bims-metlip 2023-12-24 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023‒12‒24
35 papers selected by
Sofia Costa, Matterworks

Comparison of 3-nitrophenylhydrazine, O-benzyl hydroxylamine, and 2-picolylamine derivatizations for analysis of short-chain fatty acids through liquid chromatography coupled with tandem mass spectrometry.
MSIpixel: a fully automated pipeline for compound annotation and quantitation in mass spectrometry imaging experiments.
Development of a single mobile phase for LC-IM-MS-based discovery lipidomics and metabolic phenotyping: Application to methapyrilene hepatotoxicity in the rat.
Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS.
Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography-Tandem Mass Spectrometry.
An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.
High-Throughput Microbore LC-MS Lipidomics to Investigate APOE Phenotypes.
A comprehensive LC-MS/MS method for the simultaneous measurement of 24 adrenal steroids: From research to clinical practice.
Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.
Analysis of vitamin D3-sulfate and 25-hydroxyvitamin D3-sulfate in breastmilk by LC-MS/MS.
Open access repository-scale propagated nearest neighbor suspect spectral library for untargeted metabolomics.
Development and Validation of a Method for Hydrolysis and Analysis of Amino Acids in Ruminant Feeds, Tissue, and Milk Using Isotope Dilution Z-HILIC Coupled with Electrospray Ionization Triple Quadrupole LC-MS/MS.
Development of simultaneous quantitative analytical method for three active components of Korean mint (Agastache rugosa (Fisch. & C.A.Mey.) Kuntze) extract in human plasma using ultra-high-performance liquid chromatography-tandem mass spectrometry.
Ultra-high-throughput mass spectrometry in drug discovery: fundamentals and recent advances.
Injectiondesign: web service of plate design with optimized stratified block randomization for modern GC/LC-MS-based sample preparation.
Multi-MSIProcessor: Data Visualizing and Analysis Software for Spatial Metabolomics Research.
Development and validation of a LC-MS/MS method for the quantification of phenolic compounds in human saliva after intake of a procyanidin-rich pine bark extract.
Extended automated quantification algorithm (AQuA) for targeted 1H NMR metabolomics of highly complex samples: application to plant root exudates.
Evaluating the Effect of Data Merging and Postacquisition Normalization on Statistical Analysis of Untargeted High-Resolution Mass Spectrometry Based Urinary Metabolomics Data.
Hyperpolarization-Enhanced NMR Spectroscopy of Unaltered Biofluids Using Photo-CIDNP.
A liquid chromatography-mass spectrometry method for the enantioselective multiresidue determination of nine chiral agrochemicals in urine using an enrichment procedure based on graphitized carbon black.
Trace analysis of steroid hormones in tear films via liquid chromatography-high resolution mass spectrometry.
Mycotoxins in Vegetable Oils: A Review of Recent Developments, Current Challenges and Future Perspectives in Sample Preparation, Chromatographic Determination, and Analysis of Real Samples.
Quantification of psilocin in human whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Development and validation of an LC-MS/MS method for the quantification of KRASG12C inhibitor opnurasib in several mouse matrices and its application in a pharmacokinetic mouse study.
A robust method for simultaneous determination of eight B vitamins in human serum by liquid chromatography tandem mass spectrometry.
A comprehensive LC-MS/MS method for simultaneous analysis of 65 synthetic cannabinoids in human hair samples and application to forensic investigations.
Optimization of a rapid method for screening drugs in blood by liquid chromatography tandem mass spectrometry.
Untargeted metabolomics to expand the chemical space of the marine diatom Skeletonema marinoi.
Non-targeted analysis of lipidic extracts by high-resolution mass spectrometry to characterise the chemical exposome: Comparison of four clean-up strategies applied to egg.
Improving quality of analysis by suppression of unwanted signals through band-selective excitation in NMR spectroscopy for metabolomics studies.
Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay.
Method Validation for Quantification of PFOS and PFOA in Human Plasma and a Pilot Study in Blood Donors from Thai Red Cross Society.
Spatialized Metabolomic Annotation Combining MALDI Imaging and Molecular Networks.
Liquid chromatography-mass spectrometry-based metabolomics and fluxomics reveals the metabolic alterations in glioma U87MG multicellular tumor spheroids versus two-dimensional cell cultures.

Anal Sci. 2023 Dec 19.

Comparison of 3-nitrophenylhydrazine, O-benzyl hydroxylamine, and 2-picolylamine derivatizations for analysis of short-chain fatty acids through liquid chromatography coupled with tandem mass spectrometry.

Ryosuke Nagatomo, Aoi Ichikawa, Haruki Kaneko, Koichi Inoue.

  Short-chain fatty acids (SCFAs) are metabolites derived from gut microbiota and implicated in host homeostasis. Hence, the profiling SCFAs from biological samples plays an important role in revealing the interaction between gut microbiota and pathogens. Previous studies, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with various derivatization strategies have been performed to obtain the SCFA profiles from biological samples. However, it is poor evidence to compare these derivatization regents and conditions. Thus, we present the evaluation of three major derivatization reagents, namely 3-nitrophenylhydrazine (3-NPH), O-benzylhydroxylamine (O-BHA), and 2-picolylamine (2-PA), for the analysis of eight SCFAs classified as C2-C5 isomers using LC-MS/MS. First, in a reversed-phase LC separation, 3-NPH showed good retention capacity. Although O-BHA derivatization showed higher sensitivity and good retention capacity than 2-PA, only 2-PA derivatization could successfully separate eight SCFAs. The matrix effects in human serum ranged 77.1-99.0% (RSD ≤ 3.4%, n = 6) for 3-NPH derivatives, 91.0-94.6% (RSD ≤ 5.4%, n = 6) for O-BHA derivatives, 81.6-99.5% (RSD ≤ 8.0%, n = 6) for 2-PA derivatives. These compared results showed each characteristic of 3-NPH, O-BHA, and 2-PA for SCFA derivatization based on LC-MS/MS approaches.

Keywords:  2-Picolylamine; 3-Nitrophenylhydrazine; O-benzylhydroxylamine; Short-chain fatty acids

DOI:  https://doi.org/10.1007/s44211-023-00474-7
Brief Bioinform. 2023 Nov 22. pii: bbad463. [Epub ahead of print]25(1):

MSIpixel: a fully automated pipeline for compound annotation and quantitation in mass spectrometry imaging experiments.

Lavinia Morosi, Matteo Miotto, Sara Timo, Sara Carloni, Eleonora Bruno, Marina Meroni, Elisabetta Menna, Simona Lodato, Maria Rescigno, Giuseppe Martano.

  Mass spectrometry imaging (MSI) is commonly used to map the spatial distribution of small molecules within complex biological matrices. One of the major challenges in imaging MS-based spatial metabolomics is molecular identification and metabolite annotation, to address this limitation, annotation is often complemented with parallel bulk LC-MS2-based metabolomics to confirm and validate identifications. Here we applied MSI method, utilizing data-dependent acquisition, to visualize and identify unknown molecules in a single instrument run. To reach this aim we developed MSIpixel, a fully automated pipeline for compound annotation and quantitation in MSI experiments. It overcomes challenges in molecular identification, and improving reliability and comprehensiveness in MSI-based spatial metabolomics.

Keywords:  MSIpixel; annotations; mass spectrometry imaging; metabolomics

DOI:  https://doi.org/10.1093/bib/bbad463
J Chromatogr A. 2023 Dec 10. pii: S0021-9673(23)00777-X. [Epub ahead of print]1714 464552

Development of a single mobile phase for LC-IM-MS-based discovery lipidomics and metabolic phenotyping: Application to methapyrilene hepatotoxicity in the rat.

Ian D Wilson, Corey Broeckling, Lee A Gethings, Nyasha C Munjoma, Robert Trengove, Paul D Rainville, Steven K Lai, Giorgis Isaac, Robert S Plumb.

