bims-metlip 2024-12-29 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–12–29
24 papers selected by
Sofia Costa, Matterworks

A Software Tool for Rapid and Automated Preprocessing of Large-Scale Serum Metabolomic Data by Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry.
[Progress in applications of ambient ionization mass spectrometry for lipids identification].
Development of a sequential laser microdissection tissue cuts workflow for the spatial and quantitative analysis of drugs in fresh frozen tissue sections.
Plasma bile acid profile analysis by liquid chromatography-tandem mass spectrometry and its application in healthy subjects and IBD patients.
Chromatographic Separation and Quantification of Nine Nitrosamine Genotoxic Impurities in a Single Method in Nebivolol Tablet by Using Validated Ultra-Sensitive Liquid Chromatography: Mass Spectrometry Analytical Method.
[Current advances in the analysis of free RNA modified nucleosides by high performance liquid chromatography-tandem mass spectrometry].
Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of oximes in KIKO mouse plasma.
Rapid and Robust Workflows Using Different Ionization, Computation, and Visualization Approaches for Spatial Metabolome Profiling of Microbial Natural Products in Pseudoalteromonas.
Determination of new biomarkers for diagnosis of pyridoxine dependent epilepsy in human plasma and urine by liquid chromatography-mass spectrometry.
Analyzing Various Biological Fluids with a Single Automated Proteomic Workflow for Biomarker Discovery.
[Solid-phase extraction coupled with high performance liquid chromatography-triple quadrupole mass spectrometry for simultaneous determination of seven coumarins in water samples from drinking water treatment plants].
[Recent progress in mass spectrometry imaging using nanospray desorption electrospray ionization].
Simultaneous determination of three β-Lactam/β-lactamase inhibitor combinations in critically ill patients by UPLC-MS/MS.
Ghost Peaks of Aromatic Metabolites Induced by Corona Discharge Artifacts in LC-ESI-MS/MS.
Untargeted Spatial Metabolomics and Spatial Proteomics on the Same Tissue Section.
Online native hydrophobic interaction chromatography-mass spectrometry of antibody-drug conjugates.
Highly sensitive ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method for the multiplex analysis of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma.
[Determination of 12 halogenated organic pollutants in edible fish by ultra performance liquid chromatography-high resolution mass spectrometry combined with ultrasound-assisted extraction and gel permeation chromatography purification].
[Determination of eight phthalate metabolites in urine of pregnant women and evaluation of neonatal birth outcomes based on solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry].
Evaluation of metabolite stability in dried blood spot stored at different temperatures and times.
Closing the Knowledge Gap of Post-Acquisition Sample Normalization in Untargeted Metabolomics.
Dried blood spot LC-MS/MS quantification of voclosporin in renal transplant recipients using volumetric dried blood spot sampling.
High-Throughput Free Amino Acid Quantification from Maize Tissues.
LipoCLEAN: A Machine Learning Filter to Improve Untargeted Lipid Identification Confidence.

Anal Chem. 2024 Dec 27.

A Software Tool for Rapid and Automated Preprocessing of Large-Scale Serum Metabolomic Data by Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry.

Erick Helmeczi, Zachary Kroezen, Meera Shanmuganathan, Ana Ruxandra Stanciu, Vanessa Martinez, Natasia Kurysko, Paula Normando, Inês Rugani Ribeiro de Castro, Raquel Machado Schincaglia, Gilberto Kac, Philip Britz-McKibbin.

Mass spectrometry (MS)-based metabolomics often rely on separation techniques when analyzing complex biological specimens to improve method resolution, metabolome coverage, quantitative performance, and/or unknown identification. However, low sample throughput and complicated data preprocessing procedures remain major barriers to affordable metabolomic studies that are scalable to large populations. Herein, we introduce PeakMeister as a new software tool in the R statistical environment to enable standardized processing of serum metabolomic data acquired by multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS), a high-throughput separation platform (<4 min/sample) which takes advantage of a serial injection format of 13 samples within a single analytical run. We performed a rigorous validation of PeakMeister by analyzing 47 cationic metabolites consistently measured in 5,000 serum and 420 quality control samples from the Brazilian National Survey on Child Nutrition (ENANI-2019) comprising a total of 224,983 metabolite peaks acquired in 40 days across three batches over an eight-month period. A migration time index using a panel of 11 internal standards was introduced to correct for large variations in migration times, which allowed for reliable peak annotation, peak integration, and sample position assignment for serum metabolites having two flanking internal standards or a single comigrating stable-isotope internal standard. PeakMeister accelerated data preprocessing times by 30-fold compared to manual processing of MSI-CE-MS data by an experienced analyst using vendor software, while also achieving excellent peak annotation fidelity (median accuracy >99.9%), acceptable intermediate precision (median CV = 16.0%), consistent metabolite peak integration (mean bias = -2.1%), and good mutual agreement when quantifying 16 plasma metabolites from NIST SRM-1950 (mean bias = -1.3%). Reference ranges are also reported for 40 serum metabolites in a national nutritional survey of Brazilian children under 5 years of age from the ENANI-2019 study. MSI-CE-MS in conjunction with PeakMeister allows for rapid and automated processing of large-scale metabolomic studies that tolerate nonlinear migration time shifts without complicated dynamic time warping or effective mobility scale transformations.

DOI: https://doi.org/10.1021/acs.analchem.4c03513
Se Pu. 2025 Jan;43(1): 22-32

[Progress in applications of ambient ionization mass spectrometry for lipids identification].

Xiao-Rong Wang, Yi-Yan Yin, Jin Ouyang, Na Na.

  Lipids are indispensable components of living organisms and play pivotal roles in cell-membrane fluidity, energy provision, and neurotransmitter transmission and transport. Lipids can act as potential biomarkers of diseases given their abilities to indicate cell-growth status. For example, the lipid-metabolism processes of cancer cells are distinct from those of normal cells owing to their rapid proliferation and adaptation to ever-changing biological environments. As a result, the ability to rapidly detect, identify, and monitor lipid components is critical for tracking life-related processes and may enhance cancer diagnosis and treatment efficacy. Mass spectrometry (MS) is regarded to be among the most efficient methods for directly obtaining molecular-structural information, and is distinctly advantageous for identifying lipids. Recent years have witnessed the emergence of ambient mass spectrometry (AMS), which enables direct analyte sampling and ionization without the need for sample preprocessing. These characteristics endow AMS with special advantages for identifying and monitoring lipids. Furthermore, the ongoing development of soft ionization technologies has led to the widespread use of AMS for the detection of complex and diverse lipid molecules. Electrospray ionization (ESI) is a gentle ionization method that can be used to detect medium-to-high-polarity compounds and provide detailed chemical information for lipids by producing a fine mist of charged droplets from a liquid sample. Consequently, a series of ESI-based ionization methods have been developed for fabricating different AMS systems capable of rapidly detecting lipids in a simple manner. For example, desorption electrospray ionization (DESI) is among the most extensively employed ambient ionization techniques, and has been used to detect a wide range of samples, including solids, liquids, and gases. DESI involves spraying a charged solvent onto the surface of a sample, after which the solvent is desorbed, the analyte is ionized, and the generated ions are transferred to the detector of the mass spectrometer via a gas plume. DESI can easily and precisely regulate the sampling space, thereby offering a highly effective approach for the in-situ detection of lipids from tissue samples. Additionally, single-cell lipid analysis is limited by small cell volumes, complex cellular matrices, and minimal absolute amounts of analyte. Common detection methods for single cells include flow cytometry and fluorescence microscopy, both of which require fluorescent labeling to detect specific target molecules, which limits detection selectivity and reproducibility to some extent. ESI-based single-cell mass spectrometry has emerged as a more-effective method for detecting cellular lipids owing to advantages that include high sensitivity, low sample consumption, high throughput, and multiple-detection capabilities. Moreover, lipid chemical diversity poses a significant challenge for determining structural details. Therefore, AMS-based lipid detection has been augmented with a series of chemical-treatment methods that provide more-comprehensive structural information for lipids. For example, diverse gas-phase dissociation techniques have been used to discriminate between lipid C=C-bond isomers and their sn-positions. Strategies that involve chemically modifying specific target C=C bonds prior to MS detection have also been employed. For example, the Paternò-Büchi (P-B) photochemical reaction oxidizes C=C bonds in unsaturated lipids to form oxetane structures, C=C bonds can be epoxidized to form the corresponding oxaziridines, the N-H aziridination reaction converts C=C bonds into aziridines, and the 1ΔO2 ene reaction adds an OOH group to a C=C bond. In this review, we discuss various environmental ionization techniques for lipid AMS developed over the past five years, with an emphasis on typical chemical strategies used to analyze lipid fine structures. Obtaining a high-coverage, high-sensitivity lipid-detection platform based on AMS remains challenging and requires further in-depth studies despite significant improvements in lipid MS-based detection techniques.

