bims-metlip 2022-03-27 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022–03–27
nineteen papers selected by
Sofia Costa, Icahn School of Medicine at Mount Sinai

Artifactual fatty acid dimers mimic FAHFA signals in untargeted metabolomics pipelines.
JPA: Joint Metabolic Feature Extraction Increases the Depth of Chemical Coverage for LC-MS-Based Metabolomics and Exposomics.
A rapid and robust method for amino acid quantification using a simple N-hydroxysuccinimide ester derivatization and liquid chromatography-ion mobility-mass spectrometry.
Ultrahigh-performance supercritical fluid chromatography for intraclass separation of lipids: Investigation of general principles.
High-Throughput UHPLC-MS to Screen Metabolites in Feces for Gut Metabolic Health.
Untargeted Metabolomics Study of Three Matrices: Seminal Fluid, Urine, and Serum to Search the Potential Indicators of Prostate Cancer.
Secondary or Specialized Metabolites, or Natural Products: A Case Study of Untargeted LC-QTOF Auto-MS/MS Analysis.
Microfluidic device integrating single-cell extraction and electrical lysis for mass spectrometry detection of intracellular compounds.
Metabolic Footprinting of Microbial Systems Based on Comprehensive In Silico Predictions of MS/MS Relevant Data.
Correlating Mass Spectrometry Imaging and Liquid Chromatography-Tandem Mass Spectrometry for Tissue-Based Pharmacokinetic Studies.
A High-Throughput Platform for the Rapid Screening of Vitamin D Status by Direct Infusion-Tandem Mass Spectrometry.
A Fully Automated Online SPE-LC-MS/MS Method for the Determination of 10 Pharmaceuticals in Wastewater Samples.
High Performance Liquid Chromatography (HPLC) with Fluorescence Detection for Quantification of Steroids in Clinical, Pharmaceutical, and Environmental Samples: A Review.
Quantification of the irreversible fibroblast growth factor receptor inhibitor futibatinib by UPLC-MS/MS: Application to the metabolic stability assay in human liver microsomes for the estimation of its in vitro hepatic intrinsic clearance.
Enhancing the Throughput of FT Mass Spectrometry Imaging Using Joint Compressed Sensing and Subspace Modeling.
Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish.
A rapid method for the screening of fatty acids in lipids in plasma or serum without prior extraction.
Lipid Extraction and Analysis.
Fishing for lipid lactones using selective reaction and characteristic fragmentation pattern.

J Lipid Res. 2022 Mar 18. pii: S0022-2275(22)00034-7. [Epub ahead of print] 100201

Artifactual fatty acid dimers mimic FAHFA signals in untargeted metabolomics pipelines.

Alisa B Nelson, Lisa S Chow, Curtis C Hughey, Peter A Crawford, Patrycja Puchalska.

  Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipokines with extensive structural and regional isomeric diversity that impact multiple physiological functions, including insulin sensitivity and glucose homeostasis. Due to their low molar abundance, FAHFAs are typically quantified using highly sensitive liquid chromatography and tandem mass spectrometry (LC-MS/MS) methods. Numerous relevant MS databases house in silico-spectra that allow identification and speciation of FAHFAs. These provisional chemical feature assignments provide a useful starting point, but could lead to misidentification. To address this possibility, we analyzed human serum with a commonly applied high-resolution LC-MS untargeted metabolomics platform. We found that many chemical features are putatively assigned to the FAHFA lipid class based on exact mass and fragmentation patterns matching spectral databases. Careful validation using authentic standards revealed that many investigated signals provisionally assigned as FAHFAs are in fact fatty acid dimers formed in the LC-MS pipeline. These isobaric fatty acid dimers differ structurally only by the presence of an olefinic bond. Furthermore, stable isotope-labeled oleic acid spiked into human serum at sub-physiological concentrations showed concentration-dependent formation of a diverse repertoire of fatty acid dimers that analytically mimicked FAHFAs. Conversely, validated FAHFA species did not form spontaneously in the LC-MS pipeline. Together, these findings underscore that FAHFAs are endogenous lipid species. However, non-biological fatty acid dimers forming in the setting of high concentrations of free fatty acids can be misidentified as FAHFAs. Based on these results, we assembled a fatty acid dimer database to identify non-biological fatty acid dimers in untargeted metabolomics datasets.

Keywords:  Insulin resistance; LC-MS/MS; adipose tissue; isobaric fatty acid dimers; lipidomics; lipids; lipokines; obesity; olefinic bond; spectral database

DOI:  https://doi.org/10.1016/j.jlr.2022.100201
Metabolites. 2022 Feb 26. pii: 212. [Epub ahead of print]12(3):

JPA: Joint Metabolic Feature Extraction Increases the Depth of Chemical Coverage for LC-MS-Based Metabolomics and Exposomics.