  The untargeted global profiling of endogenous metabolites and lipids has the potential to increase knowledge and understanding in many areas of biology. LC-MS/MS is a key technology for such analyses however, several different LC methodologies, using different mobile phase compositions, are required to cover the diversity in polarity and analyte structure encountered in biological samples. Most notably many lipid screening methods make use of isopropanol (IPA) as a major component of mobile phases employed for comprehensive lipidomic profiling. In order to increase laboratory efficiency, and minimize opportunities for errors, a suite of methods, based on a single acetonitrile (ACN)-aqueous buffer mobile phase combination, has been developed. This mobile phase can be used for hydrophobic interaction liquid chromatography on an amide stationary phase (for polar analytes), reversed-phase (RP) LC analysis on a C8 stationary phase (for moderately polar-non-polar compounds) and RPLC using a CSH phenyl-hexyl bonded column (for lipids). All of these sub 10 minute separations had good throughput and reproducibility with CV's of analyte response <25 % whilst eliminating the need for complex mobile phase preparation and the use of IPA as an organic modifier for lipidomics. Advantages of removing IPA and replacing it with the ACN-based method were a 58 % increase in peak capacity for lipids, with improved resolution for the di- and triglycerides and cholesterol esters compared to current methods. Compared to the IPA-containing solvent system the ACN-based mobile phase also resulted in a 61 % increase in lipid feature detection. The utility of this "universal" mobile phase approach was demonstrated by its application to a rat toxicology study investigating the consequences of methapyrilene administration through on the endogenous metabolite profiles of plasma and urine. Methapyrilene and its metabolites were also profiled in these samples.

Keywords:  Chromatography; Efficiency; LC-MS/MS; Lipidomics; Metabolomics; Mobile phase

DOI:  https://doi.org/10.1016/j.chroma.2023.464552
J Lipid Res. 2023 Dec 20. pii: S0022-2275(23)00165-7. [Epub ahead of print] 100492

Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS.

Kohta Nakatani, Yoshihiro Izumi, Hironobu Umakoshi, Maki Yokomoto-Umakoshi, Tomoko Nakaji, Hiroki Kaneko, Hiroshi Nakao, Yoshihiro Ogawa, Kazutaka Ikeda, Takeshi Bamba.

  Quantitative information on blood metabolites can be used in developing advanced medical strategies such as early detection and prevention of disease. Monitoring bioactive lipids such as steroids, bile acids, and polyunsaturated fatty acid (PUFA) metabolites could be a valuable indicator of health status. However, a method for simultaneously measuring these bioactive lipids has not yet been developed. Here, we report a liquid chromatography tandem mass spectrometry (LC/MS/MS) method that can simultaneously measure 144 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal by methanol precipitation and purification of bioactive lipids by solid-phase extraction improved the recovery of the targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods for a wide range of bioactive lipid analyses. Using the developed method, we studied the plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The developed method of bioactive lipid analysis can be applied to health monitoring and disease biomarker discovery in precision medicine.

Keywords:  bile acid; comprehensive analysis; liquid chromatography; polyunsaturated fatty acid; solid-phase extraction; steroid; tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jlr.2023.100492
Curr Issues Mol Biol. 2023 Nov 22. 45(12): 9354-9367

Application of Normal-Phase Silica Column in Hydrophilic Interaction Liquid Chromatography Mode for Simultaneous Determination of Underivatized Amino Acids from Human Serum Samples via Liquid Chromatography-Tandem Mass Spectrometry.

Krisztina Németh, Ildikó Szatmári, Viktória Tőkési, Pál Tamás Szabó.

  In neonatal screening, amino acids have a significant diagnostic role. Determination of their values may identify abnormal conditions. Early diagnosis and continuous monitoring of amino acid disorders results in a better disease outcome. An easy and simple LC-MS/MS method was developed for the quantitation of underivatized amino acids. Amino acids were separated using a normal-phase HPLC column having a totally porous silica stationary phase and using classical reversed-phase eluents. Mass spectrometry in multiple reaction monitoring mode was used for the analysis, providing high selectivity and sensitivity. A standard addition calibration model was applied for quantitation using only one isotope-labeled internal standard for all amino acids. Five calibration points were used for quantitation, and the method was successfully validated. The slopes of the calibration curves of the individual amino acids in parallel measurements were found to be similar. Since the measured slopes were reproducible, one serum sample could represent every series of serum samples of a given day. The method was tested on human serum samples and adequate results were obtained. This new method can be easily applied in clinical laboratories.

Keywords:  LC-MS/MS; amino acids; normal-phase chromatography; standard addition calibration

DOI:  https://doi.org/10.3390/cimb45120586
Clin Chem Lab Med. 2023 Dec 19.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

Tobias Schierscher, Linda Salzmann, Neeraj Singh, Janik Wild, Vanessa Fischer, Friederike Bauland, Andrea Geistanger, Lorenz Risch, Christian Geletneky, Christoph Seger, Judith Taibon.

  OBJECTIVES: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations.METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement.
RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 μg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively.
CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Keywords:  SI units; isotope dilution-liquid chromatography-tandem mass spectrometry; qNMR; reference measurement procedure; traceability; zonisamide

DOI:  https://doi.org/10.1515/cclm-2023-0736
Anal Chem. 2023 Dec 19.

High-Throughput Microbore LC-MS Lipidomics to Investigate APOE Phenotypes.

Darshak Gadara, Vratislav Berka, Zdenek Spacil.

Microflow liquid chromatography interfaced with mass spectrometry (μLC-MS/MS) is increasingly applied for high-throughput profiling of biological samples and has been proven to have an acceptable trade-off between sensitivity and reproducibility. However, lipidomics applications are scarce. We optimized a μLC-MS/MS system utilizing a 1 mm inner diameter × 100 mm column coupled to a triple quadrupole mass spectrometer to establish a sensitive, high-throughput, and robust single-shot lipidomics workflow. Compared to conventional lipidomics methods, we achieve a ∼4-fold increase in response, facilitating quantification of 351 lipid species from a single iPSC-derived cerebral organoid during a 15 min LC-MS analysis. Consecutively, we injected 303 samples over ∼75 h to prove the robustness and reproducibility of the microflow separation. As a proof of concept, μLC-MS/MS analysis of Alzheimer's disease patient-derived iPSC cerebral organoid reveals differential lipid metabolism depending on APOE phenotype (E3/3 vs E4/4). Microflow separation proves to be an environmentally friendly and cost-effective method as it reduces the consumption of harmful solvents. Also, the data demonstrate robust, in-depth, high-throughput performance to enable routine clinical or biomedical applications.

DOI: https://doi.org/10.1021/acs.analchem.3c02652
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Nov 28. pii: S1570-0232(23)00351-3. [Epub ahead of print]1232 123941

A comprehensive LC-MS/MS method for the simultaneous measurement of 24 adrenal steroids: From research to clinical practice.

Songlin Yu, Yicong Yin, Yutong Zou, Qianqian Li, Jialei Yu, Shaowei Xie, Wei Luo, Xiaoli Ma, Danchen Wang, Liling Lin, Ling Qiu.

  Steroids are essential in the differential diagnosis of congenital adrenal hyperplasia (CAH) subtypes; however, they may confuse physicians with multifarious results. In this study, we established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of 24 steroids and developed a steroid metabolite pathway-based report to aid physicians in understanding these results. Solid-phase extraction was used to concentrate and purify target plasma steroids. The linearity, precision, recovery, and matrix effects were thoroughly evaluated. PowerBuilder was used to transfer the results from LC-MS/MS to the graphic report in a laboratory information management system (LIS) and was applied to different subtypes of CAH. Twenty-four steroids were separated and analyzed in one sample preparation and two injections using LC-MS/MS. The linearity of the steroids was excellent, with coefficients of linear regression greater than 0.99. The relative recovery ranged from 90.0 to 107.1 %, whereas the intra- and total coefficient variations were 1.6 ∼ 8.7 % and 2.0 ∼ 9.9 %, respectively. Matrix effects were compensated after internal standard correction. A graphic combination report mode was established and used to effectively identify CAH subtypes. In conclusion, a useful LC-MS/MS method and graphic combination report of 24 steroids based on their metabolite pathways were established.

