bims-metlip 2021-07-18 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–07–18
nineteen papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS.
ISFrag: De Novo Recognition of In-Source Fragments for Liquid Chromatography-Mass Spectrometry Data.
MS2Planner: improved fragmentation spectra coverage in untargeted mass spectrometry by iterative optimized data acquisition.
Simultaneous 3-Nitrophenylhydrazine Derivatization Strategy of Carbonyl, Carboxyl and Phosphoryl Submetabolome for LC-MS/MS-Based Targeted Metabolomics with Improved Sensitivity and Coverage.
Applying Log-Normal Peak Fitting to Parallel Reaction Monitoring Data Analysis.
Lipid Profiling of 20 Mammalian Cells by Capillary Microsampling Combined with High-Resolution Spectral Stitching Nanoelectrospray Ionization Direct-Infusion Mass Spectrometry.
Quick-start infrastructure for untargeted metabolomics analysis in GNPS.
Rapid, sensitive, and reliable quantitation of nicotine and its main metabolites cotinine and trans-3'-hydroxycotinine by LC-MS/MS: Method development and validation for human plasma.
A pipeline for making 31P NMR accessible for small- and large-scale lipidomics studies.
Fluoride-assisted liquid chromatography-tandem mass spectrometry method for simultaneous analysis of propofol and its metabolites without derivatization in urine.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for simultaneous quantification of aldosterone, renin activity, and angiotensin II in human plasma.
Development and Validation of a Simple and Sensitive LC-MS/MS Method for Quantification of Metformin in Dried Blood Spot Its Application as an Indicator for Medication Adherence.
Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS.
Determination of camostat and its metabolites in human plasma - preservation of samples and quantification by a validated UHPLC-MS/MS method.
Suspect Screening of Fentanyl Analogs Using Matrix-Assisted Ionization and a Miniature Mass Spectrometer with a Custom Expandable Mass Spectral Library.
Exploring 20 eV electron impact ionization in gas chromatography-tandem mass spectrometry for the determination of estrogenic compounds.
Best Practices for Preparation of Staphylococcus aureus Metabolomics Samples.
Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.
Development of novel projection-type imaging mass spectrometer.

Anal Bioanal Chem. 2021 Jul 09.

Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS.

Zuzana Vaňková, Ondřej Peterka, Michaela Chocholoušková, Denise Wolrab, Robert Jirásko, Michal Holčapek.

  Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2-μm particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full-scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full-scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS-based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

Keywords:  Human plasma; Lipidomics; Lipids; Mass spectrometry; Retention behavior; Reversed-phase; Ultrahigh-performance liquid chromatography

DOI:  https://doi.org/10.1007/s00216-021-03492-4
Anal Chem. 2021 Jul 16.

ISFrag: De Novo Recognition of In-Source Fragments for Liquid Chromatography-Mass Spectrometry Data.

Jian Guo, Sam Shen, Shipei Xing, Huaxu Yu, Tao Huan.

In-source fragmentation (ISF) is a naturally occurring phenomenon during electrospray ionization (ESI) in liquid chromatography-mass spectrometry (LC-MS) analysis. ISF leads to false metabolite annotation in untargeted metabolomics, prompting misinterpretation of the underlying biological mechanisms. Conventional metabolomic data cleaning mainly focuses on the annotation of adducts and isotopes, and the recognition of ISF features is mainly based on common neutral losses and the LC coelution pattern. In this work, we recognized three increasingly important patterns of ISF features, including (1) coeluting with their precursor ions, (2) being in the tandem MS (MS2) spectra of their precursor ions, and (3) sharing similar MS2 fragmentation patterns with their precursor ions. Based on these patterns, we developed an R package, ISFrag, to comprehensively recognize all possible ISF features from LC-MS data generated from full-scan, data-dependent acquisition, and data-independent acquisition modes without the assistance of common neutral loss information or MS2 spectral library. Tested using metabolite standards, we achieved a 100% correct recognition of level 1 ISF features and over 80% correct recognition for level 2 ISF features. Further application of ISFrag on untargeted metabolomics data allows us to identify ISF features that can potentially cause false metabolite annotation at an omics-scale. With the help of ISFrag, we performed a systematic investigation of how ISF features are influenced by different MS parameters, including capillary voltage, end plate offset, ion energy, and "collision energy". Our results show that while increasing energies can increase the number of real metabolic features and ISF features, the percentage of ISF features might not necessarily increase. Finally, using ISFrag, we created an ISF pathway to visualize the relationships between multiple ISF features that belong to the same precursor ion. ISFrag is freely available on GitHub (https://github.com/HuanLab/ISFrag).