Jian Guo, Sam Shen, Min Liu, Chenjingyi Wang, Brian Low, Ying Chen, Yaxi Hu, Shipei Xing, Huaxu Yu, Yu Gao, Mingliang Fang, Tao Huan.

  Extracting metabolic features from liquid chromatography-mass spectrometry (LC-MS) data has been a long-standing bioinformatic challenge in untargeted metabolomics. Conventional feature extraction algorithms fail to recognize features with low signal intensities, poor chromatographic peak shapes, or those that do not fit the parameter settings. This problem also poses a challenge for MS-based exposome studies, as low-abundant metabolic or exposomic features cannot be automatically recognized from raw data. To address this data processing challenge, we developed an R package, JPA (short for Joint Metabolomic Data Processing and Annotation), to comprehensively extract metabolic features from raw LC-MS data. JPA performs feature extraction by combining a conventional peak picking algorithm and strategies for (1) recognizing features with bad peak shapes but that have tandem mass spectra (MS2) and (2) picking up features from a user-defined targeted list. The performance of JPA in global metabolomics was demonstrated using serial diluted urine samples, in which JPA was able to rescue an average of 25% of metabolic features that were missed by the conventional peak picking algorithm due to dilution. More importantly, the chromatographic peak shapes, analytical accuracy, and precision of the rescued metabolic features were all evaluated. Furthermore, owing to its sensitive feature extraction, JPA was able to achieve a limit of detection (LOD) that was up to thousands of folds lower when automatically processing metabolomics data of a serial diluted metabolite standard mixture analyzed in HILIC(-) and RP(+) modes. Finally, the performance of JPA in exposome research was validated using a mixture of 250 drugs and 255 pesticides at environmentally relevant levels. JPA detected an average of 2.3-fold more exposure compounds than conventional peak picking only.

Keywords:  data processing; exposomics; feature extraction; metabolite annotation; untargeted metabolomics

DOI:  https://doi.org/10.3390/metabo12030212
Anal Bioanal Chem. 2022 Mar 26.

A rapid and robust method for amino acid quantification using a simple N-hydroxysuccinimide ester derivatization and liquid chromatography-ion mobility-mass spectrometry.

Taylor M Domenick, Austin L Jones, Robin H J Kemperman, Richard A Yost.

  The vast majority of mass spectrometry (MS)-based metabolomics studies employ reversed-phase liquid chromatography (RPLC) to separate analytes prior to MS detection. Highly polar metabolites, such as amino acids (AAs), are poorly retained by RPLC, making quantitation of these key species challenging across the broad concentration ranges typically observed in biological specimens, such as cell extracts. To improve the detection and quantitation of AAs in microglial cell extracts, the implementation of a 4-dimethylaminobenzoylamido acetic acid N-hydroxysuccinimide ester (DBAA-NHS) derivatization agent was explored for its ability to improve both analyte retention and detection limits in RPLC-MS. In addition to the introduction of the DBAA-NHS labeling reagent, a uniformly (U) 13C-labeled yeast extract was also introduced during the sample preparation workflow as an internal standard (IS) to eliminate artifacts and to enable targeted quantitation of AAs, as well as untargeted amine submetabolome profiling. To improve method sensitivity and selectivity, multiplexed drift-tube ion mobility (IM) was integrated into the LC-MS workflow, facilitating the separation of isomeric metabolites, and improving the structural identification of unknown metabolites. Implementation of the U-13C-labeled yeast extract during the multiplexed LC-IM-MS analysis enabled the quantitation of 19 of the 20 common AAs, supporting a linear dynamic range spanning up to three orders of magnitude in concentration for microglial cell extracts, in addition to reducing the required cell count for reliable quantitation from 10 to 5 million cells per sample.

Keywords:  Amino acids; Derivatization; Ion mobility spectrometry; Mass spectrometry; Metabolomics; Quantitation

DOI:  https://doi.org/10.1007/s00216-022-03993-w
J Chromatogr A. 2022 Mar 15. pii: S0021-9673(22)00173-X. [Epub ahead of print]1670 462975

Ultrahigh-performance supercritical fluid chromatography for intraclass separation of lipids: Investigation of general principles.

Oleksandr Kozlov, Miroslav Lísa.