Keywords:  Congenital adrenal hyperplasia; Intelligent report; Liquid chromatography tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.jchromb.2023.123941
Anal Chem. 2023 Dec 21.

Ultrahigh-Mass Resolution Mass Spectrometry Imaging with an Orbitrap Externally Coupled to a High-Performance Data Acquisition System.

Andrej Grgic, Konstantin O Nagornov, Anton N Kozhinov, Jesse A Michael, Ian G M Anthony, Yury O Tsybin, Ron M A Heeren, Shane R Ellis.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool that enables molecular sample analysis while simultaneously providing the spatial context of hundreds or even thousands of analytes. However, because of the lack of a separation step prior to ionization and the immense diversity of biomolecules, such as lipids, including numerous isobaric species, the coupling of ultrahigh mass resolution (UHR) with MSI presents one way in which this complexity can be resolved at the spectrum level. Until now, UHR MSI platforms have been restricted to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Here, we demonstrate the capabilities of an Orbitrap-based UHR MSI platform to reach over 1,000,000 mass resolution in a lipid mass range (600-950 Da). Externally coupling the Orbitrap Q Exactive HF with the high-performance data acquisition system FTMS Booster X2 provided access to the unreduced data in the form of full-profile absorption-mode FT mass spectra. In addition, it allowed us to increase the time-domain transient length from 0.5 to 10 s, providing improvement in the mass resolution, signal-to-noise ratio, and mass accuracy. The resulting UHR performance generates high-quality MALDI MSI images and simplifies the identification of lipids. Collectively, these improvements resulted in a 1.5-fold increase in annotations, demonstrating the advantages of this UHR imaging platform for spatial lipidomics using MALDI-MSI.

DOI: https://doi.org/10.1021/acs.analchem.3c04146
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Dec 06. pii: S1570-0232(23)00364-1. [Epub ahead of print]1232 123954

Analysis of vitamin D3-sulfate and 25-hydroxyvitamin D3-sulfate in breastmilk by LC-MS/MS.

Carmen J Reynolds, Roy B Dyer, Brady A Vizenor, Nicholas J Koszewski, Ravinder J Singh, Tom D Thacher.

  Sulfated metabolites of vitamin D have been suggested to be in breastmilk, although current methods to measure sulfated vitamin D compounds in breastmilk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) have not adequately accounted for increased aqueous solubility of these sulfated metabolites. The purpose of this study was to generate a method of LC-MS/MS for measuring vitamin D3-3-sulfate (VitD3-S) and 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) specifically in human breastmilk. The resulting method uses methanol to precipitate protein and solid phase extraction to prepare the samples for LC-MS/MS. The limits of quantification for analytes in solvent were 0.23 ng/mL VitD3-S and 0.2 ng/mL 25OHD3-S. Various experiments observed concentrations ranging 0.53 to 1.7 ng/mL VitD3-S and ≤ 0.29 ng/mL 25OHD3-S. Both analytes were present in aqueous skim milk, demonstrating the enhanced aqueous solubility of these vitamin D sulfates. In conclusion, we describe an effective method for measuring VitD3-S and 25OHD3-S in breastmilk by LC-MS/MS.

Keywords:  25-Hydroxyvitamin D; Mass Spectrometry; Milk; Sulfate; Vitamin D

DOI:  https://doi.org/10.1016/j.jchromb.2023.123954
Nat Commun. 2023 Dec 20. 14(1): 8488

Open access repository-scale propagated nearest neighbor suspect spectral library for untargeted metabolomics.

Wout Bittremieux, Nicole E Avalon, Sydney P Thomas, Sarvar A Kakhkhorov, Alexander A Aksenov, Paulo Wender P Gomes, Christine M Aceves, Andrés Mauricio Caraballo-Rodríguez, Julia M Gauglitz, William H Gerwick, Tao Huan, Alan K Jarmusch, Rima F Kaddurah-Daouk, Kyo Bin Kang, Hyun Woo Kim, Todor Kondić, Helena Mannochio-Russo, Michael J Meehan, Alexey V Melnik, Louis-Felix Nothias, Claire O'Donovan, Morgan Panitchpakdi, Daniel Petras, Robin Schmid, Emma L Schymanski, Justin J J van der Hooft, Kelly C Weldon, Heejung Yang, Shipei Xing, Jasmine Zemlin, Mingxun Wang, Pieter C Dorrestein.

Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of MS/MS spectra originating from published untargeted metabolomics experiments. Entries in this library, or "suspects," were derived from unannotated spectra that could be linked in a molecular network to an annotated spectrum. Annotations were propagated to unknowns based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer's brain phenotype. The nearest neighbor suspect spectral library is openly available for download or for data analysis through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data.

DOI: https://doi.org/10.1038/s41467-023-44035-y
J Agric Food Chem. 2023 Dec 20.

Development and Validation of a Method for Hydrolysis and Analysis of Amino Acids in Ruminant Feeds, Tissue, and Milk Using Isotope Dilution Z-HILIC Coupled with Electrospray Ionization Triple Quadrupole LC-MS/MS.

Andres F Ortega, Hui Zhao, Michael E Van Amburgh.

  Current analytical methods for amino acid (AA) analysis in ruminant nutrition are time-consuming and expensive. This study aimed to develop a method for AA analysis that is faster, more efficient, rugged, and accessible. Four representative matrixes were selected for method development and validation: milk, tissue, feed, and soy flour standard reference material from National Institute of Standards and Technology. Acid and alkaline hydrolysis were used to analyze 18 AA. Separation of AA was performed using a Z-HILIC column in an 18-min run coupled to a triple quadrupole LC/MS system in positive and negative electrospray ionization for identification and quantitation. The method was evaluated for recovery, precision, calibration curve linearity, and limits of detection (LODs) and limits of quantitation (LOQs) and applied to other feed samples. Good quantitation results were achieved for all AA, with coefficients of determination (R2) over 0.995; LODs at 0.2-28.2 and LOQs at 0.7-94.1 ng/mL; intraday and interday precision <14.9% relative standard deviation; blank recovery between 75.6 and 116.2%; and sample recovery between 75.6 and 118.0%. Overall, AA concentrations were similar to literature values, and there was a tendency for higher N recovery as AA. In conclusion, an efficient and robust method was validated to routinely analyze AA for appropriate characterization in diet formulation for dairy cattle.

Keywords:  Z-HILIC; amino acids; feed chemistry; isotope dilution; triple quadrupole LC-MS

DOI:  https://doi.org/10.1021/acs.jafc.3c05266
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Dec 11. pii: S1570-0232(23)00367-7. [Epub ahead of print]1232 123957

Development of simultaneous quantitative analytical method for three active components of Korean mint (Agastache rugosa (Fisch. & C.A.Mey.) Kuntze) extract in human plasma using ultra-high-performance liquid chromatography-tandem mass spectrometry.

Urmi Rahman Rinik, Ji Eon Kim, Eunok Lee, Oran Kwon, Byung Hwa Jung.

