bims-metlip 2022-07-24 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022–07–24
eightteen papers selected by
Sofia Costa, Matterworks

Prioritize biologically relevant ions for data-independent acquisition (BRI-DIA) in LC-MS/MS-based lipidomics analysis.
Potential of atmospheric pressure ionization sources for the analysis of free fatty acids in clinical and biological samples by gas chromatography-mass spectrometry.
Combining data acquisition modes in liquid-chromatography-tandem mass spectrometry for comprehensive determination of acylcarnitines in human serum.
Mass spectrometry imaging to explore molecular heterogeneity in cell culture.
A dynamic multiple reaction monitoring strategy to develop and optimize targeted metabolomics methods: Analyzing bile acids in capecitabine-induced diarrhea.
Protomer Formation Can Aid the Structural Identification of Caffeine Metabolites.
A sensitive UPLC-MS/MS method for simultaneous quantification of one-carbon metabolites & co-factors in human plasma.
A multicenter performance evaluation of the new Elecsys Vitamin D total III assay versus reference isotope dilution liquid chromatography tandem mass spectrometry and commercially available comparators.
Simulating comprehensive two-dimensional gas chromatography mass spectrometry data with realistic run-to-run shifting to evaluate the robustness of tile-based Fisher ratio analysis.
Locality-sensitive hashing enables efficient and scalable signal classification in high-throughput mass spectrometry raw data.
Identification of pre-diagnostic metabolites associated with prostate cancer risk by untargeted mass spectrometry-based metabolomics: A case-control study nested in the Northern Sweden Health and Disease Study.
Development of an Internal Calibration Algorithm for Ultrahigh-Resolution Mass Spectra of Dissolved Organic Matter.
Optimization and automation of rapid and selective analysis of fatty acid methyl esters from aqueous samples by headspace SPME arrow extraction followed by GC-MS/MS analysis.
Real-Time Non-Invasive Monitoring of Short-Chain Fatty Acids in Exhaled Breath.
Automated and High-Throughput Urine Drug Screening using Paper Spray Mass Spectrometry.
Explaining unexplained hypoglycemia: How LC-MS/MS can help.
Development and Validation of a DLLME-GC-MS/MS Method for the Determination of Benzotriazole UV Stabilizer UV-327 and its Metabolites in Human Blood.
Non-Invasive Identification of Sex in Cultured Bovine Embryos by UHPLC-MS/MS Metabolomics.

Metabolomics. 2022 Jul 16. 18(8): 55

Prioritize biologically relevant ions for data-independent acquisition (BRI-DIA) in LC-MS/MS-based lipidomics analysis.

Likun Duan, Grace Scheidemantle, Mareca Lodge, Magdalina J Cummings, Eva Pham, Xiaoqiu Wang, Arion Kennedy, Xiaojing Liu.

INTRODUCTION: Data-dependent acquisition (DDA) is the most commonly used MS/MS scan method for lipidomics analysis on orbitrap-based instrument. However, MS instrument associated software decide the top N precursors for fragmentation, resulting in stochasticity of precursor selection and compromised consistency and reproducibility. We introduce a novel workflow using biologically relevant lipids to construct inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow.
OBJECTIVES: To ensure consistent coverage of biologically relevant lipids in LC-MS/MS-based lipidomics analysis.
METHODS: Biologically relevant ion list was constructed based on LIPID MAPS and lipidome atlas in MS-DIAL 4. Lipids were extracted from mouse tissues and used to assess different MS/MS scan workflow (DDA, BRI-DIA, and hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer.
RESULTS: DDA resulted in more MS/MS events, but the total number of unique lipids identified by three methods (DDA, BRI-DIA, and hybrid MS/MS scan mode) is comparable (580 unique lipids across 44 lipid subclasses in mouse liver). Major cardiolipin molecular species were identified by data generated using BRI-DIA and hybrid methods and allowed calculation of cardiolipin compositions, while identification of the most abundant cardiolipin CL72:8 was missing in data generated using DDA method, leading to wrong calculation of cardiolipin composition.
CONCLUSION: The method of using inclusion list comprised of biologically relevant lipids in DIA MS/MS scan is as efficient as traditional DDA method in profiling lipids, but offers better consistency of lipid identification, compared to DDA method. This study was performed using Orbitrap Exploris 480, and we will further evaluate this workflow on other platforms, and if verified by future work, this biologically relevant ion fragmentation workflow could be routinely used in many studies to improve MS/MS identification capacities.