DOI: https://doi.org/10.1021/acs.analchem.1c01644
Bioinformatics. 2021 07 12. 37(Suppl_1): i231-i236

MS2Planner: improved fragmentation spectra coverage in untargeted mass spectrometry by iterative optimized data acquisition.

Zeyuan Zuo, Liu Cao, Louis-Félix Nothia, Hosein Mohimani.

MOTIVATION: Untargeted mass spectrometry experiments enable the profiling of metabolites in complex biological samples. The collected fragmentation spectra are the metabolite's fingerprints that are used for molecule identification and discovery. Two main mass spectrometry strategies exist for the collection of fragmentation spectra: data-dependent acquisition (DDA) and data-independent acquisition (DIA). In the DIA strategy, all the metabolites ions in predefined mass-to-charge ratio ranges are co-isolated and co-fragmented, resulting in multiplexed fragmentation spectra that are challenging to annotate. In contrast, in the DDA strategy, fragmentation spectra are dynamically and specifically collected for the most abundant ions observed, causing redundancy and sub-optimal fragmentation spectra collection. Yet, DDA results in less multiplexed fragmentation spectra that can be readily annotated.
RESULTS: We introduce the MS2Planner workflow, an Iterative Optimized Data Acquisition strategy that optimizes the number of high-quality fragmentation spectra over multiple experimental acquisitions using topological sorting. Our results showed that MS2Planner increases the annotation rate by 38.6% and is 62.5% more sensitive and 9.4% more specific compared to DDA.
AVAILABILITY AND IMPLEMENTATION: MS2Planner code is available at https://github.com/mohimanilab/MS2Planner. The generation of the inclusion list from MS2Planner was performed with python scripts available at https://github.com/lfnothias/IODA_MS.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btab279
Anal Chem. 2021 Jul 16.

Simultaneous 3-Nitrophenylhydrazine Derivatization Strategy of Carbonyl, Carboxyl and Phosphoryl Submetabolome for LC-MS/MS-Based Targeted Metabolomics with Improved Sensitivity and Coverage.

Xiangjun Meng, Huanhuan Pang, Fei Sun, Xiaohan Jin, Bohong Wang, Ke Yao, LiAng Yao, Lijuan Wang, Zeping Hu.

Metabolomics is a powerful and essential technology for profiling metabolic phenotypes and exploring metabolic reprogramming, which enables the identification of biomarkers and provides mechanistic insights into physiology and disease. However, its applications are still limited by the technical challenges particularly in its detection sensitivity for the analysis of biological samples with limited amount, necessitating the development of highly sensitive approaches. Here, we developed a highly sensitive liquid chromatography tandem mass spectrometry method based on a 3-nitrophenylhydrazine (3-NPH) derivatization strategy that simultaneously targets carbonyl, carboxyl, and phosphoryl groups for targeted metabolomic analysis (HSDccp-TM) in biological samples. By testing 130 endogenous metabolites including organic acids, amino acids, carbohydrates, nucleotides, carnitines, and vitamins, we showed that the derivatization strategy resulted in significantly improved detection sensitivity and chromatographic separation capability. Metabolic profiling of merely 60 oocytes and 5000 hematopoietic stem cells primarily isolated from mice demonstrated that this method enabled routine metabolomic analysis in trace amounts of biospecimens. Moreover, the derivatization strategy bypassed the tediousness of inferring the MS fragmentation patterns and simplified the complexity of monitoring ion pairs of metabolites, which greatly facilitated the metabolic flux analysis (MFA) for glycolysis, the tricarboxylic acid (TCA) cycle, and pentose phosphate pathway (PPP) in cell cultures. In summary, the novel 3-NPH derivatization-based method with high sensitivity, good chromatographic separation, and broad coverage showed great potential in promoting metabolomics and MFA, especially in trace amounts of biospecimens.