  Agastache rugosa contains phenolic compounds and flavonoids, and has been extensively used as a traditional herbal medicine. The major components in Agastache rugosa extract (ARE) are rosmarinic acid, tilianin, and acacetin, for which several analytical techniques have been reported. However, these substances have yet to be simultaneously quantified in human plasma. In this study, we aimed to simultaneously determine the three active components of ARE in human plasma by developing a reliable quantitative analytical method using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Chromatographic separation of the plasma samples was achieved using an ACQUITY UPLC® BEH C18 column with a gradient mobile phase of water and acetonitrile containing 0.1 % formic acid. Mass spectrometric detection was performed using a triple quadrupole tandem mass spectrometer in negative electrospray ionization (ESI-) and multiple reaction monitoring (MRM) modes. The developed quantitative method was validated for the three active components. All three analytes exhibited a linear response over the ranges of 0.5-50 ng/mL for rosmarinic acid, 0.1-20 ng/mL for acacetin, and 0.5-20 ng/mL for tilianin with a weighting factor of 1/x (where x is the concentration). At three quality control (QC) concentration levels (low, medium, and high), including the lower limit of quantitation (LLOQ), acceptable accuracy (±15 %) was achieved in the intra- and interday validations. The concentration of rosmarinic acid was highest in plasma. Tilianin and acacetin appeared and were eliminated earlier in the plasma than rosmarinic acid. This study provides a successfully validated method that can be used in further clinical applications of Agastache rugosa extracts.

Keywords:  Acacetin; Agastache rugosa; Human plasma; Rosmarinic acid; Simultaneous quantitative analysis; Tilianin; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2023.123957
Expert Opin Drug Discov. 2023 Dec 19. 1-11

Ultra-high-throughput mass spectrometry in drug discovery: fundamentals and recent advances.

Jon D Williams, Fan Pu, James W Sawicki, Nathaniel L Elsen.

  INTRODUCTION: Ultra-high-throughput mass spectrometry, uHT-MS, is a technology that utilizes ionization and sample delivery technologies optimized to enable sampling from well plates at > 1 sample per second. These technologies do not need a chromatographic separation step and can be utilized in a wide variety of assays to detect a broad range of analytes including small molecules, lipids, and proteins.AREAS COVERED: This manuscript provides a brief historical review of high-throughput mass spectrometry and the recently developed technologies that have enabled uHT-MS. The report also provides examples and references on how uHT-MS has been used in biochemical and chemical assays, nuisance compound profiling, protein analysis and high throughput experimentation for chemical synthesis.
EXPERT OPINION: The fast analysis time provided by uHT-MS is transforming how biochemical and chemical assays are performed in drug discovery. The potential to associate phenotypic responses produced by 1000's of compound treatments with changes in endogenous metabolite and lipid signals is becoming feasible. With the augmentation of simple, fast, high-throughput sample preparation, the scope of uHT-MS usage will increase. However, it likely will not supplant LC-MS for analyses that require low detection limits from complex matrices or characterization of complex biotherapeutics such as antibody-drug conjugates.

Keywords:  Ultra-high-throughput; acoustic droplet ejection; acoustic mist; desorption electrospray ionization; drug discovery; electrospray; infrared matrix-assisted laser desorption electrospray ionization; mass spectrometry; matrix assisted laser desorption ionization

DOI:  https://doi.org/10.1080/17460441.2023.2293153
BMC Bioinformatics. 2023 Dec 20. 24(1): 489

Injectiondesign: web service of plate design with optimized stratified block randomization for modern GC/LC-MS-based sample preparation.

Miaoshan Lu, Hengxuan Jiang, Ruimin Wang, Shaowei An, Jiawei Wang, Changbin Yu.

  BACKGROUND: Plate design is a necessary and time-consuming operation for GC/LC-MS-based sample preparation. The implementation of the inter-batch balancing algorithm and the intra-batch randomization algorithm can have a significant impact on the final results. For researchers without programming skills, a stable and efficient online service for plate design is necessary.RESULTS: Here we describe InjectionDesign, a free online plate design service focused on GC/LC-MS-based multi-omics experiment design. It offers the ability to separate the position design from the sequence design, making the output more compatible with the requirements of a modern mass spectrometer-based laboratory. In addition, it has implemented an optimized block randomization algorithm, which can be better applied to sample stratification with block randomization for an unbalanced distribution. It is easy to use, with built-in support for common instrument models and quick export to a worksheet.
CONCLUSIONS: InjectionDesign is an open-source project based on Java. Researchers can get the source code for the project from Github: https://github.com/CSi-Studio/InjectionDesign . A free web service is also provided: http://www.injection.design .

Keywords:  Block randomization; InjectionDesign; Mass spectrometry; Metabolomics; Plate design; Stratified balancing; Web service

DOI:  https://doi.org/10.1186/s12859-023-05598-1
Anal Chem. 2023 Dec 16.

Multi-MSIProcessor: Data Visualizing and Analysis Software for Spatial Metabolomics Research.

Siwei Bi, Manjiangcuo Wang, Qianlun Pu, Jinxi Yang, Na Jiang, Xueshan Zhao, Siyuan Qiu, Ruiqi Liu, Renjie Xu, Xia Li, Chenggong Hu, Lie Yang, Jun Gu, Dan Du.

Mass spectrometry imaging (MSI) has emerged as a revolutionary analytical strategy in biomedical research for molecular visualization. By linking the characterization of functional metabolites with tissue architecture, it is now possible to reveal unknown biological functions of tissues. However, due to the complexity and high dimensionality of MSI data, mining bioinformatics-related peaks from batch MSI data sets and achieving complete spatially resolved metabolomics analysis remain a great challenge. Here, we propose novel MSI data processing software, Multi-MSIProcessor (MMP), which integrates the data read-in, MSI visualization, processed data preservation, and biomarker discovery functions. The MMP focuses on the AFADESI-MSI data platform but also supports mzXML and imzmL data input formats for compatibility with data generated by other MSI platforms such as MALDI/SIMS-MSI. MMP enables deep mining of batch MSI data and has flexible adaptability with the source code opened that welcomes new functions and personalized analysis strategies. Using multiple clinical biosamples with complex heterogeneity, we demonstrated that MMP can rapidly establish complete MSI analysis workflows, assess batch sample data quality, screen and annotate differential MS peaks, and obtain abnormal metabolic pathways. MMP provides a novel platform for spatial metabolomics analysis of multiple samples that could meet the diverse analysis requirements of scholars.

DOI: https://doi.org/10.1021/acs.analchem.3c04192
J Pharm Biomed Anal. 2023 Dec 09. pii: S0731-7085(23)00683-0. [Epub ahead of print]239 115914

Development and validation of a LC-MS/MS method for the quantification of phenolic compounds in human saliva after intake of a procyanidin-rich pine bark extract.

Jasmin Bayer, Petra Högger.

  Plant-derived phenolic compounds are regularly ingested as food compounds or as food supplements. Concentrations of individual compounds and metabolites are typically measured in serum or urine samples. This, however, allows no conclusion on the distribution into organs and tissues. An easily accessible biofluid is saliva. At this point, it was not clear yet, whether polyphenols circulating in the blood would be secreted or diffuse into saliva. The purpose of the present study was to develop and validate a method using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for analysis of phenolic compounds in human saliva. Method validation for the quantification of taxifolin, ferulic acid, caffeic acid, gallic acid, para-coumaric acid, and protocatechuic acid and the gut microbial catechin metabolite δ-(3,4-dihydroxyphenyl)-γ-valerolactone (M1) in human saliva was performed according to current guidelines for bioanalytical method validation. The lower limit of quantification ranged from 0.82 ng/ml for M1 to 8.20 ng/ml for protocatechuic acid. The method was successfully applied to an authentic saliva sample of a volunteer after swallowing of procyanidin-rich pine bark extract capsules (dietary supplement Pycnogenol®). All polyphenols except ferulic acid were quantified at concentrations ranging from 1.20 ng/ml (M1) to 10.34 ng/ml (gallic acid). Notably, in contrast to serum samples, all phenolic compounds were present without sulfate or glucuronic acid conjugation in saliva, suggesting an enzymatic deconjugation, e.g., by a β-glucuronidase activity, during compound transfer from serum to saliva. Since M1 is only produced in the gut, its presence in saliva ruled out the possibility of sample contamination by phenolic compounds residing in the oral cavity after food intake. To the best of our knowledge, this is the first time that the gut microbiota-derived metabolite M1 has been detected in saliva. To further investigate the role of phenolic compounds in saliva, the described analytical method can be applied in clinical studies investigating the biodistribution of polyphenols and their metabolites.

