bims-metlip 2024-09-22 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024‒09‒22
thirty-two papers selected by
Sofia Costa, Matterworks

Development and validation of an LC-MS/MS method for simultaneous quantification of eight drugs in plasma and brain: Application in a pharmacokinetic study in mice.
An Untargeted Lipidomics Workflow Incorporating High-Resolution Demultiplexing (HRdm) Drift Tube Ion Mobility-Mass Spectrometry.
Clearing the plate: a strategic approach to mitigate well-to-well contamination in large-scale microbiome studies.
Quantification of mycolic acids in different mycobacterial species by standard addition method through liquid chromatography mass spectrometry.
Comprehensive determination of fatty acids in real samples without derivatization by DMU-SPME-GC methods.
Simultaneous determination of canonical purine metabolism using a newly developed HILIC-MS/MS in cultured cells.
Multianalyte Approach-Including Automated Preparation of Calibrators-for Validated Quantification of 82 Drugs in Whole Blood by Liquid Chromatography-Tandem Mass Spectrometry.
Magnetic solid-phase extraction coupled with LC-MS/MS methods for the simple extraction and rapid determination of sugammadex in human plasma.
Application of a molecular networking approach using LC-HRMS combined with the MetWork webserver for clinical and forensic toxicology.
Streamlining Phenotype Classification and Highlighting Feature Candidates: A Screening Method for Non-Targeted Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS) Data.
Absolute Quantification of Glutathione Using Top-Hat Optics for IR-MALDESI Mass Spectrometry Imaging.
Correlative Imaging for Comprehensive Molecular Mapping of Individual Cell Types in Biological Tissues.
Understanding isotopes, isomers, and isobars in mass spectrometry.
Recent advances in liquid chromatography-tandem mass spectrometry for the detection of thyroid hormones and thyroglobulin in clinical samples: A review.
The optimization and validation of a gas chromatography-mass spectrometry method to analyze the concentration of acetate, propionate and butyrate in human plasma or serum.
Development and validation of a highly sensitive UPLC-MS/MS method for the determination of Huperzine A in rat plasma.
Supercritical fluid chromatography- Nanospray ionization-mass spectrometry (SFC-nSI-MS).
An interactive R-based custom quantification program for semi-quantitative analysis of triacylglycerols in bovine milk.
Simultaneous determination of glyphosate, glufosinate and their metabolites in soybeans using solid-phase analytical derivatization and LC-MS/MS determination.
Isotope-dilution-LC-MS/MS candidate reference measurement procedure for cefepime in human serum.
Bioinformatics-driven untargeted metabolomic profiling for clinical screening of methamphetamine abuse.
LipidOne 2.0: A Web Tool for Discovering Biological Meanings Hidden in Lipidomic Data.
Rapid Analytical Method for Quantification of Gamma-Hydroxybutyrate (GHB) in Hair by UPLC-MS/MS.
Faster ciguatoxin extraction methods for toxicity screening.
Digestion-Free Middle-Down Mass Spectrometry Method for Absolute Quantification of Conjugated Payload from Antibody-Drug Conjugates.
How storage post sampling influences the stability of sebum when used for mass spectrometry metabolomics analysis?
Technologies for Decoding Cancer Metabolism with Spatial Resolution.
Application of newly developed and validated UPLC-MS/MS method for pharmacokinetic study of ROS1/NTRK inhibitor taletrectinib in beagle dog plasma.
Development and validation of a green analytical method for determining fourteen bisphenols in bee pollen by ultra-high-performance liquid chromatography-tandem mass spectrometry.
UPLC-PDA factorial design assisted method for simultaneous determination of oseltamivir, dexamethasone, and remdesivir in human plasma.
A streamlined, resource-efficient immunoprecipitation-mass spectrometry method for quantifying plasma amyloid-β biomarkers in Alzheimer's disease.
The GenoPred Pipeline: A Comprehensive and Scalable Pipeline for Polygenic Scoring.

J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 12. pii: S1570-0232(24)00317-9. [Epub ahead of print]1247 124308

Development and validation of an LC-MS/MS method for simultaneous quantification of eight drugs in plasma and brain: Application in a pharmacokinetic study in mice.

Aristeidis Lentzas, Nikkie Venekamp, Jos H Beijnen, Olaf van Tellingen.

  A selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous quantitation of a cassette of 8 drugs, including docetaxel, erlotinib, loperamide, riluzole, vemurafenib, verapamil, elacridar and tariquidar. Stable isotopically labeled compounds were available for use as internal standards for all compounds, except for tariquidar for which we used elacridar-d4. Sample pre-treatment involved liquid-liquid extraction using tert-butyl-methyl ether as this resulted in good recovery and low ion suppression. Chromatographic separation was achieved using a Zorbax Extend C18 analytical column and a linear gradient from 20 % to 95 % methanol in 0.1 % (v/v) formic acid in water. MS/MS detection using multiple reaction monitoring was done in positive ionization mode. We validated this assay for human and mouse plasma and mouse brain homogenates. The calibration curves were linear over a range 1-200 nM for each drug in the mix, except for tariquidar probably due to the lack of a stable isotope labeled analog. The intra-day and inter-day accuracies were within the 85-115 % range for all compounds at low, medium and high concentrations in the three different matrices. Similarly, the precision for all compounds at three different concentration levels ranged below 15 %, with the exception of tariquidar in mouse plasma and brain homogenate and riluzole in brain homogenate. Pilot studies have confirmed that the method is suitable for the analysis of mouse plasma samples and brain homogenates following cassette dosing of this mixture in mice.

Keywords:  Brain homogenate; Cassette quantitation; Human plasma; LC-MS/MS method validation; Liquid–liquid extraction; Mouse plasma

DOI:  https://doi.org/10.1016/j.jchromb.2024.124308
J Am Soc Mass Spectrom. 2024 Sep 14.

An Untargeted Lipidomics Workflow Incorporating High-Resolution Demultiplexing (HRdm) Drift Tube Ion Mobility-Mass Spectrometry.

David C Koomen, Jody C May, Alexander J Mansueto, Todd R Graham, John A McLean.

  Global discovery lipidomics can provide comprehensive chemical information toward understanding the intricacies of metabolic lipid disorders such as dyslipidemia; however, the isomeric complexity of lipid species remains an analytical challenge. Orthogonal separation strategies, such as ion mobility (IM), can be inserted into liquid chromatography-mass spectrometry (LC-MS) untargeted lipidomic workflows for additional isomer separation and high-confidence annotation, and the emergence of high-resolution ion mobility (HRIM) strategies provides marked improvements to the resolving power (Rp > 200) that can differentiate small structural differences characteristic of isomers. One such HRIM strategy, high-resolution demultiplexing (HRdm), utilizes multiplexed drift tube ion mobility spectrometry (DTIMS) with post-acquisition algorithmic deconvolution to access high IM resolutions while retaining the measurement precision inherent to the drift tube technique; however, HRdm has yet to be utilized in untargeted studies. In this manuscript, a proof-of-concept study using ATP10D dysfunctional murine models was investigated to demonstrate the utility of HRdm-incorporated untargeted lipidomic analysis pipelines. Total lipid features were found to increase by 2.5-fold with HRdm compared to demultiplexed DTIMS as a consequence of more isomeric lipids being resolved. An example lipid, PC 36:5, was found to be significantly higher in dysfunctional ATP10D mice with two resolved peaks observed by HRdm that were absent in both the functional ATP10D mice and the standard demultiplexed DTIMS acquisition mode. The benefits of utilizing HRdm for discerning isomeric lipids in untargeted workflows have the potential to enhance our analytical understanding of lipids related to disease complexity and biologically relevant studies.

