bims-metlip 2022-11-13 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022–11–13
23 papers selected by
Sofia Costa, Matterworks

A Concise Review of Liquid Chromatography-Mass Spectrometry-Based Quantification Methods for Short Chain Fatty Acids as Endogenous Biomarkers.
Sample multiplexing for increasing throughput for quantification of estrogens in serum by LC-MS/MS.
Recent progress in mass spectrometry for single-cell metabolomics.
Alignment and Analysis of a Disparately Acquired Multibatch Metabolomics Study of Maternal Pregnancy Samples.
An automated online three-phase electro-extraction setup with machine-vision process monitoring hyphenated to LC-MS analysis.
Development and validation of a simple and rapid HILIC-MS/MS method for the quantification of low-abundant lysoglycerophospholipids in human plasma.
Molecular Networking and On-Tissue Chemical Derivatization for Enhanced Identification and Visualization of Steroid Glycosides by MALDI Mass Spectrometry Imaging.
Quantitative analysis of primaquine and its metabolites in human urine using liquid chromatography coupled with tandem mass spectrometry.
Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection.
Determination of Genotoxic Impurity N-Nitroso-N-methyl-4-aminobutyric Acid in Four Sartan Substances through Using Liquid Chromatography-Tandem Mass Spectrometry.
Congruence and Complementarity of Differential Mobility Spectrometry and NMR Spectroscopy for Plasma Lipidomics.
Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC-MS/MS analysis.
Online Pressure Change Focusing-Supercritical Fluid Selective Extraction Chromatography for Analyzing Chiral Drugs in Microliter-Scale Plasma Samples.
A Sensitive Ultrahigh-Performance Liquid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Analysis of Phytocannabinoids and Endocannabinoids in Plasma and Brain.
Comparison of LC-MS3 and LC-MRM Methods for Quantifying Amantadine and Its Application in Therapeutic Amantadine Monitoring in Human Plasma.
Boronate affinity paper spray mass spectrometry for determination of elevated levels of catecholamines in urine.
IR-MALDI Mass Spectrometry Imaging with Plasma Post-Ionization of Nonpolar Metabolites.
NMR-Based Chromatography Readouts: Indispensable Tools to "Translate" Analytical Features into Molecular Structures.
Space and Time Coherent Mapping for Subcellular Resolution of Imaging Mass Spectrometry.
Development of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Hydroxylated Polychlorinated Biphenyls in Animal-Derived Food.
Influence of data acquisition modes and data analysis approaches on non-targeted analysis of phthalate metabolites in human urine.
Easily Operable Quantification Method of 21 Plant-Derived Alkaloids in Human Serum by Automatic Sample Preparation and Liquid Chromatography-Tandem Mass Spectrometry.
A robust LC-MS/MS method to measure 8-oxoGuo, 8-oxodG, and NMN in human serum and urine.

Int J Mol Sci. 2022 Nov 03. pii: 13486. [Epub ahead of print]23(21):

A Concise Review of Liquid Chromatography-Mass Spectrometry-Based Quantification Methods for Short Chain Fatty Acids as Endogenous Biomarkers.

Neerja Trivedi, Helen E Erickson, Veenu Bala, Yashpal S Chhonker, Daryl J Murry.

  Fatty acids are widespread naturally occurring compounds, and essential constituents for living organisms. Short chain fatty acids (SCFAs) appeared as physiologically relevant metabolites for their involvement with gut microbiota, immunology, obesity, and other pathophysiological functions. This has raised the demand for reliable analytical detection methods in a variety of biological matrices. Here, we describe an updated overview of sample pretreatment techniques and liquid chromatography-mass spectrometry (LC-MS)-based methods for quantitative analysis of SCFAs in blood, plasma, serum, urine, feces and bacterial cultures. The present review incorporates various procedures and their applications to help researchers in choosing crucial parameters, such as pretreatment for complex biological matrices, and variables for chromatographic separation and detection, to establish a simple, sensitive, and robust quantitative method to advance our understanding of the role of SCFAs in human health and disease as potential biomarkers.

Keywords:  LC-MS/MS; biological samples; biomarkers; short chain fatty acids

DOI:  https://doi.org/10.3390/ijms232113486
Anal Bioanal Chem. 2022 Nov 12.

Sample multiplexing for increasing throughput for quantification of estrogens in serum by LC-MS/MS.

Tengfei Yuan.

  Estrogens are involved in many physiological processes in vivo. The accurate and rapid quantification of estrogens is required for the diagnosis and prognosis of estrogen-related diseases. To achieve high-volume assays, we developed and validated a sample-multiplexing liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of serum estrogens including estrone (E1), estradiol (E2), and estriol (E3). A total of 100 μL serum samples were extracted using ethyl acetate. After derivatization with either dansyl chloride or pyridine-3-sulfonyl chloride, derivatized samples were combined. Then we performed the second liquid-liquid extraction using hexane to purify the mixture. Finally, the reconstitution solutions were injected into LC-MS/MS. In addition, the proposed LC-MS/MS method was validated according to FDA and CLSI guidelines. Within a single run (7 min), this sample-multiplexing LC-MS/MS method could simultaneously analyze E1, E2, and E3 in 2 serum samples. Meanwhile, the method demonstrated satisfactory analytical characteristics including accuracy (87.7-110.3%), linearity (2-1000 pg/mL, R2 > 0.99), precision (intra-assay CV, 1.7-8.7%; inter-assay CV, 1.9-9.4%), and negligible interference and carry-over effect as well as acceptable matrix effect. In conclusion, this sample-multiplexing LC-MS/MS method has achieved a doubled-throughput assay for simultaneous quantification of E1, E2, and E3 without compromising analytical characteristics.