Keywords:  ambient mass spectrometry (AMS); ionization techniques; lipid; lipid fine structure; tandem mass spectrometry (MS/MS)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.06007
PLoS One. 2024 ;19(12): e0312542

Development of a sequential laser microdissection tissue cuts workflow for the spatial and quantitative analysis of drugs in fresh frozen tissue sections.

Elias Maris, Farid Jahouh, Kathleen Allaerts, Rob Vreeken.

Mass spectrometry imaging (MSI) is a well-established technique that allows to determine the distribution of small molecules, such as lipids, metabolites, and drugs, as well as large molecules in tissue sections. Because of the tissue heterogeneity, resulting in different matrix effects, and to the fact that the measured compounds are not entirely "extracted" from the tissue during the measurement, the absolute quantitative aspect of MSI is limited. To combine compound quantification with spatial information on fresh frozen unstained tissue sections, laser (capture) microdissection has been used to isolate tissue sections for compound extraction and LC-MS/MS quantification. Although this method relying on manual ROIs selection is rather sensitive compared to traditional MSI methods, it lacks the throughput needed to screen entire tissue sections. To apply a higher throughput tissue screening approach, we propose herein a workflow for performing indiscriminate and sequential LMD tissue section cuts that can cover up to 96 cuts collected in a 96 well plate on Leica LMD systems, for further extractions and LC-MS/MS analysis. Our workflow relies on the creation and implementation of 96 squares microgrid templates for the LMD cut of different area sizes (30x30 μm2, 50x50 μm2, 100x100 μm2, 200x200 μm2, 270x270 μm2 and 500x500 μm2) using 5 different magnifications (5x, 10x, 20x, 40x and 63x), on fresh frozen tissue sections. The method was applied on 20μm mouse brain and liver tissue sections. The tissue cut collection yields were evaluated visually and by the detection of the sprayed standards on the tissue sections, and found to be ranging from 78% to 91%, and the throughput of the LMD cuts and collection in a 96 well format, was measured to be from 19 to 37 minutes per tissue section, depending on the 96 squares microgrid template and the corresponding magnification lens used. Further extraction and LC-MS/MS analysis of 3 different compounds previously sprayed on a mouse liver tissue section allowed to determine the LLOQ the workflow allows to achieve when using the different templates.

DOI: https://doi.org/10.1371/journal.pone.0312542
J Pharm Biomed Anal. 2024 Dec 15. pii: S0731-7085(24)00681-2. [Epub ahead of print]255 116639

Plasma bile acid profile analysis by liquid chromatography-tandem mass spectrometry and its application in healthy subjects and IBD patients.

Xuepeng Gong, Dong Liu, Lu Liu, Guangjie Yang, Yongfang Lei, NingHong Li, Yufei Chen, Hengyi Yu, Xiping Li, Dong Xiang.

  Bile acids (BAs), not only promote the absorption of fat-soluble nutrients and regulate the metabolism of multiple substances but also have a potential role as diagnostic and prognostic indicators in a variety of diseases such as cholestasis, hepatocellular carcinoma, and diabetes mellitus. Here, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of 50 BAs was developed and validated. Sample preparation included internal standard spiking, followed by protein precipitation, centrifugation, solvent evaporation, and reconstitution. Baseline separation of all isobaric BA species was achieved on an Ultimate XS-C18 column (5 μm, 150 mm × 4.6 mm). The method showed good linearity with high regression coefficients (>0.990) with acceptable accuracy and precision for intra-day and inter-day analyses and achieved good recovery rates for representative analytes. No apparent carryover or matrix effect was observed. The analytical method was successfully applied to the determination of the plasma BA profile in healthy subjects and patients with inflammatory bowel disease (IBD). The routine instrumentation, low sample volume, simple pretreatment, wide range of BAs, and good separation make this LC-MS/MS method suitable for use as a BA profile assay in clinical and basic research studies. This method could be poised to identify possible BA biomarkers for non-invasive early diagnosis and therapeutic evaluation of IBD.

Keywords:  Bile acid profile; Inflammatory bowel disease; LC-MS/MS; Plasma

DOI:  https://doi.org/10.1016/j.jpba.2024.116639
J Chromatogr Sci. 2024 Dec 27. pii: bmae061. [Epub ahead of print]

Chromatographic Separation and Quantification of Nine Nitrosamine Genotoxic Impurities in a Single Method in Nebivolol Tablet by Using Validated Ultra-Sensitive Liquid Chromatography: Mass Spectrometry Analytical Method.

Mehul Pathak, Dhara D Patel, Dalip Kumar, Avineesh Singh, Suresh Agrawal.

N-nitrosamine impurities have been detected in a vast variety of drug substances and drug products, showing concern for regulatory aspects. To meet the regulatory requirement for the concerned impurity, a sensitive analytical method capable of quantifying these impurities at a lower level with accuracy and precision is required. This article focuses on the development and validation of an analytical method for the simultaneous detection of nine nitrosamine impurities in a single method for nebivolol drug product using liquid chromatography-mass spectrometry/mass spectrometry-atmospheric pressure chemical ionization (LC-MS/MS-APCI). The chromatographic separation was performed using the LC-MS column Allure BiPh C18 (250 × 4.6 mm), 5 μm employed a gradient mode elution program using 0.002 M Ammonium acetate buffer pH 4.5 as mobile phase A and methanol as mobile phase B. The method was challenged for accuracy, precision and linearity in accordance with International Council for Harmonization guidelines to ensure its suitability for the intended usage. The developed method was specific, accurate and linear with square of correlation coefficient (r2) found to be greater than 0.99 (0.9970-0.9992). The LOQ obtained in the range of 9.85-19.62 ppb for nine nitrosamines showed good sensitivity. The results demonstrated that method can be applied to quantify the nitrosamines in nebivolol drug products.