Keywords:  HRIM; Hadamard transform; murine dyslipidemia; peak deconvolution; untargeted lipidomics

DOI:  https://doi.org/10.1021/jasms.4c00251
mSystems. 2024 Sep 16. e0098524

Clearing the plate: a strategic approach to mitigate well-to-well contamination in large-scale microbiome studies.

Caitriona Brennan, Pedro Belda-Ferre, Simone Zuffa, Vincent Charron-Lamoureux, Ipsita Mohanty, Gail Ackermann, Celeste Allaband, Madison Ambre, Tara Boyer, MacKenzie Bryant, Kalen Cantrell, Antonio Gonzalez, Daniel McDonald, Rodolfo A Salido, Se Jin Song, Gillian Wright, Pieter C Dorrestein, Rob Knight.

  Large-scale studies are essential to answer questions about complex microbial communities that can be extremely dynamic across hosts, environments, and time points. However, managing acquisition, processing, and analysis of large numbers of samples poses many challenges, with cross-contamination being the biggest obstacle. Contamination complicates analysis and results in sample loss, leading to higher costs and constraints on mixed sample type study designs. While many researchers opt for 96-well plates for their workflows, these plates present a significant issue: the shared seal and weak separation between wells leads to well-to-well contamination. To address this concern, we propose an innovative high-throughput approach, termed as the Matrix method, which employs barcoded Matrix Tubes for sample acquisition. This method is complemented by a paired nucleic acid and metabolite extraction, utilizing 95% (vol/vol) ethanol to stabilize microbial communities and as a solvent for extracting metabolites. Comparative analysis between conventional 96-well plate extractions and the Matrix method, measuring 16S rRNA gene levels via quantitative polymerase chain reaction, demonstrates a notable decrease in well-to-well contamination with the Matrix method. Metagenomics, 16S rRNA gene amplicon sequencing (16S), and untargeted metabolomics analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the Matrix method recovers reproducible microbial and metabolite compositions that can distinguish between subjects. This advancement is critical for large-scale study design as it minimizes well-to-well contamination and technical variation, shortens processing times, and integrates with automated infrastructure for enhancing sample randomization and metadata generation.IMPORTANCE: Understanding dynamic microbial communities typically requires large-scale studies. However, handling large numbers of samples introduces many challenges, with cross-contamination being a major issue. It not only complicates analysis but also leads to sample loss and increased costs and restricts diverse study designs. The prevalent use of 96-well plates for nucleic acid and metabolite extractions exacerbates this problem due to their wells having little separation and being connected by a single plate seal. To address this, we propose a new strategy using barcoded Matrix Tubes, showing a significant reduction in cross-contamination compared to conventional plate-based approaches. Additionally, this method facilitates the extraction of both nucleic acids and metabolites from a single tubed sample, eliminating the need to collect separate aliquots for each extraction. This innovation improves large-scale study design by shortening processing times, simplifying analysis, facilitating metadata curation, and producing more reliable results.

Keywords:  cross-contamination; large-scale studies; metabolomics; microbiome; well-to-well contamination

DOI:  https://doi.org/10.1128/msystems.00985-24
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 10. pii: S1570-0232(24)00306-4. [Epub ahead of print]1247 124297

Quantification of mycolic acids in different mycobacterial species by standard addition method through liquid chromatography mass spectrometry.

Zeeshan Fatima, Meenakshi Chugh, Gaurav Nigam, Saif Hameed.

  Mycobacteria possess unique and robust lipid profile responsible for their pathogenesis and drug resistance. Mycolic acid (MA) represents an attractive diagnostic biomarker being absent in humans, inert and known to modulate host-pathogen interaction. Accurate measurement of MA is significant to design efficient therapeutics. Despite considerable advances in Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based approaches, quantification of mycobacterial lipids including MA is still challenging mainly because of ion suppression effects due to complex matrix and non-availability of suitable internal standards for MA. The current study demonstrates the use of standard addition method (SAM) to circumvent this problem and provides a reliable and exhaustive analytical method to quantify mycobacterial MA based on reversed-phase ultra-high-performance liquid chromatography- mass spectrometry data acquisition. In this method, multiple reaction monitoring (MRM) has been applied, wherein 16 MRM channels or transitions have been chosen for quantification of alpha-, methoxy- and keto-MAs with C-24 and C-26 hydrocarbon chains that are actually best suited for TB diagnostics. We found that the overall methodological limit of detection and limit of quantification were in the range 0.05-0.71 ng/µl and 0.16-2.16 ng/µl. Taken together, SAM quantitative technique could serve as promising alternative for relative concentration determination of MA to aid medical research.

Keywords:  LCMS; Multiple reaction monitoring; Mycobacterium tuberculosis; Mycolic acids; Standard addition method; Tuberculosis

DOI:  https://doi.org/10.1016/j.jchromb.2024.124297
Food Res Int. 2024 Nov;pii: S0963-9969(24)01056-1. [Epub ahead of print]195 114986

Comprehensive determination of fatty acids in real samples without derivatization by DMU-SPME-GC methods.

Yun-Jiao Ma, Ping Li, Bei-Wei Zhu, Ming Du, Xian-Bing Xu.

  The comprehensive determination of fatty acids without derivatization, including short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs) and long-chain fatty acids (LCFAs), is a big challenge but powerful for lipidomics in biology, food, and environment. Herein, the dual mode unity solid-phase microextraction (DMU-SPME) combined with gas chromatography-flame ionization detector (GC-FID) or mass spectrometry (MS) was proposed as a powerful method for the determination of comprehensive free fatty acids in real samples. Under the optimized DMU-SPME conditions, the proposed method has good linearity (R2 ≥ 0.994) and low limits of determination (0.01-0.14 mg/L). In the stability analysis, the intra-day relative standard deviation was 1.39-12.43 %, and the inter-day relative standard deviation was 2.84-10.79 %. The recoveries of selected 10 fatty acids in real samples ranged from 90.18 % to 110.75 %, indicating that the method has good accuracy. Fatty acids ranging from C2 to C22 were detected in real samples by the untargeted determination method of DMU-SPME combined with gas chromatography-mass spectrometry (GC-MS). The DMU-SPME method proposed in this study can be used for lipid metabolism analysis and free fatty acid determination in the fields of biology, food, and environment.

Keywords:  Dual mode unity solid-phase microextraction; Fatty acids; GC–MS

DOI:  https://doi.org/10.1016/j.foodres.2024.114986
J Pharm Biomed Anal. 2024 Sep 10. pii: S0731-7085(24)00508-9. [Epub ahead of print]252 116468

Simultaneous determination of canonical purine metabolism using a newly developed HILIC-MS/MS in cultured cells.

Ayinazhaer Aihemaiti, Yuqing Liu, Peichen Zou, Hongyu Liu, Liang Zhu, Yabin Tang.