Keywords:  Distribution; Human saliva; LC-ESI-MS/MS; Validation

DOI:  https://doi.org/10.1016/j.jpba.2023.115914
Metabolomics. 2023 Dec 23. 20(1): 11

Extended automated quantification algorithm (AQuA) for targeted 1H NMR metabolomics of highly complex samples: application to plant root exudates.

Elin Alexandersson, Corine Sandström, Johan Meijer, Gustav Nestor, Anders Broberg, Hanna E Röhnisch.

  INTRODUCTION: The Automated Quantification Algorithm (AQuA) is a rapid and efficient method for targeted NMR-based metabolomics, currently optimised for blood plasma. AQuA quantifies metabolites from 1D-1H NMR spectra based on the height of only one signal per metabolite, which minimises the computational time and workload of the method without compromising the quantification accuracy.OBJECTIVES: To develop a fast and computationally efficient extension of AQuA for quantification of selected metabolites in highly complex samples, with minimal prior sample preparation. In particular, the method should be capable of handling interferences caused by broad background signals.
METHODS: An automatic baseline correction function was combined with AQuA into an automated workflow, the extended AQuA, for quantification of metabolites in plant root exudate NMR spectra that contained broad background signals and baseline distortions. The approach was evaluated using simulations as well as a spike-in experiment in which known metabolite amounts were added to a complex sample matrix.
RESULTS: The extended AQuA enables accurate quantification of metabolites in 1D-1H NMR spectra with varying complexity. The method is very fast (< 1 s per spectrum) and can be fully automated.
CONCLUSIONS: The extended AQuA is an automated quantification method intended for 1D-1H NMR spectra containing broad background signals and baseline distortions. Although the method was developed for plant root exudates, it should be readily applicable to any NMR spectra displaying similar issues as it is purely computational and applied to NMR spectra post-acquisition.

Keywords:  AQuA; Automated quantification; Baseline correction; NMR; Root exudate; Targeted metabolomics

DOI:  https://doi.org/10.1007/s11306-023-02073-z
Anal Chem. 2023 Dec 19.

Evaluating the Effect of Data Merging and Postacquisition Normalization on Statistical Analysis of Untargeted High-Resolution Mass Spectrometry Based Urinary Metabolomics Data.

Fynn Brix, Tobias Demetrowitsch, Julia Jensen-Kroll, Helena U Zacharias, Silke Szymczak, Matthias Laudes, Stefan Schreiber, Karin Schwarz.

Urine is one of the most widely used biofluids in metabolomic studies because it can be collected noninvasively and is available in large quantities. However, it shows large heterogeneity in sample concentration and consequently requires normalization to reduce unwanted variation and extract meaningful biological information. Biological samples like urine are commonly measured with electrospray ionization (ESI) coupled to a mass spectrometer, producing data sets for positive and negative modes. Combining these gives a more complete picture of the total metabolites present in a sample. However, the effect of this data merging on subsequent data analysis, especially in combination with normalization, has not yet been analyzed. To address this issue, we conducted a neutral comparison study to evaluate the performance of eight postacquisition normalization methods under different data merging procedures using 1029 urine samples from the Food Chain plus (FoCus) cohort. Samples were measured with a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS). Normalization methods were evaluated by five criteria capturing the ability to remove sample concentration variation and preserve relevant biological information. Merging data after normalization was generally favorable for quality control (QC) sample similarity, sample classification, and feature selection for most of the tested normalization methods. Merging data after normalization and the usage of probabilistic quotient normalization (PQN) in a similar setting are generally recommended. Relying on a single analyte to capture sample concentration differences, like with postacquisition creatinine normalization, seems to be a less preferable approach, especially when data merging is applied.

DOI: https://doi.org/10.1021/acs.analchem.3c01380
Anal Chem. 2023 Dec 18.

Hyperpolarization-Enhanced NMR Spectroscopy of Unaltered Biofluids Using Photo-CIDNP.

Lars T Kuhn, Stefan Weber, Joachim Bargon, Teodor Parella, Míriam Pérez-Trujillo.

The direct and unambiguous detection and identification of individual metabolite molecules present in complex biological mixtures constitute a major challenge in (bio)analytical research. In this context, nuclear magnetic resonance (NMR) spectroscopy has proven to be particularly powerful owing to its ability to provide both qualitative and quantitative atomic-level information on multiple analytes simultaneously in a noninvasive manner. Nevertheless, NMR suffers from a low inherent sensitivity and, moreover, lacks selectivity regarding the number of individual analytes to be studied in a mixture of a myriad of structurally and chemically very different molecules, e.g., metabolites in a biofluid. Here, we describe a method that circumvents these shortcomings via performing selective, photochemically induced dynamic nuclear polarization (photo-CIDNP) enhanced NMR spectroscopy on unmodified complex biological mixtures, i.e., human urine and serum, which yields a single, background-free one-dimensional NMR spectrum. In doing this, we demonstrate that photo-CIDNP experiments on unmodified complex mixtures of biological origin are feasible, can be performed straightforwardly in the native aqueous medium at physiological metabolite concentrations, and act as a spectral filter, facilitating the analysis of NMR spectra of complex biofluids. Due to its noninvasive nature, the method is fully compatible with state-of-the-art metabolomic protocols providing direct spectroscopic information on a small, carefully selected subset of clinically relevant metabolites. We anticipate that this approach, which, in addition, can be combined with existing high-throughput/high-sensitivity NMR methodology, holds great promise for further in-depth studies and development for use in metabolomics and many other areas of analytical research.

DOI: https://doi.org/10.1021/acs.analchem.3c03215
Anal Bioanal Chem. 2023 Dec 18.

A liquid chromatography-mass spectrometry method for the enantioselective multiresidue determination of nine chiral agrochemicals in urine using an enrichment procedure based on graphitized carbon black.

Elena Lucci, Lorenzo Antonelli, Monica Gherardi, Chiara Fanali, Salvatore Fanali, Anita Scipioni, Paolo Lupattelli, Alessandra Gentili, Bezhan Chankvetadze.

  Many agrochemicals are chiral molecules, and most of them are marketed as racemates or diastereomeric mixtures. Stereoisomers that are not the active enantiomer have little or no pesticidal activity and can exert serious toxic effects towards non-target organisms. Thus, investigating the possible exposure to different isomers of chiral pesticides is an urgent need. The present work was aimed at developing a new enantioselective high-performance liquid chromatography-mass spectrometry method for the simultaneous determination of nine chiral pesticides in urine. Two solid-phase extraction (SPE) procedures, based on different carbon-based sorbents (graphitized carbon black (GCB) and buckypaper (BP)), were developed and compared. By using GCB, all analytes were recovered with yields ranging from 60 to 97%, while BP allowed recoveries greater than 54% for all pesticides except those with acid characteristics. Baseline separation was achieved for the enantiomers of all target agrochemicals on a Lux Cellulose-2 column within 24 min under reversed-phase mode. The developed method was then validated according to the FDA guidelines for bioanalytical methods. Besides recovery, the other evaluated parameters were precision (7-15%), limits of detection (0.26-2.21 µg/L), lower limits of quantitation (0.43-3.68 µg/L), linear dynamic range, and sensitivity. Finally, the validated method was applied to verify the occurrence of the pesticide enantiomers in urine samples from occupationally exposed workers.

Keywords:  Carbon-based sorbents; Chiral pesticides; Enantioselective separations; HPLC–MS/MS; Solid-phase extraction; Urine

DOI:  https://doi.org/10.1007/s00216-023-05098-4
Anal Methods. 2023 Dec 21.

Trace analysis of steroid hormones in tear films via liquid chromatography-high resolution mass spectrometry.