DOI: https://doi.org/10.1093/chromsci/bmae061
Se Pu. 2025 Jan;43(1): 3-12

[Current advances in the analysis of free RNA modified nucleosides by high performance liquid chromatography-tandem mass spectrometry].

Lyu-Ye Zhang, Wei-Bing Zhang, Hai-Lin Wang.

  Post-transcriptional ribonucleic acid (RNA) modifications play crucial roles in regulating gene expression, with both eukaryotic and prokaryotic RNA exhibiting more than 170 distinct and ubiquitous modifications. RNA turnover generates numerous free nucleosides, including unmodified nucleosides and a variety of modified ones. Unlike unmodified nucleosides, modified nucleosides are not further degraded or used in the salvage-synthesis pathway owing to a lack of specific enzymes, which leads to the cytosolic accumulation or cellular efflux of modified nucleosides. These modified nucleosides can act as signaling molecules that regulate downstream pathways once transported to the extracellular space; alternatively, they are metabolized in the bloodstream and excreted in urine. Metabolized modified nucleosides are altered by cellular stress responses and mediate abnormal physiological states. Changes in the urinary and blood levels of modified nucleosides associated with cancer can serve as biomarkers for disease. Therefore, identifying and accurately quantifying nucleosides is vital for understanding RNA degradation and associated patterns of nucleoside metabolism. Such analyses are helpful when studying the biological functions and potential clinical applications of modified nucleosides. In this regard, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) offers significant advantages in terms of sensitivity, selectivity, and efficiency, and has been widely used to analyze DNA and RNA nucleosides/nucleotides and their analogues. Multiple MS detection patterns and quantification methods have been established to detect nucleosides in biological samples, including cultured cells, urine, blood, and tissue samples. However, the development of an accurate HPLC-MS/MS method faces several challenges. Firstly, the presence of a complex biological matrix that contains macromolecules, small molecules, and salts can interfere with analysis. Salts and co-eluting substances in the extraction solution often affect mass-spectrometric responses for target analytes. Secondly, various nucleosides are present in vastly different abundances, with contents varying by up to four orders of magnitude; hence, accurately quantifying multiple nucleosides in a single assay is challenging. Thirdly, N-glycosidic bonds are favorably cleaved in most nucleosides during MS to produce the same characteristic fragment ions, which are often accompanied by nucleobases. This tendency poses challenges for distinguishing structural isomers and mass-analogs of modified nucleosides by MS. Post-transcriptional chemical modifications include methylation, hydroxylation, sulfur/oxygen substitution, and side-chain additions. Developing a unified method for simultaneously screening modified nucleosides is difficult owing to biochemical diversity; consequently, there is a need for advanced HPLC-MS/MS method capable of accurately quantifying such nucleosides. This review summarizes the development and applications of LC-MS technologies for analyzing endogenous nucleosides, covering sample preparation, chromatographic-separation and mass-spectrometric-detection conditions, and the development of quantification methods. Additionally, we discuss applications aimed at detecting and quantifying RNA-derived modified nucleosides in biological samples. The applications of HPLC-MS/MS technology are highlighted, the regulation and function of free modified nucleosides are discussed, and the potential functions of modified nucleosides as disease biomarkers for clinical applications are introduced.

Keywords:  RNA modifications; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); metabolite analysis; methylated nucleosides

DOI:  https://doi.org/10.3724/SP.J.1123.2024.07004
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Dec 15. pii: S1570-0232(24)00435-5. [Epub ahead of print]1251 124426

Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of oximes in KIKO mouse plasma.

Katie A Walker, Justin N Vignola, Trinity K Rudd, C Linn Cadieux, Robert C diTargiani.

  Chemical warfare nerve agents (CWNAs) are potent and irreversible inhibitors of acetylcholinesterase (AChE). Oxime reactivators are an important part of the standard treatment for CWNA exposure as they can reactivate inhibited AChE. Evaluating the oxime candidates of interest in biological samples requires analytical detection methods and oxime reactivators as a class of compounds have historically been notoriously difficult to isolate, detect and analyze in an analytical laboratory, and there are currently no sensitive or robust analytical detection methods in the literature. The goal of this study was to develop reliable and robust novel extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to detect and quantitate 2-PAM, HI-6, HLö-7, and MMB-4 in a human AChE knock-in, mouse carboxylesterase knock-out (KIKO) mouse in vivo model. This study identified an LC column that achieved retention for all four oxime compounds which is a major advancement over past oxime methods. A unique extraction and chromatographic method was developed for each oxime. The developed methods were sensitive down to 0.5 ng/mL for 2-PAM, 50 ng/mL for HI-6, and 15 ng/mL for both HLö-7 and MMB-4. These methods were validated to meet the Food and Drug Administration (FDA) bioanalytical method validation requirements under Good Laboratory Practice (GLP) conditions. The 4 methods were validated for performance by assessing linearity, sensitivity, precision, accuracy, selectivity, specificity, carryover, extraction recovery, dilution analysis, and stability.

Keywords:  2-PAM; Chemical warfare nerve agent; HI-6; HLö-7; LC-MS/MS; MMB-4

DOI:  https://doi.org/10.1016/j.jchromb.2024.124426
ACS Meas Sci Au. 2024 Dec 18. 4(6): 668-677

Rapid and Robust Workflows Using Different Ionization, Computation, and Visualization Approaches for Spatial Metabolome Profiling of Microbial Natural Products in Pseudoalteromonas.

Jian Yu, Haidy Metwally, Jennifer Kolwich, Hailey Tomm, Martin Kaufmann, Rachel Klotz, Chang Liu, J C Yves Le Blanc, Thomas R Covey, John Rudan, Avena C Ross, Richard D Oleschuk.

Ambient mass spectrometry (MS) technologies have been applied to spatial metabolomic profiling of various samples in an attempt to both increase analysis speed and reduce the length of sample preparation. Recent studies, however, have focused on improving the spatial resolution of ambient approaches. Finer resolution requires greater analysis times and commensurate computing power for more sophisticated data analysis algorithms and larger data sets. Higher resolution provides a more detailed molecular picture of the sample; however, for some applications, this is not required. A liquid microjunction surface sampling probe (LMJ-SSP) based MS platform combined with unsupervised multivariant analysis based hyperspectral visualization is demonstrated for the metabolomic analysis of marine bacteria from the genus Pseudoalteromonas to create a rapid and robust spatial profiling workflow for microbial natural product screening. In our study, metabolomic profiles of different Pseudoalteromonas species are quickly acquired without any sample preparation and distinguished by unsupervised multivariant analysis. Our robust platform is capable of automated direct sampling of microbes cultured on agar without clogging. Hyperspectral visualization-based rapid spatial profiling provides adequate spatial metabolite information on microbial samples through red-green-blue (RGB) color annotation. Both static and temporal metabolome differences can be visualized by straightforward color differences and differentiating m/z values identified afterward. Through this approach, novel analogues and their potential biosynthetic pathways are discovered by applying results from the spatial navigation to chromatography-based metabolome annotation. In this current research, LMJ-SSP is shown to be a robust and rapid spatial profiling method. Unsupervised multivariant analysis based hyperspectral visualization is proven straightforward for facile/rapid data interpretation. The combination of direct analysis and innovative data visualization forms a powerful tool to aid the identification/interpretation of interesting compounds from conventional metabolomics analysis.