  Purine metabolism acts as the core role in human metabolic network. It offers purine metabolites as raw material for building blocks in cell survival and proliferation. Purine metabolites are the most abundant metabolic substrates in organisms. There are few reports to simultaneously quantify canonical purine metabolism in cells. A novel hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS/MS) method was developed to simultaneously determine purines profile in biological samples. Chromatographic separation was achieved using a HILIC (Waters Xbridge™ Amide) column. Different optimizing chromatographic conditions and mass spectrometric parameters were tested in order to provide the best separation and the lowest limit of quantification (LLOQ) values for targeted metabolites. The validation was evaluated according to the Food and Drug Administration guidelines. The limit of determination (LOD) and the LOQ values were in the range of 0.02-8.33 ng mL-1 and 0.1-24.5 ng mL-1, respectively. All calibration curves displayed good linear relationship of with excellent correlation coefficient (r) ranging from 0.9943 to 0.9999. Both intra-day and inter-day variability were below 15 %, respectively. Trueness, expressed as relative error, was always within ±15 %. In addition, no derivatization procedure and ion-pair reagents are in need. The innovated approach demonstrates high sensitivity, strong specificity, and good repeatability, making it suitable for absolute quantitative studies of canonical purine metabolism in cultured cells.

Keywords:  Cultured cells; HILIC-MS/MS; Purine metabolism; Simultaneous determination

DOI:  https://doi.org/10.1016/j.jpba.2024.116468
Drug Test Anal. 2024 Sep 16.

Multianalyte Approach-Including Automated Preparation of Calibrators-for Validated Quantification of 82 Drugs in Whole Blood by Liquid Chromatography-Tandem Mass Spectrometry.

Andrea E Steuer, Maja Keller, Thomas Kraemer, Sandra N Poetzsch.

  Bioanalysis, such as the quantification of drugs in different matrices, is of great importance in forensic toxicology. Nowadays, mainly so-called multianalyte approaches are used given their increased speed and effectiveness. However, such multianalyte procedures can be difficult to develop and maintain with sufficient robustness in the laboratory. One aspect of this is the tedious, manual preparation of spiking solutions containing such a great number of analytes. Therefore, the current study aimed to develop and validate a fast, simple, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 82 classic drugs and to evaluate an alternative autosampler-assisted automated approach for the preparation of spiking solutions. Simple protein precipitation of 200-μL whole blood was used followed by analysis by reversed-phase LC-MS/MS in advanced scheduled multiple reaction monitoring (MRM) mode. The method was fully validated according to international guidelines, including selectivity, recovery, matrix effects, linearity, bias/imprecision, processed-sample stability, and limits. Validation criteria were fulfilled for all analytes except for buprenorphine and five benzodiazepines. In the context of a multianalyte procedure, a (multipurpose) autosampler-assisted automatic preparation of calibrator spiking solutions proved comparable to manual preparation. Thus, automated preparation can overcome the frequently performed manual, time-consuming, and error-prone steps of multianalyte approaches and still allow for customized calibration ranges. Since its introduction, more than 8000 cases have been measured with the presented method, and 35 proficiency tests have been passed.

Keywords:  LC–MS/MS; automated spiking solution preparation; drugs (of abuse); multianalyte

DOI:  https://doi.org/10.1002/dta.3794
Anal Methods. 2024 Sep 16.

Magnetic solid-phase extraction coupled with LC-MS/MS methods for the simple extraction and rapid determination of sugammadex in human plasma.

Yanan Wang, Yinyin Qu, Yi Jiang, Zhuo Li, Xiaofei Hou, Li Yang, Libo Zhao, Xin Xiong, Mao Xu.

Sugammadex (SUG) is a novel antagonist of neuromuscular blocking agents (NMBAs). The NMBA rocuronium is usually employed to obtain better surgical conditions in kidney transplant. Nevertheless, rocuronium has several disadvantages, such as an increased risk of pulmonary complications. Thus, SUG is vital to kidney-transplant surgery. However, because SUG is excreted by the kidneys in prototypes, the pharmacokinetics (PK) may be affected in patients with renal impairment. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to monitor SUG in plasma samples to investigate the PK of SUG in kidney-transplant patients. Due to the complexity and limitation of other methods of sample preparation, magnetic solid-phase extraction (MSPE) was adopted to purify samples. Chromatographic separation was obtained using a reversed-phase Polaris® C18 column and gradient elution with 0.1% formic acid (FA) in water as phase A and in methanol (MeOH) as phase B as mobile phases. The transitions 999.7 → 963.9 (m/z) and 1055.7 → 1012.2 (m/z) were used to quantify SUG and ORG26265, respectively, under negative electrospray ionization. A linear calibration curve was achieved in concentrations varying from 100 to 10 000 ng mL-1. The acceptable accuracy varied from 95.7% to 106.4%, and intra- and inter-precision did not exceed 15% (20% at the lower limit of quantitation (LLOQ)). The matrix effect, stability, dilution integrity, and carry-over were validated. This method was applied successfully for the PK study of 13 recipients and 12 donors of kidney transplant after intravenous injection of SUG (2 mL per kg bodyweight).

DOI: https://doi.org/10.1039/d4ay01185f
Heliyon. 2024 Sep 15. 10(17): e36735

Application of a molecular networking approach using LC-HRMS combined with the MetWork webserver for clinical and forensic toxicology.

Romain Magny, Yann Beauxis, Gregory Genta-Jouve, Emmanuel Bourgogne.

  Backgrounds and aims: In toxicology, LC-HRMS for untargeted screening yields a great deal of high quality spectral data. However, there we lack tools to visualize/organize the MS data. We applied molecular networking (MN) to untargeted screening interpretation. Our aims were to compare theoretical MS libraries obtained in silico with our experimental dataset in patients to broaden its application, and to use the MetWork web application for metabolite identification.Methods: Samples were analyzed using an LC-HRMS system. For MN, data was generated using MZmine, and analyzed and visualized using MetGem. MetWork annotations were ﬁltered and this ﬁle was used for annotation of the previously obtained MN.
Results: 155 compounds including drugs found in patients were recorded. Using this dataset, we confirmed in 60 patients intake of tramadol, amitriptyline bromazepam, and cocaine. The results obtained by the reference methods were confirmed by MN approaches. Eighty percent of the compounds were common to both conventional and MN approaches. Using MetWork, metabolites and parent drugs such as amitriptyline, its metabolite nortriptyline and amitriptyline glucuronide phase 2 metabolites were anticipated and proposed as putative annotations.
Conclusion: The workflow increases confidence in toxicological screening by highlighting putative structures in biological matrices in combination with CFM-ID (Competitive Fragmentation Modeling for Metabolite Identification) and MetWork to extend the annotation of potential drugs even without a reference standard.

Keywords:  Clinical toxicology; Drug identification; High-resolution mass spectrometry; In silico data analysis; Molecular network; Untargeted screening

DOI:  https://doi.org/10.1016/j.heliyon.2024.e36735
Anal Chem. 2024 Sep 18.

Streamlining Phenotype Classification and Highlighting Feature Candidates: A Screening Method for Non-Targeted Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS) Data.

Jessie R Chappel, Kaylie I Kirkwood-Donelson, James N Dodds, Jonathon Fleming, David M Reif, Erin S Baker.

Nontargeted analysis (NTA) is increasingly utilized for its ability to identify key molecular features beyond known targets in complex samples. NTA is particularly advantageous in exploratory studies aimed at identifying phenotype-associated features or molecules able to classify various sample types. However, implementing NTA involves extensive data analyses and labor-intensive annotations. To address these limitations, we developed a rapid data screening capability compatible with NTA data collected on a liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) platform that allows for sample classification while highlighting potential features of interest. Specifically, this method aggregates the thousands of IMS-MS spectra collected across the LC space for each sample and collapses the LC dimension, resulting in a single summed IMS-MS spectrum for screening. The summed IMS-MS spectra are then analyzed with a bootstrapped Lasso technique to identify key regions or coordinates for phenotype classification via support vector machines. Molecular annotations are then performed by examining the features present in the selected coordinates, highlighting potential molecular candidates. To demonstrate this summed IMS-MS screening approach, we applied it to clinical plasma lipidomic NTA data and exposomic NTA data from water sites with varying contaminant levels. Distinguishing coordinates were observed in both studies, enabling the evaluation of phenotypic molecular annotations and resulting in screening models capable of classifying samples with up to a 25% increase in accuracy compared to models using annotated data.