  Powerful chromatographic techniques are required for lipidomic analyses due to the extreme complexity of natural lipidomes. In the past few years, ultrahigh-performance supercritical fluid chromatography (UHPSFC) has proven to be a good alternative to conventional LC methods for comprehensive lipidomic analysis. The goal of this work was to study UHPSFC intraclass separation of lipids according to the fatty acyl composition. The effects of column chemistry, mobile phase composition and gradient, flow rate, back pressure, temperature, and column coupling on intraclass separation of lipids were widely investigated and discussed. In general, UHPSFC exhibited interclass selectivity together with intraclass separation of lipids according to their total number of double bonds and acyl chain lengths. Moreover, separations of diacylglycerol and lysophosphatidylcholines regioisomers were achieved in some cases. The nature of the stationary phase showed the most prominent effect on UHPSFC intraclass selectivity, while other chromatographic conditions were used for partial improvement in resolution of lipid species. An octadecyl-based stationary phase showed excellent separation of nonpolar lipid species, including triacylglycerol isobars; however, it provided poor peak shapes and limited retention time reproducibility for polar lipids. Diol- and 1-aminoanthracene-based columns provided the best inter- and intraclass resolution of most lipids. The main benefit for UHPSFC separation of complex lipid samples is the combination of the acyl chain/double bond intraclass separation of lipids with excellent lipid class selectivity, which can facilitate mass spectrometry detection and quantitation of trace species without ion suppression effects.

Keywords:  Column coupling; Intraclass separation; Lipid analysis; Lipidomics; Supercritical fluid chromatography

DOI:  https://doi.org/10.1016/j.chroma.2022.462975
Metabolites. 2022 Feb 25. pii: 211. [Epub ahead of print]12(3):

High-Throughput UHPLC-MS to Screen Metabolites in Feces for Gut Metabolic Health.

Andressa de Zawadzki, Maja Thiele, Tommi Suvitaival, Asger Wretlind, Min Kim, Mina Ali, Annette F Bjerre, Karin Stahr, Ismo Mattila, Torben Hansen, Aleksander Krag, Cristina Legido-Quigley.

  Feces are the product of our diets and have been linked to diseases of the gut, including Chron's disease and metabolic diseases such as diabetes. For screening metabolites in heterogeneous samples such as feces, it is necessary to use fast and reproducible analytical methods that maximize metabolite detection. As sample preparation is crucial to obtain high quality data in MS-based clinical metabolomics, we developed a novel, efficient and robust method for preparing fecal samples for analysis with a focus in reducing aliquoting and detecting both polar and non-polar metabolites. Fecal samples (n = 475) from patients with alcohol-related liver disease and healthy controls were prepared according to the proposed method and analyzed in an UHPLC-QQQ targeted platform in order to obtain a quantitative profile of compounds that impact liver-gut axis metabolism. MS analyses of the prepared fecal samples have shown reproducibility and coverage of n = 28 metabolites, mostly comprising bile acids and amino acids. We report metabolite-wise relative standard deviation (RSD) in quality control samples, inter-day repeatability, LOD (limit of detection), LOQ (limit of quantification), range of linearity and method recovery. The average concentrations for 135 healthy participants are reported here for clinical applications. Our high-throughput method provides a novel tool for investigating gut-liver axis metabolism in liver-related diseases using a noninvasive collected sample.

Keywords:  bile acids; fecal metabolomics; gut-liver axis; sample preparation; targeted metabolomics

DOI:  https://doi.org/10.3390/metabo12030211
Front Mol Biosci. 2022 ;9 849966

Untargeted Metabolomics Study of Three Matrices: Seminal Fluid, Urine, and Serum to Search the Potential Indicators of Prostate Cancer.

Magdalena Buszewska-Forajta, Joanna Raczak-Gutknecht, Wiktoria Struck-Lewicka, Magdalena Nizioł, Małgorzata Artymowicz, Marcin Markuszewski, Marta Kordalewska, Marcin Matuszewski, Michał J Markuszewski.

  The simultaneous determination of metabolites from biological fluids may provide more accurate information about the current body condition. So far, the metabolomics approach has been successfully applied to study the mechanism of several disorders and to search for novel biomarkers. Urine and plasma are widely accepted matrices for the evaluation of several pathologies, while prostate cancer (CaP) development is still unknown. For this reason, an alternative matrix, the seminal fluid, was proposed to expand the knowledge about the CaP pathomechanism. The main aim of this study was to develop and optimize the sample preparation protocol to ensure the highest coverage of the metabolome of ejaculate samples. Parameters like the type and composition of the solvent mixture, time of extraction, and applied volume of the solvent were tested. The optimized method was applied for the untargeted metabolomics profiling of seminal fluid samples obtained from CaP patients. Moreover, urine and serum samples were also prepared for untargeted metabolomics analysis. Analyses were carried out with the use of two complementary analytical techniques: GC-EI-QqQ/MS and LC-ESI-TOF/MS. Finally, the metabolic signature of seminal fluid (n = 7), urine (n = 7), and plasma (n = 7) samples was compared. Furthermore, the hypothesis of the increased level of metabolites in ejaculate samples related to the CaP development was evaluated. The results indicated that the developed and optimized sample preparation protocol for seminal fluid may be successfully applied for metabolomics study. Untargeted analysis of ejaculate enabled to determine the following classes of compounds: fatty acids, sphingolipids, phospholipids, sugars, and their derivatives, as well as amino acids. Finally, a comparison of the three tested matrices was carried out. To our best knowledge, it is the first time when the metabolic profile of the three matrices, namely, urine, plasma, and seminal fluid, was compared. Based on the results, it can be pointed out that ejaculate comprises the metabolic signature of both matrices (polar compounds characteristic for urine, and non-polar ones present in plasma samples). Compared to plasma, semen samples revealed to have a similar profile; however, determined levels of metabolites were lower in case of ejaculate. In case of urine samples, compared to semen metabolic profiles, the levels of detected metabolites were decreased in the latter ones.