DOI: https://doi.org/10.1021/acs.analchem.1c00767
J Proteome Res. 2021 Jul 14.

Applying Log-Normal Peak Fitting to Parallel Reaction Monitoring Data Analysis.

Christoph Stingl, Theo M Luider.

  Chromatographic separation is often an important part of mass-spectrometry-based proteomic analysis. It reduces the complexity of the initial samples before they are introduced to mass-spectrometric detection and chromatographic characteristics (such as retention time) add analytical features to the analyte. The acquisition and analysis of chromatographic data are thus of great importance, and specialized software is used for the extraction of quantitative information in an efficient and optimized manner. However, occasionally, automatic peak picking and correct peak boundary setting is challenged by, for instance, aberration of peak shape, peak truncation, and peak tailing, and a manual review of a large number of peaks is frequently required. To support this part of the analysis, we present here a software tool, Peakfit, that fits acquired chromatographic data to the log-normal peak equation and reports the calculated peak parameters. The program is written in R and can easily be integrated into Skyline, a popular software packages that is frequently used for proteomic parallel reaction monitoring applications. The program is capable of processing large data sets (>10 000 peaks) and detecting sporadic outliers in peak boundary selection performed, for instance, in Skyline. In an example data set, available via ProteomeXchange with identifier PXD026875, we demonstrated the capability of the program to characterize chromatographic peaks and showed an example of its ability to objectively and reproducibly detect and solve problematic peak-picking situations.

Keywords:  data processing; parallel reaction monitoring; peak fitting; peak modeling; software tool

DOI:  https://doi.org/10.1021/acs.jproteome.1c00371
Anal Chem. 2021 Jul 16.

Lipid Profiling of 20 Mammalian Cells by Capillary Microsampling Combined with High-Resolution Spectral Stitching Nanoelectrospray Ionization Direct-Infusion Mass Spectrometry.

Tianrun Xu, Hang Li, Disheng Feng, Peng Dou, Xianzhe Shi, Chunxiu Hu, Guowang Xu.

Studies of cellular metabolism can provide profound insights into the underlying molecular mechanisms and metabolic function. To date, the majority of cellular metabolism studies based on chromatography-mass spectrometry (MS) require population cells to obtain informative metabolome. These methods are not only time-consuming but also not suitable for amount-limited cell samples such as circulating tumor cells, stem cells, and neurons. Therefore, it is extremely essential to develop analytical methods enabling to detect metabolome from tens of cells in a high-throughput and high-sensitivity way. In this work, a novel platform for rapid and sensitive detection of lipidome in 20 mammalian cells was proposed using capillary microsampling combined with high-resolution spectral stitching nanoelectrospray ionization direct-infusion MS. It can be used to collect cells rapidly and accurately via the capillary microprobe, extract lipids directly in a 96-well plate using a spray solvent, and detect more than 500 lipids covering 19 lipid subclasses within 3 min. This novel platform was successfully applied to study the lipid features of different cancer cell types and subtypes as well as target cells from tissue samples. This study provides a strategy for determining the lipid species with rich information in tens of cells and demonstrates great potential for clinical applications.

DOI: https://doi.org/10.1021/acs.analchem.1c00373
Nat Metab. 2021 Jul 09.

Quick-start infrastructure for untargeted metabolomics analysis in GNPS.