Keywords:  Derivatization; Estrogen; LC–MS/MS; Sample multiplexing

DOI:  https://doi.org/10.1007/s00216-022-04423-7
Curr Opin Chem Biol. 2022 Nov 05. pii: S1367-5931(22)00111-9. [Epub ahead of print]71 102226

Recent progress in mass spectrometry for single-cell metabolomics.

Xingyu Pan, Huan Yao, Sichun Zhang, Xinrong Zhang.

  Metabolites are the end products of cellular vital activities and can reflect the state of cellular to a certain extent. Rapid change of metabolites and the low abundance of signature metabolites cause difficulties in single-cell detection, which is a great challenge in single-cell metabolomics analysis. Mass spectrometry (MS) is a powerful tool that uniquely suited to detect intracellular small-molecule metabolites and has shown good application in single-cell metabolite analysis. In this mini-review, we describe three types of emerging technologies for MS-based single-cell metabolic analysis in recent years, including nano-ESI-MS based single-cell metabolomics analysis, high-throughput analysis via flow cytometry, and cellular metabolic imaging analysis. These techniques provide a large amount of single-cell metabolic data, allowing the potential of MS in single-cell metabolic analysis is gradually being explored and is of great importance in disease and life science research.

Keywords:  Cytometry; ESI; MADLI; Mass Spectrometry; Nano-ESI; SIMS; Single-cell metabolomics

DOI:  https://doi.org/10.1016/j.cbpa.2022.102226
J Proteome Res. 2022 Nov 11.

Alignment and Analysis of a Disparately Acquired Multibatch Metabolomics Study of Maternal Pregnancy Samples.

Hani Habra, Maureen Kachman, Vasantha Padmanabhan, Charles Burant, Alla Karnovsky, Jennifer Meijer.

  Untargeted liquid chromatography-mass spectrometry metabolomics studies are typically performed under roughly identical experimental settings. Measurements acquired with different LC-MS protocols or following extended time intervals harbor significant variation in retention times and spectral abundances due to altered chromatographic, spectrometric, and other factors, raising many data analysis challenges. We developed a computational workflow for merging and harmonizing metabolomics data acquired under disparate LC-MS conditions. Plasma metabolite profiles were collected from two sets of maternal subjects three years apart using distinct instruments and LC-MS procedures. Metabolomics features were aligned using metabCombiner to generate lists of compounds detected across all experimental batches. We applied data set-specific normalization methods to remove interbatch and interexperimental variation in spectral intensities, enabling statistical analysis on the assembled data matrix. Bioinformatics analyses revealed large-scale metabolic changes in maternal plasma between the first and third trimesters of pregnancy and between maternal plasma and umbilical cord blood. We observed increases in steroid hormones and free fatty acids from the first trimester to term of gestation, along with decreases in amino acids coupled to increased levels in cord blood. This work demonstrates the viability of integrating nonidentically acquired LC-MS metabolomics data and its utility in unconventional metabolomics study designs.

Keywords:  LC-HRMS; alignment; metabolomics; normalization; partial correlation; plasma; pregnancy

DOI:  https://doi.org/10.1021/acs.jproteome.2c00371
Anal Chim Acta. 2022 Dec 01. pii: S0003-2670(22)01092-3. [Epub ahead of print]1235 340521

An automated online three-phase electro-extraction setup with machine-vision process monitoring hyphenated to LC-MS analysis.

Yupeng He, Paul Miggiels, Nicolas Drouin, Peter W Lindenburg, Bert Wouters, Thomas Hankemeier.

  Sample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample preparation should ideally be hyphenated to downstream on-line analysis such as liquid chromatography-mass spectrometry (LC-MS). In this study, an automated three-phase electro-extraction (EE) method coupled to machine vision was developed, integrated with a robotic autosampler hyphenated to LC-MS. Eight model compounds, i.e. amitriptyline, clemastine, clomipramine, haloperidol, loperamide, propranolol, oxeladin, and verapamil were utilized for the optimization and evaluation of the automated EE setup. The stability of automated EE was evaluated by monitoring the acceptor droplet size by machine vision and recording the current during EE. A Design of Experiment approach (Box-Behnken design) was utilized to optimize the critical parameters of the EE method, i.e., the ratio of formic acid in the sample to acceptor phase, extraction voltage, and extraction time. The developed quadratic models showed good fitness (p < 0.001, R2 > 0.95). Automated EE could be achieved in less than 2 min with enrichment factors (EF) up to 387 and extraction recoveries (ER) up to 97% for academic samples. Finally, the optimized EE method was successfully applied to both spiked human urine and plasma samples with low-concentration (50 ng mL-1) analytes and a low starting sample volume of 20 μL of plasma and urine in 10-fold diluted samples. The developed automated EE setup is easy to operate, provides a fast extraction method for analytes from volume-limited biological samples, and is hyphenated with on-line LC-MS analysis. Therefore, this method can provide fast and automated sample preparation to solve bottlenecks in high-throughput bioanalysis workflows.

Keywords:  Automation; Bioanalysis; Electro-extraction; Machine vision; Sample preparation

DOI:  https://doi.org/10.1016/j.aca.2022.340521
Anal Bioanal Chem. 2022 Nov 12.

Development and validation of a simple and rapid HILIC-MS/MS method for the quantification of low-abundant lysoglycerophospholipids in human plasma.