DOI: https://doi.org/10.1007/s11306-022-01913-8
Anal Bioanal Chem. 2022 Jul 18.

Potential of atmospheric pressure ionization sources for the analysis of free fatty acids in clinical and biological samples by gas chromatography-mass spectrometry.

Paul E Görs, Pia Wittenhofer, Juan F Ayala-Cabrera, Sven W Meckelmann.

  Because of the central role of fatty acids in biological systems, their accurate quantification is still important. However, the impact of the complex matrix of biologically and clinically relevant samples such as plasma, serum, or cells makes the analysis still challenging, especially, when free non-esterified fatty acids have to be quantified. Here we developed and characterized a novel GC-MS method using pentafluorobenzyl bromide as a derivatization agent and compared different ionization techniques such as atmospheric pressure chemical ionization (APCI), atmospheric pressure chemical photoionization (APPI), and negative ion chemical ionization (NICI). The GC-APCI-MS showed the lowest limits of detection from 30 to 300 nM for a broad range of fatty acids and a similar response for various fatty acids from a chain length of 10 to 20 carbon atoms. This allows the number of internal standards necessary for accurate quantification to be reduced. Moreover, the use of pentafluorobenzyl bromide allows the direct derivatization of free fatty acids making them accessible for GC-MS analysis without labor-intense sample pretreatment.

Keywords:  Fatty acids; Gas chromatography; Lipidomics; Non-esterified fatty acids; Pentafluorobenzyl bromide; Systems biology

DOI:  https://doi.org/10.1007/s00216-022-04223-z
Metabolomics. 2022 Jul 21. 18(8): 59

Combining data acquisition modes in liquid-chromatography-tandem mass spectrometry for comprehensive determination of acylcarnitines in human serum.

D Luque-Córdoba, M Calderón-Santiago, F Priego-Capote.

  Acylcarnitines (ACs) are metabolites involved in fatty acid β-oxidation and organic acid metabolism. Metabolic disorders associated to these two processes can be evaluated by determining the complete profile of ACs. In this research, we present an overall strategy for identification, confirmation, and quantitative determination of acylcarnitines in human serum. By this strategy we identified the presence of 47 ACs from C2 to C24 with detection of the unsaturation degree by application of a data-independent acquisition (DIA) liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Complementary, quantitative determination of ACs is based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC-MS/MS in data-dependent acquisition (DDA) to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL-1 level, the intra-day and between-day variability were below 15-20%, respectively; and the accuracy, expressed as bias, was always within ± 25%. The proposed method was tested with 40 human volunteers to determine the relative concentration of ACs in serum and identify predominant forms. Significant differences were detected by comparing the ACs profile of obese versus non-obese individuals.

Keywords:  Acylcarnitines; Data-dependent acquisition; Data-independent acquisition; Multiple reaction monitoring; SPE–LC–MS/MS; Serum

DOI:  https://doi.org/10.1007/s11306-022-01916-5
Proc Natl Acad Sci U S A. 2022 Jul 19. 119(29): e2114365119

Mass spectrometry imaging to explore molecular heterogeneity in cell culture.

Tanja Bien, Krischan Koerfer, Jan Schwenzfeier, Klaus Dreisewerd, Jens Soltwisch.

  Molecular analysis on the single-cell level represents a rapidly growing field in the life sciences. While bulk analysis from a pool of cells provides a general molecular profile, it is blind to heterogeneities between individual cells. This heterogeneity, however, is an inherent property of every cell population. Its analysis is fundamental to understanding the development, function, and role of specific cells of the same genotype that display different phenotypical properties. Single-cell mass spectrometry (MS) aims to provide broad molecular information for a significantly large number of cells to help decipher cellular heterogeneity using statistical analysis. Here, we present a sensitive approach to single-cell MS based on high-resolution MALDI-2-MS imaging in combination with MALDI-compatible staining and use of optical microscopy. Our approach allowed analyzing large amounts of unperturbed cells directly from the growth chamber. Confident coregistration of both modalities enabled a reliable compilation of single-cell mass spectra and a straightforward inclusion of optical as well as mass spectrometric features in the interpretation of data. The resulting multimodal datasets permit the use of various statistical methods like machine learning-driven classification and multivariate analysis based on molecular profile and establish a direct connection of MS data with microscopy information of individual cells. Displaying data in the form of histograms for individual signal intensities helps to investigate heterogeneous expression of specific lipids within the cell culture and to identify subpopulations intuitively. Ultimately, t-MALDI-2-MSI measurements at 2-µm pixel sizes deliver a glimpse of intracellular lipid distributions and reveal molecular profiles for subcellular domains.