Tiago F Leao, Chase M Clark, Anelize Bauermeister, Emmanuel O Elijah, Emily C Gentry, Makhai Husband, Michelli F Oliveira, Nuno Bandeira, Mingxun Wang, Pieter C Dorrestein.

DOI: https://doi.org/10.1038/s42255-021-00429-0
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 May 05. pii: S1570-0232(21)00216-6. [Epub ahead of print]1179 122736

Rapid, sensitive, and reliable quantitation of nicotine and its main metabolites cotinine and trans-3'-hydroxycotinine by LC-MS/MS: Method development and validation for human plasma.

Nadja Mallock, Andrea Rabenstein, Peter Laux, Tobias Rüther, Christoph Hutzler, Maria Kristina Parr, Andreas Luch.

  New nicotine delivery products are gaining market share. For evaluation of their characteristics, toxicokinetic investigations are in current research focus. For reliable determination of blood plasma levels of nicotine and its main metabolites cotinine and trans-3'-hydroxycotinine, a quantitation method based on LC-ESI-MS/MS was developed and validated. Addition of isotope labeled internal standards prior to rapid sample preparation using protein precipitation with methanol was chosen for sample preparation. Different stationary phases were tested and phenyl-hexyl separation was found to be superior to HILIC, C18, and C8 stationary phases. Ion suppression effects caused by hydrophilic early eluting matrix were eliminated by the adjustment of an adequate retention utilizing a phenyl-hexyl separation stationary phase. Exchange of acetonitrile as organic mobile phase by methanol and elevation of pH value of aqueous mobile phase containing 5 mM NH4Ac to 4.50 improved the chromatographic resolution. The limits of quantitation for nicotine, cotinine, and hydroxycotinine were 0.15, 0.30, and 0.40 ng/mL, respectively. Linearity was proven by matrix matched calibration for the whole working range from 0.50 ng/mL to 35.0 ng/mL for nicotine and from 6.00 to 420 ng/mL for cotinine and hydroxycotinine (Mandel's fitting test with R2 > 0.995). Quality control samples at four different levels (0.50, 1.50, 17.5, 28.0 ng/mL for nicotine and 6.00, 18.0, 210, 336 ng/mL for cotinine and hydroxycotinine) in plasma were analyzed six times on three days. Mean accuracies ranged from 87.7% to 105.8% for nicotine, from 90.3% to 102.9% for cotinine, and from 99.9% to 109.9% for hydroxycotinine. Intra- and inter-day precisions (RSD %) were below 15% for all analytes (<20% for LLOQ). As proof of concept, the method was successfully applied to a real plasma sample from a cigarette smoking volunteer.

Keywords:  Cotinine; Hydroxycotinine; Nicotine; Phenyl-hexyl column; Protein precipitation

DOI:  https://doi.org/10.1016/j.jchromb.2021.122736
Anal Bioanal Chem. 2021 Jul 12.

A pipeline for making 31P NMR accessible for small- and large-scale lipidomics studies.

Samuel Furse, Huw E L Williams, Adam J Watkins, Samuel Virtue, Antonio Vidal-Puig, Risha Amarsi, Marika Charalambous, Albert Koulman.

  Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways, organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of tools and techniques to acquire lipidomics data. 31P NMR for lipidomics offers well-resolved head group/lipid class analysis, structural data that can be used to inform and strengthen interpretation of mass spectrometry data and part of a priori structural determination. In the present study, we codify the use of 31P NMR for lipidomics studies to make the technique more accessible to new users and more useful for a wider range of questions. The technique can be used in isolation (phospholipidomics) or as a part of determining lipid composition (lipidomics). We describe the process from sample extraction to data processing and analysis. This pipeline is important because it allows greater thoroughness in lipidomics studies and increases scope for answering scientific questions about lipid-containing systems.