Keywords:  HPLC; metabolites; metabolomics; plasma; prostate cancer; seminal fluid; urine

DOI:  https://doi.org/10.3389/fmolb.2022.849966
Cells. 2022 Mar 17. pii: 1025. [Epub ahead of print]11(6):

Secondary or Specialized Metabolites, or Natural Products: A Case Study of Untargeted LC-QTOF Auto-MS/MS Analysis.

Franz Hadacek.

  The large structural diversity of specialized metabolites represents a substantial challenge in untargeted metabolomics. Modern LC-QTOF instruments can provide three- to four-digit numbers of auto-MS/MS spectra from sample sets. This case study utilizes twelve structurally closely related flavonol glycosides, characteristic specialized metabolites of plant tissues, some of them isomeric and isobaric, to illustrate the possibilities and limitations of their identification. This process requires specific software tools that perform peak picking and feature alignment after spectral deconvolution and facilitate molecular structure base searching with subsequent in silico fragmentation to obtain initial ideas about possible structures. The final assignment of a putative identification, so long as spectral databases are not complete enough, requires structure searches in a chemical reference database, such as SciFindern, in attempts to obtain additional information about specific product ions of a metabolite candidate or check its feasibility. The highlighted problems in this process not only apply to specialized metabolites in plants but to those occurring in other organisms as well. This case study is aimed at providing guidelines for all researchers who obtain data from such analyses but are interested in deeper information than just Venn diagrams of the feature distribution in their sample groups.

Keywords:  auto-MS/MS spectra; identification; specialized metabolites; untargeted metabolomics

DOI:  https://doi.org/10.3390/cells11061025
Electrophoresis. 2022 Mar 22.

Microfluidic device integrating single-cell extraction and electrical lysis for mass spectrometry detection of intracellular compounds.

Tomáš Václavek, František Foret.

  Analysis of cellular composition and metabolism at a single cell resolution allows gaining more information about complex relationships of cells within tissues or whole living organisms by resolving the variance stemming from the cellular heterogeneity. Mass spectrometry is a perfect analytical tool satisfying the demanding requirements of detecting and identifying compounds present in such ultra-low-volume samples of high chemical complexity. However, the method of sampling and sample ionization is crucial in obtaining relevant information. In this work, we present a microfluidic sampling platform that integrates single-cell extraction from MS-incompatible media with electrical cell lysis and nanoESI-MS analysis of human erythrocytes. Hemoglobin alpha and beta chains (300 amol/cell) were successfully identified in mass spectra of single erythrocyte lysates. This article is protected by copyright. All rights reserved.

Keywords:  bioanalysis; hemoglobin; mass spectrometry; microfluidics; single-cell analysis

DOI:  https://doi.org/10.1002/elps.202100379
Metabolites. 2022 Mar 17. pii: 257. [Epub ahead of print]12(3):

Metabolic Footprinting of Microbial Systems Based on Comprehensive In Silico Predictions of MS/MS Relevant Data.

Alexander Reiter, Jian Asgari, Wolfgang Wiechert, Marco Oldiges.

  Metabolic footprinting represents a holistic approach to gathering large-scale metabolomic information of a given biological system and is, therefore, a driving force for systems biology and bioprocess development. The ongoing development of automated cultivation platforms increases the need for a comprehensive and rapid profiling tool to cope with the cultivation throughput. In this study, we implemented a workflow to provide and select relevant metabolite information from a genome-scale model to automatically build an organism-specific comprehensive metabolome analysis method. Based on in-house literature and predicted metabolite information, the deduced metabolite set was distributed in stackable methods for a chromatography-free dilute and shoot flow-injection analysis multiple-reaction monitoring profiling approach. The workflow was used to create a method specific for Saccharomyces cerevisiae, covering 252 metabolites with 7 min/sample. The method was validated with a commercially available yeast metabolome standard, identifying up to 74.2% of the listed metabolites. As a first case study, three commercially available yeast extracts were screened with 118 metabolites passing quality control thresholds for statistical analysis, allowing to identify discriminating metabolites. The presented methodology provides metabolite screening in a time-optimised way by scaling analysis time to metabolite coverage and is open to other microbial systems simply starting from genome-scale model information.

