bims-metlip 2022-01-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022‒01‒23
forty-four papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

Defining Blood Plasma and Serum Metabolome by GC-MS.
Isomer-selective analysis of inositol phosphates with differential isotope labelling by phosphate methylation using liquid chromatography with tandem mass spectrometry.
Development of a high-throughput differential mobility separation-tandem mass spectrometry (DMS-MS/MS) method for clinical urine drug testing.
Development of a fast, online three-phase electroextraction hyphenated to fast liquid chromatography-mass spectrometry for analysis of trace-level acid pharmaceuticals in plasma.
Identification and Distribution of Sterols, Bile Acids, and Acylcarnitines by LC-MS/MS in Humans, Mice, and Pigs-A Qualitative Analysis.
Simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry.
Twins labeling derivatization-based LC-MS/MS strategy for absolute quantification of paired prototypes and modified metabolites.
Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.
Annotation of complex mass spectra by multi-layered analysis.
Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of androgens, estrogens, glucocorticoids and progestagens in human serum.
A hydrophilic interaction liquid chromatography - Tandem mass spectrometry method for the determination of phenylephrine in dried blood spots from preterm infants.
Opening the Random Forest Black Box of the Metabolome by the Application of Surrogate Minimal Depth.
Application of a Target-Guided Data Processing Approach in Saturated Peak Correction of GC×GC Analysis.
Separation of phosphorothioated oligonucleotide diastereomers using multiplexed drift tube ion mobility mass spectrometry.
Simultaneous analysis of derivatized allyl isothiocyanate together with its phase II metabolites by UHPLC-MS/MS.
Spatially Mapping the Baseline and Bisphenol-A Exposed Daphnia magna Lipidome Using Desorption Electrospray Ionization-Mass Spectrometry.
Data-Driven Soft Independent Modeling of Class Analogy in Paper Spray Ionization Mass Spectrometry-Based Metabolomics for Rapid Detection of Prostate Cancer.
Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma.
Derivatization-assisted LC-MS/MS method for simultaneous quantification of endogenous gamma-hydroxybutyric acid and its metabolic precursors and products in human urine.
Determination of Parabens, Bisphenol A and Its Analogs, Triclosan, and Benzophenone-3 Levels in Human Urine by Isotope-Dilution-UPLC-MS/MS Method Followed by Supported Liquid Extraction.
High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry.
ptairMS: real-time processing and analysis of PTR-TOF-MS data for biomarker discovery in exhaled breath.
A Checklist for Reproducible Computational Analysis in Clinical Metabolomics Research.
Analysis of cycloheximide in rat specimens using liquid chromatography with tandem massspectrometry detection.
Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine-Chromatographic Studies.
Self-supervised clustering of mass spectrometry imaging data using contrastive learning.
A new platform for untargeted UHPLC-HRMS data analysis to address the time-shift problem.
Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics.
Evaluating the Accuracy of the QCEIMS Approach for Computational Prediction of Electron Ionization Mass Spectra of Purines and Pyrimidines.
Analysis of hydrophilic per- and polyfluorinated sulfonates including trifluoromethanesulfonate using solid phase extraction and mixed-mode liquid chromatography-tandem mass spectrometry.
Two-Dimensional Gas Chromatographic and Mass Spectrometric Characterization of Lipid-Rich Biological Matrices-Application to Human Cerumen (Earwax).
High throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.
massNet: integrated processing and classification of spatially resolved mass spectrometry data using deep learning for rapid tumor delineation.
Improved Thyreostatic Drug Detection in Animal Tissues Using Liquid Chromatography-High-Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry.
Reduced Hemoglobin Signal and Improved Detection of Endogenous Proteins in Blood-Rich Tissues for MALDI Mass Spectrometry Imaging.
Quantification of allantoin and other metabolites of the purine degradation pathway in human plasma samples using a newly developed HILIC-LC-MS/MS method.
Optimisation of saliva volumes for lipidomic analysis by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry.
Discovery and quantification of lipoamino acids in bacteria.
Optimised Method for Short-Chain Fatty Acid Profiling of Bovine Milk and Serum.
Screening of new psychoactive substances in human plasma by magnetic solid phase extraction and LC-QTOF-MS.
Hydrophilic interaction LC-MS/MS method to avoid endogenous interference in the analysis of 4-hydroxy isoleucine from dietary supplementation of fenugreek.
Stability of Wheat Floret Metabolites during Untargeted Metabolomics Studies.
In Vivo Identification of Adducts from the New Hypoxia-Activated Prodrug CP-506 Using DNA Adductomics.
Integration of a liquid-phase ion trap with a miniature mass spectrometer.

Metabolites. 2021 Dec 24. pii: 15. [Epub ahead of print]12(1):

Defining Blood Plasma and Serum Metabolome by GC-MS.

Olga Kiseleva, Ilya Kurbatov, Ekaterina Ilgisonis, Ekaterina Poverennaya.

  Metabolomics uses advanced analytical chemistry methods to analyze metabolites in biological samples. The most intensively studied samples are blood and its liquid components: plasma and serum. Armed with advanced equipment and progressive software solutions, the scientific community has shown that small molecules' roles in living systems are not limited to traditional "building blocks" or "just fuel" for cellular energy. As a result, the conclusions based on studying the metabolome are finding practical reflection in molecular medicine and a better understanding of fundamental biochemical processes in living systems. This review is not a detailed protocol of metabolomic analysis. However, it should support the reader with information about the achievements in the whole process of metabolic exploration of human plasma and serum using mass spectrometry combined with gas chromatography.

Keywords:  gas chromatography-mass spectrometry (GC-MS); metabolomics; multi-omics; plasma; serum

DOI:  https://doi.org/10.3390/metabo12010015
Anal Chim Acta. 2022 Jan 25. pii: S0003-2670(21)01112-0. [Epub ahead of print]1191 339286

Isomer-selective analysis of inositol phosphates with differential isotope labelling by phosphate methylation using liquid chromatography with tandem mass spectrometry.

Peng Li, Meinrad Gawaz, Madhumita Chatterjee, Michael Lämmerhofer.

  Inositol phosphates belong to a family of structurally diverse signaling molecules playing crucial role in Ca2+ release from intracellular storage vesicles. There are many possibilities of phosphorylation, including their degree and position. Inositol (1,4,5) trisphosphate has been well recognized as the most important second messenger among this family. It remains a challenge to analyse the entire inositol phosphate metabolite family due to its structural complexity, high polarity, and high phosphate density. In this study, we have established an improved UHPLC-ESI-MS/MS method based on a differential isotope labelling methylation strategy. An SPE extraction kit composed of TiO2 and PTFE filter was employed for sample preparation which provided good extraction performance. Samples were methylated (light label) to neutralize the phosphate groups and give better performance in liquid chromatography. Regioisomers and inositol phosphates differing in their number of phosphate residues were successfully separated after optimization on a core-shell cholesterylether-bonded RP-type column (Cosmocore 2.6Cholester) using methanol as organic modifier. Triple quadrupole MS detection was based on selected reaction monitoring (SRM) acquisition with characteristic fragments. Stable isotope labeling methylation was performed to generate internal standards (heavy label). Limits of quantification from 0.32 to 0.89 pmol on column was achieved. This method was validated to be suitable for inositol phosphate profiling in biological samples. After application in cultured HeLa cells, NIST SRM1950 plasma, and human platelets, distinct inositol profiles were obtained. This newly established method exhibited improved analytical performance, holding the potential to advance the understanding of inositol phosphate signaling.

Keywords:  Derivatization; Inositol phosphate; Isotope labeling; Metal oxide-based affinity chromatography; Solid-phase extraction; Targeted metabolomics

DOI:  https://doi.org/10.1016/j.aca.2021.339286
J Mass Spectrom Adv Clin Lab. 2022 Jan;23 50-57

Development of a high-throughput differential mobility separation-tandem mass spectrometry (DMS-MS/MS) method for clinical urine drug testing.

Shirin Hooshfar, Simone Tchu, Cassandra Yun, Kara L Lynch.

  Introduction: Differential mobility separation (DMS) is an analytical technique used for rapid separation of ions and isomers based on gas phase mobility prior to entering a mass spectrometer for analysis. The entire DMS process is accomplished in fewer than 20 ms and can be used as a rapid alternative to chromatographic separation.Objective: The primary objective was to evaluate the utility of DMS-tandem mass spectrometry (DMS-MS/MS) as a replacement for immunoassay-based clinical toxicology testing.
Methods: A sensitive DMS-MS/MS method was developed and validated for simultaneous identification of 33 drugs and metabolites in human urine samples. After DMS optimization, the method was validated and used to screen 56 clinical urine samples. These results were compared to results obtained by immunoassay.
Results: The DMS-MS/MS method achieved limits of detection ranging from 5 to 100 ng/mL. Moreover, the total analysis time was 2 min per sample. For the method performance evaluation, DMS-MS/MS results were compared with previously obtained urine toxicology immunoassay results. DMS-MS/MS showed higher sensitivity and identified 20% more drugs in urine, which were confirmed by LC-MS/MS.
Conclusion: The DMS-MS/MS as applied in our lab demonstrated the capability for rapid drug screening and provided better analytical performance than immunoassay.