DOI: https://doi.org/10.1021/acs.analchem.4c03256
J Mass Spectrom. 2024 Oct;59(10): e5091

Absolute Quantification of Glutathione Using Top-Hat Optics for IR-MALDESI Mass Spectrometry Imaging.

Emily R Bruce, Russell R Kibbe, Emily C Hector, David C Muddiman.

  Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) uses an infrared laser to desorb neutral biomolecules with postionization via ESI at atmospheric pressure. The Gaussian profile of the laser with conventional optics results in the heating of adjacent nonablated tissue due to the energy profile being circular. A diffractive optical element (DOE) was incorporated into the optical train to correct for this disadvantage. The DOE produces a top-hat beam profile and square ablation spots, which have uniform energy distributions. Although beneficial to mass spectrometry imaging (MSI), it is unknown how the DOE affects the ability to perform quantitative MSI (qMSI). In this work, we evaluate the performance of the DOE optical train against our conventional optics to define the potential advantages of the top-hat beam profile. Absolute quantification of glutathione (GSH) was achieved by normalizing the analyte of interest to homoglutathione (hGSH), spotting a dilution series of stable isotope labeled glutathione (SIL-GSH), and analyzing by IR-MALDESI MSI with either the conventional optical train or with the DOE incorporated. Statistical comparison indicates that there was no significant difference between the quantification of GSH by the two optical trains as evidenced by similar calibration curves. Results support that both optical trains can be used for qMSI without a change in the ability to carry out absolute quantification but providing the benefits of the top-hat optical train (i.e., flat energy profile and square ablation spots)-for future qMSI studies.

Keywords:  diffractive optical element; mass spectrometry imaging; qMSI; top‐hat IR‐MALDESI

DOI:  https://doi.org/10.1002/jms.5091
bioRxiv. 2024 Sep 04. pii: 2024.09.04.611280. [Epub ahead of print]

Correlative Imaging for Comprehensive Molecular Mapping of Individual Cell Types in Biological Tissues.

Manxi Yang, Mushfeqa Iqfath, Frederick Nguele Meke, Zihan Qu, Emerson L Hernly, Pei Su, Zhong-Yin Zhang, Julia Laskin.

Mass spectrometry imaging (MSI) is a powerful technique for label-free spatial mapping of multiple classes of biomolecules in tissue sections. However, differences in desorption and ionization efficiency of different classes of molecules make it challenging to simultaneously map biomolecules at each omics layer in the same tissue sample. Herein, we present a correlative imaging method using nanospray desorption electrospray ionization (nano-DESI) MSI, which enables the spatial mapping of lipids, metabolites, peptides, and proteins with cellular-level spatial resolution in a single tissue section. We demonstrate the molecular profiling of specific cell types and identify truncated peptides in mouse pancreatic tissue. Distinct chemical gradients of peptides and lipids extending from endocrine cells to exocrine cells indicate their different roles in endocrine-exocrine crosstalk and intracellular signaling. The results underscore the power of the developed imaging approach for spatial multi-omics analysis that provides deep insights into cellular diversity and the intricate molecular interactions that occur within heterogenous biological tissues.

DOI: https://doi.org/10.1101/2024.09.04.611280
J Mass Spectrom Adv Clin Lab. 2024 Aug;33 49-54

Understanding isotopes, isomers, and isobars in mass spectrometry.

Katharina Habler, Arber Rexhaj, Manuela Adling-Ehrhardt, Michael Vogeser.

  Mass spectrometry (MS) is a versatile analytical tool used in various fields such as biochemistry, pharmacology, omics, and clinical analysis for determining and quantifying compounds based on their molecular mass and structure through the mass-to-charge ratio. While MS offers high specificity and selectivity, it encounters challenges including matrix effects, in-source fragmentation, and other interferences caused by natural isotopic abundance, as well as isomeric and isobaric compounds. These challenges can impede accurate qualitative and quantitative analysis. Visual aids such as graphical illustrations can help elucidate the chemical differences and similarities among isotopes, isomers, and isobaric compounds.

Keywords:  Interference; Isobar; Isomer; Isotope; Mass spectrometry

DOI:  https://doi.org/10.1016/j.jmsacl.2024.08.002
J Sep Sci. 2024 Sep;47(18): e2400466

Recent advances in liquid chromatography-tandem mass spectrometry for the detection of thyroid hormones and thyroglobulin in clinical samples: A review.

Yuting Jin, Taiyu Zhai, Ying Wang, Jiuyan Li, Tingting Wang, Jing Huang.

  Thyroid hormones (THs), including triiodothyronine (T3), thyroxine (T4), and their metabolites, are essential for regulating development, growth, and energy metabolism. Thyroglobulin (Tg) produced by thyroid follicular cells acts as an essential substrate for TH synthesis. The combination of THs with Tg is a widely used serological laboratory test for thyroid function assessment. Early detection and timely intervention are significant for preventing and managing thyroid disease. In recent years, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for the precise detection of small molecular analytes and steroid hormones in clinical practice as a result of its high sensitivity and specificity. While LC-MS/MS has been increasingly used for detecting THs and Tg recently, its application in clinical practice is still in its early stages. Recent advances in the assessment of thyroid metabolism using LC-MS/MS in clinical samples published during 2004-2023 were reviewed, with a special focus on the use of this technique for quantifying molecules involved in thyroid diseases.

Keywords:  clinical laboratory; liquid chromatography‐tandem mass spectrometry; thyroglobulin; thyroid disease; thyroid hormones

DOI:  https://doi.org/10.1002/jssc.202400466
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 07. pii: S1570-0232(24)00308-8. [Epub ahead of print]1247 124299

The optimization and validation of a gas chromatography-mass spectrometry method to analyze the concentration of acetate, propionate and butyrate in human plasma or serum.

Greet Vandermeulen, Riet Rosseel, Georgia Chatonidi, Lise Deroover, Eef Boets, Kristin Verbeke.

  Fermentation-derived short-chain fatty acids (SCFA)4 are potential mediators of the health benefits associated with dietary fiber intake. SCFA affect physiological processes locally in the gut and on distant organs via the systemic circulation. Since SCFA are used as energy source for colonocytes and substrate for the liver metabolism, their concentrations in the systemic circulation are low. Therefore, quantification of systemic SCFA requires sensitive analytical techniques. This article covers the optimization and validation of a gas chromatography-mass spectrometry method to measure systemic SCFA concentrations following derivatization with 2,4-difluoroaniline (DFA)5 and extraction in ethyl acetate. Sample preparation was optimized by varying the amount of DFA, coupling agent 1,3-dicyclohexylcarbodiimide, ethyl acetate and sodium bicarbonate, which is used to quench derivatization. In addition, evaporation of the samples using a vacuum concentrator resulted in less contamination, notably of acetate, compared to drying with N2 gas. The method showed excellent linearity with coefficient of variation (R2) > 0.99 and a good precision (relative standard deviation < 20 %) and accuracy. Finally, systemic concentrations of SCFA in human plasma samples could successfully be determined.