Keywords:  31P NMR; Lipid profiling; Lipidomics

DOI:  https://doi.org/10.1007/s00216-021-03430-4
J Chromatogr A. 2021 Jun 27. pii: S0021-9673(21)00484-2. [Epub ahead of print]1652 462360

Fluoride-assisted liquid chromatography-tandem mass spectrometry method for simultaneous analysis of propofol and its metabolites without derivatization in urine.

Kang Mi Lee, Sang Moon Han, Seunghwa Lee, Tae Young Jeong, Ho Jun Kim, Hophil Min, Ki Hun Kim, Sangwon Cha, Won Keun Oh, Jaeick Lee.

  The misuse of propofol for recreational purposes has become a serious social issue. Accordingly, practical and sensitive analytical methods to investigate the chronic abuse and toxicity of propofol are required. However, current propofol determination methods using liquid chromatography-mass spectrometry (LC-MS/MS) suffer from problems associated with loss in sample preparation due to its volatility and its poor ionization efficiency and collision-induced dissociation in mass spectrometry. Herein, we have developed a sensitive and accurate fluoride-assisted LC-MS/MS method combined with direct-injection for propofol determination. Ionization via fluoride-ion attachment/induced deprotonation, effected by ammonium fluoride in the mobile phase, was found to dramatically improve the sensitivity of propofol without derivatization. Furthermore, direct injection without derivatization enables the simultaneous analysis of propofol and its phase II metabolites without analyte loss. The optimal concentration of ammonium fluoride in the mobile phase was found to be 1 mM under methanol conditions. The linearity is good (R2 ≥ 0.999) and the intra- and inter-day precisions for propofol determination are between 1.9 and 8.7%. The accuracies range from 87.5% to 105.4% and the limits of detection and quantitation for propofol in urine are 0.15 and 0.44 ng mL-1, respectively. The present method was successfully applied to human urine and showed a sufficient sensitivity to determine propofol and five phase II metabolites over 48 h in human urine after administration. Consequently, the fluoride-assisted LC-MS/MS method was demonstrated to be sensitive, accurate, and practical for the determination of propofol and its metabolites.

Keywords:  Electrospray ionization; Fluoride; Mass spectrometry; Propofol

DOI:  https://doi.org/10.1016/j.chroma.2021.462360
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jul 03. pii: S1570-0232(21)00220-8. [Epub ahead of print]1179 122740

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for simultaneous quantification of aldosterone, renin activity, and angiotensin II in human plasma.

Fangjun Chen, Ziyun Cheng, Yingfei Peng, Zhenxin Wang, Chao Huang, Dan Liu, Beili Wang, Baishen Pan, Wei Guo.

  Accurate quantification of plasma aldosterone (ALD) and renin activity (PRA)is critical for the diagnosis of primary aldosteronism (PA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the "gold standard" method for the determination of ALDand PRA. The aim of this study is to develop a new LC-MRM/MS assay for quantifying plasma ALD, PRA, and angiotensin II (Ang II) simultaneously and validate its effectiveness. To be more specific, plasmasamples were prepared by solid-phase extraction and separated in an ultra-performance reversed-phase column. MS detection was performed via a triple quadrupole mass spectrometer containing both positive and negative ion monitoring modes. The developed assay was then validated according to the standard guidelines and the influence of sample incubation on ALD and Ang II concentration was evaluated. In addition, the variation of endogenous Ang I was explored. The proposed LC-MRM/MS method was compared another LC-MS/MS method, which detects ALD, Ang I, and Ang II separately. Analyteswere separated and quantified within 5 min. The assay wasvalidated to be linear up to 5000 pg/ml for ALD and Ang II and 33.3 ng/ml/h for PRA.The lower limit of quantification (LLOQ) was 15 pg/ml, 15 pg/ml, and 0.1 ng/ml/hfor ALD, Ang II, and PRArespectively. Specificity, precision, accuracy, and stability were tested to meet the requirements of the guidelines. Significant changes were not found in ALD and Ang II concentrations over the 3 h-incubation. In addition, it was demonstratedthat the resultof PRA was not stronglyinfluenced by the endogenous Ang I. Comparison with another LC-MS/MS method was performed using the same apparatusand the proposed method was proved to be in good coincidence with the correlation coefficients rangingfrom 0.955to0.996. A sensitive and reliable method for simultaneousquantification of ALD, PRA, and Ang II has been developed and this study will significantly promote laboratory workflow efficiency and throughput.