Keywords:  automation; bioprocess development; digitalisation; dilute and shoot; flow-injection analysis; mass spectrometry; metabolomics; method development; prediction; yeast extract

DOI:  https://doi.org/10.3390/metabo12030257
Metabolites. 2022 Mar 18. pii: 261. [Epub ahead of print]12(3):

Correlating Mass Spectrometry Imaging and Liquid Chromatography-Tandem Mass Spectrometry for Tissue-Based Pharmacokinetic Studies.

Andreas Dannhorn, Emine Kazanc, Gregory Hamm, John G Swales, Nicole Strittmatter, Gareth Maglennon, Richard J A Goodwin, Zoltan Takats.

  Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a standard tool used for absolute quantification of drugs in pharmacokinetic (PK) studies. However, all spatial information is lost during the extraction and elucidation of a drugs biodistribution within the tissue is impossible. In the study presented here we used a sample embedding protocol optimized for mass spectrometry imaging (MSI) to prepare up to 15 rat intestine specimens at once. Desorption electrospray ionization (DESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) were employed to determine the distributions and relative abundances of four benchmarking compounds in the intestinal segments. High resolution MALDI-MSI experiments performed at 10 µm spatial resolution allowed to determine the drug distribution in the different intestinal histological compartments to determine the absorbed and tissue bound fractions of the drugs. The low tissue bound drug fractions, which were determined to account for 56-66% of the total drug, highlight the importance to understand the spatial distribution of drugs within the histological compartments of a given tissue to rationalize concentration differences found in PK studies. The mean drug abundances of four benchmark compounds determined by MSI were correlated with the absolute drug concentrations. Linear regression resulted in coefficients of determination (R2) ranging from 0.532 to 0.926 for MALDI-MSI and R2 values ranging from 0.585 to 0.945 for DESI-MSI, validating a quantitative relation of the imaging data. The good correlation of the absolute tissue concentrations of the benchmark compounds and the MSI data provides a bases for relative quantification of compounds within and between tissues, without normalization to an isotopically labelled standard, provided that the compared tissues have inherently similar ion suppression effects.

Keywords:  DESI; DMPK; MALDI; drug absorption; mass spectrometry imaging; tissue imaging

DOI:  https://doi.org/10.3390/metabo12030261
J Lipid Res. 2022 Mar 22. pii: S0022-2275(22)00037-2. [Epub ahead of print] 100204

A High-Throughput Platform for the Rapid Screening of Vitamin D Status by Direct Infusion-Tandem Mass Spectrometry.

Erick Helmeczi, Eric Fries, Lauren Perry, Karen Choong, Katie O'Hearn, Dayre McNally, Philip Britz-McKibbin.

  Vitamin D is an important fat-soluble prohormone with pleiotropic effects on human health, such as immunomodulation of the innate and adaptive immune system. There is an unmet clinical need for a rapid screening platform for 25-hydroxyvitamin D (25OH-D) determination without chromatographic separation that offers better precision and accuracy than immunoassays. Here, we introduce a high-throughput method for assessing vitamin D status from blood specimens based on direct infusion-tandem mass spectrometry (DI-MS/MS) following click derivatization using 2-nitrosopyridine. We developed an optimized liquid-phase extraction protocol to minimize ion suppression when directly infusing serum or plasma extracts via a capillary electrophoresis system for quantitative determination of 25OH-D. Acceptable reproducibility (mean CV = 10.9%, n=412), recovery (mean = 102% at 15, 30, and 45 nmol/L), and linearity (R2 > 0.998) were achieved for 25OH-D with lower detection limits (LOD ∼ 1.2 nmol/L, S/N ∼ 3), greater throughput (∼ 3 min/sample), and less bias than a commercial chemiluminescence immunoassay prone to batch effects. There was mutual agreement in 25OH-D concentrations from reference blood samples measured by DI-MS/MS as compared to LC-MS/MS (mean bias = 7.8%, n=18). We also demonstrate that this method could reduce immunoassay misclassification of vitamin D deficiency in a cohort of critically ill children (n=30). In conclusion, DI-MS/MS offers a viable alternative to LC-MS/MS for assessment of vitamin D status in support of large-scale studies in nutritional epidemiology, as well as clinical trials to rapidly screen individual patients who may benefit from vitamin D supplementation.