DOI: https://doi.org/10.1021/acsmeasuresciau.4c00035
Clin Chim Acta. 2024 Dec 25. pii: S0009-8981(24)02364-7. [Epub ahead of print]567 120111

Determination of new biomarkers for diagnosis of pyridoxine dependent epilepsy in human plasma and urine by liquid chromatography-mass spectrometry.

Roberta Damiano, Maria Della Bona, Elena Procopio, Serena Gasperini, Renzo Guerrini, Alessandra Bettiol, Giancarlo la Marca.

   BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is a rare inborn error of lysine metabolism. To date, diagnosis of PDE relies on the quantification of α-AminoAdipic SemiAldehyde (α- AASA), Piperideine-6-Carboxylate (P6C) and Pipecolic acid (PA) in urine or plasma from patients with overt symptoms. However, these biomarkers are not specific, and their biochemical analysis is challenged by their instability and technical limitations. We set-up and validated a method for the quantification of two new biomarkers of PDE (2S,6S- and 2S,6R-oxopropylpiperidine-2-carboxylic acid, 2-OPP, and 6-oxopiperidine2-carboxylic acid, 6-oxoPIP) on human urine and plasma by LC-MS/MS, to overcome the diagnostic and technical challenges of old biomarkers.
METHODS: We analysed urine and plasma samples by LC-MS/MS, and validated the method in both biological matrices.
RESULTS: We performed the biomarkers extraction from a 10 µL aliquot of urine or plasma in around 15 min using water 100 % for urine, and a solution of water/methanol 50 % for plasma, respectively. The analytical method was validated and gave good linearity (r2 > 0.999) in the range 0-15 µmol/L for 2-OPP and 0-25 µmol/L for 6-oxoPIP. In both matrices, the biomarkers were stable at different storage temperatures tested.
CONCLUSIONS: We set-up and validated a reliable method and confirmed its clinical applicability on real samples from PDE patients. This method could be used as routine test for the diagnosis and monitoring of PDE.

Keywords:  2S,6S-/2S,6R-Oxopropylpiperidine-2-Carboxylic Acid; 6-Oxopiperidine-2-Carboxylic Acid; High pressure liquid chromatography; Plasma; Pyridoxine dependent epilepsy; Tandem mass spectrometry; Urine

DOI:  https://doi.org/10.1016/j.cca.2024.120111
Methods Mol Biol. 2025 ;2884 157-178

Analyzing Various Biological Fluids with a Single Automated Proteomic Workflow for Biomarker Discovery.

Ane Laura Pedersen, Marion Ernest, Michael Affolter, Loïc Dayon.

  Protein biomarker discovery in human biological fluids has greatly developed over the past two decades thanks to technological advances allowing deeper proteome coverage and higher sample throughput, among others. While blood samples are most commonly investigated due to their moderate ease of collection and high information content, other biological fluids such as cerebrospinal fluid (CSF) and urine are highly relevant for specific pathologies, such as brain and urologic diseases, respectively. Independently of the biofluid of interest, platforms that can robustly handle a large number of samples are essential in the discovery phase of a clinical study.We have previously described a scalable automated proteomic pipeline (ASAP2) for the sample preparation of hundreds to thousands of blood plasma, serum, and CSF samples before liquid chromatography (LC)-mass spectrometry (MS) analysis. Here, we describe how the workflow was further adapted to milk and urine samples, with small modifications at the beginning of the workflow. For blood and CSF samples, an optional immuno-affinity depletion step for abundant-protein removal constitutes the first step of the workflow. In the analysis of milk, a defatting step is incorporated before the samples are further processed with ASAP2, while acetone precipitation is used for the analysis of urine samples. The main sample preparation steps then remain identical for all sample types, are automated on a liquid handling workstation, and include reduction of disulfide bridges, alkylation of free thiols, protein digestion, isobaric labeling of peptides, sample pooling, and purifications. The workflow is completed by LC-MS analysis of the samples and subsequent data processing.

Keywords:  Automation; Biofluid; CSF; Clinical research; Human; Isobaric tagging; Mass spectrometry; Milk; Plasma; Proteomics; Serum; Urine

DOI:  https://doi.org/10.1007/978-1-0716-4298-6_11
Se Pu. 2025 Jan;43(1): 78-86

[Solid-phase extraction coupled with high performance liquid chromatography-triple quadrupole mass spectrometry for simultaneous determination of seven coumarins in water samples from drinking water treatment plants].

Wen-Mei Jiao, Jing-Ming Yang, Ce Xu, Fu-Kang Gao, Lu-Yao Shen, Yu-Bo Yuan, Zhi-Fen Guo, Guang Huang.

  Chlorinated coumarins, which are as cytotoxic as highly toxic halobenzoquinones toward CHO-K1 cells, have recently been identified as disinfection byproducts in drinking water disinfection processes. Therefore, detecting coumarins in water samples collected at various stages from drinking water treatment plants helps assess the formation of chlorinated coumarins in drinking water. Hence, a simple, rapid, accurate, and sensitive method for quantifying coumarins in water samples is required. In this study, a method was developed based on solid-phase extraction coupled with high performance liquid chromatography-triple-quadrupole mass spectrometry for analyzing seven coumarins in water samples from drinking water treatment plants, including 6,7-dihydroxycoumarin, 7-hydroxycoumarin, 6-hydroxy-4-methylcoumarin, 8-chloro-7-hydroxycoumarin, coumarin, 7-chloro-6-hydroxy-4-methylcoumarin, and 3,8-dichloro-7-hydroxycoumarin. Sample pretreatment involved solid-phase extraction using HLB columns, followed by elution with water and methanol, each containing 0.25% formic acid. The extracted solution was separated via gradient elution using a Phenomenex Luna C18 column (100 mm×2.0 mm, 3 mm) with 0.1% formic acid aqueous solution and methanol as the mobile phases, with analytes detected by triple-quadrupole mass spectrometry equipped with electrospray ionization source in multiple reaction monitoring mode. The matrix effect, precision, and accuracy of the developed method were investigated using raw and treated water as matrices. Matrix effects of 0.84-1.12 were recorded for the detection of 6,7-dihydroxycoumarin, 7-hydroxycoumarin, 6-hydroxy-4-methylcoumarin, and coumarin in raw water, while values of 0.67-0.70 were recorded for 8-chloro-7-hydroxycoumarin, 7-chloro-6-hydroxy-4-methylcoumarin, and 3,8-dichloro-7-hydroxycoumarin in finished water. The three chlorinated coumarins exhibited matrix effects above 0.80 after the finished water matrix had been diluted four times. These results suggest that only simple solid-phase extraction or sample dilution is required to accurately determine the seven coumarins in drinking water from treatment plants. In addition, these coumarins exhibited good linear relationships at their respective mass concentrations. The precision and accuracy of the method were evaluated using raw and treated water as matrices. The seven coumarins exhibited good linearities by triple-quadrupole mass spectrometry in a certain range, with correlation coefficients (r) greater than 0.99 and method detection limits (MDLs) of 0.67-1.12 ng/L. The seven coumarins exhibited recoveries of 61.4%-91.5% at three spiked levels (20, 50, 100 ng/L) with relative standard deviations (RSDs, n=6)≤11.2%. The developed method can be used to analyze water samples from various treatment stages of a drinking-water treatment plant. 7-Hydroxycoumarin, 6,7-dihydroxycoumarin, and coumarin were detected at levels of 0.21-27.9 ng/L in 100% of the samples, while 6-hydroxy-4-methylcoumarin was not detected in raw water, post-coagulated water, and post-carbon-filtered water, but was found in sand-filtered water and finished water, with higher levels recorded for the latter (4.69 ng/L) than the former (1.79 ng/L). 8-Chloro-7-hydroxycoumarin was only detected in treated water (0.07 ng/L). This method is highly precise and accurate, provides results in short analysis times, and can be used to effectively monitor coumarins in real water samples and assess their removal during drinking-water treatment.