Keywords:  6-MAM, 6-Monoacetylmorphine; AMPH, amphetamines/ecstasy; BENZ, benzodiazepines; BUPR, buprenorphine; CE, Collision energy; COV, compensation voltage; CXP, collision cell exit potential; DAPPI, atmospheric pressure photo ionization; DART, direct analysis in real time; DC, direct current; DESI, desorption electrospray ionization; DMO, DMS offse; DMS, differential mobility separation; DP, declustering potential; DR, DMS resolution enhancement; DT, DMS cell temperature; Differential mobility separation; Drugs of abuse; EDDP, 2-ethylidene1,5-dimethyl-3,3-diphenylpyrrolidine; EP, entrance potential; FAIMS, field asymmetric waveform ion mobility spectrometry; FSI, fiber spray ionization; GC-MS or LC-MS, gas chromatography- or liquid chromatography-mass spectrometry; GS1, ion source gas 1; GS2, ion source gas 2; IMS, ion mobility spectrometry, IS, internal standards, LOD, limit of detection, MD, modifier, MDC, modifier composition, ME, matrix effects; MRM, multiple reaction monitoring; MS/MS, tandem mass spectrometry; Mass spectrometry; OPI, opiates; OXY, oxycodone/oxmorphone; QCs, quality controls; SRM, selected reaction monitoring; SV, separation voltage; Urine drug screening; WT-ESI, wooden-tip electrospray ionization

DOI:  https://doi.org/10.1016/j.jmsacl.2021.12.008
Anal Chim Acta. 2022 Feb 01. pii: S0003-2670(21)01190-9. [Epub ahead of print]1192 339364

Development of a fast, online three-phase electroextraction hyphenated to fast liquid chromatography-mass spectrometry for analysis of trace-level acid pharmaceuticals in plasma.

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

  Sample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while removing the interferents to reduce the matrix effect and improve both sensitivity and quantification. In this study, a three-phase electroextraction (EE) method hyphenated to fast online liquid chromatography-mass spectrometry (LC-MS) was developed. Four model acidic drugs of relevance for drug monitoring in plasma, i.e. naproxen, fenoprofen, flurbiprofen, and ibuprofen, were utilized for the optimization and evaluation of the method. A Design of Experiment approach (Box-Behnken design) was used to optimize the critical parameters of the method, i.e., the type of organic solvent, pH of the sample and acceptor phase, and the extraction voltage and time. Good fitness (P < 0.02, R2 > 0.95) was observed for the developed quadratic model. Extraction could be achieved in less than 2 min (115 s) with enrichment factors (EF) up to 190 and extraction recoveries (ER) up to 38% for academic samples. Additionally, the optimized three-phase EE method was successfully applied to spiked plasma samples with low-abundant (50 ng mL-1) analytes and a low sample volume of 15 μL plasma in 10-fold diluted samples. Finally, two crucial contributors to the matrix effect of three-phase EE application on plasma samples were determined. Specifically, the ion-suppression effect in the MS source was reduced by the fast LC separation, and the matrix effect during extraction was negligible for the diluted protein-precipitated plasma samples. The developed three-phase EE method is easy to operate and provides fast and online extraction of trace-level acidic analytes from volume-limited biological samples. Therefore, this method can provide a potential solution for sample-preparation bottlenecks in high-throughput bioanalysis workflows.

Keywords:  Bioanalysis; Electroextraction; Human plasma; Online fast LC-MS; Sample preparation

DOI:  https://doi.org/10.1016/j.aca.2021.339364
Metabolites. 2022 Jan 07. pii: 49. [Epub ahead of print]12(1):

Identification and Distribution of Sterols, Bile Acids, and Acylcarnitines by LC-MS/MS in Humans, Mice, and Pigs-A Qualitative Analysis.

Ambrin Farizah Babu, Ville Mikael Koistinen, Soile Turunen, Gloria Solano-Aguilar, Joseph F Urban, Iman Zarei, Kati Hanhineva.

  Sterols, bile acids, and acylcarnitines are key players in human metabolism. Precise annotations of these metabolites with mass spectrometry analytics are challenging because of the presence of several isomers and stereoisomers, variability in ionization, and their relatively low concentrations in biological samples. Herein, we present a sensitive and simple qualitative LC-MS/MS (liquid chromatography with tandem mass spectrometry) method by utilizing a set of pure chemical standards to facilitate the identification and distribution of sterols, bile acids, and acylcarnitines in biological samples including human stool and plasma; mouse ileum, cecum, jejunum content, duodenum content, and liver; and pig bile, proximal colon, cecum, heart, stool, and liver. With this method, we detected 24 sterol, 32 bile acid, and 27 acylcarnitine standards in one analysis that were separated within 13 min by reversed-phase chromatography. Further, we observed different sterol, bile acid, and acylcarnitine profiles for the different biological samples across the different species. The simultaneous detection and annotation of sterols, bile acids, and acylcarnitines from reference standards and biological samples with high precision represents a valuable tool for screening these metabolites in routine scientific research.

Keywords:  LC–MS/MS; acylcarnitine; annotation; bile acid; sterol

DOI:  https://doi.org/10.3390/metabo12010049
Leg Med (Tokyo). 2021 Dec 30. pii: S1344-6223(21)00175-9. [Epub ahead of print]55 102011

Simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry.

Takao Sakurai, Toshiaki Irii, Kimiharu Iwadate.

  In forensic analysis, the identification of urine or human urine among unknown liquids plays an important role. Urea, uric acid, and creatinine are major organic compounds found in human urine. Previous studies have reported that the concentration quotients of these three compounds can be used as an index for the identification of human urine. Here we describe a method for the simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry (LC/MS), with the aim of forensic identification of human urine. Separation of the three analytes was achieved by hydrophilic interaction chromatography, using a TSK gel Amide-80 column with a mobile phase composed of acetonitrile and ammonium acetate aqueous solution, coupled with detection using a mass spectrometer. For quantification, melamine and violuric acid were used as internal standards. Human urine samples were pretreated for LC/MS analysis by dilution with LC mobile phase, followed by centrifugation and filtration. The analytes and internal standards were separated within 9 min. The linear ranges were 2.0-40.0, 0.10-1.60, and 0.13-2.00 mg/mL for urea, uric acid, and creatinine, respectively, with correlation coefficients > 0.99. The intra- and inter-day accuracies of the analytes were - 10.6% to 7.4%, and the precision was within 7.6%. For all analytes, no significant matrix effects were observed and recoveries ranged from 95.4% to 104.6%. Quantitative results of 3 analytes were obtained within their linear range from 10 human urine samples and the quotients, UA/UN × 20 and UA/Cre, were calculated based on previous reports.

Keywords:  Creatinine; LC/MS; Urea; Uric acid; Urine

DOI:  https://doi.org/10.1016/j.legalmed.2021.102011
Anal Chim Acta. 2022 Feb 08. pii: S0003-2670(21)01225-3. [Epub ahead of print]1193 339399

Twins labeling derivatization-based LC-MS/MS strategy for absolute quantification of paired prototypes and modified metabolites.

Wei Li, Pei Zhang, Xiaoying Hou, Tian Tang, Siqi Li, Ruiqi Sun, Zunjian Zhang, Fengguo Xu.

  Modified metabolites play significant roles in disease occurrence, progression and diagnosis. Sensitive and accurate analytical methods for the quantification of these metabolites are therefore of great importance. In this study, a liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous measurement of 13 pairs of prototypes and their modified forms covering nucleobases, nucleosides and amino acids. In order to improve the quantification sensitivity and accuracy, two structure analogs named N-dimethyl-amino naphthalene-1-sulfonyl chloride (Dns-Cl) and N-diethyl-amino naphthalene-1-sulfonyl chloride (Dens-Cl) were introduced for twins labeling derivatization. Dns-labeling was utilized to react with target analytes while the Dens-labeling of standard compounds provided one-to-one internal standards. With the introduce of naphthalene and easily ionizable moiety tertiary ammonium, chromatography retention and separation of these polar metabolites were notably improved on C18 columns and the detection sensitivity was increased up to 400 folds. The method is sensitive with the lower limit of quantification (LLOQ) values of 0.002-0.5 μg/mL. Comparisons of the performance of twins labeling derivatization and traditional chemical isotope labeling (CIL) derivatization verified the ability of our method in the absolute quantification. The established method was applied to human lung adenocarcinoma cell line A549 and its cisplatin resistant derivative A549/DDP. Significant shifts in 12 metabolites as well as 9 modified-to-prototypical ratios in A549/DDP were observed, demonstrating the utility of our method and the potential role of modified metabolites in mediating anticancer drug resistance. The method can be easily extended to determine other types of modified metabolites in various biological matrices, which will greatly expand our knowledge on these metabolites.

Keywords:  Cisplatin; Drug resistance; LC-MS/MS; Metabolomics; Modified metabolites; Twins labeling derivatization

DOI:  https://doi.org/10.1016/j.aca.2021.339399
Scand J Clin Lab Invest. 2022 Jan 20. 1-13

Development of quantitative assay for simultaneous measurement of purine metabolites and creatinine in biobanked urine by liquid chromatography-tandem mass spectrometry.

Dmitri Svistounov, Marit D Solbu, Trond G Jenssen, Ulla Dorte Mathisen, Terkel Hansen, Katja Benedikte Prestø Elgstøen, Svetlana N Zykova.