Keywords:  cellular heterogeneity; lipidomics; single-cell mass spectrometry; t-MALDI-2-MSI

DOI:  https://doi.org/10.1073/pnas.2114365119
J Pharm Biomed Anal. 2022 Jul 14. pii: S0731-7085(22)00359-4. [Epub ahead of print]219 114938

A dynamic multiple reaction monitoring strategy to develop and optimize targeted metabolomics methods: Analyzing bile acids in capecitabine-induced diarrhea.

Zhipeng Wang, Hanglin Li, Yunlei Yun, Hongsen Wang, Bosu Meng, Yuhui Mu, Shouhong Gao, Xia Tao, Wansheng Chen.

   OBJECTIVE: We sought to develop and optimize a targeted bile acids (BAs) metabolomics method based on a dynamic multiple reaction monitoring (dMRM) strategy and explored the dynamic alterations of BAs in diarrhea induced by capecitabine in a mouse model.
METHOD: The targeted metabolomics method was developed using an Agilent 6460A triple quadrupole mass spectrometer, and 41 types of BAs were monitored in negative ionization mode. The mass spectrometer detection was optimized using dMRM to enhance the responses, separation, and peak shape and to shorten the analysis time. A mouse model of diarrhea was established by multiple administration of capecitabine, and plasma samples were collected at baseline and the end of drug administration for subsequent BAs analysis.
RESULTS: The targeted BA metabolomics method achieved shorter chromatographic separation time (10 min) for 41 BAs, with good peak shapes and response increases of 3- to 10-fold after application of dMRM. The mouse model of capecitabine-induced diarrhea was established, and the three BAs 23-norcholic acid, isolithocholic acid, and isodeoxycholic acid in the baseline samples contributed the most to differentiating mice with diarrhea from those without diarrhea. For mice that ultimately developed diarrhea, apocholic acid, isodeoxycholic acid, and 7-ketodeoxycholic acid exhibited the largest change in concentrations compared with their baseline concentrations.
CONCLUSION: The dMRM strategy has obvious advantages compared with common MRM. The results in model mice showed that a differentiated profile of BAs in the baseline may indicate biomarkers of diarrhea induced by capecitabine, and disturbed homeostasis may explain the metabolomic mechanism of diarrhea occurrence.

Keywords:  Capecitabine; Diarrhea; Dynamic multiple reaction monitoring; Liquid chromatography tandem mass spectrometry; Targeted bile acids metabolomics

DOI:  https://doi.org/10.1016/j.jpba.2022.114938
Anal Chem. 2022 Jul 21.

Protomer Formation Can Aid the Structural Identification of Caffeine Metabolites.

Helen Sepman, Sofja Tshepelevitsh, Henrik Hupatz, Anneli Kruve.

The structural annotation of isomeric metabolites remains a key challenge in untargeted electrospray ionization/high-resolution mass spectrometry (ESI/HRMS) metabolomic analysis. Many metabolites are polyfunctional compounds that may form protomers in electrospray ionization sources and therefore yield multiple peaks in ion mobility spectra. Protomer formation is strongly structure-specific. Here, we explore the possibility of using protomer formation for structural elucidation in metabolomics on the example of caffeine, its eight metabolites, and structurally related compounds. It is observed that two-thirds of the studied compounds formed high- and low-mobility species in high-resolution ion mobility. Structures in which proton hopping was hindered by a methyl group at the purine ring nitrogen (position 3) yielded structure-indicative fragments with collision-induced dissociation (CID) for high- and low-mobility ions. For compounds where such a methyl group was not present, a gas-phase equilibrium could be observed for tautomeric species with two-dimensional ion mobility. We show that the protomer formation and the gas-phase properties of the protomers can be related to the structure of caffeine metabolites and facilitate the identification of the structural isomers.

DOI: https://doi.org/10.1021/acs.analchem.2c00257
J Pharm Biomed Anal. 2022 Jul 16. pii: S0731-7085(22)00365-X. [Epub ahead of print]219 114944

A sensitive UPLC-MS/MS method for simultaneous quantification of one-carbon metabolites & co-factors in human plasma.