Keywords:  Critical Care; Direct Infusion-MS/MS; Infectious Disease; Interlaboratory Performance; Mass Spectrometry; Method Validation; Nutrition; Proficiency Testing; Vitamin D; Vitamin D Deficiency

DOI:  https://doi.org/10.1016/j.jlr.2022.100204
Toxics. 2022 Feb 23. pii: 103. [Epub ahead of print]10(3):

A Fully Automated Online SPE-LC-MS/MS Method for the Determination of 10 Pharmaceuticals in Wastewater Samples.

Masho Hilawie Belay, Ulrich Precht, Peter Mortensen, Emilio Marengo, Elisa Robotti.

  The increasing use of pharmaceuticals, their presence in the aquatic environment, and the associated toxic effects, have raised concerns in recent years. In this work, a new multi-residue analytical method was developed and validated for the determination of 10 pharmaceuticals in wastewaters using online solid-phase extraction (online SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds included in the method were antineoplastics (cabazitaxel, docetaxel, doxorubicin, etoposide, irinotecan, methotrexate, paclitaxel, and topotecan), renin inhibitors (aliskiren), and antidepressants (maprotiline). The method was developed through several experiments on four online SPE cartridges, three reversed phase chromatography columns, and four combinations of mobile phase components. Under optimal conditions, very low limits of detection (LODs) of 1.30 to 10.6 ng L-1 were obtained. The method was repeatable, with relative standard deviations (RSD, %) for intraday and interday precisions ranged from 1.6 to 7.8 and from 3.3 to 13.2, respectively. Recovery values ranged from 78.4 to 111.4%, indicating the reproducibility of the method. Matrix effects were mainly presented as signal suppression, with topotecan and doxorubicin being the two most affected compounds (31.0% signal suppression). The proposed method was successfully applied to hospital effluents, detecting methotrexate (4.7-9.3 ng L-1) and maprotiline (11.2-23.1 ng L-1). Due to the shorter overall run time of 15 min, including sample preparation, and reduced sample volume (0.9 mL), this on-line SPE-LC-MS/MS method was extremely convenient and efficient in comparison to the classical off-line SPE method. The proposed method was also highly sensitive and can be used for ultratrace quantification of the studied pharmaceuticals in wastewaters, providing useful data for effective environmental monitoring.

Keywords:  LC-MS/MS; anticancer drugs; antidepressant; antineoplastic agents; method validation; online SPE; pharmaceuticals; renin inhibitor; wastewater

DOI:  https://doi.org/10.3390/toxics10030103
Molecules. 2022 Mar 10. pii: 1807. [Epub ahead of print]27(6):

High Performance Liquid Chromatography (HPLC) with Fluorescence Detection for Quantification of Steroids in Clinical, Pharmaceutical, and Environmental Samples: A Review.

Fatima Hameedat, Sahar Hawamdeh, Soraya Alnabulsi, Aref Zayed.

  Steroids are compounds widely available in nature and synthesized for therapeutic and medical purposes. Although several analytical techniques are available for the quantification of steroids, their analysis is challenging due to their low levels and complex matrices of the samples. The efficiency and quick separation of the HPLC combined with the sensitivity, selectivity, simplicity, and cost-efficiency of fluorescence, make HPLC coupled to fluorescence detection (HPLC-FLD) an ideal tool for routine measurement and detection of steroids. In this review, we covered HPLC-FLD methods reported in the literature for the steroids quantification in clinical, pharmaceutical, and environmental applications, focusing on the various approaches of fluorescent derivatization. The aspects related to analytical methodology including sample preparation, derivatization reagents, and chromatographic conditions will be discussed.

Keywords:  HPLC; derivatization; fluorescence; quantification; steroids

DOI:  https://doi.org/10.3390/molecules27061807
J Pharm Biomed Anal. 2022 Mar 17. pii: S0731-7085(22)00152-2. [Epub ahead of print]214 114731

Quantification of the irreversible fibroblast growth factor receptor inhibitor futibatinib by UPLC-MS/MS: Application to the metabolic stability assay in human liver microsomes for the estimation of its in vitro hepatic intrinsic clearance.

Lloyd Wei Tat Tang, Eric Chun Yong Chan.