  Purine metabolism is essential for all known living creatures, including humans in whom elevated serum concentration of purine break-down product uric acid (UA) is probably an independent risk factor for mortality, type 2 diabetes and cardiovascular events. An automated multiplex assay that measures several purine metabolites could therefore prove useful in many areas of medical, veterinary and biological research. The aim of the present work was to develop a sensitive LC-MS/MS method for simultaneous quantitation of xanthine, hypoxanthine, UA, allantoin, and creatinine in biobanked urine samples. This article describes details and performance of the new method studied in 55 samples of human urine. Archival sample preparation and effect of storage conditions on stability of the analytes are addressed. The intra-day and inter-day coefficients of variation were small for all the analytes, not exceeding 1% and 10%, respectively. Measurements of UA and creatinine in biobanked urine showed good agreement with values obtained using routine enzymatic assays on fresh urine. Spearman's correlation coefficients were 0.869 (p < .001) for creatinine and 0.964 (p < .001) for UA. Conclusion: the newly developed LC-MS/MS method allows reliable quantitative assessment of xanthine, hypoxanthine, allantoin, UA and creatinine. The proposed pre-analytical processing makes the method suitable for both fresh and biobanked urine stored frozen at -80 °C for at least 5.5 years.

Keywords:  Liquid chromatography; allantoin; biological specimen bank; creatinine; hydrophilic interaction; hypoxanthine; mass spectroscopy; purines; reference ranges; solubility; uric acid; urine; xanthine

DOI:  https://doi.org/10.1080/00365513.2021.2015799
Anal Chim Acta. 2022 Feb 08. pii: S0003-2670(21)01143-0. [Epub ahead of print]1193 339317

Annotation of complex mass spectra by multi-layered analysis.

Ron Bonner, Gérard Hopfgartner.

  Annotating electrospray small molecule mass spectra remains a challenging problem due to the multiple processes occurring during ionization. Although an [M+H]+ is often present, ions can be formed by reactions with other cations, background compounds and co-eluting species, and by in-source fragmentation. Even single analytes can produce multiple ion forms, many of which remain unidentified and may appear to be different species, affecting reproducibility, quantification and precursor selection in DDA experiments. Annotation usually compares differences between peaks to known adducts and losses but fails if key peaks are missing or if the peaks are from unexpected adducts. Further, isotopes are often assumed to be due to 13C and removed prior to analysis which can leave 'orphan' peaks if unusual elements are present. Here we describe an alternative multi-layered approach (MLA) which successively matches spectra to calculated target ion lists and reprocesses the residual ions. This allows the analyst to focus on the unknown ions and to progressively increase target list complexity since explained ions are removed. Target ion lists can be calculated from expected or observed masses and potential adducts or can be pre-defined lists, for example common contaminants. Using this approach on spectra of known standards we identified adducts with Ca, Al, Fe, Ba and possibly Mg and Sr. We also detected several compounds and adducts in a spectrum of co-eluting species from an LC-MS analysis.

Keywords:  Adducts; Data reduction. metabolomics; Electrospray; LC-MS; Software; Spectra annotation

DOI:  https://doi.org/10.1016/j.aca.2021.339317
Biomed Chromatogr. 2022 Jan 22. e5344

Development and validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of androgens, estrogens, glucocorticoids and progestagens in human serum.

Michele Iannone, Anna Pia Dima, Francesca Sciarra, Francesco Botrè, Andrea M Isidori.

We present a liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 16 endogenous steroids (androgens, estrogens, glucocorticoids, and progestogens) in human serum. Samples (250 μL of matrix) were extracted with t-butylmethyl ether prior to LC-MS/MS analysis. The chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. The HPLC was coupled to a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source with acquisition in multiple reaction mode. The method was validated using surrogated matrices and human serum samples. Specificity of the method was confirmed for all the considered steroids; linearity was also assessed (R2 > 0.99, lack-of-fit test) in the ranges of concentrations investigated. The lower limits of quantification were in the range 10 - 400 pg/mL depending on the target steroid. Accuracy was in the range 85 - 115% for all target steroids except for LLOQ levels where is 80 - 120%; the extraction recovery was always higher than 65%; no significant matrix effects were observed. To test the reliability of the method, the analysis of serum samples collected from ten healthy subjects (5M/5F) was performed. The presented method can be used to identify trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes.

DOI: https://doi.org/10.1002/bmc.5344
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 06. pii: S1570-0232(21)00565-1. [Epub ahead of print]1190 123084

A hydrophilic interaction liquid chromatography - Tandem mass spectrometry method for the determination of phenylephrine in dried blood spots from preterm infants.

E Boutou, C Virgiliou, A K Seliniotaki, M Lithoxopoulou, A Mataftsi, N Ziakas, E Diamanti, N Raikos, H Gika.

  A sensitive and accurate hydrophilic interaction liquid chromatography - tandem mass spectrometry method (HILIC-MS/MS) was developed and validated for the determination of phenylephrine concentration in Dried Blood Spot (DBS) samples from preterm infants, after ocular administration of an ophthalmic solution with phenylephrine. Sample preparation involved the extraction of the analyte from an 85 μL DBS sample with methanol - acetonitrile (50:50, v/v). Chromatographic separation was achieved on an ACQUITY UPLC BEH AMIDE column, under isocratic conditions within a 5 min run. Detection was achieved with a triple quadrupole MS applying electrospray ionization in positive mode. The method was fully validated and proved precise and accurate with in a linear range of 0.59-3.53 ng/ml in blood. The method was developed to provide insights on the level of exposure of infant population to phenylephrine after ocular administration.

Keywords:  DBS; Drug; HILIC; Pharmaceutical; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2021.123084
Metabolites. 2021 Dec 21. pii: 5. [Epub ahead of print]12(1):

Opening the Random Forest Black Box of the Metabolome by the Application of Surrogate Minimal Depth.

Soeren Wenck, Marina Creydt, Jule Hansen, Florian Gärber, Markus Fischer, Stephan Seifert.

  For the untargeted analysis of the metabolome of biological samples with liquid chromatography-mass spectrometry (LC-MS), high-dimensional data sets containing many different metabolites are obtained. Since the utilization of these complex data is challenging, different machine learning approaches have been developed. Those methods are usually applied as black box classification tools, and detailed information about class differences that result from the complex interplay of the metabolites are not obtained. Here, we demonstrate that this information is accessible by the application of random forest (RF) approaches and especially by surrogate minimal depth (SMD) that is applied to metabolomics data for the first time. We show this by the selection of important features and the evaluation of their mutual impact on the multi-level classification of white asparagus regarding provenance and biological identity. SMD enables the identification of multiple features from the same metabolites and reveals meaningful biological relations, proving its high potential for the comprehensive utilization of high-dimensional metabolomics data.

Keywords:  LC-MS; characterization; chemometrics; classification; feature relations; feature selection; machine learning; metabolomics; random forest; surrogate minimal depth; white asparagus

DOI:  https://doi.org/10.3390/metabo12010005
Anal Chem. 2022 Jan 20.

Application of a Target-Guided Data Processing Approach in Saturated Peak Correction of GC×GC Analysis.

Penghan Zhang, Silvia Carlin, Pietro Franceschi, Fulvio Mattivi, Urska Vrhovsek.

Detector and column saturations are problematic in comprehensive two-dimensional gas chromatography (GC×GC) data analysis. This limits the application of GC×GC in metabolomics research. To address the problems caused by detector and column saturations, we propose a two-stage data processing strategy that will incorporate a targeted data processing and cleaning approach upstream of the "standard" untargeted analysis. By using the retention time and mass spectrometry (MS) data stored in a library, the annotation and quantification of the targeted saturated peaks have been significantly improved. After subtracting the nonperfected signals caused by saturation, peaks of coelutes can be annotated more accurately. Our research shows that the target-guided method has broad application prospects in the data analysis of GC×GC chromatograms of complex samples.

DOI: https://doi.org/10.1021/acs.analchem.1c02719
Anal Chim Acta. 2022 Jan 25. pii: S0003-2670(21)01123-5. [Epub ahead of print]1191 339297

Separation of phosphorothioated oligonucleotide diastereomers using multiplexed drift tube ion mobility mass spectrometry.

Alice Demelenne, Gwenael Nys, Cindy Nix, John C Fjeldsted, Jacques Crommen, Marianne Fillet.

  Hydrophilic interaction liquid chromatography (HILIC) coupled to drift tube ion mobility spectrometry (DTIMS) was used to separate diastereomers of five-unit oligonucleotides containing 0, 1, 2 or 3 phosphorothioate (PS) linkages. Multiplexed DTIMS (where ions are pulsed into the drift tube according to a pre-encoded sequence) and post-acquisition processing using an innovative demultiplexing tool were investigated. The electric field inside the drift tube was optimized to achieve the highest resolving power. The entrance voltage providing the best two-peak resolution was -1000V with 3-bit multiplexing. Under optimized conditions, the eight diastereomers of an oligonucleotide with three PS linkages (5'-TC∗G∗T∗G-3') could be separated unambiguously. Indeed, those diastereomers differed in their collision cross section (CCS) values. The minimal CCS values difference between two adjacent diastereomers was 0.9% with maximal RSD on CCS values of 0.3%. The use of multiplexed ion mobility and the novel high-resolution demultiplexing tool represents a real breakthrough for resolution enhancement of diastereomers in linear DTIMS.

Keywords:  CCS; Drift tube; Ion mobility; Multiplexing; Resolution

DOI:  https://doi.org/10.1016/j.aca.2021.339297
Rapid Commun Mass Spectrom. 2022 Jan 17. e9257

Simultaneous analysis of derivatized allyl isothiocyanate together with its phase II metabolites by UHPLC-MS/MS.