Ping Chen, Yun Tang, Qiangqiang He, Lishun Liu, Ziyi Zhou, Yun Song, Nan Zhang, Binyan Wang, Houqing Zhou, Hanping Shi, Jie Jiang.

  One-carbon metabolism is an important metabolic pathway involved in many diseases, such as congenital malformations, tumours, cardiovascular diseases, anaemia, depression, cognitive diseases and liver disease. However, the current methods have specific defects in detecting and qualifying the related compounds of one-carbon metabolism. In this study, a validated method was established to simultaneously quantify 22 one-carbon metabolites & co-factors in human plasma and applied to the study of correlation between one-carbon metabolism and colorectal cancer in human plasma samples, which were from 44 healthy subjects and 55 colorectal cancer patients. The method used ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UPLC-MS/MS), and the analytes included betaine, L-carnitine, L-cystathionine, L-cysteine, dimethylglycine, DL-homocysteic acid, homocysteine, methionine, pyridoxal hydrochloride, pyridoxamine dihydrochloride, pyridoxine dihydrochloride, S-(5'-Adenosyl)-L-homocysteine, serine, choline chloride, folic acid, glycine, pyridoxal phosphate monohydrate, riboflavin, taurine, 5-methyltetrahydrofolate, S-(5'-adenosyl)-L-methionine disulfate salt, trimethylamine oxide. The developed method was successfully applied to the quantification of 22 one-carbon metabolites & co-factors in human plasma from colorectal cancer patients and healthy individuals. The plasma concentrations of dimethylglycine was significantly decreased in the patients compared with the healthy individuals, while L-cystathionine was increased.

Keywords:  Folic acid; Homocysteine; Human plasma; LC-MS/MS; One-carbon metabolism

DOI:  https://doi.org/10.1016/j.jpba.2022.114944
J Clin Lab Anal. 2022 Jul 19. e24610

A multicenter performance evaluation of the new Elecsys Vitamin D total III assay versus reference isotope dilution liquid chromatography tandem mass spectrometry and commercially available comparators.

Peter Findeisen, Marta Leis, Garnet Bendig, Jill Grimme, Elizabeth Moser, Christian Vogl, Robert Christenson.

   BACKGROUND: Vitamin D deficiency/insufficiency and toxicity are worldwide issues; thus, accurate diagnostic assays are required to measure vitamin D. We evaluated the performance of the new Elecsys® Vitamin D total III assay (Roche Diagnostics International Ltd).
METHODS: Repeatability and intermediate precision of the Elecsys Vitamin D total III assay (cobas e 601 analyzer) were evaluated at three sites using five human serum pools (HSPs) and two PreciControls (five-day model, one reagent lot [CLSI-EP05-A3]) and compared against prespecified acceptance criteria. A serum verification panel, with reference isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) values, was used for comparator assay/concordance studies at two sites, assessed using unweighted Deming regression. Testing of serum vs. plasma on the Elecsys assay was conducted at one site using samples from healthy adults; assessed using Passing-Bablok regression.
RESULTS: Repeatability (HSP1 [16.8-18.4 ng/ml], SD 0.87-1.07; HSP5 [94.5-98.0 ng/ml], CV 1.58%-2.76%) and intermediate precision (HSP1, SD 1.14-1.77; HSP5, CV 2.00%-4.13%) met acceptance criteria across sites. Agreement was observed between the Elecsys assay and (i) the ID-LC-MS/MS verification panel (slope, 0.936-1.01; Pearson's r, 0.960-0.986) and (ii) comparator assays (slope, 0.921-1.15; Pearson's r, 0.958-0.982). The Elecsys assay correctly assigned the highest combined percentage of samples to deficient (100%) and insufficient (89.5%) vitamin D categories vs. comparator assays and demonstrated comparable performance in serum and plasma (y = 0.103 + 0.984x).
CONCLUSIONS: The Elecsys Vitamin D total III assay demonstrated good analytical performance and compared favorably with other assays, supporting its use in clinical practice.

Keywords:  analytical performance; assay; method comparison; vitamin D

DOI:  https://doi.org/10.1002/jcla.24610
J Chromatogr A. 2022 Jul 10. pii: S0021-9673(22)00514-3. [Epub ahead of print]1677 463321

Simulating comprehensive two-dimensional gas chromatography mass spectrometry data with realistic run-to-run shifting to evaluate the robustness of tile-based Fisher ratio analysis.