Keywords:  2,4-difluoroaniline; Blood; Derivatization; Systemic circulation; gas chromatography-mass spectrometry (GC–MS); short-chain fatty acids (SCFA)

DOI:  https://doi.org/10.1016/j.jchromb.2024.124299
Biomed Chromatogr. 2024 Sep 19. e6011

Development and validation of a highly sensitive UPLC-MS/MS method for the determination of Huperzine A in rat plasma.

Kejun Zhang, Haizhou Wang.

  Huperzine A is a reversible and selective cholinesterase inhibitor and has been approved for the treatment of Alzheimer's diseases. In this study, we developed a highly sensitive and specific ulta-high-performance liquid chromatography-tandem mass spectrometry method for the determination of Huperzine A in rat plasma. An aliquot of 50 μL of rat plasma sample was pretreated with 200 μL of acetonitrile-methanol (v/v; 1:1) containing 0.2% formic acid followed by solid phase extraction. The resulting sample was separated on a Waters ACQUITY BEH C18 column using acetonitrile and water containing 0.2% formic acid as mobile phase, at a flow rate of 0.3 mL/min. Multiple-reaction monitoring (MRM) mode was used for quantitative analysis of Huperzine A in positive electrospray ionization. In the concentration range of 0.01-10 ng/mL, Huperzine A showed excellent linearity with correlation coefficient > 0.998. The intra- and inter-day RSD% were less than 9.7%, while the RE% ranged from -6.7% to 10.0%. The mean recovery was >84.5%. The validated method was demonstrated to be selective, sensitive, and reliable, which has been successfully applied to pharmacokinetic study of Huperzine A in rat plasma. Huperzine A displayed a long half-life in rat plasma and high oral bioavailability.

Keywords:  Huperzine A; UPLC‐MS/MS; pharmacokinetics; solid phase extraction

DOI:  https://doi.org/10.1002/bmc.6011
J Chromatogr A. 2024 Sep 13. pii: S0021-9673(24)00751-9. [Epub ahead of print]1736 465377

Supercritical fluid chromatography- Nanospray ionization-mass spectrometry (SFC-nSI-MS).

Mahmoud Elhusseiny Mostafa, James P Grinias, James L Edwards.

  A nanospray emitter coupled to a supercritical fluid chromatograph (SFC-nSI-MS) for mass spectrometric (MS) analysis of fatty acids (FA) positional isomers is introduced. The experimental setup uses conventional bore columns before the SF back-pressure regulator (pre-BPR). The flow is then split and nanosprayed using a short emitter post-BPR. A C18 column was used to resolve positional isomers of unsaturated FA with a 5 min gradient. Chromatographic resolution of the nSFC was compared to a LC-MS system with superior resolving power demonstrated in the nSFC MS system. This system has proven quantitative performance for analyzing pharmaceutical effects on FA composition in a complex biological matrix like E coli lysate.

Keywords:  Capillary LC; Duty cycle; Escherichia coli; Isomers; Metabolomics; NSFC; Nanospray; Optimization; Rapid chromatography; Resolution; SFC

DOI:  https://doi.org/10.1016/j.chroma.2024.465377
Anal Bioanal Chem. 2024 Sep 17.

An interactive R-based custom quantification program for semi-quantitative analysis of triacylglycerols in bovine milk.

Wm Craig Byrdwell, Kenneth F Kalscheur.

  The R programming language, RStudio, and open-source software solutions for analysis of liquid chromatography-mass spectrometry (LC-MS) data have been used with user-written R-based custom quantification programs (CQP) for semi-quantification of triacylglycerols (TAGs) in bovine milk lipid extracts. Using the peak-finding capabilities of the package "xcms" in RStudio, peaks were integrated, and retention times aligned, normalized, and then used for semi-quantitative analysis of a custom set of four extraction internal standards (EISs) and 29 TAG regioisomers using the choice of four analytical internal standards (AISs). Alternating stereospecific numbering (sn) 1,3 TAG regioisomers (standards 1, 3, and 5 of six calibration standards) and sn-1,2 TAG regioisomers (standards 2, 4, and 6 of six standards) were used to make a set of six calibration standards, which were used for quantification using a linear fit model, polynomial fit model, power fit model, level-bracketed linear fit, replicate-bracketed polynomial fit, replicate-bracketed power fit, and replicate- and level-bracketed linear fit and response factors. For example, the linear fit for EIS1 gave an unacceptable coefficient of determination (CoD), r2 = 0.9616, whereas the polynomial fit gave r2 = 0.9908 and the power fit gave r2 = 0.9928, while the double-bracketed linear fit gave CoDs of r2 = 0.9960, 0.9848, and 0.9781 for the three brackets, yet gave the least % difference to known calibration concentrations. For unparalleled transparency, the CQP produced webpages that allowed every step in the data processing and quantification sequence to be verified and reproduced, and contained interactive figures. The data are publicly available using a digital object identifier (DOI). The R code can be downloaded and used with the downloadable data to reproduce the results, to modify the code and further customize the results, or to copy and paste and adapt the code to other quantification applications.

Keywords:  Bovine milk; ESI–MS; Lipids; RStudio; Triacylglycerols; Xcms

DOI:  https://doi.org/10.1007/s00216-024-05528-x
Food Chem X. 2024 Dec 30. 24 101806

Simultaneous determination of glyphosate, glufosinate and their metabolites in soybeans using solid-phase analytical derivatization and LC-MS/MS determination.

Ryoichi Sasano, Junpei Sekizawa, Isao Saito, Mikihisa Harano, Kyoka Katsumoto, Rie Ito, Yusuke Iwasaki, Takaaki Taguchi, Tomoaki Tsutsumi, Hiroshi Akiyama.

  Glyphosate and glufosinate are the most widely used herbicides worldwide. We developed a simple and rapid analytical method for detecting glyphosate, glufosinate, and their metabolites (N-acetyl glyphosate: Gly-A, N-acetyl glufosinate: Glu-A, and 3-(hydroxymethylphosphinyl)propanoic acid: MPPA) in soybeans. The method involved extraction with water, trapping in a mini-column containing polymer-based resin with strong anion exchange groups, dehydration with acetonitrile, and solid-phase analytical derivatization at ambient temperature for 1 min using N-(tert-butyldimethylsilyl)-N-methyl trifluoroacetamide (MTBSTFA), followed by Liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. This method offers a straightforward and rapid analysis, using on-solid phase dehydration and rapid derivatization at an ambient temperature with MTBSTFA, yielding reliable results for glyphosate, glufosinate, and their metabolites. The method was applied to both domestic and imported soybean samples. Glyphosate, glufosinate, and Glu-A were detected in imported feed soybeans and processed soybean meal for feed use, reflecting the current conditions of GM soybean cultivation.

Keywords:  Derivatization; Glufosinate (PubChem CID: 4794); Glyphosate; Glyphosate (PubChem CID: 3496); LC-MS/MS; MPPA (3-(hydroxymethylphosphinyl)propanoic acid PubChem CID: 84788); N-(tert-butyldimethylsilyl)-N-methyl trifluoroacetamide; N-acetyl glufosinate; N-acetyl glufosinate (PubChem CID: 129650895); N-acetyl glyphosate; N-acetyl glyphosate (PubChem CID: 23510850); Solid-phase analytical derivatization

DOI:  https://doi.org/10.1016/j.fochx.2024.101806
J Mass Spectrom Adv Clin Lab. 2024 Aug;33 43-48

Isotope-dilution-LC-MS/MS candidate reference measurement procedure for cefepime in human serum.