Keywords:  Aldosterone; Angiotensin II; Liquid chromatography-tandem mass spectrometry; Renin activity

DOI:  https://doi.org/10.1016/j.jchromb.2021.122740
Int J Gen Med. 2021 ;14 3225-3233

Development and Validation of a Simple and Sensitive LC-MS/MS Method for Quantification of Metformin in Dried Blood Spot Its Application as an Indicator for Medication Adherence.

Bushra T ALquadeib, Nouf M Aloudah, Alanood S Almurshedi, Iman M ALfagih, Basmah N ALdosari, Adim S ALmeleky, Nour M Almubyedh.

   Objective: Metformin (MET), an oral biguanide agent, can improve insulin resistance and decrease hepatic glucose production, leading to a reduction in blood-sugar levels. The objective of the present study was to develop and validate simple and rapid LC-MS/MS method for analysis of MET in dried blood spot (DBS) sample for patient monitoring studies purposes (drug adherence).
Methods: The chromatographic separation was achieved with Waters HSS-T3 column using gradient elution of mobile phases of two solvents: 1) solvent A, consisted of 10mM ammonium formate, 0.2% formic acid 1%; and 2) acetonitrile solvent B, contained 0.2% formic acid in acetonitrile at a flow rate of 0.2 mL/min. The total run time was 3.0 min. The effectiveness of chromatographic conditions was optimized, and afatinib was used as the internal standard. The assay method was validated using USP 26 and the ICH guidelines.
Results: The method showed good linearity in the range 8-48 ng/mL for MET with correlation coefficient (r) >0.9907. The intra- and inter‑day precision values for MET met the acceptance criteria as per regulatory guidelines. MET was stable during the stability studies at ambient temperature 25 °C, at refrigerator 4 °C, at 10 °C autosampler, freeze/thaw cycles and 30 days storage in a freezer at -30 ± 0.5 °C.
Conclusion: This method has successfully fulfilled all validation requirements referring to EMA and FDA guidelines, and successfully can be applied for MET adherence study. All the six studied patients were approved to metformin adherence.

Keywords:  dried blood spot; liquid chromatography–tandem mass spectrometry; medication adherence; metformin; method validation

DOI:  https://doi.org/10.2147/IJGM.S312633
Clin Chem Lab Med. 2021 Jul 09.

Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS.

Ivan Hartling, Alessio Cremonesi, Ester Osuna, Phing-How Lou, Eliana Lucchinetti, Michael Zaugg, Martin Hersberger.

   OBJECTIVES: Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma.
METHODS: Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion.
RESULTS: We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2).
CONCLUSIONS: Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice.

Keywords:  AA; AEA; ALOX12; COX; CYP; D-series resolvin; DGLA; DHA; DHEA; DPA; DiHETE; EPA; EPEA; EpDPA; EpEDE; EpETE; EpOME; HDHA; HEPE; HETE; HETrE; HODE; LA; LC-MS/MS; LOX; LTB4; LXA4; LXB4; MaR; PD1; PDX; PGD2; PGE2; PGF1α; PUFA; RvD; SPE; SPM; TPN; TXB2; UHPLC-MS/MS; arachidonate 12-lipoxygenase; arachidonic acid; arachidonoyl ethanolamide; cyclooxygenase; cytochrome P450 monooxygenase; dihomo-γ linoleic acid; dihydroxyeicosatetraenoic acid; docosahexaenoic acid; docosahexaenoyl ethanolamide; docosapentaenoic acid; eicosanoids; eicosapentaenoic acid; eicosapentaenoyl ethanolamide; epoxydocosapentaenoic acid; epoxyeicosadienoic acid; epoxyeicosatetraenoic acid; epoxyoctadecenoic acid; hydroxydocosahexaenoic acid; hydroxyeicosapentaenoic acid; hydroxyeicosatetraenoic acid; hydroxyeicosatrienoic acid; hydroxyoctadecadienoic acid; leukotriene B4; linoleic acid; lipid mediators; lipoxin A4; lipoxin B4; lipoxygenase; maresin; oxylipins; polyunsaturated fatty acid; prostaglandin D2; prostaglandin E2; prostaglandin F1α; protectin D1; protectin DX; reference value; solid phase extraction; specialized pro-resolving mediator; thromboxane B2; total parenteral nutrition; ultra-high-performance liquid chromatography-tandem mass spectrometry; α-linolenic acid; αLA