Timothy J Trinklein, Robert E Synovec.

  Untargeted analysis of comprehensive two-dimensional (2D) gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) data has the potential to be hindered by run-to-run retention time shifting. To address this challenge, tile-based Fisher ratio (F-ratio) analysis (FRA) has been developed, which utilizes a supervised, untargeted approach involving a chromatographic segmentation routine termed "tiling" combined with the ANOVA F-ratio statistic to discover class-distinguishing analytes while minimizing false positives arising from shifting. The tiling algorithm is designed to account for retention shifting in both separation dimensions. Although applications of FRA have been reported, there remains a need to thoroughly evaluate the robustness of FRA for different levels of run-to-run retention shifting in order to broaden the scope of its application. To this end, a novel method of simulating GC×GC-TOFMS chromatograms with realistic run-to-run shifting is presented by random generation of low-frequency "shift functions". The dimensionless retention-time precision, <δr>, which is four times the standard deviation in retention time normalized to the peak width-at-base is used as a key modeling variable along with the 2D chromatographic saturation, αe,2D, and within-class relative standard deviation in peak area, RSDwc. We demonstrate that all three of these variables operate together to impact true positive discovery. To quantify the "success" of true positive discovery, GC×GC-TOFMS datasets for various combinations of <δr>, αe,2D, and RSDwc were simulated and then analyzed by FRA using a wide range of relative tile areas (RTA), which is a dimensionless measure of tile size. Since each hit in the FRA hit list was known a priori as either a true or false positive based on the simulation inputs, receiver operating characteristic (ROC) curves were readily constructed. Then, the area under the ROC curve (AUROC) was used as a metric for discovery "success" for various combinations of the modeling variables. Based on the results of this study, recommendations for tile size selection and experimental design are provided, and further supported by comparison to previous tile-based FRA applications. For instance, values for <δr>, αe,2D, and RSDwc obtained from a GC×GC-TOFMS dataset of yeast metabolites suggested an optimum RTA of 6.25, corresponding closely to the RTA of 4.00 employed in the study, implying the simulation results obtained here can be generalized to real datasets.

Keywords:  GC × GC-TOFMS; Run-to-run retention shifting; Simulated realistic 2D chromatograms; Tile-based Fisher-ratio analysis

DOI:  https://doi.org/10.1016/j.chroma.2022.463321
BMC Bioinformatics. 2022 Jul 20. 23(1): 287

Locality-sensitive hashing enables efficient and scalable signal classification in high-throughput mass spectrometry raw data.

Konstantin Bob, David Teschner, Thomas Kemmer, David Gomez-Zepeda, Stefan Tenzer, Bertil Schmidt, Andreas Hildebrandt.

   BACKGROUND: Mass spectrometry is an important experimental technique in the field of proteomics. However, analysis of certain mass spectrometry data faces a combination of two challenges: first, even a single experiment produces a large amount of multi-dimensional raw data and, second, signals of interest are not single peaks but patterns of peaks that span along the different dimensions. The rapidly growing amount of mass spectrometry data increases the demand for scalable solutions. Furthermore, existing approaches for signal detection usually rely on strong assumptions concerning the signals properties.
RESULTS: In this study, it is shown that locality-sensitive hashing enables signal classification in mass spectrometry raw data at scale. Through appropriate choice of algorithm parameters it is possible to balance false-positive and false-negative rates. On synthetic data, a superior performance compared to an intensity thresholding approach was achieved. Real data could be strongly reduced without losing relevant information. Our implementation scaled out up to 32 threads and supports acceleration by GPUs.
CONCLUSIONS: Locality-sensitive hashing is a desirable approach for signal classification in mass spectrometry raw data.
AVAILABILITY: Generated data and code are available at https://github.com/hildebrandtlab/mzBucket . Raw data is available at https://zenodo.org/record/5036526 .

Keywords:  Locality-sensitive hashing; Mass spectrometry; Signal processing

DOI:  https://doi.org/10.1186/s12859-022-04833-5
Int J Cancer. 2022 Jul 22.

Identification of pre-diagnostic metabolites associated with prostate cancer risk by untargeted mass spectrometry-based metabolomics: A case-control study nested in the Northern Sweden Health and Disease Study.