  Futibatinib (FUT) is a potent irreversible inhibitor of fibroblast growth factor receptor 1-4 currently under clinical investigation for the treatment of cholangiocarcinoma. However, there remains a paucity of information pertaining to its hepatic metabolism. In this study, our overarching aims were to systematically develop and validate a novel ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analytical method to quantify FUT for the subsequent application to the metabolic stability assay. Chromatographic separation was achieved on a C18 column and a gradient elution system comprising 0.1% formic acid in water (A) and acetonitrile (B). Positive electrospray ionization in conjunction with multiple reaction monitoring (MRM) mode was harnessed for the selective and sensitive quantification of FUT (m/z 419.2 → 296.0) and erdafitinib (m/z 447.0 → 362.0; internal standard). The retention time was 1.49 min for FUT and 1.29 min for erdafitinib. The calibration curve was linear from 0.003 to 3 µM (r2 > 0.99) and the lower limit of quantification was 0.003 µM. The intra-day and inter-day precision (% RSD) and accuracy (% bias) were all < 11.4% and < 11.3% respectively. Quality control samples were determined to be stable under several conditions routinely employed in sample preparation and UPLC-MS/MS analyses. Moreover, the liver microsomal matrix did not adversely affect the quantification of FUT. Following which, the in vitro microsomal intrinsic clearance (CLint) of FUT was calculated from our metabolic stability assay to be 29.3 µL/min/mg, thereby suggesting that it was a medium clearance drug. Finally, extrapolating the CLint with human scaling factors yielded an estimated in vitro hepatic intrinsic clearance value of 2075 mL/min. Our study reports the first UPLC-MS/MS method and offers a specific, sensitive and rapid means of determining FUT human liver microsomal stability.

Keywords:  Futibatinib; Human liver microsomes; Intrinsic clearance; Metabolic stability; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2022.114731
Anal Chem. 2022 Mar 24.

Enhancing the Throughput of FT Mass Spectrometry Imaging Using Joint Compressed Sensing and Subspace Modeling.

Yuxuan Richard Xie, Daniel C Castro, Stanislav S Rubakhin, Jonathan V Sweedler, Fan Lam.

Mass spectrometry imaging (MSI) allows for untargeted mapping of the chemical composition of tissues with attomole detection limits. MSI using Fourier transform (FT)-based mass spectrometers, such as FT-ion cyclotron resonance (FT-ICR), grants the ability to examine the chemical space with unmatched mass resolution and mass accuracy. However, direct imaging of large tissue samples using FT-ICR is slow. In this work, we present an approach that combines the subspace modeling of ICR temporal signals with compressed sensing to accelerate high-resolution FT-ICR MSI. A joint subspace and spatial sparsity constrained model computationally reconstructs high-resolution MSI data from the sparsely sampled transients with reduced duration, allowing a significant reduction in imaging time. Simulation studies and experimental implementation of the proposed method in investigation of brain tissues demonstrate a 10-fold enhancement in throughput of FT-ICR MSI, without the need for instrumental or hardware modifications.

DOI: https://doi.org/10.1021/acs.analchem.1c05279
Mar Drugs. 2022 Feb 26. pii: 173. [Epub ahead of print]20(3):

Optimization and Validation of a High Throughput UHPLC-MS/MS Method for Determination of the EU Regulated Lipophilic Marine Toxins and Occurrence in Fresh and Processed Shellfish.

Teresa D'Amore, Sonia Lo Magro, Valeria Vita, Aurelia Di Taranto.

  Under the name of lipophilic marine toxins, there are included more than 1000 toxic secondary metabolites, produced by phytoplankton, with the common chemical property of lipophilicity. Due to toxicological effects and geographical distribution, in European legislation relevant compounds are regulated, and their determination is accomplished with the reference liquid chromatography-tandem mass spectrometry method. In this study a modified ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the identification and quantification of EU-regulated lipophilic toxins. The method optimization included a refinement of SPE-C18 clean-up, in order to reduce matrix interferences. Improved LC conditions and upgraded chromatographic ammonia-based gradient ensured the best separation of all analytes and, in particular, of the two structural isomers (OA and DTX2). Also, different MS parameters were tested, and confirmation criteria finally established. The validation studies confirmed that all parameters were satisfactory. The requirements for precision (RSD% < 11.8% for each compound), trueness (recoveries from 73 to 101%) and sensitivity (limits of quantification in the range 3-8 µg kg-1) were fulfilled. The matrix effect, ranging from -9 to 19%, allowed the use of a calibration curve in solvent (3-320 µg kg-1 in matrix) for quantification of real samples. Method relative uncertainty ranged from 12 to 20.3%. Additionally, a total of 1000 shellfish samples was analysed, providing a first preliminary surveillance study that may contribute to the knowledge of lipophilic marine toxins contamination. Increase in algae proliferation events and intoxication cases, EFSA suggestions for modification of maximum permitted levels and toxicity equivalency factors, and new studies of important toxic effects underline that implementation of reference methods still represents an important task for health and food safety laboratories.