Keywords:  coumarins; disinfection byproducts; drinking water; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); solid-phase extraction (SPE)

DOI:  https://doi.org/10.3724/SP.J.1123.2024.06014
Se Pu. 2025 Jan;43(1): 43-49

[Recent progress in mass spectrometry imaging using nanospray desorption electrospray ionization].

Jing-Bo Wang, Xiao-Lan Li, Rui-Xia Fan, Ping Lü, Rui-Chuan Yin.

  Ambient mass spectrometry imaging (MSI) enables hundreds of analytes in tissue sections to be directly mapped at atmospheric pressure with minimal sample preparation. This field is currently experiencing rapid growth, with numerous reported ambient ionization techniques resulting in a "hundred flowers bloom" situation. Nanospray desorption electrospray ionization (nano-DESI), developed by the Laskin group in 2010, is a widely used liquid-extraction-based ambient ionization technique that was first used for mass spectrometry imaging of tissue in 2012. The nano-DESI probe comprises a primary capillary and a nanospray capillary, with the latter efficiently transferring analyte-containing droplets via a tiny liquid bridge formed between the probe and sample surface, thereby enabling nanoelectrospray ionization (nano-ESI) in front of the inlet of a mass spectrometer. The advantages of nano-DESI MSI include minimal sample preparation, high spatial resolution, and high sensitivity. These features are well-suited for imaging various sample types, including frozen tissue sections, microbial communities, and environmental samples. A PubMed-database search using the "nano-DESI" keyword revealed 72 related articles in the 2010-2024 period, with 34 of them published between 2021 and 2024, which indicates that nano-DESI has rapidly developed as an ambient ionization technique over recent years. Herein, we briefly introduce key nano-DESI-MSI research progress reported in the past three years with the aim of better understanding and facilitating the use of this technology. We first discuss advances in ion-source development. Since no commercial nano-DESI source exists, designing and constructing ion sources remain technical challenges that limit its development. Nano-DESI has been successfully coupled with various types of mass spectrometer, including LTQ Orbitrap, quadrupole-Orbitrap (Q Exactive), 6560 IM QTOF, timsTOF Pro2, triple quadrupole, and FTICR. These couplings have significantly expanded the applications range of the nano-DESI technique. Secondly, lipid analysis is a major nano-DESI-MSI applications area. While the complexities of lipid structures present great challenges for nano-DESI MSI, new nano-DESI coupling techniques have enabled the identification and imaging of fine lipid structures. Several novel imaging methods have recently been introduced to address difficulties associated with identifying lipid structures, such as distinguishing carbon-carbon double bonds (C=C) and sn-positional isomers. We finally highlight recent research progress in the nano-DESI MSI of intact protein assembles and proteoforms, which is a growing hotspot in the field. Unlike small lipid molecules, large protein molecules are very challenging to image and consequently demand higher instrumental performance (e.g., ionization efficiency, mass range, and sensitivity). In a similar manner to the ESI technique, nano-DESI tends to generate multiply charged molecular ions, which endows it with a significant advantage when imaging large protein molecules. Recent years have witnessed important nano-DESI-MSI progress for studying protein-ligand interactions and identifying and imaging endogenous proteoforms. In summary, this article focuses on nano-DESI research progress in terms of ion-source development, lipid-structure analysis, and spatial proteomics over the past three years and discusses key challenges that need to be addressed in the field.

Keywords:  ambient mass spectrometry imaging; nanospray desorption electrospray ionization; spatial proteomics; structural characterization of lipid

DOI:  https://doi.org/10.3724/SP.J.1123.2024.07013
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Dec 20. pii: S1570-0232(24)00440-9. [Epub ahead of print]1251 124431

Simultaneous determination of three β-Lactam/β-lactamase inhibitor combinations in critically ill patients by UPLC-MS/MS.

Xiaoyang Liu, Bo Li, Shu Li, Xiaoxue Wang, Xudong Kong, Yue Chen, Qian Zhang, Jun Duan, Wenqian Chen, Pengmei Li.

  β-Lactam/β-lactamase inhibitors (BL/BLIs) are widely used in critically ill patients. Recent research has shown the importance of therapeutic drug monitoring (TDM) of BLs, but few studies have highlighted the importance of detecting BLIs in critically ill patients. In our laboratory, we have developed and validated a simple and robust method for the determination of ceftazidime, cefoperazone, piperacillin, avibactam, sulbactam and tazobactam in human plasma by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Sample preparation was by protein precipitation of 100 µL of sample, followed by chromatographic separation on an ACQUITY UPLC® BEH C18 column (2.1 × 50 mm, 1.7 µm) and mass spectrometric detection using a SHIMADZU 8050CL in multiple reaction monitoring (MRM) mode. The method was fully validated for selectivity, carry-over, linearity, lower limit of quantification, matrix effect, extraction recovery, stability and dilution integrity. The results of the TDM could provide feedback to clinicians and allow timely adjustment of dosing regimens in critically ill patients. The method is suitable for routine TDM and has been successfully applied to the clinical determination of 81 plasma concentrations in 44 patients.

Keywords:  Antibiotics; Therapeutic drug monitoring; UPLC–MS/MS; β-Lactam/β-lactamase inhibitors

DOI:  https://doi.org/10.1016/j.jchromb.2024.124431
J Mass Spectrom. 2025 Jan;60(1): e5102

Ghost Peaks of Aromatic Metabolites Induced by Corona Discharge Artifacts in LC-ESI-MS/MS.

Yayoi Hongo, Daisuke Fukuyama, Lee Chuin Chen, Kanako Sekimoto, Hiroshi Watanabe.

  LC-ESI-MS/MS is a preferred method for detecting and identifying metabolites, including those that are unpredictable from the genome, especially in basal metazoans like Cnidaria, which diverged earlier than bilaterians and whose metabolism is poorly understood. However, the unexpected appearance of a "ghost peak" for dopamine, which exhibited the same m/z value and MS/MS product ion spectrum during an analysis of Nematostella vectensis, a model cnidarian, complicated its accurate identification. Understanding the mechanism by which "ghost peaks" appear is crucial to accurately identify the monoamine repertoire in early animals so as to avoid misassignments. Verification experiments showed that in-source oxidation of tyramine, which produced an intense signal, was responsible for this "ghost peak." This artifact commonly occurs among aromatic compounds with high signal intensities and appears at the same m/z as their respective in vivo oxidized metabolites. In metabolomics, spectra contain diverse signals from complex biological mixtures, making it difficult to recognize artifact peaks. To prevent misassignments, despite +16 Da differences, adequate chromatographic separation of metabolites from their respective in vivo oxidation precursors is necessary. Whereas both electrolysis and gas-phase corona discharge can cause in-source oxidation in ESI, corona discharge proved to be the dominant factor. Additionally, the presence of multiple oxygen atom sources was suggested by the voltage-dependent mass shift of +16 Da to +18 Da of the "ghost peak" when using 18O-labeled water as a solvent. Accurate metabolite identification using LC-ESI-MS/MS requires accounting for in-source products that can mimic in vivo products.