DOI:  https://doi.org/10.1515/cclm-2021-0644
Clin Biochem. 2021 Jul 09. pii: S0009-9120(21)00196-X. [Epub ahead of print]

Determination of camostat and its metabolites in human plasma - preservation of samples and quantification by a validated UHPLC-MS/MS method.

Lambert K Sørensen, Jørgen B Hasselstrøm, Jesper D Gunst, Ole S Søgaard, Mads Kjolby.

   OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected.
DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds.
RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 hours of storage at room temperature or 4 months of storage at -20°C and -80°C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ± 3% at concentrations of 1-800 ng/mL.
CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Keywords:  COVID-19; Camostat; FOY-251; FOY-305; GBA; GBPA; LC-MS/MS; esterase inhibitors

DOI:  https://doi.org/10.1016/j.clinbiochem.2021.07.007
Anal Chem. 2021 Jul 13.

Suspect Screening of Fentanyl Analogs Using Matrix-Assisted Ionization and a Miniature Mass Spectrometer with a Custom Expandable Mass Spectral Library.

Xiangyu Guo, Yuhan Shang, Yueguang Lv, Hua Bai, Qiang Ma.

The reliable identification of fentanyl and its analogs is of great significance for public security. However, with the growing prevalence of fentanyl compounds, current analytical strategies cannot fully meet the need for fast and high-throughput detection. In this study, a simple, rapid, and on-site analytical protocol was developed based on a miniature mass spectrometer. A dramatically simplified workflow was implemented using matrix-assisted ionization, bypassing complex sample pretreatment and chromatographic separation. The tandem mass spectrometry (MS/MS) capability afforded by the miniature ion trap mass spectrometer facilitated the investigation of fragmentation patterns for 49 fentanyl analogs during collision-induced dissociation, revealing valuable information on marker fragment ions and characteristic neutral loss. Calculations on Laplacian bond order values further verified the mass spectrometric behavior. A computation-assisted expandable mass spectral library was constructed in-house for fentanyl compounds. Smart suspect screening was carried out based on the full-scan MS and MS/MS data. The present study demonstrates an appealing potential for forensic applications, enabling streamlined screening for the presence of illicit fentanyl compounds at the point of seizures of suspect samples.

DOI: https://doi.org/10.1021/acs.analchem.1c01117
J Chromatogr A. 2021 Jun 26. pii: S0021-9673(21)00483-0. [Epub ahead of print]1652 462359

Exploring 20 eV electron impact ionization in gas chromatography-tandem mass spectrometry for the determination of estrogenic compounds.

Alex Glineur, Marco Beccaria, Giorgia Purcaro.