Haonan Li, Yiqun Han, Teng Wang, Wu Chen, Yifan Xu, Ke Gao, Yanwen Wang, Jicheng Gong, Weiju Li, Hongyin Zhang, Junxia Wang, Xinghua Qiu, Tong Zhu.

  Lysoglycerophospholipids (Lyso-GPLs) are an essential class of signaling lipids with potential roles in human diseases, such as cancer, central nervous system diseases, and atherosclerosis. Current methods for the quantification of Lyso-GPLs involve complex sample pretreatment, long analysis times, and insufficient validation, which hinder the research of Lyso-GPLs in human studies, especially for Lyso-GPLs with low abundance in human plasma such as lysophosphatidic acid (LPA), lysophosphatidylinositol (LPI), lysophosphatidylglycerol (LPG), lysophosphatidylserine (LysoPS), lyso-platelet-activating factor (LysoPAF), and cyclic phosphatidic acid (cPA). Herein, we report the development and validation of a simple and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of Lyso-GPLs with low abundance in plasma. Protein precipitation using MeOH for Lyso-GPL extraction, quick separation (within 18 min) based on hydrophilic interaction liquid chromatography (HILIC), and sensitive MS detection under dynamic multiple reaction monitoring (dMRM) mode enabled efficient quantification of 22 Lyso-GPLs including 2 cPA, 4 LPG, 11 LPA, 2 LysoPS, and 3 LysoPAF in 50 μL of human plasma. The present method showed good linearity (goodness of fit, 0.99823-0.99995), sensitivity (lower limit of quantification, 0.03-14.06 ng/mL), accuracy (73-117%), precision (coefficient of variation ≤ 28%), carryover (≤ 17%), recovery (80-110%), and stability (83-123%). We applied the method in an epidemiological study and report concentrations of 18 Lyso-GPLs in 567 human plasma samples comparable to those of previous studies. Significant negative associations of LysoPAF C18, LysoPAF C18:1, and LysoPAF C16 with homeostatic model assessment for insulin resistance (HOMA-IR) level were observed; this indicates possible roles of LysoPAF in glucose homeostasis. The application of the present method will improve understanding of the roles of circulating low-abundant Lyso-GPLs in health and diseases.

Keywords:  Diabetes; High-throughput; Lipidomics; Lysophospholipids

DOI:  https://doi.org/10.1007/s00216-022-04421-9
Anal Chem. 2022 Nov 08.

Molecular Networking and On-Tissue Chemical Derivatization for Enhanced Identification and Visualization of Steroid Glycosides by MALDI Mass Spectrometry Imaging.

Domenic Dreisbach, Sven Heiles, Dhaka R Bhandari, Georg Petschenka, Bernhard Spengler.

Spatial metabolomics describes the spatially resolved analysis of interconnected pathways, biochemical reactions, and transport processes of small molecules in the spatial context of tissues and cells. However, a broad range of metabolite classes (e.g., steroids) show low intrinsic ionization efficiencies in mass spectrometry imaging (MSI) experiments, thus restricting the spatial characterization of metabolic networks. Additionally, decomposing complex metabolite networks into chemical compound classes and molecular annotations remains a major bottleneck due to the absence of repository-scaled databases. Here, we describe a multimodal mass-spectrometry-based method combining computational metabolome mining tools and high-resolution on-tissue chemical derivatization (OTCD) MSI for the spatially resolved analysis of metabolic networks at the low micrometer scale. Applied to plant toxin sequestration in Danaus plexippus as a model system, we first utilized liquid chromatography (LC)-MS-based molecular networking in combination with artificial intelligence (AI)-driven chemical characterization to facilitate the structural elucidation and molecular identification of 32 different steroidal glycosides for the host-plant Asclepias curassavica. These comprehensive metabolite annotations guided the subsequent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) analysis of cardiac-glycoside sequestration in D. plexippus. We developed a spatial-context-preserving OTCD protocol, which improved cardiac glycoside ion yields by at least 1 order of magnitude compared to results with untreated samples. To illustrate the potential of this method, we visualized previously inaccessible (sub)cellular distributions (2 and 5 μm pixel size) of steroidal glycosides in D. plexippus, thereby providing a novel insight into the sequestration of toxic metabolites and guiding future metabolomics research of other complex sample systems.

DOI: https://doi.org/10.1021/acs.analchem.2c02694
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 01. pii: S1570-0232(22)00422-6. [Epub ahead of print]1213 123517

Quantitative analysis of primaquine and its metabolites in human urine using liquid chromatography coupled with tandem mass spectrometry.

Washim Khan, Yan-Hong Wang, N P Dhammika Nanayakkara, H M T Bandara Herath, Narayan D Chaurasiya, Babu L Tekwani, Mahmoud A ElSohly, James D McChesney, Ikhlas A Khan, Larry A Walker.