Keywords:  Cnidaria; corona discharge; electrolysis in ESI; ghost peak; metabolomics

DOI:  https://doi.org/10.1002/jms.5102
Anal Chem. 2024 Dec 21.

Untargeted Spatial Metabolomics and Spatial Proteomics on the Same Tissue Section.

Gregory W Vandergrift, Marija Veličković, Le Z Day, Brittney L Gorman, Sarah M Williams, Bindesh Shrestha, Christopher R Anderton.

An increasing number of spatial multiomic workflows have recently been developed. Some of these approaches have leveraged initial mass spectrometry imaging (MSI)-based spatial metabolomics to inform the region of interest (ROI) selection for downstream spatial proteomics. However, these workflows have been limited by varied substrate requirements between modalities or have required analyzing serial sections (i.e., one section per modality). To mitigate these issues, we present a new multiomic workflow that uses desorption electrospray ionization (DESI)-MSI to identify representative spatial metabolite patterns on-tissue prior to spatial proteomic analyses on the same tissue section. This workflow is demonstrated here with a model mammalian tissue (coronal rat brain section) mounted on a poly(ethylene naphthalate)-membrane slide. Initial DESI-MSI resulted in 160 annotations (SwissLipids) within the METASPACE platform (≤20% false discovery rate). A segmentation map from the annotated ion images informed the downstream ROI selection for spatial proteomics characterization from the same sample. The unspecific substrate requirements and minimal sample disruption inherent to DESI-MSI allowed for an optimized, downstream spatial proteomics assay, resulting in 3888 ± 240 to 4717 ± 48 proteins being confidently directed per ROI (200 μm × 200 μm). Finally, we demonstrate the integration of multiomic information, where we found ceramide localization to be correlated with SMPD3 abundance (ceramide synthesis protein), and we also utilized protein abundance to resolve metabolite isomeric ambiguity. Overall, the integration of DESI-MSI into the multiomic workflow allows for complementary spatial- and molecular-level information to be achieved from optimized implementations of each MS assay inherent to the workflow itself.

DOI: https://doi.org/10.1021/acs.analchem.4c04462
MAbs. 2025 Dec;17(1): 2446304

Online native hydrophobic interaction chromatography-mass spectrometry of antibody-drug conjugates.

Trevor Kempen, Lance Cadang, Yuchen Fan, Kelly Zhang, Tao Chen, Bingchuan Wei.

  Hydrophobic interaction chromatography (HIC) is commonly used to determine the drug-to-antibody ratio (DAR) and drug load distribution of antibody-drug conjugates (ADCs). However, identifying various DAR species separated by HIC is challenging due to the traditional use of mobile phases that are incompatible with mass spectrometry (MS). Existing approaches used to couple HIC with MS often encounter issues, such as complex instrumentation, compromised separation efficiency, and reduced MS sensitivity. In this study, we introduce a 22-min online native HIC-MS method for the separation and characterization of different DAR species in ADCs, addressing these challenges. The key novelty of this method is the use of ammonium tartrate, a kosmotropic and thermally decomposable salt, as the salt of HIC mobile phase, ensuring both excellent HIC separation and MS compatibility. Additionally, an ultrashort size exclusion chromatography step is integrated for online sample cleaning, enhancing MS sensitivity. This platform native HIC-MS method offers a rapid, sensitive, and robust solution for comprehensive profiling of DAR species in ADCs with a simple and cost-effective instrumental setup.

Keywords:  Antibody-drug conjugates; critical quality attributes; drug-to-antibody ratio; hydrophobic interaction chromatography; multi-dimensional liquid chromatography; native mass spectrometry

DOI:  https://doi.org/10.1080/19420862.2024.2446304
J Pharm Biomed Anal. 2024 Dec 03. pii: S0731-7085(24)00654-X. [Epub ahead of print]255 116612

Highly sensitive ultra-high-performance liquid chromatography coupled with tandem mass spectrometry method for the multiplex analysis of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma.

Stéphane Bertin, François Versace, Thomas Mercier, Amarande Murisier, Geraldine Sauvain, David Haefliger, François R Girardin, Maria-Helena Perez, Raphaël Giraud, Antoine Schneider, Thierry Buclin, Laurent A Decosterd, Eva Choong, Françoise Livio.

  Levosimendan is a positive inotrope and vasodilator used in patients with acute and chronic decompensated heart failure. It is metabolized into OR-1855 (inactive metabolite), which is further acetylated into OR-1896 (active metabolite having a prolonged half-life, hence a sustained effect). Levosimendan represents a valuable alternative to traditional inotropes with broad clinical applications in critically ill patients with cardiogenic shock, advanced heart failure and post-cardiac surgery. However, while levosimendan demonstrates dose-dependent hemodynamic effects, its pharmacokinetics has not yet been investigated in adult critically ill patients, a vulnerable population characterized by complex and unstable conditions that may significantly alter drug disposition. Therefore, pharmacokinetics studies of levosimendan and metabolites in critically ill patients require a reliable and sensitive quantification method. We developed and validated a highly sensitive method using ultra-high-performance liquid-chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the quantification of levosimendan, OR-1855 and OR-1896 in human plasma. To achieve the required analytical sensitivity, plasma sample preparation included protein precipitation with acetonitrile, subsequent supernatant's evaporation to dryness under nitrogen, and reconstitution of the solid residues with a solution of H2O:MeOH 4:1, followed by a 40 µL-aliquot injection into the LC column. Chromatographic separation of the three analytes was achieved in a 6-minute run in gradient mode, using an Acquity UPLC BEH C18 1.7 µm, 2.1 × 150 mm column. The method was extensively validated according to international bioanalytical assay guidelines, on a clinically relevant concentration range of 0.1-100 ng/mL, for each analyte. Considering these very low concentrations, the assay showed excellent performances in terms of trueness (94.3-105.3 %), repeatability (1.9-7.2 %) and intermediate fidelity (2.3-9.7 %). Of note, during our ex vivo studies on whole blood samples stability, acetylation of the metabolite OR-1855 into the active OR-1896 metabolite was observed in the presence of red blood cells. The UHPLC method is being applied for a prospective observational pharmacokinetics study of levosimendan in patients undergoing extracorporeal membrane oxygenation support. The benefit of levosimendan therapeutic drug monitoring in intensive care patients remains to be assessed.

Keywords:  ECMO; Intensive care medicine; Levosimendan; OR-1855; OR-1896; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jpba.2024.116612
Se Pu. 2025 Jan;43(1): 68-77

[Determination of 12 halogenated organic pollutants in edible fish by ultra performance liquid chromatography-high resolution mass spectrometry combined with ultrasound-assisted extraction and gel permeation chromatography purification].