Judith Schäffler, Michael Vogeser, Katharina Habler.

  Background: Reference measurement procedures are an essential element in the standardization and comparability of analytical measurement results in laboratory medicine. No LC-MS/MS-based reference measurement procedure for cefepime in serum has been published previously.Materials and methods: An isotope-dilution based two-dimensional LC-MS/MS reference measurement procedure for cefepime concentrations in human serum was developed and tested. The value assignment of unknown samples is based on a defined measurement series validation. Six unknown samples can be measured per series. Pass criteria for the run and the samples were determined empirically based on a performance evaluation. For this purpose, a between-run determination of five runs of the defined measurement series with six cefepime samples was carried out and evaluated. The goal was to define rigorous, realistic target limits and minimize measurement uncertainty. The final defined target limits are used for series-based validation and value assignment. The results for the six unknown samples are provided with the associated measurement uncertainty for this series.
Results: The developed and extensively studied measurement procedure for the quantification of cefepime in serum was found to be practicable and fit for its purpose. The between-run mean imprecision of the six cefepime samples was ≤ 2.0 %, for the QCs it was ≤ 2.3 % and the between-run mean inaccuracy of the QCs was within ± 1.1 %.
Conclusion: The novel isotope-dilution-LC-MS/MS measurement procedure in accordance to ISO 15193 can be recommended as candidate reference measurement procedure for the value assignment of cefepime concentrations in human serum.

Keywords:  Cefepime; Isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS); Metrological traceability; Reference measurement procedures; Standardization

DOI:  https://doi.org/10.1016/j.jmsacl.2024.08.001
Forensic Toxicol. 2024 Sep 18.

Bioinformatics-driven untargeted metabolomic profiling for clinical screening of methamphetamine abuse.

Elif Kesmen, Hızır Asliyüksek, Ahmet Nezih Kök, Cem Şenol, Semih Özli, Onur Senol.

  PURPOSE: Amphetamine-type stimulants are very common, and their usage is becoming a very big social problem all over the world. Thousands of addicts encounter several health problems including mental, metabolic, behavioral and neurological disorders. In addition to these, there are several reports about the elevated risk of tendency on committing criminal cases by addicted persons. Hence, methamphetamine addiction is not only an individual health problem but also a social problem. In our study, we aimed to investigate the pathogenesis of chronic usage of methamphetamine via untargeted metabolomics approach.METHODS: 38 plasma samples were carefully collected and extracted for untargeted metabolomics assay. A liquid-liquid extraction was performed to get as much metabolite as possible from the samples. After the extraction procedure, samples were transferred into vials and they were evaluated via time of flight mass spectrometry instrument.
RESULTS: Significantly, altered metabolites were identified by the fold analysis and Welch's test between the groups. 42 different compounds were annotated regarding to data-dependent acquisition method. Pathway analysis were also performed to understand the hazardous effect of methamphetamine on human body.
CONCLUSION: It has been reported that drug exposure may affect several metabolic pathways for amino acids, fats, energy metabolism and vitamins. An alternative bioinformatic model was also developed and validated in order to predict the chronic methamphetamine drug users in any criminal cases. This generated model passes the ROC curve analysis and permutation test and classify the controls and drug users correctly by evaluating the metabolic alterations between the groups.

Keywords:  Bioinformatics; Criminal cases; Metabolomics; Methamphetamine; Q-TOF MS/MS

DOI:  https://doi.org/10.1007/s11419-024-00703-2
Curr Protoc. 2024 Sep;4(9): e70009

LipidOne 2.0: A Web Tool for Discovering Biological Meanings Hidden in Lipidomic Data.

Husam B R Alabed, Dorotea Frongia Mancini, Sandra Buratta, Eleonora Calzoni, Danika Di Giacomo, Carla Emiliani, Sabata Martino, Lorena Urbanelli, Roberto Maria Pellegrino.

  LipidOne 2.0 (https://lipidone.eu) is a new web bioinformatic tool for the analysis of lipidomic data. It facilitates the exploration of the three structural levels of lipids: classes, molecular species, and lipid building blocks (acyl, alkyl, or alkenes chains). The tool's flexibility empowers users to seamlessly include or exclude experimental groups and lipid classes at any stage of the analysis. LipidOne 2.0 offers a range of mono- and multivariate statistical analyses, specifically tailored to each structural level. This includes a novel lipid biomarker identification function, integrating four diverse statistical parameters. LipidOne 2.0 incorporates Lipid Pathway analysis across all three structural levels of lipids. Users can identify lipid-involved reactions through case-control comparisons, generating lists of genes/enzymes and their activation states based on Z scores. Accessible without the need for registration, LipidOne 2.0 provides a user-friendly and efficient platform for exploring and analyzing lipidomic data. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Dataset preparation for LipidOne 2.0 Support Protocol: Lipid nomenclature from spectrometric experiments Basic Protocol 2: Uploading a dataset into LipidOne 2.0 Basic Protocol 3: Data mining of lipidomic dataset by LipidOne 2.0.

Keywords:  R Core Team (2024); data mining; lipid pathway; lipidomics; system biology

DOI:  https://doi.org/10.1002/cpz1.70009
Drug Test Anal. 2024 Sep 15.

Rapid Analytical Method for Quantification of Gamma-Hydroxybutyrate (GHB) in Hair by UPLC-MS/MS.

Miriam Blanco-Ces, Ana de-Castro-Rios, Angela Lopez-Rabuñal, Maria Cobo-Golpe, Angelines Cruz, Elena Lendoiro.

  Gamma-hydroxybutyrate (GHB), an endogenous compound related to the neurotransmitter gamma-aminobutyric acid (GABA), is used as a therapeutic and recreational drug and as a "weapon" in drug-facilitated crimes. The very short window of detection of GHB in conventional matrices (blood and urine) makes necessary the use of alternative matrices like hair. Hair has a long window of detection and the possibility to perform segmental analysis, which makes it very useful for proving GHB intake. In the present work, a method for quantification of GHB in hair was developed and validated. Hair (10 mg) was washed twice with dichloromethane and then incubated at room temperature with Milli-Q water in an ultrasound bath for 30 min. Analysis was performed by UPLC-MS/MS using a CORTECS UPLC HILIC (1.6 μm), 2.1 × 100-mm column, and a gradient with acetonitrile and ammonium acetate (10 mM) at pH 6.0, with a total run-time of 10 min. For detection, a triple quadrupole mass spectrometer in ESI negative mode was used. The method was validated, following the criteria established in the "AAFS Standard Practices for Method Validation in Forensic Toxicology" guideline, obtaining satisfactory results for linearity (0.5-50 ng/mg), accuracy (95.0%-103.2%), imprecision (< 10.2%), limit of detection (0.1 ng/mg) and quantification (0.5 ng/mg), exogenous selectivity (no interferences), matrix effect (less than -44.2%), extraction efficiency (> 86.4%), process efficiency (> 46.1%), and autosampler stability (< 4.3%). The method was used for the analysis of 26 authentic hair samples, 25 from non-drug users, obtaining values between < LOQ and 6.25 ng/mg of endogenous GHB and 1 from a former GHB chronic user to prove abstinence.

Keywords:  GHB; UPLC‐MS/MS; alternative matrix; drug‐facilitated crime; hair

DOI:  https://doi.org/10.1002/dta.3798
Sci Rep. 2024 09 17. 14(1): 21715

Faster ciguatoxin extraction methods for toxicity screening.

