mM) with mefenamic acid (0-3000 nM) for 24 hours. Cells were also exposed to 0.5 mM p-cresol or indole with/without 30 nM mefenamic acid (nontoxic and half-inhibitory concentration) to characterize the effects on lactate dehydrogenase (LDH) release.
RESULTS: The validated assay exhibited high sensitivity and wide calibration ranges covering known human concentrations (i.e. 103.39 nM 53,134.96 nM). HepaRG cells exposed to p-cresol or indole generated physiologically-relevant maximum concentrations of pCS (29.98 ± 2.29 µM), pCG (479.95 ± 10.10 µM), IxS (8.43 ± 1.22 µM), and IG (10.23 ± 2.48 µM). Mefenamic acid inhibited pCS formation in a concentration-dependent manner without affecting pCG, IxS, or IG. Mefenamic acid also reduced LDH release from p-cresol (by 50.12 ± 5.86%) or indole (56.26 ± 3.58%) exposure.
CONCLUSIONS: This is a novel assay capable of quantifying these uremic metabolites in HepaRG cells. Our novel findings suggest that mefenamic acid can be utilized therapeutically to attenuate pCS-associated toxicities, but additional experiments are required to establish causality and dosing strategies.

Keywords:  Indoxyl glucuronide; indoxyl sulfate; liquid chromatography tandem-mass spectrometry; mefenamic acid; p-cresol glucuronide; p-cresol sulfate

DOI:  https://doi.org/10.1080/17425255.2024.2409257
Analyst. 2024 Sep 27.

Storing liquid chromatographic separations on surface energy traps: decoupling the LC and the mass spectrometer.

Timothy T Salomons, David Simon, Richard Oleschuk.

We report a micro-fractionation device for high performance liquid chromatography-mass spectrometry to archive chromatographic separations on an array of optimized surface energy traps (SETs). The method has the potential to significantly alter nanoflow LC-MS workflow, decoupling separation and analysis. The wetting characteristics of the SETs cause the HPLC eluent stream to spontaneously split into droplet microfractions. The droplet mirofractions are then dried down to enable facile storage and transport of the archived separation. Discontinuously dewetting array parameters were explored to maximize array volume and resolution using a combination of SET design, shape, size, and spacing. Mass spectrometry analysis is performed utilizing a liquid micro-junction surface sampling probe to extract dried analytes from the surface of the SETs followed by electrospray ionisation. A reverse phase separation of pharmaceutical compounds is "recorded" using the micro-fractionation device followed by "reading" the chromatographic trace with a mass spectrometer 24 hours after the separation was performed/archived, demonstrating a true decoupling of LC, and MS. Additionally, we demonstrate the ability to collect microfractions with sub-one-second integration time, approaching the scan time of a mass spectrometer or UV-Vis detector. With further improvements to the device, sub-1-second micro-fractionation may enable seamless reconstruction of archived chromatograms indistinguishable from online LC-MS data, while also providing the benefits of easy storage and transport of archived separations.

DOI: https://doi.org/10.1039/d4an00828f
mSystems. 2024 Sep 24. e0115224

Single-colony MALDI mass spectrometry imaging reveals spatial differences in metabolite abundance between natural and cultured Trichodesmium morphotypes.

Karl J Romanowicz, Futing Zhang, Siyuan Wang, Dušan Veličković, Rosalie K Chu, Yeala Shaked, Rene M Boiteau.

  Trichodesmium, a globally significant N2-fixing marine cyanobacterium, forms extensive surface blooms in nutrient-poor ocean regions. These blooms consist of a dynamic assemblage of Trichodesmium species that form distinct colony morphotypes and are inhabited by diverse microorganisms. Trichodesmium colony morphotypes vary in ecological niche, nutrient uptake, and organic molecule release, differentially impacting ocean carbon and nitrogen biogeochemical cycles. Here, we assessed the poorly studied spatial abundance of metabolites within and between three morphologically distinct Trichodesmium colonies collected from the Red Sea. We also compared these results with two morphotypes of the cultivable Trichodesmium strain IMS101. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) coupled with liquid extraction surface analysis (LESA) tandem mass spectrometry (MS2), we identified and localized a wide range of small metabolites associated with single-colony Trichodesmium morphotypes. Our untargeted MALDI-MSI approach revealed 80 unique features (metabolites) shared between Trichodesmium morphotypes. Discrimination analysis showed spatial variations in 57 shared metabolites, accounting for 62% of the observed variation between morphotypes. The greatest variations in metabolite abundance were observed between the cultured morphotypes compared to the natural colony morphotypes, suggesting substantial differences in metabolite production between the cultivable strain IMS101 and the naturally occurring colony morphotypes that the cultivable strain is meant to represent. This study highlights the variations in metabolite abundance between natural and cultured Trichodesmium morphotypes and provides valuable insights into metabolites common to morphologically distinct Trichodesmium colonies, offering a foundation for future targeted metabolomic investigations.IMPORTANCEThis work demonstrates that the application of spatial mass spectrometry imaging at single-colony resolution can successfully resolve metabolite differences between natural and cultured Trichodesmium morphotypes, shedding light on their distinct biochemical profiles. Understanding the morphological differences between Trichodesmium colonies is crucial because they impact nutrient uptake, organic molecule production, and carbon and nitrogen export, and subsequently influence ocean biogeochemical cycles. As such, our study serves as an important initial assessment of metabolite differences between distinct Trichodesmium colony types, identifying features that can serve as ideal candidates for future targeted metabolomic studies.