Yi-Zhe Zhu, Rui-Fen Zheng, Zi-Hao Fan, Ling Liu, Jing-Yao Ye, Kai Wang, Cai-Ming Tang.

  Halogenated organic pollutants (HOPs) have attracted considerable attention owing to their persistence, bioaccumulation, and toxicity. The development of methods to detect HOPs in fish is challenging owing to the compositional complexity of fish matrices, which contain high levels of lipids and relatively low concentrations of HOPs. In addition, the lipophilicity of most HOPs renders their extraction difficult. Moreover, the simultaneous determination of multiple HOPs to achieve the high-throughput screening of these analytes is complex. In this study, a reliable and efficient pretreatment method based on ultrasound-assisted extraction, gel permeation chromatography purification, and ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) was developed for the determination of 12 HOPs in edible fish. The procedures of sample extraction and purification and LC-HRMS detection parameters were optimized to improve the performance of the method. Fresh fish samples were thoroughly rinsed with water, and non-edible parts, including the skin, bones, and phosphorus, were removed. The fish were weighed, cut into small pieces, and vacuum freeze-dried for 48 h. Subsequently, a freeze grinder was used to grind the dried fish into a fine powder. Exactly 2 g of the fish powder was weighed, fortified with isotope-labeled internal standards of the HOPs, and allowed to stand for 5 min. Methanol-acetonitrile (1∶1, v/v) was then added, followed by vortex mixing and ultrasonication. After centrifugation, the supernatant was transferred to a fresh tube. The extraction process was repeated twice and all extracts were combined. The extract was evaporated under a gentle nitrogen flow and redissolved in a mixture of ethyl acetate-cyclohexane (1∶1, v/v). The sample mixture was cleaned using gel permeation chromatography, and the eluate was collected and concentrated under a nitrogen flow. Sample residuals were reconstituted with water-methanol (1∶1, v/v) prior to instrumental analysis. Chromatographic separation was performed using an ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm). Water containing 2 mmol/L NH4Ac and acetonitrile were used as the mobile phases, and an optimized gradient elution program was applied. Isotope dilution and an internal standard method were used to quantify the HOPs. An electrospray ionization source operated in negative mode was applied to ionize the HOPs, and a full scan together with data-dependent acquisition (DDA) was applied for HRMS. Excellent linearities (R2>0.99) were obtained for all HOPs in the quantification range of 1.0-1000.0 ng/mL. The limits of quantification were 0.5 ng/g. The analytical method was validated using pooled fish samples fortified with HOP standards (4, 40, and 400 ng/g). The recoveries of the HOPs were in the range of 67.6%-133.8%, and the corresponding RSDs were 0.5%-15.6%. A total of 27 commercially available fish samples were analyzed using the developed method, and the results revealed the presence of HOPs in the fish, indicating the practicability of the method for real-world samples. The developed method is rapid, accurate, precise, and suitable for detecting HOPs in fish. This study provides a useful approach for environmental monitoring and food safety assurance by enabling the accurate and efficient analysis of HOPs in commonly consumed fish. Given increasing global concerns over HOPs, the method developed in this study will provide practical technical support for consumers aiming to reduce their exposure to and the adverse impacts of HOPs via fish.

Keywords:  edible fish; gel permeation chromatography (GPC); halogenated organic pollutants (HOPs); ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)

DOI:  https://doi.org/10.3724/SP.J.1123.2023.12028
Se Pu. 2025 Jan;43(1): 60-67

[Determination of eight phthalate metabolites in urine of pregnant women and evaluation of neonatal birth outcomes based on solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry].

Zi-Hao Wang, Meng-Fei Xu, Bei-Ni Li, Ping Wu, Wei Wu.

  Phthalates (PAEs) are endocrine-disrupting chemicals that are widely present in everyday life and enter the human body through various pathways. The release of PAEs into the environment through pathways that include leaching, evaporation, abrasion, and the use of personal care products exposes humans to PAEs via ingestion, inhalation, and dermal absorption. Pregnant women, as a particularly vulnerable population, risk adverse newborn growth and development when exposed to PAEs. While the concentrations of PAEs in urine reflect recent exposure levels in humans, urinary levels of phthalate metabolites (mPAEs) are commonly used as biomarkers of internal exposure owing to the relatively short biological half-lives of PAEs (<24 h). In this study, we developed a solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-HPLC-MS/MS) method for simultaneously detecting eight mPAEs in the urine of pregnant women. Urine samples were enzymatically hydrolyzed with β-glucosidase and then purified using the Bond Elut Plexa SPE column, with subsequent elution, concentration, and redissolved performed prior to HPLC-MS/MS. Separation was achieved using an Agilent Eclipse Plus C18 column (100 mm×3 mm, 3.5 μm), with gradient elution performed using 0.1% acetic acid aqueous solution and 0.1% acetic acid acetonitrile as mobile phases. Multiple reaction monitoring (MRM) mode was used for detection, with quantification performed using the internal-standard method. Good linearities were obtained in the range of 0.1-200 ng/mL for the eight mPAEs, with limits of detection (LODs) and quantification (LOQs) of 0.015-0.048 and 0.050-0.160 ng/mL, respectively. The eight mPAEs exhibited recoveries of 80.2%-99.7% at three spiked levels (1, 10, and 50 ng/mL). This method was subsequently used to analyze the eight mPAEs levels in urine samples of 497 pregnant women from the Ezhou Maternity and Child Health Care Hospital. The participants exhibited widespread exposure to PAEs, with monobutyl phthalate (MBP) showing the highest median level of 104.46 ng/mL, and monobenzyl phthalate (MBzP) showing the lowest (0.22 ng/mL). In addition, this study assessed neonatal birth outcomes. Linear regression modeling revealed that gestational age decreased by 0.11 weeks (95% confidence interval (CI): -0.18--0.03) for every natural-log (ln) increase in the level of monoethyl phthalate (MEP) in urine samples of pregnant woman. Moreover, the birth weight decreased by 39.28 g (95% CI: -76.48--2.09) and 39.62 g (95% CI: -73.73--5.52), for every ln increase in mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono(2-ethylhexyl) phthalate (MECPP) levels, respectively. The developed method is characterized by its simplicity, low LODs, high accuracy, and precision. This study provides clear evidence that PAE exposure during pregnancy negatively affects newborn growth and development by measuring the levels of eight mPAEs in the urine of pregnant women and linking these findings to neonatal outcomes. Further large-scale cohort studies are needed to validate these findings, along with mechanistic studies using animal models or in-vitro systems that elucidate the biological pathways through which mPAEs contribute to adverse birth outcomes.

Keywords:  birth outcomes; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); phthalate metabolites (mPAEs); urine samples of pregnant women

DOI:  https://doi.org/10.3724/SP.J.1123.2023.12032
Sci Rep. 2024 Dec 28. 14(1): 30964

Evaluation of metabolite stability in dried blood spot stored at different temperatures and times.

Hui-Na Cui, Fei Shi, Guihong Huang, Yun He, Shuqi Yu, Li Liu, Yan Li, He Wen.