Hiu-Lok Ngan, Tao Wang, Yi Ru, Yanyan Chen, Charlie Li, Zongwei Cai.

RATIONALE: Allyl isothiocyanate (AITC) in food products such as wasabi is commonly analyzed by GC/MS, while LC/MS is more suitable for the polar metabolites. The development of an effective method for the simultaneous determination of AITC and its phase II metabolites is needed for supporting drug metabolism and pharmacokinetics (DMPK) study.METHODS: Derivatization of AITC by reacting with N-(tert-butoxycarbonyl)-L-cysteine methyl ester (t BocCysME) was used for UHPLC/ESI-MS analysis, allowing the simultaneous determination with its phase II metabolites. Under room temperature, reaction conditions have been optimized to maximize AITC reacted with t BocCysME quantitively. Reaction selectivity was examined on the presence of AITC metabolite. Quantification of AITC was performed by multiple reaction monitoring (MRM) with an external calibration method and applied on the serum of C57BL/6J mouse for DMPK study.
RESULTS: LOQ was determined to be 0.842 nM from the derivatization method and by UHPLC-MS/MS analysis. The method accuracy in the mouse serum samples was 75 ± 2% with the RSD of 3.0% of method variation. The UHPLC-MS/MS analysis gave its RSD of 0.2% and 1.8% for intra-day and inter-day variations. With the newly developed method, AITC could be detected in mouse serum upon oral administration for the first time.
CONCLUSIONS: An analytical method involving the rapid derivatization pre-treatment and quantitative UHPLC-ESI-QqQ-MS/MS analysis is developed for biological samples assay of AITC, enabling selective and sensitive detection of the AITC derivative and AITC metabolites simultaneously. The method developed could be applied to the DMPK study of AITC as well.

DOI: https://doi.org/10.1002/rcm.9257
Metabolites. 2022 Jan 01. pii: 33. [Epub ahead of print]12(1):

Spatially Mapping the Baseline and Bisphenol-A Exposed Daphnia magna Lipidome Using Desorption Electrospray Ionization-Mass Spectrometry.

Matthew J Smith, Ralf J M Weber, Mark R Viant.

  Untargeted lipidomics has previously been applied to the study of daphnids and the discovery of biomarkers that are indicative of toxicity. Typically, liquid chromatography-mass spectrometry is used to measure the changes in lipid abundance in whole-body homogenates of daphnids, each only ca. 3 mm in length which limits any biochemical interpretation of site-specific toxicity. Here, we applied mass spectrometry imaging of Daphnia magna to combine untargeted lipidomics with spatial resolution to map the molecular perturbations to defined anatomical regions. A desorption electrospray ionization-mass spectrometry (DESI-MS) method was optimized and applied to tissue sections of daphnids exposed to bisphenol-A (BPA) compared to unexposed controls, generating an untargeted mass spectrum at each pixel (35 µm2/pixel) within each section. First, unique lipid profiles from distinct tissue types were identified in whole-body daphnids using principal component analysis, specifically distinguishing appendages, eggs, eye, and gut. Second, changes in the lipidome were mapped over four stages of normal egg development and then the effect of BPA exposure on the egg lipidome was characterized. The primary perturbations to the lipidome were annotated as triacylglycerides and phosphatidylcholine, and the distributions of the individual lipid species within these classes were visualized in whole-body D. magna sections as ion images. Using an optimized DESI-MS workflow, the first ion images of D. magna tissue sections were generated, mapping both their baseline and BPA-perturbed lipidomes.

Keywords:  Daphnia magna; bisphenol-A; cryosectioning; desorption electrospray ionization; egg development; ion images; lipidome; mass spectrometry imaging

DOI:  https://doi.org/10.3390/metabo12010033
Anal Chem. 2022 Jan 21.

Data-Driven Soft Independent Modeling of Class Analogy in Paper Spray Ionization Mass Spectrometry-Based Metabolomics for Rapid Detection of Prostate Cancer.

Frederico G Pinto, Iqbal Mahmud, Vanessa Y Rubio, Ademar Domingos Viagem Máquina, Anízia Fausta Furtado Durans, Waldomiro Borges Neto, Timothy J Garrett.

Sensitive, rapid, and meaningful diagnostic tools for prostate cancer (PC) screening are urgently needed. Paper spray ionization mass spectrometry (PSI-MS) is an emerging rapid technology for detecting biomarker and disease diagnoses. Due to lack of chromatography and difficulties in employing tandem MS, PSI-MS-based untargeted metabolomics often suffers from increased ion suppression and subsequent feature detection, affecting chemometric methods for disease classification. This study first evaluated the data-driven soft independent modeling of class analogy (DD-SIMCA) model to analyze PSI-MS-based global metabolomics of a urine data matrix to classify PC. The efficiency of DD-SIMCA was analyzed based on the sensitivity and specificity parameters that showed 100% correct classification of the training set, based on only PC and test set samples, based on normal and PC. This analytical methodology is easy to interpret and efficient and does not require any prior information from the healthy individual. This new application of DD-SIMCA in PSI-MS-based metabolomics for PC disease classification could also be extended to other diseases and opens a rapid strategy to discriminate against health problems.

DOI: https://doi.org/10.1021/acs.analchem.1c04004
Antioxidants (Basel). 2022 Jan 07. pii: 134. [Epub ahead of print]11(1):

Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma.

Gwendolyn van Gorkom, Birgit Gijsbers, Erik-Jan Ververs, Ahmed El Molla, Cindy Sarodnik, Celine Riess, Will Wodzig, Gerard Bos, Catharina Van Elssen.

  Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood.

Keywords:  ascorbic acid; peripheral blood mononuclear cells; vitamin C

DOI:  https://doi.org/10.3390/antiox11010134
Anal Chim Acta. 2022 Feb 15. pii: S0003-2670(21)01227-7. [Epub ahead of print]1194 339401

Derivatization-assisted LC-MS/MS method for simultaneous quantification of endogenous gamma-hydroxybutyric acid and its metabolic precursors and products in human urine.

Suji Kim, Min Seo Lee, Mingyu Kim, Beom Jun Ko, Hye Suk Lee, Sooyeun Lee.

  The accurate, precise, and robust quantification of endogenous biomarkers is a challenging task because of the presence of significantly low levels of endogenous compounds in biological samples, the absence of analyte-free matrix-matched calibrators, and sample instability due to in-vitro production or degradation of the analytes. Gamma-hydroxybutyric acid (GHB), a compound often used in drug-facilitated crimes, is a human neurotransmitter produced during both the biosynthesis and metabolism of gamma-aminobutyric acid (GABA). Evidently, proving GHB intoxication through the quantification of GHB and its metabolites in biological samples is not straightforward. This study aimed to develop a sensitive and accurate quantitative method for the simultaneous determination of endogenous GHB and its metabolic precursors and products (glutamic acid, GABA, succinic acid, 2,4-dihydroxybutyric acid, 3,4-dihydroxybutyric acid, glycolic acid, and succinylcarnitine) in human urine using LC-MS/MS. For this purpose, chemical derivatization with benzoyl chloride was employed to improve the sensitivity to glutamic acid and GABA. Synthetic urine was used to prepare calibrators, and the validity of this approach was fully demonstrated, particularly focusing on the instability issues. The validation results proved the method to be selective, sensitive, accurate, and precise, with acceptable linearity within calibration ranges. Moreover, our results regarding the in-vitro production or degradation of metabolites highlight the effects of handling and storage conditions of urine samples. Finally, this effective analytical method is expected to be useful in studying the relationship between GHB intoxication and metabolic alterations and, thus, discovering practical biomarkers for GHB ingestion.

Keywords:  Biomarker metabolites; Chemical derivatization; Drug-facilitated crimes; Gamma-hydroxybutyric acid; Instability; LC-MS/MS

DOI:  https://doi.org/10.1016/j.aca.2021.339401
Toxics. 2022 Jan 06. pii: 21. [Epub ahead of print]10(1):

Determination of Parabens, Bisphenol A and Its Analogs, Triclosan, and Benzophenone-3 Levels in Human Urine by Isotope-Dilution-UPLC-MS/MS Method Followed by Supported Liquid Extraction.

Hsin-Chang Chen, Jung-Wei Chang, Yi-Chen Sun, Wan-Ting Chang, Po-Chin Huang.

  The development of a rapid analytical approach for determining levels of antibacterial agents, plasticizers, and ultraviolet filters in biosamples is crucial for individual exposure assessment. We developed an analytical method to determine the levels of four parabens-bisphenols A (BPA) and its analogs, triclosan (TCS), triclocarban, and benzophenone-3 (BP-3)-in human urine. We further measured the levels of these chemicals in children and adolescents. We used a supported liquid extraction (SLE) technique coupled with an isotope-dilution ultraperformance liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) method to assess the detection performance for these chemicals. Forty-one urine samples from 13 children and 28 adolescents were assessed to demonstrate the capability and feasibility of our method. An acceptable recovery (75.6-102.4%) and matrix effect (precision < 14.2%) in the three-level spiked artificial urine samples were achieved, and good performance of the validated ID-UPLC-MS/MS method regarding linearity, limits of detection, and quantitation was achieved. The within-run and between-run accuracy and precision also demonstrated the sensitivity and stability of this analytical method, applied after SLE. We concluded that the ID-UPLC-MS/MS method with SLE pretreatment is a valuable analytical method for the investigation of urinary antibacterial agents, plasticizers, and ultraviolet filters in humans, useful for human biomonitoring.