Christopher R Loeffler, Astrid Spielmeyer.

  Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis.

Keywords:  (max. 6, American spelling): marine biotoxins; Ciguatera; Extraction; Fish poisoning; Mass spectrometry; Neuro-2a bioassay

DOI:  https://doi.org/10.1038/s41598-024-72708-1
Anal Chem. 2024 Sep 19.

Digestion-Free Middle-Down Mass Spectrometry Method for Absolute Quantification of Conjugated Payload from Antibody-Drug Conjugates.

Jiaqi Yuan, Hui Yin Tan, Yue Huang, Anton I Rosenbaum.

Antibody-drug conjugate (ADC) is a therapeutic modality that aims to improve payload delivery specificity and reduce systemic toxicity. Considering the complex structure of ADCs, various bioanalytical methods by liquid chromatography coupled with mass spectrometry (LC-MS), ligand binding assay (LBA) and hybrid LBA-LC-MS approaches have been established for ADC characterization and quantification. LCMS-based assays enable drug-antibody ratio (DAR) sensitive quantification of the conjugated payload. Typically, for quantitative, DAR-sensitive, assessment by LC-MS/MS,the conjugated payload is enzymatically liberated and quantified. Despite recent advances in ADC bioanalytical methods, the DAR-sensitive quantification of noncleavable linker ADCs by LC-MS/MS remains challenging. Thus, we developed a novel digestion-free middle-down mass spectrometry (DF-MDMS) using a collision-induced dissociation approach for absolute quantification of conjugated payload from four different ADCs in a biological matrix with minimum sample preparation. These results demonstrate that ADCs with different linker-payload structures can be quantified, including a noncleavable linker ADC, trastuzumab emtansine. It also shows that the assay sensitivity is comparable to the conventional ADC quantification method by linker-payload cleavage using enzyme, while the assay dynamic range depends on factors including payload ionization and dissociation efficiency, DAR and its distribution, and species abundance. By demonstrating absolute quantification of both cleavable and noncleavable linker ADCs, this novel middle-down ADC approach demonstrates its potential application in bioanalysis and analytical characterization, especially for early discovery where high-throughput screening is required as the new approach saves time and resources by not requiring enzymatic digestion for cleavable ADCs or development of anti-payload antibodies for noncleavable linker ADCs.

DOI: https://doi.org/10.1021/acs.analchem.4c03383
Sci Rep. 2024 09 17. 14(1): 21707

How storage post sampling influences the stability of sebum when used for mass spectrometry metabolomics analysis?

Caitlin Walton-Doyle, Eleanor Sinclair, Humayra Begum, Katherine A Hollywood, Drupad K Trivedi, Perdita Barran.

Sebum is a biofluid excreted by sebaceous glands in the skin. In recent years sebum has been shown to contain endogenous metabolites diagnostic of disease, with remarkable results for Parkinson's Disease. Given that sebum sampling is facile and non-invasive, its potential for use in clinical biochemistry diagnostic assays should be explored including the parameters for standard operating procedures around collection, transport, and storage. To this aim we have here investigated the reproducibility of mass spectrometry data from sebum in relation to both storage temperature and length of storage. Sebum samples were collected from volunteers and stored for up to four weeks at a range of temperatures: ambient (circa 17 °C), -20 °C and -80 °C. Established extraction protocols were employed and samples were analysed by two chromatographic mass spectrometry techniques and data investigated using PCA, PLS-DA and ANOVA. We cannot discriminate samples as a function of storage temperature or time stored in unsupervised analysis using data acquired via TD-GC-MS and LC-IM-MS, although the sampling of volatiles was susceptible to batch effects. This study indicates that the requirements for storage and transport of sebum samples that may be used in clinical assays are less stringent than for liquid samples and indicate that sebum is suitable for remote and at home sampling prior to analysis.

DOI: https://doi.org/10.1038/s41598-024-71598-7
Cold Spring Harb Perspect Med. 2024 Sep 16. pii: a041553. [Epub ahead of print]

Technologies for Decoding Cancer Metabolism with Spatial Resolution.

Walter W Chen, Michael E Pacold, David M Sabatini, Naama Kanarek.

It is increasingly appreciated that cancer cells adapt their metabolic pathways to support rapid growth and proliferation as well as survival, often even under the poor nutrient conditions that characterize some tumors. Cancer cells can also rewire their metabolism to circumvent chemotherapeutics that inhibit core metabolic pathways, such as nucleotide synthesis. A critical approach to the study of cancer metabolism is metabolite profiling (metabolomics), the set of technologies, usually based on mass spectrometry, that allow for the detection and quantification of metabolites in cancer cells and their environments. Metabolomics is a burgeoning field, driven by technological innovations in mass spectrometers, as well as novel approaches to isolate cells, subcellular compartments, and rare fluids, such as the interstitial fluid of tumors. Here, we discuss three emerging metabolomic technologies: spatial metabolomics, single-cell metabolomics, and organellar metabolomics. The use of these technologies along with more established profiling methods, like single-cell transcriptomics and proteomics, is likely to underlie new discoveries and questions in cancer research.

DOI: https://doi.org/10.1101/cshperspect.a041553
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 14. pii: S1570-0232(24)00314-3. [Epub ahead of print]1247 124305

Application of newly developed and validated UPLC-MS/MS method for pharmacokinetic study of ROS1/NTRK inhibitor taletrectinib in beagle dog plasma.

Yunfeng Zhu, Fangkai Wang, Xin Wang, Ya Cheng, Xingyu Wang, Ali Fan, Jiawei Chang.

  Taletrectinib is a potent selective ROS and pan-NTRK tyrosine kinase inhibitor (TKI) and has been developed to treat non-small cell lung cancer (NSCLC). To facilitate pharmacokinetic and toxicokinetic studies of taletrectinib, we developed a procedure for ultra-high-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to detect the plasma level of taletrectinib in dogs. This assay procedure was validated in compliance with FDA guidance. The dog plasma samples were spiked with internal standard (IS), followed by protein precipitation, and analyzed using a Waters ACQUITY BEH C18 column coupled to a Thermo triple quadrupole mass spectrometer. Separation was executed using the acetonitrile-0.1 % formic acid solution with gradient elution, at a flow rate of 0.4 mL/min. Taletrectinib and IS were monitored by multiple reaction monitoring (MRM) with m/z 406.2 > 349.2 and m/z 441.2 > 138.1, respectively. The procedure demonstrated excellent linearity with a correlation coefficient greater than 0.999 within the concentration range of 0.2-200 ng/mL. The inter- and intra-day accuracy ranged from -5.25 % to 5.26 %, and the precision was below 6.39 %. Acetonitrile-mediated protein precipitation showed high extraction efficiency and a recovery above 85 %. The procedure was then applied to quantify taletrectinib in beagle dog plasma after oral and intravenous doses and achieved success. The obtained pharmacokinetic parameters indicated high bioavailability of taletrectinib (>85 %) and extensive tissue distribution (>40 L/kg).

Keywords:  Pharmacokinetics; ROS1/NTRK inhibitor; Taletrectinib; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124305
Food Res Int. 2024 Nov;pii: S0963-9969(24)01025-1. [Epub ahead of print]195 114955

Development and validation of a green analytical method for determining fourteen bisphenols in bee pollen by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Ana M Ares, Lucía Alcaide, José Bernal, Silvia Valverde.