  Primaquine (PQ), a prototype 8-aminoquinoline (8-AQ) drug used to treat malaria, is rapidly metabolized into different inactive and active metabolites. Due to the hemolytic toxicity, the uses of PQ have been confined. To understand its overall metabolism and its relation to drug efficacy and toxicity, profiling of urine for the parent drug and its metabolites is important. The current study presents a convenient and rapid method for simultaneously quantifying primaquine (PQ) and its metabolites in human urine. A simple liquid-liquid extraction followed by chromatographic separation and quantification through ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated to quantify PQ and its eleven metabolites in the urine of healthy human volunteers who received a single oral dose of PQ. The developed method separated fourteen analytes, including internal standards, within nine minutes of run time. The linearity of all analytes was suitable in the range of 1-500 ng/mL. The extraction recovery for all concentrations of analytes from urine was ranged from 90.1 to 112.9 %. The relative standard deviation for intra- and inter-day precision were < 9.8 and < 10.7 %, respectively. Along with PQ, its different metabolites were detected in urine. Primaquine-5,6-orthoquinone, the N-carbamoylglucuronide conjugate of PQ and carboxyprimaquine were the major metabolites found in urine. Significant enantiomeric differences in the urinary excretion profiles for PQ and metabolites were observed. This analytical method can be implemented in the pharmacokinetic analysis of PQ to explain its toxicity and clinical decision making.

Keywords:  PQ metabolites; Pharmacokinetic; Primaquine; UPLC-MS/MS; Urine analysis

DOI:  https://doi.org/10.1016/j.jchromb.2022.123517
J Chromatogr A. 2022 Nov 01. pii: S0021-9673(22)00823-8. [Epub ahead of print]1685 463632

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection.

Xiao Gu, Lishi Yang, Qiaofeng Tao, Jie Ai, Chenxiao Yan, Jinqi Zheng, Liya Hong.

  The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Keywords:  Calcium gluconate injection; Heart-cutting 2D-LC; Highly polar impurity; IPRP-HILIC-MS; Make-up flow module

DOI:  https://doi.org/10.1016/j.chroma.2022.463632
Molecules. 2022 Nov 03. pii: 7498. [Epub ahead of print]27(21):

Determination of Genotoxic Impurity N-Nitroso-N-methyl-4-aminobutyric Acid in Four Sartan Substances through Using Liquid Chromatography-Tandem Mass Spectrometry.

Bin Xie, Dong Guo, Binliang Mai, Jun Fan.

  N-nitroso-N-methyl-4-aminobutyric acid (NMBA) is the third N-nitrosamine impurity found in sartans. Herein, a sensitive and stable LC-MS/MS method with multiple reactions monitoring mode has been developed for the quantitative determination of NMBA in four sartan substances. The effective separation of NMBA and sartan substances was achieved on a C18 column under gradient elution conditions. The mass spectrometry method of the atmospheric pressure chemical ionization source and internal standard method was selected as the quantitative analysis method of NMBA. Then, this proposed LC-MS/MS analysis method was validated in terms of specificity, sensitivity, linearity, accuracy, precision and stability. Good linearity with correlation coefficient over 0.99 was obtained at the NMBA concentration of 3-45 ng/mL, and the limit of quantification was 3 ng/mL. Additionally, the recoveries of NMBA in four sartan substances ranged from 89.9% to 115.7%. The intra-day and inter-day relative standard deviation values were less than 5.0%. In conclusion, this developed determination method for NMBA through liquid chromatography-tandem mass spectrometry showed the characteristics of good sensitivity, high accuracy and precision, which will be of great help for the quantitative analysis of NMBA in sartan products.

Keywords:  LC-MS/MS quantitative determination; N-nitrosamine genotoxic impurity; N-nitroso-N-methyl-4-aminobutyric acid; multiple reactions monitoring; sartan substance

DOI:  https://doi.org/10.3390/molecules27217498
Metabolites. 2022 Oct 27. pii: 1030. [Epub ahead of print]12(11):

Congruence and Complementarity of Differential Mobility Spectrometry and NMR Spectroscopy for Plasma Lipidomics.

Mohan Ghorasaini, Konstantina Ismini Tsezou, Aswin Verhoeven, Yassene Mohammed, Panayiotis Vlachoyiannopoulos, Emmanuel Mikros, Martin Giera.

  The lipid composition of lipoprotein particles is determinative of their respective formation and function. In turn, the combination and correlation of nuclear magnetic resonance (NMR)-based lipoprotein measurements with mass spectrometry (MS)-based lipidomics is an appealing technological combination for a better understanding of lipid metabolism in health and disease. Here, we developed a combined workflow for subsequent NMR- and MS-based analysis on single sample aliquots of human plasma. We evaluated the quantitative agreement of the two platforms for lipid quantification and benchmarked our combined workflow. We investigated the congruence and complementarity between the platforms in order to facilitate a better understanding of patho-physiological lipoprotein and lipid alterations. We evaluated the correlation and agreement between the platforms. Next, we compared lipid class concentrations between healthy controls and rheumatoid arthritis patient samples to investigate the consensus among the platforms on differentiating the two groups. Finally, we performed correlation analysis between all measured lipoprotein particles and lipid species. We found excellent agreement and correlation (r > 0.8) between the platforms and their respective diagnostic performance. Additionally, we generated correlation maps detailing lipoprotein/lipid interactions and describe disease-relevant correlations.

Keywords:  differential mobility spectrometry; lipid; lipidomics; lipoprotein; mass spectrometry; nuclear magnetic resonance

DOI:  https://doi.org/10.3390/metabo12111030
Anal Bioanal Chem. 2022 Nov 07.

Stability of sample extracts of vitamin D3 metabolites after chemical derivatization for LC-MS/MS analysis.

Anastasia Alexandridou, Dietrich A Volmer.

  Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is widely used to determine vitamin D3 metabolites in biological samples. The ionization efficiencies of these metabolites, however, are poor under electrospray ionization conditions. Moreover, the chromatographic separation of multiple vitamin D metabolites and their epimers can be challenging. For these reasons, chemical derivatization reagents are often used to improve sensitivity and selectivity of analysis. While the derivatization schemes have been proven to be very effective, one missing aspect is the investigation of the stability of the chemical derivatization products in stored sample extracts. In this study, we investigated the long-term stability of several vitamin D3 metabolites after 1 and 3 months of storage at - 20 °C. Five vitamin D3 metabolites were examined after derivatization with seven different derivatization reagents. Generally, Amplifex products were the most stable in the long term in our study with 11-20% degraded after 1 month of storage and 14-35% after 3 months. The stabilities for some of the metabolites' 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyl]-1,2,4-triazoline-3,5-dione (DMEQ-TAD), 2-fluoro-1-methylpyridinium p-toluenesulfonate (FMP-TS), isonicotinoyl chloride (INC) and 4-phenyl-1,2,4-triazoline-3,5-dione acetylated (PTAD-Ac) products were also acceptable after 1 month of storage. Other derivatized metabolites, however, degraded extensively already after 1 month of storage, such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) (54-72% degradation) and 2-nitrosopyridine (PyrNO) (32-100% degradation). Importantly, for every metabolite, there was an optimum derivatization reagent that met the criteria of stability proposed by international regulatory bodies after 1 month of storage. Some derivatives were stable for even up to 3 months of storage, with degradation of less than 15%.

Keywords:  25-Hydroxyvitamin D3; Chemical derivatization; LC–MS/MS; Stability; Vitamin D3 metabolites

DOI:  https://doi.org/10.1007/s00216-022-04409-5
Anal Chem. 2022 Nov 10.

Online Pressure Change Focusing-Supercritical Fluid Selective Extraction Chromatography for Analyzing Chiral Drugs in Microliter-Scale Plasma Samples.

Jieqing Feng, Qisheng Zhong, Ting Zhou.

The online coupling technique of sample preparation with chromatography is a frontier topic in analytical chemistry since it minimizes errors caused by sample loss, shortens analysis time, and reduces solvent consumption. An online pressure change focusing-supercritical fluid selective extraction chromatography (PCF-SFSEC) technique was developed in this study, realizing extraction, purification, separation, and detection in a single run with only microliter-scale samples. The pressure change focusing strategy achieved column-head stacking by decreasing the dissolving capacity of the supercritical fluid, enabling the large volume introduction of extractants into supercritical fluid chromatography without causing peak broadening or distortion. All the extracts could be directly loaded into the chromatography system without split flow. Based on the supercritical fluid selective extraction (SFSE) strategy, the sorbents removed interferences and water from samples, effectively alleviating matrix effects and realizing the direct aqueous sample analysis. The efficiency of online PCF-SFSEC was demonstrated by the enantioselective analysis of 22 chiral drugs in rat plasma, covering eight categories with different pharmacological effects. The entire analysis took 25 min, consuming only 5 μL samples. All analytes in PCF-SFSEC obtained sharp and symmetrical peaks with resolutions higher than 1.0, and 86% had resolutions higher than 1.5. Limits of quantification (LOQs) ranged from 0.0600 to 32.1 μg/L. Recoveries were in the range of 75.8-117.2%. In addition, the developed approach obtained more satisfactory repeatability and significantly reduced matrix effects than conventional methods. The newly established online PCF-SFSEC technique is believed to be a green and powerful tool for the chiral analysis of complex samples.

DOI: https://doi.org/10.1021/acs.analchem.2c03892
Cannabis Cannabinoid Res. 2022 Nov 11.

A Sensitive Ultrahigh-Performance Liquid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Analysis of Phytocannabinoids and Endocannabinoids in Plasma and Brain.

Faizy Ahmed, Alexa Torrens, Stephen V Mahler, Francesca Ferlenghi, Marilyn A Huestis, Daniele Piomelli.

  Introduction: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are major chemical constituents of cannabis, which may interact either directly or indirectly with the endocannabinoid and endocannabinoid-like ("paracannabinoid") systems, two lipid-based signaling complexes that play important roles in physiology. Legislative changes emphasize the need to understand how THC and CBD might impact endocannabinoid and paracannabinoid signaling, and to develop analytical approaches to study such impact. In this study, we describe a sensitive and accurate method for the simultaneous quantification of THC, its main oxidative metabolites [11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)], CBD, and a representative set of endocannabinoid [anandamide and 2-arachidonoyl-sn-glycerol (2-AG)] and paracannabinoid [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)] compounds. Analyte separation relies on the temperature-dependent shape selectivity properties of polymerically bonded C18 stationary phases. Materials and Methods: Analytes are extracted from tissues using acetonitrile precipitation followed by phospholipid removal. The ultrahigh-performance liquid chromatography/tandem mass spectrometry protocol utilizes a commercially available C18 polymeric-bonded phase column and a simple gradient elution system. Results: Ten-point calibration curves show excellent linearity (R2>0.99) over a wide range of analyte concentrations (0.02-500 ng/mL). Lowest limits of quantification are 0.05 ng/mL for anandamide, 0.1 ng/mL for 11-OH-THC and OEA, 0.2 ng/mL for THC and CBD, 0.5 ng/mL for 11-COOH-THC, 1.0 ng/mL for 2-AG, and 2.0 ng/mL for PEA. The lowest limits of detection are 0.02 ng/mL for anandamide, 0.05 ng/mL for 11-OH-THC and OEA, 0.1 ng/mL for THC and CBD, 0.2 ng/mL for 11-COOH-THC, 0.5 ng/mL for 2-AG, and 1.0 ng/mL for PEA. Conclusions: An application of the method is presented, which showed that phytocannabinoid administration elevates endocannabinoid levels in plasma and brain of adolescent male and female mice.