Keywords:  biomonitoring; bisphenols; isotope-dilution UPLC-MS/MS; parabens; triclosan

DOI:  https://doi.org/10.3390/toxics10010021
J Chromatogr A. 2022 Jan 02. pii: S0021-9673(22)00001-2. [Epub ahead of print]1665 462803

High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry.

Guadalupe Espadas, Julia Morales-Sanfrutos, Rebeca Medina, Morghan C Lucas, Eva Maria Novoa, Eduard Sabidó.

  Over 170 post-transcriptional RNA modifications have been described and are common in all kingdoms of life. These modifications range from methylation to complex chemical structures, with methylation being the most abundant. RNA modifications play a key role in RNA folding and function and their dysregulation in humans has been linked to several diseases such as cancer, metabolic diseases or neurological disorder. Nowadays, liquid chromatography-tandem mass spectrometry is considered the gold standard method for the identification and quantification of these modifications due to its sensitivity and accuracy. However, the analysis of modified ribonucleosides by mass spectrometry is complex due to the presence of positional isomers. In this scenario, optimal separation of these compounds by highly sensitive liquid chromatography combined with the generation of high-information spectra is critical to unequivocally identify them, especially in high-complex mixtures. Here we present an analytical method that comprises a new type of mixed-mode nano-flow liquid chromatography column combined with high- and low-collision energy data-independent mass spectrometric acquisition for the identification and quantitation of modified ribonucleosides. The method produces content-rich spectra and combines targeted and screening capabilities thus enabling the identification of a variety of modified nucleosides in biological matrices by single-shot liquid chromatographic analysis coupled to mass spectrometry.

Keywords:  Chromatography; Data-independent acquisition; Epitranscriptomics; Mass spectrometry; RNA; Ribonucleosides

DOI:  https://doi.org/10.1016/j.chroma.2022.462803
Bioinformatics. 2022 Jan 19. pii: btac031. [Epub ahead of print]

ptairMS: real-time processing and analysis of PTR-TOF-MS data for biomarker discovery in exhaled breath.

Camille Roquencourt, Stanislas Grassin-Delyle, Etienne A Thévenot.

MOTIVATION: Analysis of Volatile Organic Compounds (VOCs) in exhaled breath by Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-TOF-MS) is of increasing interest for real-time, non-invasive diagnosis, phenotyping and therapeutic drug monitoring in the clinics. However, there is currently a lack of methods and software tools for the processing of PTR-TOF-MS data from cohorts and suited for biomarker discovery studies.RESULTS: We developed a comprehensive suite of algorithms that process raw data from patient acquisitions and generate the table of feature intensities. Notably, we included an innovative 2D peak deconvolution model based on penalized splines signal regression for accurate estimation of the temporal profile and feature quantification, as well as a method to specifically select the VOCs from exhaled breath. The workflow was implemented as the ptairMS software, which contains a graphical interface to facilitate cohort management and data analysis. The approach was validated on both simulated and experimental data sets, and we showed that the sensitivity and specificity of the VOC detection reached 99% and 98.4%, respectively, and that the error of quantification was below 8.1% for concentrations down to 19 ppb.
AVAILABILITY: The ptairMS software is publicly available as an R package on Bioconductor (doi:10.18129/B9.bioc.ptairMS), as well as its companion experiment package ptairData (doi:10.18129/B9.bioc.ptairData).
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btac031
Metabolites. 2022 Jan 17. pii: 87. [Epub ahead of print]12(1):

A Checklist for Reproducible Computational Analysis in Clinical Metabolomics Research.

Xinsong Du, Juan J Aristizabal-Henao, Timothy J Garrett, Mathias Brochhausen, William R Hogan, Dominick J Lemas.

  Clinical metabolomics emerged as a novel approach for biomarker discovery with the translational potential to guide next-generation therapeutics and precision health interventions. However, reproducibility in clinical research employing metabolomics data is challenging. Checklists are a helpful tool for promoting reproducible research. Existing checklists that promote reproducible metabolomics research primarily focused on metadata and may not be sufficient to ensure reproducible metabolomics data processing. This paper provides a checklist including actions that need to be taken by researchers to make computational steps reproducible for clinical metabolomics studies. We developed an eight-item checklist that includes criteria related to reusable data sharing and reproducible computational workflow development. We also provided recommended tools and resources to complete each item, as well as a GitHub project template to guide the process. The checklist is concise and easy to follow. Studies that follow this checklist and use recommended resources may facilitate other researchers to reproduce metabolomics results easily and efficiently.

Keywords:  checklist; clinical research; metabolomics; reproducibility; reproducible workflow; reusable data

DOI:  https://doi.org/10.3390/metabo12010087
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Jan 08. pii: S1570-0232(22)00016-2. [Epub ahead of print]1190 123112

Analysis of cycloheximide in rat specimens using liquid chromatography with tandem massspectrometry detection.

Hamdah M Al Nebaihi, Tyson S Le, Neal M Davies, Dion R Brocks.

  The development of a selective and sensitive high-performance liquid chromatographic tandem mass spectrometric method for the determination of cycloheximide (CHX) in rat blood and plasma is described. The extraction of CHX and colchicine as internal standard from blood fluid (0.1 mL) was achieved using n-hexane: dichloromethane: isopropanol (20:10:1 v/v/v). The mobile phase, a combination of methanol:10 mM ammonium acetate (85:15, v/v), was pumped at 0.2 mL/min through a C18 analytical column with a run time of 3.5 min. Detection was carried out by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring (MRM) mode. The assay exhibited excellent linearity (r2 > 0.999) in peak area response over the concentration ranges of 2-1000 ng CHX /mL blood fluid. The mean absolute recoveries for 20, 100 and 500 ng/mL CHX in blood fluid using the present extraction procedure were > 97%. The intra- and inter-day coefficients of variation in the plasma and blood and mean error were < 13% at different concentrations. Samples had limited stability at room temperature, and speedy processing is needed. After intravenous administration, rats had measurable concentrations of CHX for up to 24 h after dosing with 1 mg/kg of cycloheximide. The method displayed a high caliber of sensitivity and selectivity for detecting very low concentrations of CHX in rats.

Keywords:  Cycloheximide; Liquid–liquid extraction; Low dose; Pharmacokinetics; Preclinical; Small volume

DOI:  https://doi.org/10.1016/j.jchromb.2022.123112
Int J Mol Sci. 2022 Jan 06. pii: 598. [Epub ahead of print]23(2):

Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine-Chromatographic Studies.

Justyna Piechocka, Natalia Litwicka, Rafał Głowacki.

  It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography-mass spectrometry (GC-MS) based method was elaborated to identify and quantify TCA in human urine. The GC-MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1-50 µmol L-1 range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC-MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.

Keywords:  1,3-thiazinane-4-carboxylic acid; derivatization; formaldehyde; gas chromatography–mass spectrometry; homocysteine; homocysteine thiolactone; human urine; isobutyl chloroformate; liquid–liquid extraction

DOI:  https://doi.org/10.3390/ijms23020598
Chem Sci. 2021 Dec 22. 13(1): 90-98

Self-supervised clustering of mass spectrometry imaging data using contrastive learning.

Hang Hu, Jyothsna Padmakumar Bindu, Julia Laskin.

Mass spectrometry imaging (MSI) is widely used for the label-free molecular mapping of biological samples. The identification of co-localized molecules in MSI data is crucial to the understanding of biochemical pathways. One of key challenges in molecular colocalization is that complex MSI data are too large for manual annotation but too small for training deep neural networks. Herein, we introduce a self-supervised clustering approach based on contrastive learning, which shows an excellent performance in clustering of MSI data. We train a deep convolutional neural network (CNN) using MSI data from a single experiment without manual annotations to effectively learn high-level spatial features from ion images and classify them based on molecular colocalizations. We demonstrate that contrastive learning generates ion image representations that form well-resolved clusters. Subsequent self-labeling is used to fine-tune both the CNN encoder and linear classifier based on confidently classified ion images. This new approach enables autonomous and high-throughput identification of co-localized species in MSI data, which will dramatically expand the application of spatial lipidomics, metabolomics, and proteomics in biological research.

DOI: https://doi.org/10.1039/d1sc04077d
Anal Chim Acta. 2022 Feb 08. pii: S0003-2670(21)01219-8. [Epub ahead of print]1193 339393

A new platform for untargeted UHPLC-HRMS data analysis to address the time-shift problem.

Juan-Juan Zhao, Yang Zhang, Xing-Cai Wang, Xuan Wang, Qian Zhang, Peng Lu, Ping-Ping Liu, Yong-Jie Yu, Lu Han, Hui-Na Zhou, Qing-Xia Zheng, Hai-Yan Fu.

  Substantial deviations in retention times among samples pose a great challenge for the accurate screening and identifying of metabolites by ultrahigh-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). In this study, a coarse-to-refined time-shift correction methodology was proposed to efficiently address this problem. Metabolites producing multiple fragment ions were automatically selected as landmarks to generate pseudo-mass spectra for a coarse time-shift correction. Refined peak alignment for extracted ion chromatograms was then performed by using a moving window-based multiple-peak alignment strategy. Based on this novel coarse-to-refined time-shift correction methodology, a new comprehensive UHPLC-HRMS data analysis platform was developed for UHPLC-HRMS-based metabolomics. Original datasets were employed as inputs to automatically extract and register features in the dataset and to distinguish fragment ions from metabolites for chemometric analysis. Its performance was further evaluated using complex datasets, and the results suggest that the new platform can satisfactorily resolve the time-shift problem and is comparable with commonly used UHPLC-HRMS data analysis tools such as XCMS Online, MS-DIAL, Mzmine2, and Progenesis QI. The new platform can be downloaded from: http://www.pmdb.org.cn/antdas2tsc.