Johnny R Östman, Rui C Pinto, Timothy Md Ebbels, Elin Thysell, Göran Hallmans, Ali A Moazzami.

  Prostate cancer (PCa) is the most common cancer form in males in many European and American countries, but there are still open questions regarding its etiology. Untargeted metabolomics can produce an unbiased global metabolic profile, with the opportunity for uncovering new plasma metabolites prospectively associated with risk of PCa, providing insights into disease etiology. We conducted a prospective untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics analysis using pre-diagnostic fasting plasma samples from 752 PCa case-control pairs nested within the Northern Sweden Health and Disease Study (NSHDS). The pairs were matched by age, BMI, and sample storage time. Discriminating features were identified by a combination of orthogonal projection to latent structures-effect projections (OPLS-EP) and Wilcoxon signed-rank tests. Their prospective associations with PCa risk were investigated by conditional logistic regression. Subgroup analyses based on stratification by disease aggressiveness and baseline age were also conducted. Various free fatty acids and phospholipids were positively associated with overall risk of PCa and in various stratification subgroups. Aromatic amino acids were positively associated with overall risk of PCa. Uric acid was positively, and glucose negatively, associated with risk of PCa in the older subgroup. This is the largest untargeted LC-MS based metabolomics study to date on plasma metabolites prospectively associated with risk of developing PCa. Different subgroups of disease aggressiveness and baseline age showed different associations with metabolites. The findings suggest that shifts in plasma concentrations of metabolites in lipid, aromatic amino acid, and glucose metabolism are associated with risk of developing PCa during following two decades. This article is protected by copyright. All rights reserved.

Keywords:  Liquid chromatography-mass spectrometry; Nested case-control study; Prostate cancer; Risk biomarkers; Untargeted metabolomics

DOI:  https://doi.org/10.1002/ijc.34223
Anal Chem. 2022 Jul 21.

Development of an Internal Calibration Algorithm for Ultrahigh-Resolution Mass Spectra of Dissolved Organic Matter.

Qing-Long Fu, Manabu Fujii, Eunsang Kwon.

In order to obtain a spectrum with high mass accuracy, an internal calibration of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is inevitable. This in turn is critical for subsequent data processing and is generally performed using the commercial instrument software DataAnalysis in the benchmark calibration mode. However, no methodological study has systemically addressed the automated internal calibration of FTICR-MS spectra for dissolved organic matter (DOM) from different sources such as terrestrial and aquatic environments. In this study, a new piecewise algorithm, FTMSCalibrate, was developed to automatically calibrate FTICR-MS spectra in both positive and negative ion modes. FTMSCalibrate was found to reproduce 91.7% ± 4.4% (referred to as the true positive ratio) of the chemical formulas obtained by calibration using manual DataAnalysis. In addition to significantly reducing the mass error, FTMSCalibrate is more accurate in terms of the molecular formula assignment for low m/z peaks than Formularity and MFAssignR. FTMSCalibrate was compatible with deprotonated ions for FTICR-MS spectra in the negative ion mode as well as protonated and adduct ions, including Na- and K-adducts, for FTICR-MS spectra in the positive ion mode. These results suggest that FTMSCalibrate publicly available herein is a robust alternative for the internal calibration of FTICR-MS spectra during postdata processing and will facilitate DOM analysis by FTICR-MS.

DOI: https://doi.org/10.1021/acs.analchem.2c01342
Anal Bioanal Chem. 2022 Jul 19.

Optimization and automation of rapid and selective analysis of fatty acid methyl esters from aqueous samples by headspace SPME arrow extraction followed by GC-MS/MS analysis.

Lucie K Tintrop, Maik A Jochmann, Thomas Beesley, Marco Küppers, Ruth Brunstermann, Torsten C Schmidt.

  The analysis of fatty acid methyl esters (FAMEs) is of high relevance for monitoring and control of various industrial processes and biological systems. In this study, a novel, green analytical approach for the determination of 24 FAMEs from aqueous samples is proposed, which is based on a headspace solid-phase microextraction (SPME) arrow followed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). The method was substantially accelerated to a run time of 44 min per sample by thorough optimization and automation of the relevant parameters. The limiting parameters, mostly based on expediting equilibrium attainment, were found to be parameters of extraction: material, pH, time, and temperature, which were optimized to divinylbenzene polydimethylsiloxane (DVB-PDMS), pH 2, 20 min, and 70 °C, respectively. The optimization and automation of the method led to low method detection limits (9-437 ng L-1) and high selectivity. Evaluation of the method on real samples was done by analyzing the aqueous phase of a bioreactor, whereby the matrix effect could be greatly reduced due to dilution and headspace sampling. The rapid, sensitive, selective, and matrix-reduced approach is found to be not only a novel method for water analysis but is promising for further applications, e.g., with solid and gaseous samples containing FAMEs.