Zhu Huang.

In this study, we developed an ultra-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) method for the analysis of seven steroid hormones in human tears. Tear samples were collected using Schirmer strips and extracted with methanol. The analytes were then subjected to a "one-step" clean-up process using solid phase extraction, and subsequently separated on a C18 column by UPLC. Detection was performed using an Orbitrap MS detector, operated at a resolution of 17 500 FWHM in parallel reaction monitoring mode with an HESI ion source under positive ionization. Our data showed the sensitivity with limits of detection for steroid hormones in tears ranging from 0.12 to 0.86 pg μL-1, and high correlation coefficients in the corresponding concentration range exceeding 0.99. The results also had high accuracy with spiking recoveries for spiked tear samples ranging from 78.2% to 96.7% and relative deviations of less than 15%. Furthermore, we successfully applied our method to detect the pg μL-1 level of steroid hormones in real human tear samples. Our findings showed the potential of this UPLC-Q-Orbitrap-MS method for the accurate and sensitive determination of steroid hormones in human tears, providing a valuable tool for ophthalmic and endocrine research.

DOI: https://doi.org/10.1039/d3ay01583a
Crit Rev Anal Chem. 2023 Dec 22. 1-14

Mycotoxins in Vegetable Oils: A Review of Recent Developments, Current Challenges and Future Perspectives in Sample Preparation, Chromatographic Determination, and Analysis of Real Samples.

Basil K Munjanja, Philiswa N Nomngongo, Nomvano Mketo.

  Mycotoxins are toxic compounds that are formed as secondary metabolites by some fungal species that contaminate crops during pre- and postharvest stages. Exposure to mycotoxins can lead to adverse health effects in humans, such as carcinogenicity, mutagenicity, and teratogenicity. Hence, there is a need to develop analytical methods for their determination in vegetable oils that possess high sensitivity and selectivity. In the current review (116 references), the recent developments, current challenges, and perspectives in sample preparation techniques and chromatographic determination are summarized. It is impressive that current sample preparation techniques such as dispersive liquid-liquid microextraction (DLLME), quick, easy, cheap, rugged, and safe method (QuEChERS) and solid phase extraction (SPE) have exhibited high extraction recoveries and minimal matrix effects. However, a few studies have reported signal suppression or enhancement. Regarding chromatographic techniques, high sensitivity and selectivity have been reported by liquid chromatography coupled to fluorescence detection, tandem mass spectrometry, or high-resolution mass spectrometry. Furthermore, current challenges and perspectives in this field are tentatively proposed.

Keywords:  Mycotoxins; chromatographic separation; real samples; sample preparation; sorbent materials; vegetable oils

DOI:  https://doi.org/10.1080/10408347.2023.2286642
J Forensic Sci. 2023 Dec 22.

Quantification of psilocin in human whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Munchelou M Gomonit, Britni Skillman, Madeleine J Swortwood.

  There has been burgeoning interest in psilocybin-use for the treatment of various neurological and neurodegenerative diseases. Psilocybin is mistakenly perceived as the principal pharmacologically active compound due to its high concentrations found in magic mushrooms; however, it is the prodrug of psilocin. Despite the expanding body of clinical research seeking to understand the pharmacodynamic/pharmacokinetic properties of psilocin, and its role in inducing dramatic changes to cognitive function, there has not been a corresponding increase in the development of sensitive analytical methods that can quantify psilocin in different biological fluids. Existing analytical methods have been developed using plasma, serum, and urine as the matrix of choice, but with the unknown blood-to-plasma ratio of psilocin, any pharmacokinetic conclusions drawn solely on plasma data may be misleading. Thus, the main objective of this study is to develop the first analytical method that utilizes SPE and LC-MS/MS to quantify psilocin in human whole blood. The SPE procedure yielded a high recovery efficiency (≥89%) with minimal matrix effects. The method was validated according to ANSI/ASB 036 guidelines. Linearity was between 0.7-200 ng/mL and encompassed previously reported ranges found in plasma/serum. Bias, within- and between-run precision for all quality controls met ANSI/ASB 036 acceptability criteria. Endogenous/exogenous interferences and carryover were negligible. Psilocin stability was assessed at 4°C over 48 h and was considered stable. Although a proof-of-concept study will need to be performed to characterize the method, this analytical workflow was able to detect and quantify psilocin in human whole blood at low limits of quantification.

Keywords:  forensic toxicology; hallucinogen; liquid chromatography-tandem mass spectrometry and LC-MS/MS; psilocin in human whole blood; psilocybin

DOI:  https://doi.org/10.1111/1556-4029.15454
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Dec 09. pii: S1570-0232(23)00374-4. [Epub ahead of print]1232 123964

Development and validation of an LC-MS/MS method for the quantification of KRASG12C inhibitor opnurasib in several mouse matrices and its application in a pharmacokinetic mouse study.

Irene A Retmana, Nefise Çelebi, Jamie Rijmers, Alfred H Schinkel, Jos H Beijnen, Rolf W Sparidans.

  Opnurasib (JDQ-443) is a highly potent and promising KRASG12C inhibitor that is currently under clinical investigation. Results of the ongoing clinical research demonstrated the acceptable safety profile and clinical activity of this drug candidate as a single agent for patients with NSCLC harboring KRASG12C mutations. In this early stage of development, a deeper insight into pharmacokinetic properties in both preclinical and clinical investigations of this drug is very important. Thus, a reliable quantification method is required. To date, no quantitative bioanalytical assay of opnurasib was publicly available. In this study we present a validated assay to quantify opnurasib in mouse plasma and eight mouse tissue-related matrices utilizing liquid chromatography-tandem mass spectrometry. Erlotinib was used as internal standard and acetonitrile was utilized to treat 10 µl of the sample with protein precipitation in a 96-well plate format. Separation and detection were achieved using a BEH C18 column under basic chromatographic conditions and a triple quadrupole mass spectrometer, respectively. We have fully validated this assay for mouse plasma and partially for eight mouse tissue-related matrices over the range of 2-2000 ng/ml. The accuracy and precision of the assay fulfilled international guidelines (EMA & U.S. FDA) over the validated range. The method was proven selective and sensitive to quantify opnurasib down to 2 ng/ml in all investigated matrices. The recoveries of both analyte and internal standard in mouse plasma were ∼100 % with no significant matrix effect in any of the matrices. Opnurasib in mouse plasma was stable up to 12 h at room temperature, and up to 8 h at room temperature in tissue homogenates (except for kidney up to 4 h). This presented method has been successfully applied to quantify opnurasib in preclinical samples from a mouse study and demonstrated its usability to support preclinical pharmacokinetic studies.

Keywords:  Bioanalysis; KRAS(G12C) inhibitor; LC-MS/MS; Mouse matrices; Opnurasib

DOI:  https://doi.org/10.1016/j.jchromb.2023.123964
J Sep Sci. 2023 Dec 20. e2300576

A robust method for simultaneous determination of eight B vitamins in human serum by liquid chromatography tandem mass spectrometry.

Yu-Mei Huo, Shang-Qing Zhang, Gao-Ping Wu, Hong-Bo Shan, Chao Pan.

  The level of vitamin B group in human serum is an important index of human health. Among B vitamins, cyanocobalamin in serum is unstable and its content is extremely low. Rapid and simultaneous detection of multiple B vitamins including cyanocobalamin is a challenge. Herein, we have developed a rapid and stable method that can realize the determination of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin simultaneously in 6 min. The method was established based on protein precipitation with methanol and then chromatographic separation was achieved using Waters acquity ultra-high-performance liquid chromatography high strength silica T3 column, which was stable and sensitive especially for cyanocobalamin. Limit of quantification, precision, trueness, and matrix effect were validated according to the European Medicines Agency and United States Food and Drug guidelines and Clinical and Laboratory Standards Institute guidelines on bioanalytical method. The limit of quantification for thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin was 0.4, 0.4, 0.8, 2.0, 0.4, 0.1, 0.4, and 0.04 ng/mL separately, respectively. Intra- and interday precisions were 1.1%-12.4% and 2.0%-13.5%, respectively. The relative errors were between 0.3% and 13.3%, and the matrix effects were between 2.6% and 10.4%.