Keywords:  2-arachidonoyl-sn-glycerol; anandamide; cannabidiol; endocannabinoid; shape selectivity; ultrahigh-performance liquid chromatography/tandem mass spectrometry; Δ9-tetrahydrocannabinol

DOI:  https://doi.org/10.1089/can.2022.0216
Molecules. 2022 Nov 07. pii: 7619. [Epub ahead of print]27(21):

Comparison of LC-MS3 and LC-MRM Methods for Quantifying Amantadine and Its Application in Therapeutic Amantadine Monitoring in Human Plasma.

Qiang Sun, Haiwei Cao, Yong Liu, Yanyan Li, Jing Huang.

  A simple sample preprocessing method was developed for the quantitative determination of amantadine (AMT) in human plasma by liquid chromatography-tandem mass spectrometry cubed (LC-MS3). The LC-MS3 system comprised a Shimadzu Exion LC-20AD HPLC pump coupled with a QTRAP 5500 mass spectrometer. First, the plasma samples were pretreated using acetonitrile as the extracting solution to precipitate protein. Next, amantadine and amantadine-d15 (AMT-d15) were separated on an Agilent Poroshell 120 SB-C18 column (4.6 mm × 50 mm, 2.7 μm) using isocratic elution with solvent A (70% 0.1% formic acid) and solvent B (30% acetonitrile) at a flow rate of 0.8 mL/min. The total run time for each sample was 3 min. The system used triple-stage fragmentation transitions at m/z 152.2→135.3→107.4 for AMT quantification in the positive ion mode and m/z 167.0→150.3→118.1 for AMT-d15 quantification. The LC-MS3 assay was linear (r > 0.995) with a concentration range of 50-1500 ng/mL. The lower limit of quantification (LLOQ) was 50 ng/mL, and the intra-day and inter-day accuracies and precisions were less than 8.0% at all concentrations. In addition, the recoveries and matrix effect for AMT in human plasma were within acceptable limits. In terms of stability, AMT had no significant degradation under all conditions. All the results met the requirements of the guidelines of the Food and Drug Administration (FDA) for biological method validation. The novelty of the MS3 assay was that it presented a methodology with higher selectivity and sensitivity. This method was successfully applied to 44 human plasma samples, and the obtained quantitative results were compared with another liquid chromatography-multiple reaction monitoring (LC-MRM) method. The Passing-Bablok regression coefficients and Bland-Altman plot revealed no difference between the LC-MS3 and LC-MRM methods, implying that the developed LC-MS3 method is a reliable and accurate assay for AMT determination in human plasma. These results are also a proof of concept for determining chemicals in biological samples by the LC-MS3 strategy.

Keywords:  LC-MS3; amantadine; therapeutic drug monitoring

DOI:  https://doi.org/10.3390/molecules27217619
Anal Chim Acta. 2022 Dec 01. pii: S0003-2670(22)01079-0. [Epub ahead of print]1235 340508

Boronate affinity paper spray mass spectrometry for determination of elevated levels of catecholamines in urine.

Wei Luo, Teris A van Beek, Bo Chen, Han Zuilhof, Gert Ij Salentijn.

  The analysis of catecholamines, such as dopamine, epinephrine and norepinephrine in urine can be used in the diagnosis of certain pathologies, such as hormone-producing tumors. Here, a fast and simple quantitative boronate affinity paper spray tandem mass spectrometric (PS-MS/MS) method is established, which can improve selectivity and reduce ion suppression without needing any instrumental chromatography. We use here the property of boronic acids, which can selectively bind ortho-diol-containing compounds under alkaline conditions. Paper tip modification and catechol enrichment protocols were developed to selectively bind, clean up and subsequently desorb such catecholamines. Standard catecholamine solutions, as well as human urine samples were analyzed with the PS-MS(/MS) method, which is fast, cheap and easy-to-operate compared to HPLC-MS/MS. Despite its high simplicity, boronate affinity PS-MS/MS exhibits good performance compared to HPLC-MS/MS in human urine analysis in terms of precision (2.1%-7.2% vs. 1.1%-2.9%) and accuracy (-10.2%-9.3% vs. -4.8%-5.1%), and a physiologically relevant limit of detection (0.027-0.12 μg mL-1). The boronate affinity PS-MS/MS clearly achieved the detection limits that would allow the fast analysis of urine samples for clinical purposes, such as screening for pheochromocytoma (exceeding 0.5 μg mL-1).

Keywords:  Boronate affinity; Catecholamines; Paper modification; Paper spray mass spectrometry; Selective enrichment; Urine analysis

DOI:  https://doi.org/10.1016/j.aca.2022.340508
Anal Chem. 2022 Nov 10.

IR-MALDI Mass Spectrometry Imaging with Plasma Post-Ionization of Nonpolar Metabolites.

Julian Schneemann, Karl-Christian Schäfer, Bernhard Spengler, Sven Heiles.