Keywords:  Bioreactor; Fatty acid methyl esters; GC–MS/MS; SPME arrow; Water

DOI:  https://doi.org/10.1007/s00216-022-04204-2
Front Chem. 2022 ;10 853541

Real-Time Non-Invasive Monitoring of Short-Chain Fatty Acids in Exhaled Breath.

Joris Meurs, Evangelia Sakkoula, Simona M Cristescu.

  Short-chain fatty acids (SCFAs) are important metabolites produced by the gut microbiome as a result of the fermentation of non-digestible polysaccharides. The most abundant SCFAs are acetic acid, propionic acid, and butyric acid which make up 95% of this group of metabolites in the gut. Whilst conventional analysis SCFAs is done using either blood or fecal samples, SCFAs can also be detected in exhaled breath using proton transfer reaction-time-of-flight- mass spectrometry (PTR-ToF-MS) using H3O+ for ionization. However, no investigation has been performed to characterize the reactions of SCFAs with H3O+ and with other reagent ions, such as O2 + and NO+. Gas-phase samples of acetic acid, propionic acid, and butyric acid were analyzed with SRI/PTR-ToF-MS under dry and humid conditions. The ions generated and their distribution was determined for each reagent ion. It was found the humidity did not influence the product ion distribution for each SCFA. Using H3O+ as a reagent ion, SRI/PTR-ToF-MS analysis of an exhaled breath sample was performed in real-time to demonstrate the methodology. The presence of SCFAs in exhaled breath was confirmed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). Breath sampling repeatability was within acceptable limits (<15%) for an analytical methodology for each investigated SCFA. Nutritional intervention studies could potentially benefit from real-time monitoring of exhaled SCFAs as an alternative to measuring SCFAs invasively in blood or fecal samples since it is non-invasive, and requires minimal time investment from participants.

Keywords:  SRI/PTR-ToF-MS; exhaled breath; gut micobiome; non-invasive monitoring; short-chain fatly acids

DOI:  https://doi.org/10.3389/fchem.2022.853541
J Anal Toxicol. 2022 Jul 22. pii: bkac053. [Epub ahead of print]

Automated and High-Throughput Urine Drug Screening using Paper Spray Mass Spectrometry.

Magnus Rydberg, Sarah Dowling, Nicholas E Manicke.

  Paper spray mass spectrometry (PS-MS/MS) has been shown to be a rapid, simple, and inexpensive alternative to traditional forensic drug screening methods. It can address the limitations of both immunoassays and chromatography-based techniques due to its non-reliance on sample preparation and its ability to rapidly screen for a wide array of compounds. In this study, an automated PS-MS/MS system was employed to semi-quantitatively screen for 40 commonly abused drugs and metabolites in urine after a 15-minute glucuronidase reaction. The target compounds included common prescription opioids, fentanyl and norfentanyl, stimulants including methamphetamine and cocaine, benzodiazepines, and anti-depressants. The enzyme, buffer, and internal standard solution were combined in one spiking solution to minimize sample handling. Analysis was carried out using a commercially available automated paper spray system coupled to a triple quadrupole mass spectrometer. This method may prove useful for clinical and forensic toxicology laboratories as it allows for automated screening of complex samples for drugs without extraction, separation, and sample cleanup.

Keywords:  ambient ionization; buprenorphine; forensic science; forensic toxicology; illicit drugs; metabolites; methadone; opiates; selected reaction monitoring; urine

DOI:  https://doi.org/10.1093/jat/bkac053
Pract Lab Med. 2022 Aug;31 e00291

Explaining unexplained hypoglycemia: How LC-MS/MS can help.

M T Ackermans, J Hopman, A C Heijboer, S E Siegelaar.