  Dried blood spot (DBS) sampling offers significant advantages over conventional blood collection methods, such as reduced sample volume, minimal invasiveness, suitability for home-based sampling, and ease of transport. However, understanding the effects of variable storage temperatures and times on metabolite stability is crucial due to varying intervals and delivery conditions between sample collection and metabolomics analysis. To minimize biological variances, all samples were collected from the same individual simultaneously and stored at three different temperatures (4 °C, 25 °C, and 40 °C) for diverse time points (3, 7, 14, and 21 days). Metabolic profiling was conducted an untargeted gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)-based multi-platform metabolomics. Principal component analysis (PCA) showed alterations in metabolite stability at different temperatures, with phosphatidylcholines (PCs) and triglycerides (TAGs) as the first principal component (PC1). Specifically, we identified 69 metabolites that remained stable across all three temperatures over the 21-day period, while 78 metabolites exhibited instability. Furthermore, linear correlations between metabolite intensity and storage time were observed. Overall, our study elucidated the influence of storage temperature and time on specific metabolite stability in DBS samples, providing valuable insights for study design, biomarker selection, and data improvement.

Keywords:  DBS; Metabolite stability; Metabolomics; Storage temperature and time

DOI:  https://doi.org/10.1038/s41598-024-82041-2
ACS Meas Sci Au. 2024 Dec 18. 4(6): 702-711

Closing the Knowledge Gap of Post-Acquisition Sample Normalization in Untargeted Metabolomics.

Brian Low, Yukai Wang, Tingting Zhao, Huaxu Yu, Tao Huan.

Sample normalization is a crucial step in metabolomics for fair quantitative comparisons. It aims to minimize sample-to-sample variations due to differences in the total metabolite amount. When samples lack a specific metabolic quantity to accurately represent their total metabolite amounts, post-acquisition sample normalization becomes essential. Despite many proposed normalization algorithms, understanding remains limited of their differences, hindering the selection of the most suitable one for a given metabolomics study. This study bridges this knowledge gap by employing data simulation, experimental simulation, and real experiments to elucidate the differences in the mechanism and performance among common post-acquisition sample normalization methods. Using public datasets, we first demonstrated the dramatic discrepancies between the outcomes of different sample normalization methods. Then, we benchmarked six normalization methods: sum, median, probabilistic quotient normalization (PQN), maximal density fold change (MDFC), quantile, and class-specific quantile. Our results show that most normalization methods are biased when there is unbalanced data, a phenomenon where the percentages of up- and downregulated metabolites are unequal. Notably, unbalanced data can be sourced from the underlying biological differences, experimental perturbations, and metabolic interference. Beyond normalization algorithms and data structure, our study also emphasizes the importance of considering additional factors contributed by data quality, such as background noise, signal saturation, and missingness. Based on these findings, we propose an evidence-based normalization strategy to maximize sample normalization outcomes, providing a robust bioinformatic solution for advancing metabolomics research with a fair quantitative comparison.

DOI: https://doi.org/10.1021/acsmeasuresciau.4c00047
J Pharm Biomed Anal. 2024 Dec 20. pii: S0731-7085(24)00689-7. [Epub ahead of print]255 116647

Dried blood spot LC-MS/MS quantification of voclosporin in renal transplant recipients using volumetric dried blood spot sampling.

E Metscher, S Meziyerh, E J Arends, Y K O Teng, A P J de Vries, J J Swen, D J A R Moes.

  Voclosporin is a potent immunosuppressive agent currently approved for treating active lupus nephritis. Based on its potential antiviral activity, it has also been investigated as immunosuppressive agent in an investigator-initiated study in SARS-CoV2 positive kidney transplant recipients. As with many immunosuppressive agents, optimizing dosing regimens to achieve therapeutic efficacy while minimizing toxicity remains a critical challenge in clinical practice. To prevent organ rejection as well as infections, the prescribed immunosuppression needs to be well balanced. Dried blood spot (DBS) sampling has enabled development of remote voclosporin therapeutic drug monitoring. Here, we report on the development and analytical validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantification of voclosporin in dried blood spots. Method development was based on previously developed assays for the quantification of tacrolimus, everolimus, sirolimus, cyclosporin, mycophenolic acid, creatinine and iohexol in DBS and voclosporin in whole blood using LC-MS/MS. HemaXis™ volumetric blood spot devices were used for sample collection. The sample purification was based on the extraction of voclosporin from the DBS samples. Stable isotopically labeled voclosporin-D4 was used as an internal standard prior to sample purification. Bland Altman and Passing bablok analysis were performed for cross validation between whole blood and DBS samples. The method was successfully validated following the current ICH M10 guidelines. The dynamic range for the analyte was 10-600 µg/L with an excellent mean coefficient of correlation of 0.9978. The within run and between run precision and accuracy were both within the acceptance criteria. The cross-validation against the whole blood method shows that the quantified voclosporin results are promising. This developed dried blood spot LC-MS/MS method was successfully validated and provides an easy, efficient workflow for therapeutic drug monitoring in kidney transplant patients or remote pharmacokinetic studies in lupus nephritis patients treated with voclosporin.

Keywords:  Dried blood spots; LC-MS/MS; Microsampling; Therapeutic Drug Monitoring; Voclosporin

DOI:  https://doi.org/10.1016/j.jpba.2024.116647
Cold Spring Harb Protoc. 2024 Dec 23.

High-Throughput Free Amino Acid Quantification from Maize Tissues.

Huda Ansaf, Abou Yobi, Ruthie Angelovici.

Amino acids in maize can exist in both a free and protein-bound state. While most amino acids are part of a protein backbone, a small percentage of them remain free and play important biological roles, serving as signaling molecules, nitrogen transporters, osmolytes, and precursors for multiple primary and secondary metabolites. Their levels vary widely especially in maize leaves, depending on the developmental stage and in response to environmental conditions. Therefore, accurate and reliable quantification of free amino acids (FAAs) is vital in any effort aimed at studying their response to developmental and environmental cues. In this protocol, we describe a robust, high-throughput method that quantifies the 20 proteogenic amino acids (i.e., those that can be incorporated into proteins) that are found in the free form in maize tissue. This method consists of three major parts: first, aqueous extraction of FAAs from maize tissue; second, separation, detection, and quantification of all 20 proteogenic amino acids using ultraperformance liquid chromatography-tandem mass spectrometry; and third, data analysis and processing using the MassLynx data analysis software, TargetLynx.

DOI: https://doi.org/10.1101/pdb.prot108631
Anal Chem. 2024 Dec 23.

LipoCLEAN: A Machine Learning Filter to Improve Untargeted Lipid Identification Confidence.

Steven L Tavis, Matthew J Keller, Andrew J Stai, Tomás A Rush, Robert L Hettich.

In untargeted lipidomics experiments, putative lipid identifications generated by automated analysis software require substantial manual filtering to arrive at usable high-confidence data. However, identification software tools do not make full use of the available data to assess the quality of lipid identifications. Here, we present a machine-learning-based model to provide coherent, holistic quality scores based on multiple lines of evidence. Underutilized metrics such as isotope ratios and chromatographic behavior allow for much higher accuracy of identification confidence. We find that approximately 50% of tandem mass spectrometry-based automated lipid identifications are incorrect but that multidimensional rescoring reduces false discoveries to only 7% while retaining 80% of true positives. Our method works with most chromatography methods and generalizes across a family of MS instruments. LipoCLEAN is available at https://github.com/stavis1/LipoCLEAN.

DOI: https://doi.org/10.1021/acs.analchem.4c04040