  A new analytical method was developed and validated to determine fourteen bisphenols (A, B, C, E, F, M, P, S, Z, AF, AP, BP, FL, PH) in bee pollen using ultra-high-performance liquid chromatography-tandem mass spectrometry. Two different sample treatments were proposed and evaluated: one based on the QuEChERS (quick, easy, cheap, effective, rugged & safe) approach and the other utilizing microextraction with a supramolecular solvent (SUPRAS). In both cases, average analyte recovery ranged between 71 % and 114 %, and the matrix effect was between -45 % and +5 %, although it was not significant when using the QuEChERS-based method (<±20 %). The environmental impact of both sample treatments was assessed using different analytical metrics, with both procedures classified as environmentally friendly, though slightly better results were obtained for SUPRAS. The method was fully validated, showing that the QuEChERS approach had better overall performance, particularly regarding sensitivity and matrix effect. Consequently, the QuEChERS methodology was applied to determine bisphenols in thirty bee pollen samples from different Spanish regions. Residues of three bisphenols (M, P, and S) were detected, although only bisphenol S was quantified in several samples at low concentration levels (<7 μg kg-1), which is below the established specific migration limit (SML; 50 μg kg-1). However, regarding human health, the estimated daily intake, target hazard quotient, and hazard index assessed were higher than acceptable limits, suggesting a potential risk for human consumers.

Keywords:  Bee pollen; Bisphenols; Food Safety; Food analysis; Green analytical chemistry; Method validation; Risk assessment, Sample treatment; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.foodres.2024.114955
Sci Rep. 2024 09 18. 14(1): 21758

UPLC-PDA factorial design assisted method for simultaneous determination of oseltamivir, dexamethasone, and remdesivir in human plasma.

Hanan I El-Shorbagy, Mona A Mohamed, Alaa El-Gindy, Ghada M Hadad, Fathalla Belal.

  A green and simple UPLC method was developed and optimized, adopting a factorial design for simultaneous determination of oseltamivir phosphate and remdesivir with dexamethasone as a co-administered drug in human plasma and using daclatasvir dihydrochloride as an internal standard within 5 min. The separation was established on UPLC column BEH C18 1.7 μm (2.1 × 100.0 mm) connected to UPLC pre-column BEH 1.7 μm (2.1 × 5.0 mm) at 50 °C with an injection volume of 10 μL. The photodiode array detector (PDA) was set at three wavelengths of 220, 315, and 245 nm for oseltamivir phosphate, the internal standard, and both dexamethasone and remdesivir, respectively. The mobile phase consisted of methanol and ammonium acetate solution (40 mM) adjusted to pH 4 in a ratio of 61.5:38.5 (v/v) with a flow rate of 0.25 mL min-1. The calibration curves were linear over 500.0-5000.0 ng mL-1 for oseltamivir phosphate, over 10.0-500.0 ng mL-1 and 500.0-5000.0 ng mL-1 for dexamethasone, and over 20.0-500 ng mL-1 and 500.0-5000.0 ng mL-1 for remdesivir. The Gibbs free energy and Van't Hoff plots were used to investigate the effect of column oven temperatures on retention times. Fluoride-EDTA anticoagulant showed inhibition activity on the esterase enzyme in plasma. The proposed method was validated according to the M10 ICH, FDA, and EMA's bioanalytical guidelines. According to Eco-score, GAPI, and AGREE criteria, the proposed method was considered acceptable green.

Keywords:  Fluoride-EDTA plasma; Gibbs free energy; Green analysis; UPLC-PDA

DOI:  https://doi.org/10.1038/s41598-024-71413-3
Res Sq. 2024 Sep 02. pii: rs.3.rs-4947448. [Epub ahead of print]

A streamlined, resource-efficient immunoprecipitation-mass spectrometry method for quantifying plasma amyloid-β biomarkers in Alzheimer's disease.

Thomas Karikari, Yijun Chen, Xuemei Zeng, Marcos Olvera-Rojas, Anuradha Sehrawat, Tara Lafferty, Tharick Pascoal, Victor Villemagne, Patricio Solis-Urra, Eva Triviño-Ibañez, Manuel Gómez-Rí, Ann Cohen, Milos Ikonomovic, Irene Esteban-Cornejo, Kirk Erickson, Oscar Lopez, Nathan Yates.

High-performance, resource-efficient methods for plasma amyloid-β (Aβ) quantification in Alzheimer's disease are lacking; existing mass spectrometry-based assays are resource- and time-intensive. We developed a streamlined mass spectrometry method with a single immunoprecipitation step, an optimized buffer system, and ≤75% less antibody requirement. Analytical and clinical performances were compared with an in-house reproduced version of a well-known two-step assay. The streamlined assay showed high dilution linearity (r²>0.99) and precision (< 10% coefficient of variation), low quantification limits (Aβ1-40: 12.5 pg/ml; Aβ1-42: 3.125 pg/ml), and high signal correlation (r²~0.7) with the two-step immunoprecipitation assay. The novel single-step assay showed more efficient recovery of Aβ peptides via fewer immunoprecipitation steps, with significantly higher signal-to-noise ratios, even at plasma sample volumes down to 50 µl. Both assays had equivalent performances in distinguishing non-elevated vs. elevated brain Aβ-PET individuals. The new method enables simplified yet robust evaluation of plasma Aβ biomarkers in Alzheimer's disease.

DOI: https://doi.org/10.21203/rs.3.rs-4947448/v1
Bioinformatics. 2024 Sep 18. pii: btae551. [Epub ahead of print]

The GenoPred Pipeline: A Comprehensive and Scalable Pipeline for Polygenic Scoring.

Oliver Pain, Ammar Al-Chalabi, Cathryn M Lewis.

MOTIVATION: Polygenic scoring is an approach for estimating an individual's likelihood of a given outcome. Polygenic scores are typically calculated from genome-wide association study (GWAS) summary statistics and individual-level genotype data for the target sample. Going from genotype to interpretable polygenic scores involves many steps and there are many methods available, limiting the accessibility of polygenic scores for research and clinical application. Additional challenges exist for studies in ancestrally diverse populations. We have implemented the leading polygenic scoring methodologies within an easy-to-use pipeline called GenoPred.RESULTS: Here we present the GenoPred pipeline, an easy-to-use, high-performance, reference-standardised and reproducible workflow for polygenic scoring. It requires minimal inputs and offers various configuration options to cater to a range of use-cases. GenoPred implements a comprehensive set of analyses, including genotype and GWAS quality control, target sample ancestry inference, polygenic score file generation using a range of leading methods, and target sample scoring. GenoPred standardises the polygenic scoring process using reference genetic data, providing interpretable polygenic scores. The pipeline is applicable to GWAS and target data from any population within the reference, facilitating studies of diverse ancestry. GenoPred is a Snakemake pipeline with associated Conda software environments, ensuring reproducibility. We apply the pipeline to UK Biobank data demonstrating the pipeline's simplicity, efficiency, and performance. The GenoPred pipeline provides a novel resource for polygenic scoring, integrating a range of complex processes within an easy-to-use framework. GenoPred widens access of the leading polygenic scoring methodology and their application to studies of diverse ancestry.
AVAILABILITY AND IMPLEMENTATION: Freely available on the web at https://github.com/opain/GenoPred.
SUPPLEMENTARY INFORMATION: Supplementary information are available at Bioinformatics online. Further information is also provided on the GenoPred pipeline website: Https://opain.github.io/GenoPred/pipeline_overview.html.

DOI: https://doi.org/10.1093/bioinformatics/btae551