Explaining hypoglycaemia, especially in patients without diabetes mellitus, is challenging. Here we present a case, where the added value for clinical diagnosis of insulin determination with liquid chromatography-mass spectrometry (LC-MS/MS) is shown. By the use of LC-MS/MS the different insulin analogues can be identified. The confirmation of an insulin analogue present during hypoglycaemia facilitated in our case the discussion with the patient and his family about what happened.

DOI: https://doi.org/10.1016/j.plabm.2022.e00291
J Anal Toxicol. 2022 Jul 21. pii: bkac050. [Epub ahead of print]

Development and Validation of a DLLME-GC-MS/MS Method for the Determination of Benzotriazole UV Stabilizer UV-327 and its Metabolites in Human Blood.

Corinna Fischer, Thomas Göen.

  2-(5-chloro-benzotriazol-2-yl)-4,6-di-(tert-butyl)phenol (UV-327) is used as an ultraviolet (UV) absorber in plastic materials and coatings. To investigate its metabolism and to assess human exposure, analytical methods are necessary for the determination of UV-327 and its metabolites in human biological specimens. The method thus presented targets the determination of UV-327 and several of its predicted metabolites in blood using protein precipitation, dispersive liquid-liquid microextraction (DLLME), and derivatization. The trimethylsilylated analytes and internal standards are separated by gas chromatography and analyzed with tandem mass spectrometry (GC-MS/MS). The DLLME procedure was optimized with respect to the type and volume of disperser and extraction solvents, the pH value of the sample solution, and the addition of salt. During method development, an effective ex vivo lactone/hydroxyl carboxylic acid interconversion was observed for two metabolites, each containing a carboxyl group adjacent to the phenolic hydroxyl group. The analytes resulting from interconversion enabled a more sensitive and reliable determination of the metabolites compared to their native structures. Method validation revealed limits of detection (LODs) between 0.02 and 0.36 µg/l. The mean relative recovery rates ranged from 91 to 118%. Precision and repeatability were demonstrated by relative standard deviations in the range of 0.6-14.2% and 1.1-13.7%, respectively. The presently described procedure enables the sensitive and robust analysis of UV-327 and its metabolites in human blood and allows the elucidation of the human UV-327 metabolism as well as the assessment of exposure in potentially exposed individuals.

Keywords:   ex vivo interconversion; Benzotriazole UV stabilizer; DLLME; GC-MS/MS; UV-327; blood; dispersive liquid-liquid microextraction; gas chromatography-tandem mass spectrometry; human metabolism

DOI:  https://doi.org/10.1093/jat/bkac050
Metabolomics. 2022 Jul 16. 18(8): 53

Non-Invasive Identification of Sex in Cultured Bovine Embryos by UHPLC-MS/MS Metabolomics.

Isabel Gimeno, Pablo García-Manrique, Susana Carrocera, Cristina López-Hidalgo, Marta Muñoz, Luis Valledor, David Martín-González, Enrique Gómez.

   INTRODUCTION: Different gene expression between male and female bovine embryos leads to metabolic differences.
OBJECTIVE: We used UHPLC-MS/MS to identify sex metabolite biomarkers in embryo culture medium (CM).
METHODS: Embryos were produced in vitro under highly variable conditions, i.e., fertilized with 7 bulls, two breeds, and cultured with BSA or BSA + serum until Day-6. On Day-6, embryos were cultured individually for 24 h. CM of Day-7 embryos (86 female and 81 male) was collected, and Day-6 and Day-7 embryonic stages recorded.
RESULTS: A study by sample subsets with fixed factors (culture, bull breed, and Day-6 and Day-7 stages) tentatively identified 31 differentially accumulated metabolites through 182 subsets. Day-6 and Day-7 stage together affected 13 and 11 metabolites respectively, while 19 metabolites were affected by one or another stage and/or day. Culture supplements and individual bull changed 19 and 15 metabolites, respectively. Single bull exerted the highest influence (20 metabolites with the significantly highest p values). Lipid (93 subsets; 11 metabolites) and amino acid (55 subsets; 13 metabolites) were the most relevant classes for sex identification.
CONCLUSIONS: Single biomarker led to inefficient sex diagnosis, while metabolite combinations accurately identified sex. Our study is a first in non-invasive sex identification in cattle by overcoming factors that induce metabolic variation.

Keywords:  Bovine; Embryo; Liquid-chromatography; Mass-spectrometry; Metabolomics; Sex

DOI:  https://doi.org/10.1007/s11306-022-01910-x