Keywords:  Automatic data analysis; Chemometrics; Time-shift correction; UHPLC-HRMS; Untargeted metabolomics

DOI:  https://doi.org/10.1016/j.aca.2021.339393
Metabolites. 2022 Jan 08. pii: 52. [Epub ahead of print]12(1):

Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics.

Julia M Malinowska, Taina Palosaari, Jukka Sund, Donatella Carpi, Gavin R Lloyd, Ralf J M Weber, Maurice Whelan, Mark R Viant.

  Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated sample preparation of the low biomass samples typically used for in vitro screening. Building on previous work, we have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics of HepaRG cells in 96-well microplates using a Biomek i7 Hybrid Workstation (Beckman Coulter) and Orbitrap Elite (Thermo Scientific) high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS), across polar metabolites and lipids. The experimental conditions evaluated included the day of metabolite extraction, order of extraction of samples in 96-well microplates, position of the 96-well microplate on the instrument's deck and well location within a microplate. By using the median relative standard deviation (mRSD (%)) of spectral features, we have demonstrated good repeatability of the workflow (final mRSD < 30%) with a low percentage of features outside the threshold applied for statistical analysis. To improve the quality of the automated workflow further, small method modifications were made and then applied to a large cohort study (4860 sample infusions across three nESI-DIMS assays), which confirmed very high repeatability of the whole workflow from cell culturing to metabolite measurements, whilst providing a significant improvement in sample throughput. It is envisioned that the automated in vitro metabolomics workflow will help to advance the application of metabolomics (as a part of NAMs) in chemical safety, primarily as an approach for high throughput screening and prioritisation.

Keywords:  automation; direct infusion mass spectrometry; high-throughput screening; in vitro metabolomics; sample preparation

DOI:  https://doi.org/10.3390/metabo12010052
Metabolites. 2022 Jan 12. pii: 68. [Epub ahead of print]12(1):

Evaluating the Accuracy of the QCEIMS Approach for Computational Prediction of Electron Ionization Mass Spectra of Purines and Pyrimidines.

Jesi Lee, Tobias Kind, Dean Joseph Tantillo, Lee-Ping Wang, Oliver Fiehn.

  Mass spectrometry is the most commonly used method for compound annotation in metabolomics. However, most mass spectra in untargeted assays cannot be annotated with specific compound structures because reference mass spectral libraries are far smaller than the complement of known molecules. Theoretically predicted mass spectra might be used as a substitute for experimental spectra especially for compounds that are not commercially available. For example, the Quantum Chemistry Electron Ionization Mass Spectra (QCEIMS) method can predict 70 eV electron ionization mass spectra from any given input molecular structure. In this work, we investigated the accuracy of QCEIMS predictions of electron ionization (EI) mass spectra for 80 purine and pyrimidine derivatives in comparison to experimental data in the NIST 17 database. Similarity scores between every pair of predicted and experimental spectra revealed that 45% of the compounds were found as the correct top hit when QCEIMS predicted spectra were matched against the NIST17 library of >267,000 EI spectra, and 74% of the compounds were found within the top 10 hits. We then investigated the impact of matching, missing, and additional fragment ions in predicted EI mass spectra versus ion abundances in MS similarity scores. We further include detailed studies of fragmentation pathways such as retro Diels-Alder reactions to predict neutral losses of (iso)cyanic acid, hydrogen cyanide, or cyanamide in the mass spectra of purines and pyrimidines. We describe how trends in prediction accuracy correlate with the chemistry of the input compounds to better understand how mechanisms of QCEIMS predictions could be improved in future developments. We conclude that QCEIMS is useful for generating large-scale predicted mass spectral libraries for identification of compounds that are absent from experimental libraries and that are not commercially available.

Keywords:  QCEIMS; in silico prediction; mass spectra; quantum chemistry

DOI:  https://doi.org/10.3390/metabo12010068
J Chromatogr A. 2022 Jan 08. pii: S0021-9673(22)00015-2. [Epub ahead of print]1664 462817

Analysis of hydrophilic per- and polyfluorinated sulfonates including trifluoromethanesulfonate using solid phase extraction and mixed-mode liquid chromatography-tandem mass spectrometry.

Xi-Zhi Niu, Leif Abrell, Reyes Sierra-Alvarez, Jim A Field, Jon Chorover.

  Ultra-hydrophilic per- and polyfluorinated sulfonates (PFSA) are increasingly scrutinized in recent years due to their ubiquitous occurrence, persistence, and aqueous mobility in the environment, yet analysis remains a challenge. This study developed methods for the analysis of trifluoromethanesulfonate, perfluorobutanesulfonate, 10-camphorsulfonate, and a di-fluorinated sulfonate utilizing mixed-mode liquid chromatography, where all analytes were adequately retained and separated. Chromatography and electrospray ionization parameters were optimized; instrumental limits of quantification for the anionic target analytes were in the range of 4.3 - 16.1 ng L-1. Solid phase extraction (SPE) methods were developed using Oasis WAX cartridges; SPE recoveries for the analytes ranged from 86% to 125%. Salinity and total organic carbon both impaired the SPE performance to different extents, depending on the respective analyte. Utilizing widely accessible instrumentation and materials, this is a single method to simultaneously analyze conceivably the most hydrophilic PFAS chemical, i.e., trifluoromethanesulfonate, and moderately hydrophobic PFSAs.

Keywords:  10-Camphorsulfonate; Mass spectrometry; Mixed-mode liquid chromatography; Perfluorobutanesulfonate; Solid phase extraction; Trifluoromethanesulfonate

DOI:  https://doi.org/10.1016/j.chroma.2022.462817
ACS Omega. 2022 Jan 11. 7(1): 230-239

Two-Dimensional Gas Chromatographic and Mass Spectrometric Characterization of Lipid-Rich Biological Matrices-Application to Human Cerumen (Earwax).

Allix M Coon, A John Dane, Gavin Setzen, Robert B Cody, Rabi A Musah.

Earwax is a readily accessible biological matrix that has the potential to be used in disease diagnostics. However, its semisolid nature and high chemical complexity have hampered efforts to investigate its potential to reveal disease markers. This is because more conventional methods of analysis such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry yield unsatisfactory results due to the presence of many nonvolatile and/or coeluting compounds, which in some cases have very similar mass spectrometric profiles. In addition, these routine methods often require the sample to be saponified, which dramatically increases the complexity of the analysis and makes it difficult to determine which compounds are actually present versus those that are produced by saponification. In this study, two-dimensional GC mass spectrometry (GC × GC-MS) was successfully applied for the characterization of the chemical components of earwax from healthy donors using nonpolar (primary) and midpolar (secondary) columns without saponification. Over 35 of the compounds that were identified are reported for the first time to be detected in unsaponified earwax. The resulting GC × GC-MS contour plots revealed visually recognizable compound class clusters of previously reported groups including alkanes, alkenes, fatty acids, esters, triglycerides, and cholesterol esters, as well as cholesterol and squalene. The application of GC × GC-MS revealed results that provide a foundation upon which future studies aimed at comparing healthy donor earwax to that from individuals exhibiting various disease states can be accomplished.

DOI: https://doi.org/10.1021/acsomega.1c04535
J Sep Sci. 2022 Jan 19.

High throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography.

Lukas Naumann, Patrick Schlossbauer, Florian Klingler, Friedemann Hesse, Kerstin Otte, Christian Neusüß.

  The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight MS or Orbitrap MS. Both separation methods offer fast separation (10-15 min) and the capability to prevent the separated cell supernatant matrix to enter the MS by post-separation valving. Both MS instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 μg/mL (CE-nanoCEasy-Orbitrap MS) and 5 μg/mL (LC-Orbitrap MS). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis, while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation. This article is protected by copyright. All rights reserved.

Keywords:  antibody; glycosylation; high throughput screening; mass spectrometry

DOI:  https://doi.org/10.1002/jssc.202100865
Bioinformatics. 2022 Jan 18. pii: btac032. [Epub ahead of print]

massNet: integrated processing and classification of spatially resolved mass spectrometry data using deep learning for rapid tumor delineation.

Walid M Abdelmoula, Sylwia A Stopka, Elizabeth C Randall, Michael Regan, Jeffrey N Agar, Jann N Sarkaria, William M Wells, Tina Kapur, Nathalie Y R Agar.

MOTIVATION: Mass spectrometry imaging (MSI) provides rich biochemical information in a label-free manner and therefore holds promise to substantially impact current practice in disease diagnosis. However, the complex nature of MSI data poses computational challenges in its analysis. The complexity of the data arises from its large size, high dimensionality, and spectral non-linearity. Preprocessing, including peak picking, has been used to reduce raw data complexity, however peak picking is sensitive to parameter selection that, perhaps prematurely, shapes the downstream analysis for tissue classification and ensuing biological interpretation.RESULTS: We propose a deep learning model, massNet, that provides the desired qualities of scalability, non-linearity, and speed in MSI data analysis. This deep learning model was used, without prior preprocessing and peak picking, to classify MSI data from a mouse brain harboring a patient-derived tumor. The massNet architecture established automatically learning of predictive features, and automated methods were incorporated to identify peaks with potential for tumor delineation. The model's performance was assessed using cross-validation, and the results demonstrate higher accuracy and a substantial gain in speed compared to the established classical machine learning method, support vector machine.
AVAILABILITY AND IMPLEMENTATION: https://github.com/wabdelmoula/massNet.
AVAILABILITY OF DATA: The data underlying this article are available in the NIH Common Fund's National Metabolomics Data Repository (NMDR) Metabolomics Workbench under project id (PR001292) with http://dx.doi.org/10.21228/M8Q70T.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btac032
J Agric Food Chem. 2022 Jan 21.