Ambient mass spectrometry imaging (MSI) methods come with the advantage of visualizing biomolecules from tissues with no or minimal sample preparation and operation under atmospheric-pressure conditions. Similar to all other MSI methodologies, however, ambient MSI modalities suffer from a pronounced bias toward either polar or nonpolar analytes due to the underlying desorption and ionization mechanisms of the ion source. In this study, we present the design, construction, testing, and application of an in-capillary dielectric barrier discharge (DBD) module for post-ionization of neutrals desorbed by an ambient infrared matrix-assisted laser desorption/ionization (IR-MALDI) MSI source. We demonstrate that the DBD device enhances signal intensities of nonpolar compounds by up to 104 compared to IR-MALDI without affecting transmission of IR-MALDI ions. This allows performing MSI experiments of mouse tissue and Danaus plexippus caterpillar tissue sections, visualizing the distribution of sterols, fatty acids, monoglycerides, and diglycerides that are not detected in IR-MALDI MSI experiments. The pronounced signal enhancement due to IR-MALDI-DBD compared to IR-MALDI MSI enables mapping of nonpolar analytes with pixel resolutions down to 20 μm in mouse brain tissue and to discern the spatial distribution of sterol lipids characteristic for histological regions of D. plexippus.

DOI: https://doi.org/10.1021/acs.analchem.2c03247
Cells. 2022 Nov 07. pii: 3526. [Epub ahead of print]11(21):

NMR-Based Chromatography Readouts: Indispensable Tools to "Translate" Analytical Features into Molecular Structures.

Christoph Seger, Sonja Sturm.

  Gaining structural information is a must to allow the unequivocal structural characterization of analytes from natural sources. In liquid state, NMR spectroscopy is almost the only possible alternative to HPLC-MS and hyphenating the effluent of an analyte separation device to the probe head of an NMR spectrometer has therefore been pursued for more than three decades. The purpose of this review article was to demonstrate that, while it is possible to use mass spectrometry and similar methods to differentiate, group, and often assign the differentiating variables to entities that can be recognized as single molecules, the structural characterization of these putative biomarkers usually requires the use of NMR spectroscopy.

Keywords:  HPLC; HPLC-NMR; HPLC-SPE-NMR; HPLC–MS/MS; NMR spectroscopy; capNMR; liquid chromatography; mass spectrometry; metabolomics; structure elucidation

DOI:  https://doi.org/10.3390/cells11213526
Cells. 2022 Oct 26. pii: 3382. [Epub ahead of print]11(21):

Space and Time Coherent Mapping for Subcellular Resolution of Imaging Mass Spectrometry.

Jun Aoki, Masako Isokawa, Michisato Toyoda.

  Space and time coherent mapping (STCM) is a technology developed in our laboratory for improved matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) imaging mass spectrometry (IMS). STCM excels in high spatial resolutions, which probe-based scanning methods cannot attain in conventional MALDI IMS. By replacing a scanning probe with a large field laser beam, focusing ion optics, and position-sensitive detectors, STCM tracks the entire flight trajectories of individual ions throughout the ionization process and visualizes the ionization site on the sample surface with a subcellular scale of precision and a substantially short acquisition time. Results obtained in thinly sectioned leech segmental ganglia and epididymis demonstrate that STCM IMS is highly suited for (1) imaging bioactive lipid messengers such as endocannabinoids and the mediators of neuronal activities in situ with spatial resolution sufficient to detail subcellular localization, (2) integrating resultant images in mass spectrometry to optically defined cell anatomy, and (3) assembling a stack of ion maps derived from mass spectra for cluster analysis. We propose that STCM IMS is the choice over a probe-based scanning mass spectrometer for high-resolution single-cell molecular imaging.

Keywords:  Hirudo Verbana; cluster analysis; endocannabinoid; epididymis; microscope-mode IMS; neuron; silver nanoparticle PALDI

DOI:  https://doi.org/10.3390/cells11213382
Molecules. 2022 Oct 25. pii: 7247. [Epub ahead of print]27(21):

Development of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Hydroxylated Polychlorinated Biphenyls in Animal-Derived Food.

Wenyu Zhao, Na Cui, Yuan Huang, Zihao Wang, Peilong Wang, Haijun Liang, Ruiguo Wang, Xiaoou Su.

  Hydroxylated polychlorinated biphenyls (OH-PCBs) are a group of metabolites biotransformed from polychlorinated biphenyls by animals with higher toxicities than their parent compounds. The present work developed and validated an analytical method for determinating penta-, hexa-, and hepta-chlorine substituted OH-PCBs in animal-derived food based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with isotope-dilution. The target analytes were extracted with a 50% n-hexane/dichloromethane (v/v), purified by sulfuric acid-silica gel, and separated by 5% hydrated silica gel, achieving a final concentration of 100 times before injection to LC-MS/MS. The limits of detection (LOD) and quantification (LOQ) for target OH-PCBs were within the ranges of 0.003-0.010 μg/kg and 0.009-0.030 μg/kg, respectively. Average recoveries ranged between 76.7% and 116.5%, with relative standard deviations of less than 18.4%. The proposed method is simple, time-saving, sensitive, and accurate, making it a powerful tool for risk monitoring of OH-PCBs in animal-derived food.

Keywords:  UPLC–MS/MS; determination; food; hydroxylated polychlorinated biphenyls; metabolite

DOI:  https://doi.org/10.3390/molecules27217247
Anal Bioanal Chem. 2022 Nov 08.

Influence of data acquisition modes and data analysis approaches on non-targeted analysis of phthalate metabolites in human urine.

Yong-Lai Feng, Anca Baesu.