Keywords:  simultaneous determination; ultra-high-performance liquid chromatography; vitamin B

DOI:  https://doi.org/10.1002/jssc.202300576
J Forensic Leg Med. 2023 Dec 15. pii: S1752-928X(23)00154-3. [Epub ahead of print]101 102636

A comprehensive LC-MS/MS method for simultaneous analysis of 65 synthetic cannabinoids in human hair samples and application to forensic investigations.

Yiqi Yang, Buyi Xu, Daoxia Li, Qifu Zhang, Jing Zhang, Lin Yang, Yi Ye.

  Synthetic cannabinoids (SCs) represent a diverse class of new psychoactive substances characterized by extensive substance variety and severe abuse implications. The current situation of synthetic cannabinoid abuse in China is getting worse, with an increasing number of SC variants emerging. Therefore, it is imperative to improve synthetic cannabinoid detecting methods to align with the prevalent abuse situation in the region. In this study, a reliable and validated liquid chromatography-tandem mass spectrometry method was developed for the qualitative and quantitative analysis of 65 SC analogues in human hair samples. The validation results demonstrated satisfactory linearity (r ≥ 0.99) within the range of 25-2500 pg/mg for each SC analogue. The method exhibited limits of detection ranging from 10 to 15 pg/mg and limits of quantification ranging from 25 to 40 pg/mg. The relative standard deviations of intra-day precision and inter-day precision were below 15 %. Furthermore, negligible matrix effects were observed, with recovery rates ranging from 85.70 % to 119.43 %. Analysis of abuser demographics revealed that the primary group engaged in SC analogue abuse consisted of adolescents, predominantly males, accounting for 79.5 % of cases. Among the suspected individuals, ADB-BUTINACA and MDMB-4en-PINACA were the most frequently detected substances. The present study develops a highly sensitive analytical method and provides a comprehensive overview of the prevalence of SC abuse in the eastern region of China.

Keywords:  Hair sample; LC-MS/MS; Quantitative analysis; Synthetic cannabinoids

DOI:  https://doi.org/10.1016/j.jflm.2023.102636
Adv Lab Med. 2023 Dec;4(4): 365-371

Optimization of a rapid method for screening drugs in blood by liquid chromatography tandem mass spectrometry.

Alba M Rodrigo Valero, Oscar Quintela Jorge, Begoña Bravo Serrano, Sara Ayuso Tejedor.

  Objectives: In the recent years, liquid chromatography with tandem mass spectrometry has gained popularity in laboratories. This technique has a higher specificity, detects different analytes from a single specimen, measures analytes in distinct matrices, and substantially reduce analytical interference, with respect to immunoassay. The processing and preparation of biological samples are crucial in chromatography. Interferences in blood testing are usually caused by the presence of phospholipids and proteins. The main objective of this study was to improve analytical processes for drug screening by LC-MS/MS using a novel blood sample preparation method based on protein precipitation and removal of phospholipids.Methods: An evaluation was performed of a new method for the preparation of blood samples based on protein precipitation and removal of phospholipids by LC-Q-q-LIT.
Results: Limit of detection, limit of quantification and measurement range were determined for 56 molecules. The results of 11 cases were compared with those obtained using standard blood collection methods and instruments.
Conclusions: The novel blood preparation and testing method based on LC-Q-q-LIT, a more sensitive technique, has demonstrated to yield comparable results to traditional methods. In addition, this new technique reduces turnaround time and costs.

Keywords:  blood; mass spectrometry; protein precipitation; removal of phospholipids; screening

DOI:  https://doi.org/10.1515/almed-2023-0154
Front Microbiol. 2023 ;14 1295994

Untargeted metabolomics to expand the chemical space of the marine diatom Skeletonema marinoi.

Mahnoor Zulfiqar, Daniel Stettin, Saskia Schmidt, Vera Nikitashina, Georg Pohnert, Christoph Steinbeck, Kristian Peters, Maria Sorokina.

  Diatoms (Bacillariophyceae) are aquatic photosynthetic microalgae with an ecological role as primary producers in the aquatic food web. They account substantially for global carbon, nitrogen, and silicon cycling. Elucidating the chemical space of diatoms is crucial to understanding their physiology and ecology. To expand the known chemical space of a cosmopolitan marine diatom, Skeletonema marinoi, we performed High-Resolution Liquid Chromatography-Tandem Mass Spectrometry (LC-MS2) for untargeted metabolomics data acquisition. The spectral data from LC-MS2 was used as input for the Metabolome Annotation Workflow (MAW) to obtain putative annotations for all measured features. A suspect list of metabolites previously identified in the Skeletonema spp. was generated to verify the results. These known metabolites were then added to the putative candidate list from LC-MS2 data to represent an expanded catalog of 1970 metabolites estimated to be produced by S. marinoi. The most prevalent chemical superclasses, based on the ChemONT ontology in this expanded dataset, were organic acids and derivatives, organoheterocyclic compounds, lipids and lipid-like molecules, and organic oxygen compounds. The metabolic profile from this study can aid the bioprospecting of marine microalgae for medicine, biofuel production, agriculture, and environmental conservation. The proposed analysis can be applicable for assessing the chemical space of other microalgae, which can also provide molecular insights into the interaction between marine organisms and their role in the functioning of ecosystems.

Keywords:  Skeletonema; Skeletonema marinoi; chemical classification; diatom; metabolome annotation; untargeted metabolomics

DOI:  https://doi.org/10.3389/fmicb.2023.1295994
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Dec 12. pii: S1570-0232(23)00373-2. [Epub ahead of print]1232 123963

Non-targeted analysis of lipidic extracts by high-resolution mass spectrometry to characterise the chemical exposome: Comparison of four clean-up strategies applied to egg.

Antonin Padioleau, Ronan Cariou, Ingrid Guiffard, Bruno Le Bizec, Beate I Escher, Jean-Philippe Antignac, Gaud Dervilly.

  Biota samples are used to monitor chemical stressors and their impact on the ecosystem and to describe dietary chemical exposure. These complex matrices require an extraction step followed by clean-up to avoid damaging sensitive analytical instruments based on chromatography coupled to mass spectrometry. While interest for non-targeted analysis (NTA) is increasing, there is no versatile or generic sample preparation for a wide range of contaminants suitable for a diversity of biotic matrices. Among the contaminants' variety, persistent contaminants are mostly hydrophobic (mid- to non-polar) and bio-magnify through the lipidic fraction. During their extraction, lipids are generally co-extracted, which may cause matrix effect during the analysis such as hindering the acquired signal. The aim of this study was to evaluate the efficacy of four clean-up methods to selectively remove lipids from extracts prior to NTA. We evaluated (i) gel permeation chromatography (GPC), (ii) Captiva EMR-lipid cartridge (EMR), (iii) sulphuric acid degradation (H2SO4) and (iv) polydimethyl siloxane (PDMS) for their efficiency to remove lipids from hen egg extracts. Gas and liquid chromatography coupled with high-resolution mass spectrometry fitted with either electron ionisation or electrospray ionisation sources operating in positive and negative modes were used to determine the performances of the clean-up methods. A set of 102 chemicals with a wide range of physico-chemical properties that covers the chemical space of mid- to non-polar contaminants, was used to assess and compare recoveries and matrix effects. Matrix effects, that could hinder the mass spectrometer signal, were lower for extracts cleaned-up with H2SO4 than for the ones cleaned-up with PDMS, EMR and GPC. The recoveries were satisfactory for both GPC and EMR while those determined for PDMS and H2SO4 were low due to poor partitioning and degradation/dissociation of the compounds, respectively. The choice of the clean-up methods, among those assessed, should be a compromise that takes into account the matrix under consideration, the levels and the physico-chemical properties of the contaminants.