Improved Thyreostatic Drug Detection in Animal Tissues Using Liquid Chromatography-High-Field Asymmetric Waveform Ion Mobility Spectrometry-Mass Spectrometry.

Randy W Purves, Kim Souster, Michelle West, Azhar M Huda, Caleb M E Fisher, Michael W Belford, Bryn O Shurmer.

  Thyreostatic drugs (thyreostats) interfere with thyroid function and have been used illegally in animals slaughtered for food. Thyreostat use leads to poorer quality meat, and the drug residues can cause adverse effects in humans. These drugs, with the exception of thiouracil, do not occur naturally and require sensitive methodologies for their detection in animal tissues. Because thyreostats are low-molecular-weight polar analytes, liquid chromatography-mass spectrometry (LC-MS) is typically used for detection and, in particular, triple quadrupole mass spectrometry with selective reaction monitoring (i.e., LC-SRM). However, LC-SRM thyreostat methods suffer from chemical background noise and endogenous interferences arising from the complex tissue matrix. An improved high-field asymmetric waveform ion mobility spectrometry interface (FAIMS Pro), which separates ions based on differential ion mobility, was combined with LC-SRM to minimize these interferences. Using the same samples and conditions, LC-FAIMS-SRM showed improvements in the signal-to-noise ratio (S/N) of up to 50 times compared with our validated LC-SRM method. In addition, wider linear ranges, including substantial improvements in the lower limit of quantification (approximately an order of magnitude for tapazole and methylthiouracil), were observed with LC-FAIMS-SRM.

Keywords:  FAIMS; FAIMS Pro; LC−FAIMS−MS; differential ion mobility; drug residues; thiouracil; thyreostats

DOI:  https://doi.org/10.1021/acs.jafc.1c06937
J Am Soc Mass Spectrom. 2022 Jan 21.

Reduced Hemoglobin Signal and Improved Detection of Endogenous Proteins in Blood-Rich Tissues for MALDI Mass Spectrometry Imaging.

Monica Lin, Livia S Eberlin, Erin H Seeley.

  Mass spectrometry imaging provides a powerful approach for the direct analysis and spatial visualization of molecules in tissue sections. Using matrix-assisted laser desorption/ionization mass spectrometry, intact protein imaging has been widely investigated for biomarker analysis and diagnosis in a variety of tissue types and diseases. However, blood-rich or highly vascular tissues present a challenge in molecular imaging due to the high ionization efficiency of hemoglobin, which leads to ion suppression of endogenous proteins. Here, we describe a protocol to selectively reduce hemoglobin signal in blood-rich tissues that can easily be integrated into mass spectrometry imaging workflows.

Keywords:  MALDI imaging; hemoglobin removal; mass spectrometry imaging; protein imaging; sample preparation; tissue imaging

DOI:  https://doi.org/10.1021/jasms.1c00300
Electrophoresis. 2022 Jan 20.

Quantification of allantoin and other metabolites of the purine degradation pathway in human plasma samples using a newly developed HILIC-LC-MS/MS method.

Asmin Andries, Alan Feyaerts, Djalila Mekahli, Ann Van Schepdael.

  The development of a simple HILIC-LC-MS/MS method to quantify the plasma levels of allantoin, inosine, hypoxanthine and adenosine, using stripped plasma for the bioanalytical method validation, was the purpose of this study. Chromatographic separation conducted using an XBridge BEH Amide column (2.1×150 mm, 3.5 μm) was achieved under gradient elution with two mobile phases; 0.1% formic acid - ACN (5:95) and 0.1% formic acid - ACN (50:50). Multiple reaction monitoring MS detection was performed using a triple quadrupole. The method validation experiments were performed according to the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) guidelines. The LLOQ was 50 nM, 5 nM, 20 nM and 2 nM for allantoin, inosine, hypoxanthine and adenosine, respectively. The recovery was repeatable and stable. The intra-day precision ranged from 1.6% to 6.5% while the inter-day precision ranged from 3.4% to 58.7%. Therefore, it is necessary to make a matrix-matched calibration curve each day to overcome this issue. Since the QC samples' stability did not always comply with the guidelines, the samples need to be analyzed soon after collection. This article is protected by copyright. All rights reserved.

Keywords:  HILIC-MS/MS; charcoal stripped plasma; metabolites; plasma; purine catabolic pathway

DOI:  https://doi.org/10.1002/elps.202100265
Anal Chim Acta. 2022 Feb 08. pii: S0003-2670(21)01144-2. [Epub ahead of print]1193 339318

Optimisation of saliva volumes for lipidomic analysis by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry.

Gwang Bin Lee, Ayse Caner, Myeong Hee Moon.

  Saliva is a readily accessible and clinically useful biofluid that can be used to develop disease biomarkers because of a variety of biologically active molecules in it that are also found in blood. However, even though saliva sampling is simple and non-invasive, few studies have investigated the use of salivary lipids as biomarkers, and the extraction of lipids from saliva needs to be examined thoroughly. In the present study, methods (i.e., saliva sample volume, 0.1-1.0 mL) for the extraction and analysis of salivary lipids by nanoflow ultrahigh performance liquid chromatography-tandem mass spectrometry (nUHPLC-ESI-MS/MS) were evaluated according to the matrix effect, extraction recovery, and number of quantifiable lipids. A total of 780 lipids were identified in a pooled saliva sample from 20 healthy volunteers, and 372 lipids without differentiating acyl chain structures were quantified, along with comprehensive information on salivary lipid composition and individual lipid levels. Even though extraction recovery was maintained at saliva sample volumes as low as 0.2 mL, the matrix effect and limit of detection (LOD) were relatively large with 1.0 mL. Considering the matrix effect, LOD, and number of quantifiable lipids (>limit of quantitation), the minimum volume of saliva sufficient for lipidomic analysis using nUHPLC-ESI-MS/MS was determined to be 0.5 mL.

Keywords:  Lipidomic analysis; Mass spectrometry; Saliva; Saliva volume; nUHPLC-ESI-MS/MS

DOI:  https://doi.org/10.1016/j.aca.2021.339318
Anal Chim Acta. 2022 Feb 08. pii: S0003-2670(21)01142-9. [Epub ahead of print]1193 339316

Discovery and quantification of lipoamino acids in bacteria.

Amandine Hueber, Camille Petitfils, Pauline Le Faouder, Geoffrey Langevin, Alexandre Guy, Jean-Marie Galano, Thierry Durand, Jean-François Martin, Jean-Claude Tabet, Nicolas Cenac, Justine Bertrand-Michel.

  Improving knowledge about metabolites produced by the microbiota is a key point to understand its role in human health and disease. Among them, lipoamino acid (LpAA) containing asparagine and their derivatives are bacterial metabolites which could have an impact on the host. In this study, our aim was to extend the characterization of this family. We developed a semi-targeted workflow to identify and quantify new candidates. First, the sample preparation and analytical conditions using liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) were optimized. Using a theoretical homemade database, HRMS raw data were manually queried. This strategy allowed us to find 25 new LpAA conjugated to Asn, Gln, Asp, Glu, His, Leu, Ile, Lys, Phe, Trp and Val amino acids. These metabolites were then fully characterized by MS2, and compared to the pure synthesized standards to validate annotation. Finally, a quantitative method was developed by LC coupled to a triple quadrupole instrument, and linearity and limit of quantification were determined. 14 new LpAA were quantified in gram positive bacteria, Lactobacilus animalis, and 12 LpAA in Escherichia coli strain Nissle 1917.

Keywords:  Lipidomic; Lipoaminoacids; Mass spectrometry; Microbiota; Suspect screening

DOI:  https://doi.org/10.1016/j.aca.2021.339316
Molecules. 2022 Jan 10. pii: 436. [Epub ahead of print]27(2):

Optimised Method for Short-Chain Fatty Acid Profiling of Bovine Milk and Serum.

Cheng Li, Zhiqian Liu, Carolyn Bath, Leah Marett, Jennie Pryce, Simone Rochfort.

  Short-chain fatty acids (SCFA, C2-C5) in milk and serum are derived from rumen bacterial fermentation and, thus, have the potential to be used as biomarkers for the health status of dairy cows. Currently, there is no comprehensive and validated method that can be used to analyse all SCFAs in both bovine serum and milk. This paper reports an optimised protocol, combining 3-nitrophenylhydrazine (3-NPH) derivatisation and liquid chromatography-mass spectrometry (LC-MS) analysis for quantification of SCFA and β-hydroxybutyric acid (BHBA) in both bovine milk and bovine serum. This method is sensitive (limit of detection (LOD) ≤ 0.1 µmol/L of bovine milk and serum), accurate (recovery 84-115% for most analytes) and reproducible (relative standard deviation (RSD) for repeated analyses below 7% for most measurements) with a short sample preparation step. The application of this method to samples collected from a small cohort of animals allowed us to reveal a large variation in SCFA concentration between serum and milk and across different animals as well as the strong correlation of some SCFAs between milk and serum samples.