Keywords:  SPE; UHPLC-MS/MS; azaspiracid; biotoxins; okadaic acid; pectenotoxin; yessotoxin

DOI:  https://doi.org/10.3390/md20030173
Prostaglandins Leukot Essent Fatty Acids. 2022 Mar 17. pii: S0952-3278(22)00028-X. [Epub ahead of print]178 102416

A rapid method for the screening of fatty acids in lipids in plasma or serum without prior extraction.

Ge Liu, Maria Makrides, Penelope Coates, Khoa Lam, Enzo Ranieri, Emilie Mas, Robert A Gibson.

  Serum or plasma are the commonly used blood fractions to determine the relationship between dietary and circulating fatty acids in health and disease. Most methods available for the measurement of fatty acids in serum or plasma (referred to as serum henceforth) require prior extraction with organic solvents. We have determined that it is possible to directly convert the lipids in aqueous biological samples to fatty acid methyl esters (FAME) without prior extraction, providing that the ratio of serum to transmethylation solvent does not exceed 10%. Our in-vial transmethylation system uses 50uL serum pipetted into 2 mL screw top GC vials containing 1 mL of 1% H2SO4 in methanol at 50 °C and subsequent FAME extracted in the same vial into 300uL heptane. The system yields both compositional and quantitative analysis of the fatty acids of serum identical to conventional standard methods. Evaluation of our new serum assay confirms significant correlations between the fatty acid measures and those obtained from conventional standard assay for all fatty acids (r > 0.99, P<0.0001), including the n-6 (r = 0.998, P<0.0001) and n-3 long chain polyunsaturated fatty acids (r = 0.993, P<0.0001). There were high levels of agreement between methods on Bland -Altman analysis, indicating the interchangeability of the methods. These results establish our new method as reliable for the assessment of fatty acid composition of small volumes of serum useful for high throughput situations that limits the volume of organic solvents and technical input.

Keywords:  Docosahexaenoic acid; FAME; High throughput; Plasma; Serum; n-3 fatty acids

DOI:  https://doi.org/10.1016/j.plefa.2022.102416
Methods Mol Biol. 2022 ;2468 271-281

Lipid Extraction and Analysis.

Henry H Harrison, Jennifer L Watts.

  Lipids are major components of cellular membranes and energy stores. Lipids contribute vital structural, energetic, and signaling functions. We have optimized methods to extract and analyze lipids from the nematode Caenorhabditis elegans based on standard methods. Here we describe a method to extract total lipids from C. elegans larvae, adults, or embryos. We describe a thin-layer chromatography method to separate major lipid classes and a gas chromatography method to analyze fatty acid composition from lipid extracts, lipid fractions, or directly from nematode larvae, adults, or embryos.

Keywords:  Fatty acid methyl esters; Gas chromatography; Lipid extraction; Neutral lipids; Phospholipids; Thin-layer chromatography

DOI:  https://doi.org/10.1007/978-1-0716-2181-3_14
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Mar 09. pii: S1570-0232(22)00105-2. [Epub ahead of print]1197 123201

Fishing for lipid lactones using selective reaction and characteristic fragmentation pattern.

Elana A Slutsky Smith, Soliman Khatib, Andrea Szuchman-Sapir.

  Extensive research has been invested in developing sensitive methods to identify lipid mediators (LMs) from multiple biological matrices. Previous studies point to the existence of a potential family of lactone-containing metabolites generated from eicosanoid families, isoprostanes, and prostanoid-like compounds that may function as LMs. However, targeted lipidomic studies do not routinely include lactone-containing lipids due to their low ionizability and instability under some common sample preparation conditions. Thus, the discovery of lactone-containing LM is limited. Herein we describe a method for selective identification of lipid lactones from within biological matrices. This method is based on a selective reaction of lactones with 1-(3-aminopropyl)imidazole, followed by cation exchange solid phase extraction and the identification of characteristic fragmentation patterns unique to reaction products of lactones in LC/MS/MS. NMR and LC/MS results indicated that saturated and unsaturated aliphatic ɣ and δ lactone model compounds mixed with human serum were successfully detected. MS/MS analyses of the reaction products revealed a unique pattern for the lactones, resulting from common neutral losses and fragmentation. When applied to esters and free fatty acids, some reaction products were observed. However, these reaction products' MS/MS fragmentation did not match the specific fragmentation of the lactones' reaction products. Confirming that lactones can be detected in a highly selective manner from within complex biological matrices when using the presented method. Thus, the presented method can selectively analyze lactones and may further complement existing lipidomic approaches to discover new LMs.

Keywords:  LC-MS/MS; Lactones; Lipid mediators; Lipidomics

DOI:  https://doi.org/10.1016/j.jchromb.2022.123201