Keywords:  Clean-up; High-resolution mass spectrometry; Lipid removal; Matrix effects; Non-targeted analysis; Recoveries

DOI:  https://doi.org/10.1016/j.jchromb.2023.123963
Metabolomics. 2023 Dec 19. 20(1): 7

Improving quality of analysis by suppression of unwanted signals through band-selective excitation in NMR spectroscopy for metabolomics studies.

Upendra Singh, Ruba Al-Nemi, Fatimah Alahmari, Abdul-Hamid Emwas, Mariusz Jaremko.

  INTRODUCTION: Nuclear Magnetic Resonance (NMR) spectroscopy stands as a preeminent analytical tool in the field of metabolomics. Nevertheless, when it comes to identifying metabolites present in scant amounts within various types of complex mixtures such as plants, honey, milk, and biological fluids and tissues, NMR-based metabolomics presents a formidable challenge. This predicament arises primarily from the fact that the signals emanating from metabolites existing in low concentrations tend to be overshadowed by the signals of highly concentrated metabolites within NMR spectra.OBJECTIVES: The aim of this study is to tackle the issue of intense sugar signals overshadowing the desired metabolite signals, an optimal pulse sequence with band-selective excitation has been proposed for the suppression of sugar's moiety signals (SSMS). This sequence serves the crucial purpose of suppressing unwanted signals, with a particular emphasis on mitigating the interference caused by sugar moieties' signals.
METHODS: We have implemented this comprehensive approach to various NMR techniques, including 1D 1H presaturation (presat), 2D J-resolved (RES), 2D 1H-1H Total Correlation Spectroscopy (TOCSY), and 2D 1H-13C Heteronuclear Single Quantum Coherence (HSQC) for the samples of dates-flesh, honey, a standard stock solution of glucose, and nine amino acids, and commercial fetal bovine serum (FBS).
RESULTS: The outcomes of this approach were significant. The suppression of the high-intensity sugar signals has considerably enhanced the visibility and sensitivity of the signals emanating from the desired metabolites.
CONCLUSION: This, in turn, enables the identification of a greater number of metabolites. Additionally, it streamlines the experimental process, reducing the time required for the comparative quantification of metabolites in statistical studies in the field of metabolomics.

Keywords:  Band-selective excitation; Fingerprint; Metabolomics; NMR; Sugars

DOI:  https://doi.org/10.1007/s11306-023-02069-9
Anal Bioanal Chem. 2023 Dec 23.

Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay.

Noriyuki Tomiyasu, Masatomo Takahashi, Kenji Toyonaga, Sho Yamasaki, Takeshi Bamba, Yoshihiro Izumi.

  C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors.

Keywords:  Immune receptor; LC-HRMS/MS; Lipid ligand; Lipidomics; Microfractionation; Reporter cell assay

DOI:  https://doi.org/10.1007/s00216-023-05111-w
Toxics. 2023 Dec 13. pii: 1015. [Epub ahead of print]11(12):

Method Validation for Quantification of PFOS and PFOA in Human Plasma and a Pilot Study in Blood Donors from Thai Red Cross Society.

Teerapong Lertassavakorn, Nanthanit Pholphana, Nuchanart Rangkadilok, Tawit Suriyo, Punthip Teeyapant, Jutamaad Satayavivad.

  Information regarding per- and polyfluorinated substances concentrations in biological samples from the Thai population was still lacking. A sensitive bioanalytical method was developed and validated for the quantification of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) levels in human plasma. Simple protein precipitation and LC-MS/MS techniques were used with stable isotope internal standards of 13C8-PFOS and 13C8-PFOA. The validated method followed the ICH bioanalytical validation guideline, and the results showed good accuracy, precision, and reproducibility. The validated analytical method was then applied to determine PFOS and PFOA concentrations in 50 human plasma samples from the National Blood Center, Thai Red Cross Society. The concentrations were found to be in ranges of <0.91-6.27 ng/mL for PFOS and <0.49-2.72 ng/mL for PFOA. PFOS was also measured separately for its isomers, and the geometric means of the linear isomer (L-PFOS) and branched isomer (br-PFOS) in plasma samples were at 1.85 and 0.41 ng/mL, respectively. Both PFOS and PFOA concentrations were lower in comparison to previous reports from other countries. The present study showed the application of our reliable method to determine PFOS and PFOA in biological samples in order to monitor the human exposure of both chemicals in Thailand.

Keywords:  LC–MS/MS; PFOA; PFOS; human plasma

DOI:  https://doi.org/10.3390/toxics11121015
Anal Chem. 2023 Dec 22.

Spatialized Metabolomic Annotation Combining MALDI Imaging and Molecular Networks.

Marceau Levasseur, Edith Nicol, Nicolas Elie, Emeline Houël, Véronique Eparvier, David Touboul.

MALDI mass spectrometry imaging has gained major interest in the field of chemical imaging. This technique makes it possible to locate tens to hundreds of ionic signals on the sample surface without any a priori. One of the current challenges is still the limited ability to annotate signals in order to convert m/z values into probable chemical structures. At the same time, data obtained by LC-MS/MS have benefited from the development of numerous chemoinformatics tools, in particular molecular networks, for their efficient annotation. For the first time, we present here the combination of MALDI-FT-ICR imaging with molecular networks from MALDI-MS/MS data directly acquired on plant tissue sections. Annotation improvements are demonstrated, paving the way for new annotation pipelines for MALDI imaging.

DOI: https://doi.org/10.1021/acs.analchem.3c03482
Rapid Commun Mass Spectrom. 2024 Jan 30. 38(2): e9670

Liquid chromatography-mass spectrometry-based metabolomics and fluxomics reveals the metabolic alterations in glioma U87MG multicellular tumor spheroids versus two-dimensional cell cultures.

Shanjing Wen, Xinyi Tu, Qingce Zang, Ying Zhu, Limei Li, Ruiping Zhang, Zeper Abliz.

RATIONALE: Multicellular tumor spheroids (MCTSs) that reconstitute the metabolic characteristics of in vivo tumor tissue may facilitate the discovery of molecular biomarkers and effective anticancer therapies. However, little is known about how cancer cells adapt their metabolic changes in complex three-dimensional (3D) microenvironments. Here, using the two-dimensional (2D) cell model as control, the metabolic phenotypes of glioma U87MG multicellular tumor spheroids were systematically investigated based on static metabolomics and dynamic fluxomics analysis.METHODS: A liquid chromatography-mass spectrometry-based global metabolomics and lipidomics approach was adopted to survey the cellular samples from 2D and 3D culture systems, revealing marked molecular differences between them. Then, by means of metabolomic pathway analysis, the metabolic pathways altered in glioma MCTSs were found using 13 C6 -glucose as a tracer to map the metabolic flux of glycolysis, the tricarboxylic acid (TCA) cycle, de novo nucleotide synthesis, and de novo lipid biosynthesis in the MCTS model.
RESULTS: We found nine metabolic pathways as well as glycerolipid, glycerophospholipid and sphingolipid metabolism to be predominantly altered in glioma MCTSs. The reduced nucleotide metabolism, amino acid metabolism and glutathione metabolism indicated an overall lower cellular activity in MCTSs. Through dynamic fluxomics analysis in the MCTS model, we found that cells cultured in MCTSs exhibited increased glycolysis activity and de novo lipid biosynthesis activity, and decreased the TCA cycle and de novo purine nucleotide biosynthesis activity.
CONCLUSIONS: Our study highlights specific, altered biochemical pathways in MCTSs, emphasizing dysregulation of energy metabolism and lipid metabolism, and offering novel insight into metabolic events in glioma MCTSs.

DOI: https://doi.org/10.1002/rcm.9670