Keywords:  bovine milk; liquid chromatography-mass spectrometry; serum; short-chain fatty acids

DOI:  https://doi.org/10.3390/molecules27020436
Forensic Sci Int. 2022 Jan 05. pii: S0379-0738(22)00006-8. [Epub ahead of print]332 111176

Screening of new psychoactive substances in human plasma by magnetic solid phase extraction and LC-QTOF-MS.

Jinwoo Hwang, Miri Han, Sora An, Jung Hyun Moon, Geunae Shim, Heesun Chung.

  The emergence of new psychoactive substances (NPSs) is an increasing challenge in forensic toxicology. There are many extraction methods in use to isolate NPSs in biological fluids, including protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE). However, there is a need to develop an effective extraction method with a short extraction time and low consumption of solvent. To meet these requirements, magnetic solid phase extraction (m-SPE) was attempted to isolate 40 NPSs in human plasma in this study. Forty NPSs (13 synthetic cannabinoids, 13 phenethylamines, 4 tryptamines, 4 other substances, 3 aminoindanes, 2 piperazines, 1 phencyclidine-type substance) were spiked in plasma and analyzed by m-SPE using COOH-functionalized multi-walled carbon nanotubes with magnetic nanoparticles (COOH-mMWCNTs). A liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) method was used for screening and identification of 40 target compounds. Method validation including limits of detection, recovery, matrix effect, and precision was performed for all 40 -target compounds. The limits of detection (LOD) of the 40 analytes were between 0.002 and 0.084 mg/L. Extraction recovery ranged from 36.9% to 110.6% (average 87%). Matrix effects ranged from -29.0% (ion suppression) to 9.8% (ion enhancement). Both intra- and inter-day precision values were less than 27.5% (RSD%). The accurate mass of QTOF-MS enabled the identification of analytes by exact monoisotopic mass and isotopic pattern. m-SPE was applied to extract 40 NPSs, and revealed less time-consuming and laborious than conventional SPE. This method proved to be an advantageous procedure to extract NPSs from biological fluids.

Keywords:  Human plasma; LC-QTOF-MS; Magnetic solid phase extraction; Method validation; New psychoactive substances

DOI:  https://doi.org/10.1016/j.forsciint.2022.111176
J Sep Sci. 2022 Jan 19.

Hydrophilic interaction LC-MS/MS method to avoid endogenous interference in the analysis of 4-hydroxy isoleucine from dietary supplementation of fenugreek.

Lakshminarayanan Gowtham, Das Ujjalkumar, Laxmi Moksha, Roy Kumar Tapas, Halder Nabanita, Thirumurthy Velpandian.

  4-Hydroxy isoleucine is one of the potent hypoglycaemic active constituents of fenugreek seeds. A method capable of reducing biological interferences is required for bio-availability studies. An isocratic separation of 4-hydroxy isoleucine from endogenous interferences was achieved in ZIC-cHILIC column using 0.1% formic acid in water and acetonitrile (20:80, % v/v) pumped at 0.5 mL/min. Quantification was performed in multiple reaction monitoring mode using the transitions of m/z 148.1→102.1 and m/z 276.1→142.2 for 4-hydroxy isoleucine and homatropine (as internal standard), respectively. After full method validation, 4-Hydroxy isoleucine levels in human plasma and commercial fenugreek formulations were determined. This method showed good linearity in the range of 50-2000 ng/mL. Intra-day and inter-day accuracy were in the range of 90.64-109.0% and precision was <4.82 % CV. The mean (SD) plasma concentration of 4-hydroxy isoleucine in healthy individuals at 2 hrs post oral administration of fenugreek tablet was found to be 1590 (260) ng/mL. Half of marketed formulations were found to contain < 0.05 % of 4-Hydroxy isoleucine content. We developed a rapid hydrophilic interaction LC-MS/MS method for analysis of 4-hydroxy isoleucine in human plasma. This method can be applied directly to conduct the clinical pharmacokinetics studies of 4-hydroxy isoleucine in human population. This article is protected by copyright. All rights reserved.

Keywords:  4-hydroxy isoleucine; Diabetes; Fenugreek seeds; Hydrophilic interaction liquid chromatography; Mass spectrometry

DOI:  https://doi.org/10.1002/jssc.202100894
Metabolites. 2022 Jan 11. pii: 62. [Epub ahead of print]12(1):

Stability of Wheat Floret Metabolites during Untargeted Metabolomics Studies.

Kristin Whitney, Gerardo Gracia-Gonzalez, Senay Simsek.

  A typical metabolomic analysis consists of a multi-step procedure. Variation can be introduced in any analysis segment if proper care in quality assurance is not taken, thus compromising the final results. Sample stability is one of those factors. Although sophisticated studies addressing sample decay over time have been performed in the medical field, they are emerging in plant metabolomics. Here, we focus on the stability of wheat floret extracts on queue inside an auto-injector held at 25 °C. The objective was to locate an analytical time window from extraction to injection with no significant difference occurring in the sample. Total ion current chromatograms, principal component analysis, and volcano plots were used to measure changes in the samples. Results indicate a maximum work window time of 7:45 h for Steele-ND wheat methanolic extractions in an auto-sampler at 25 °C. Comparisons showed a significant gradual increase in the number and intensity of compounds observed that may be caused by the degradation of other molecules in the sample extract. The approach can be applied as preliminary work in a metabolite profiling study, helping to set the appropriate workload to produce confident results.

Keywords:  data acquisition; liquid chromatography-mass spectrometry/quadrapol time of flight (LC-MS/QTOF); mycotoxins; sample stability; targeted metabolomics; wheat

DOI:  https://doi.org/10.3390/metabo12010062
Chem Res Toxicol. 2022 Jan 20.

In Vivo Identification of Adducts from the New Hypoxia-Activated Prodrug CP-506 Using DNA Adductomics.

Morwena J Solivio, Alessia Stornetta, Julie Gilissen, Peter W Villalta, Sofie Deschoemaeker, Arne Heyerick, Ludwig Dubois, Silvia Balbo.

Many chemotherapeutic drugs exert their cytotoxicity through the formation of DNA modifications (adducts), which interfere with DNA replication, an overactive process in rapidly dividing cancer cells. Side effects from the therapy are common, however, because these drugs also affect rapidly dividing noncancerous cells. Hypoxia-activated prodrugs (HAPs) have been developed to reduce these side effects as they preferentially activate in hypoxic environments, a hallmark of solid tumors. CP-506 is a newly developed DNA-alkylating HAP designed to exert strong activity under hypoxia. The resulting CP-506-DNA adducts can be used to elucidate the cellular and molecular effects of CP-506 and its selectivity toward hypoxic conditions. In this study, we characterize the profile of adducts resulting from the reaction of CP-506 and its metabolites CP-506H and CP-506M with DNA. A total of 39 putative DNA adducts were detected in vitro using our high-resolution/accurate-mass (HRAM) liquid chromatography tandem mass spectrometry (LC-MS3) adductomics approach. Validation of these results was achieved using a novel strategy involving 15N-labeled DNA. A targeted MS/MS approach was then developed for the detection of the 39 DNA adducts in five cancer cell lines treated with CP-506 under normoxic and hypoxic conditions to evaluate the selectivity toward hypoxia. Out of the 39 DNA adducts initially identified, 15 were detected, with more adducts observed from the two reactive metabolites and in cancer cells treated under hypoxia. The presence of these adducts was then monitored in xenograft mouse models bearing MDA-MB-231, BT-474, or DMS114 tumors treated with CP-506, and a relative quantitation strategy was used to compare the adduct levels across samples. Eight adducts were detected in all xenograft models, and MDA-MB-231 showed the highest adduct levels. These results suggest that CP-506-DNA adducts can be used to better understand the mechanism of action and monitor the efficacy of CP-506 in vivo, as well as highlight a new role of DNA adductomics in supporting the clinical development of DNA-alkylating drugs.

DOI: https://doi.org/10.1021/acs.chemrestox.1c00329
Anal Chim Acta. 2022 Feb 08. pii: S0003-2670(21)01141-7. [Epub ahead of print]1193 339315

Integration of a liquid-phase ion trap with a miniature mass spectrometer.

Wanzhen Han, Shiyu Du, Caowei Tang, Yiling Li, Ting Jiang, Wei Xu.

  Liquid-phase ion trap was recently developed in our group, which has simple configuration and could realize ion manipulations in solution. In this study, a simplified liquid-phase ion trap was integrated with a home-built miniature mass spectrometer for enhanced analytical performances. Ion enrichment and separation could be realized in the liquid-phase ion trap before mass spectrometry analyses. As a result, detection sensitivity of the miniature mass spectrometer could be improved by ∼10 folds. Besides ion intensity enrichment, this liquid-phase ion trap was also capable of separating ions according to their hydrodynamic radii and effective charges. This capability was demonstrated by separating small molecules in protein background and isobaric peptide ions. Furthermore, target analytes in salt solutions and urine were also enriched and detected by the miniature mass spectrometer. Results suggest that the coupling of liquid phase ion trap with miniature mass spectrometer is a powerful technique, which could be beneficial for low abundant target molecule analysis in practical applications.

Keywords:  Ion separation method; Liquid-phase ion trap; Miniature mass spectrometer

DOI:  https://doi.org/10.1016/j.aca.2021.339315