  Humans are often exposed to phthalates and their alternatives, on account of their widespread use in PVC as plasticizers, which are associated with harmful human effects. While targeted biomonitoring provides quantitative information for exposure assessment, only a small portion of phthalate metabolites has been targeted. This results in a knowledge gap in human exposure to other unknown phthalate compounds and their metabolites. Although the non-targeted analysis (NTA) approach is capable of screening a broad spectrum of chemicals, there is a lack of harmonized workflow in NTA to generate reproducible data within and between different laboratories. The objective of this study was to compare two different NTA data acquisition modes, the data-dependent (DDA) and independent (DIA) acquisition (DDA), as well as two data analysis approaches, based on diagnostic ions and Compound Discoverer software for the prioritization of candidate precursors and identification of unknown compounds in human urine. Liquid chromatography coupled to high-resolution mass spectrometry was used for sample analysis. The combination of three-diagnostic-ion extraction and DDA data acquisition was able to improve data filtering and data analysis for prioritizing phthalate metabolites. With DIA, 25 molecular features were identified in human urine, while 32 molecular features were identified in the same urine samples using DDA data. The number of molecular features identified with level 1 confidence was 11 and 9 using DIA and DDA data, respectively. The study demonstrated that besides sample preparation, the impact of data acquisition must be taken into account when developing a NTA method and a consistent protocol for evaluating such an impact is necessary.

Keywords:  Data acquisition mode; Data analysis approach; Human urine; Non-targeted analysis (NTA); Phthalate metabolites

DOI:  https://doi.org/10.1007/s00216-022-04407-7
Chromatographia. 2022 Oct 29. 1-13

Easily Operable Quantification Method of 21 Plant-Derived Alkaloids in Human Serum by Automatic Sample Preparation and Liquid Chromatography-Tandem Mass Spectrometry.

Masaru Taniguchi, Naoki Takamura, Tsutomu Watanabe, Reiko Ishimaru, Satoshi Chinaka, Akihiro Miki, Hitoshi Miyazaki, Hitoshi Tsuchihashi, Kei Zaitsu.

  In this study, we developed an easily operable quantification method for 21 plant-derived alkaloids in human serum by automatic sample preparation and liquid chromatography-tandem mass spectrometry. We designed to perform parallel sample preparation by a developed apparatus, which increased sample throughput. We conducted an automatic sample preparation through de-proteinization with 0.1% formic acid in methanol and achieved recovery rates of 89-107% (2.0-14% RSD) for all targeted analytes, demonstrating its high repeatability. The method validation results were satisfactory as follows: the linearity (r 2) of each calibration curve ranged from 0.978 to 1.000; the inter- and intra-day accuracies were 89.0-125% and 82.1-110%, respectively; the inter- and intra-day precisions were below 13% and 10%, respectively. Additionally, the lower limits of detection and quantification were 0.0044-0.047 and 0.013-0.14 ng/mL, respectively. Finally, the developed method was applied to pseudo-protoveratrine A poisoning serum and pseudo-colchicine poisoning serum, which were prepared by diluting acute-poisoning mice serum with human serum. Our method successfully quantitated protoveratrine A (0.15-0.25 ng/mL) and colchicine (4.8-6.0 ng/mL). Thus, our method is essential for prompt clinical treatment and critical care on patient in acute intoxication cases caused by plant-derived alkaloids.
Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04212-5.

Keywords:  Automatic sample preparation; Food poisoning; Human serum; LC/MS/MS; Plant-derived alkaloids; Quantification

DOI:  https://doi.org/10.1007/s10337-022-04212-5
Anal Biochem. 2022 Oct 29. pii: S0003-2697(22)00430-4. [Epub ahead of print]660 114970

A robust LC-MS/MS method to measure 8-oxoGuo, 8-oxodG, and NMN in human serum and urine.

Yutong Zou, Xiaoli Ma, Yueming Tang, Liling Lin, Jialei Yu, Jian Zhong, Danchen Wang, Xinqi Cheng, Jing Gao, Songlin Yu, Ling Qiu.

   OBJECTIVE: To establish and validate a robust LC-MS/MS method for simultaneously measuring 8-oxoGuo, 8-oxodG, and NMN in serum and urine to evaluate the oxidative stress status.
METHODS: A Waters TQ-XS triple quadrupole mass spectrometer system coupled with an Acquity UPLC Primer HSS T3 column was chosen. The clinical performance was verified according to the CLSI C62-A and EP-15 guidelines. Furthermore, matched serum and urine samples from 22 apparently healthy check-ups, 20 patients with atherosclerosis, and 18 individuals with dementia were evaluated.
RESULTS: The recovery for serum 8-oxoGuo, urine 8-oxoGuo, serum 8-oxodG, urine 8-oxodG, serum NMN, and urine NMN was 88.8-112.4%, 102.4-114.1%, 88.5-107.7%, 94.9-102.6%, 98.4-108.9%, and 88.5-108.6%, respectively. Based on the inter-assay results, total coefficient of variation, matrix effect, and carryover, the LC-MS/MS method was deemed robust. The limit of quantification was 0.017, 0.018, and 0.150 nmol/L for 8-oxoGuo, 8-oxodG, and NMN, respectively, which are suitable for accurate measurements in human serum and urine samples. Higher 8-oxoGuo and 8-oxodG levels and lower NMN levels, indicative of significantly higher oxidative stress status, were found in patients with dementia compared to healthy subjects.
CONCLUSION: We established and validated a robust LC-MS/MS method to simultaneously measure 8-oxoGuo, 8-oxodG, and NMN in serum and urine.

Keywords:  8-oxo-7,8-dihydro-2′-deoxyguanosine; 8-oxo-7,8-dihydroguanosine; LC-MS/MS; Nicotinamide mononucleotide

DOI:  https://doi.org/10.1016/j.ab.2022.114970