Keywords:  Trichodesmium; colony morphotypes; liquid extraction surface analysis (LESA); mass spectrometry imaging (MSI); matrix-assisted laser desorption/ionization (MALDI); metabolite abundance; untargeted metabolomics

DOI:  https://doi.org/10.1128/msystems.01152-24
J Sep Sci. 2024 Sep;47(18): e202400418

Boric Acid Functionalized Hypercrosslinked Polymers for Selective Extraction of Trace Catecholamines and Their Metabolites in Rat Serum.

Zhiyu Zhang, Cheng Yang, Di Zhao, Yuanqing Zhao, Lixin Li, Zhonghua Li, Zhenqiang Zhang, Kai Hu.

  Abnormal levels of catecholamine (CA) neurotransmitters and their metabolites in biological fluids can lead to various neurological disorders. Herein, a boric acid-functionalized hypercrosslinked polymer was prepared and utilized as a sorbent for the dispersive solid-phase extraction of CAs and their metabolites in rat serum. By combination with a high-performance liquid chromatography-fluorescence detector, the extraction parameters for the seven target analytes were optimized. Under the optimal extraction condition, the methodology for the quantitative analysis of CAs and their metabolites in rat serum samples was established. The limits of detection and limits of quantification were found to be in the ranges of 0.010-0.015 and 0.033-0.050 ng/mL, respectively. The results demonstrated satisfactory recoveries, with values ranging from 88.02% to 113.27%, accompanied by relative standard deviations within the range of 2.69%-9.59%. In addition, the method showed good anti-interference ability (matrix effect ranged from 2.64% to 18.07%). The developed method was validated for the determination of CAs and their metabolites in normal and Alzheimer's disease model rats' serum, which proved the promising application of the method for CAs neurotransmitter analysis in biological samples.

Keywords:  catecholamine and metabolites; dispersive solid‐phase extraction; hypercrosslinked polymer; rat serum

DOI:  https://doi.org/10.1002/jssc.202400418
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 12. pii: S1570-0232(24)00312-X. [Epub ahead of print]1247 124303

Identification of fentanyl analogs and potential biomarkers in urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole/time of flight mass spectrometry (LC-Q/TOF-MS).

Madison Schackmuth, Sarah Kerrigan.

  Novel synthetic opioids are a class of drugs abused for their potent analgesic effect and are responsible for many fatal intoxications, particularly within the United States. A targeted assay was developed and validated using LC-MS/MS, capable of identifying nineteen fentalogs. Solid phase extraction was used to isolate analytes of interest from urine. Limits of detection ranged from 0.05 to 0.1 ng/mL and the limit of quantitation was 0.5 ng/mL. Extraction efficiencies using the optimized procedure were 77-88 % for all targeted species. Bias, precision, matrix effects and interferences were within acceptable thresholds for all analytes. The validated assay was used to identify analytes of interest from thirty-seven individuals that had used fentanyl and related substances. In addition to quantitative analyses, a non-targeted liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS) assay was also used to identify additional substances and potential biomarkers. Additional N-oxide and N-dealkylated species were identified using this approach, and the potential for biomarker use is presented, given the stability of some analytes within this class.

Keywords:  Analogs; Biomarkers; Fentanyl; LC-MS/MS; N-oxide; Opioids; Urine

DOI:  https://doi.org/10.1016/j.jchromb.2024.124303
J Chromatogr A. 2024 Sep 08. pii: S0021-9673(24)00734-9. [Epub ahead of print]1736 465360

Quantitation of trace polyfunctional thiols in wine by liquid chromatography quadrupole Orbitrap high-resolution mass spectrometry in parallel reaction monitoring.

Liang Chen, Emilio De Longhi, Marine Gammacurta, Axel Marchal, Philippe Darriet.

  Polyfunctional thiols are key contributors to wine aroma due to their extremely low odor thresholds, and their quantitative analysis remains challenging as a result of their ultratrace concentrations and high reactivity. This work presents the first method based on ultra-high-performance liquid chromatography (UHPLC) coupled to quadrupole Orbitrap high-resolution mass spectrometry (HRMS) in parallel reaction monitoring (PRM) mode for quantifying thiols at nanograms per liter (ng/L) levels in wine. Thiols in wine were derivatized using 4,4'-dithiodipyridine and isolated by liquid-liquid extraction. This protocol allowed rapid sample preparation with minimum labor input and low consumable expenses. Instrumental analysis was conducted using UHPLC-quadrupole Orbitrap HRMS in PRM mode. Twenty thiol analytes, including literature-known, recently identified, and novel thiols were selected and validated by the optimized method in three wine matrices. The overall analytical performances demonstrated by this method were equivalent, and in most cases, greater than many previously developed GC-MS or LC-MS methods. The validated method was applied to analyze a selection of wines in which 12 thiols were quantified.

Keywords:  Orbitrap; Parallel reaction monitoring; Quantitation; Thiols; Wine

DOI:  https://doi.org/10.1016/j.chroma.2024.465360