  In electron ionization mass spectrometry (MS), the generation of characteristic fragmentation patterns allows reliable and sensitive identification of compounds. However, loss or a less intense signal of the molecular ion (or more diagnostic ions) can often be observed, which can be detrimental for identification and/or sensitivity, even when MS/MS approaches are applied for quantification. The benefits of applying lower ionization energy (i.e., 20 eV compared to 70 eV) using a gas chromatography (GC) - tandem MS (MS/MS) instrument were investigated in the detection of three estrogenic compounds, namely estrone (E1), 17β-estradiol (E2), and 17α-ethynylestradiol (EE2), emerging aquatic pollutants included in the European Commission Watch List. As expected, the relative intensity of molecular ions (M+.) or high-mass fragments closely related (M+.-CH3) increased significantly at 20 eV compared to 70 eV (from 4.6 % to 35.0 % for EE2, from 22.5 % to 87.3 % for E2, and from 76 % to 100 % for E1). This change in the spectrum profile led to an overall increase in the sensitivity of the compounds when detected using the multiple reaction monitoring mode. These results were compared with the instrumental limit of quantification obtained in liquid chromatography - MS/MS showing a limit of quantification of about 100-folds lower for GC-MS/MS and a completely neglectable matrix effect, thus posing the base for the development of a miniaturized sample preparation method (with an overall lower concentration factor) to achieve the challenging low limits of detection required by the EU regulation for estrogenic compounds.

Keywords:  Electron ionization (EI); Estrogenic compounds; Gas chromatography (GC); Mass spectrometry (MS); Milder ionization

DOI:  https://doi.org/10.1016/j.chroma.2021.462359
Methods Mol Biol. 2021 ;2341 103-116

Best Practices for Preparation of Staphylococcus aureus Metabolomics Samples.

Kimberly L James, Kelly C Rice.

  Developments in mass spectrometry have made it possible to identify individual biomolecules in complex samples. This has led to advances in the detection and quantification of both extracellular and intracellular metabolites, such as amino acids, organic acids, fatty acids, nucleotides, and CoA-esters from growth media and cellular extracts. However, the reproducibility of metabolite data can be problematic if the concentrations and/or stability of metabolites fluctuate during culture harvesting and processing. Herein we describe a standardized and efficient collection protocol and best practices for preservation and harvesting of Staphylococcus aureus cellular and supernatant samples to improve reproducibility, reliability, and consistency in mass-spectrometry-based metabolite data sets.

Keywords:  Mass spectrometry; Metabolite profiles; Metabolome; Nucleotides; Organic acid concentrations; Staphylococcus aureus

DOI:  https://doi.org/10.1007/978-1-0716-1550-8_13
J Sep Sci. 2021 Jul 13.

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

Serina Fukui, Eiji Sugiyama, Hajime Mizuno, Iwao Sakane, Daiki Asakawa, Kazumi Saikusa, Yuki Nishiya, Yuri Amano, Kentaro Takahara, Daisuke Higo, Toshimasa Toyo'oka, Kenichiro Todoroki.

  2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires single analytical instrument and less than 1 s for the separation. We compared three derivatization methods and found that a method using (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine enables the separation. In addition, we were able to detect dl-2-hydroxyglutaric acid in standard solution at lower concentrations than that previously reported for the serum. These results show the potential of the method to be used in clinical analysis. This article is protected by copyright. All rights reserved.

Keywords:  2-hydroxyglutaric acid; chiral derivatizing reagent; chiral separation; ion mobility spectrometry; mass spectrometry

DOI:  https://doi.org/10.1002/jssc.202100350
Rev Sci Instrum. 2021 May 01. 92(5): 053706

Development of novel projection-type imaging mass spectrometer.

J Aoki, M Toyoda.

We developed a novel imaging mass spectrometer based on our accumulating technology for projection-type imaging mass spectrometry, the simulation of an accurate ion trajectory, and the theory for ion optics. The newly developed apparatus yields high spatial resolution with a substantially shorter image-acquisition time compared with conventional scanning-type imaging mass spectrometers. In order to maintain a high mass resolution, a multi-turn time-of-flight mass spectrometer is combined with post-extraction differential acceleration methods. Consequently, a mass resolution of m/Δm ∼ 10 000 and a spatial resolution of 1 μm were achieved simultaneously in this study. Application of our newly established apparatus to biological samples accomplished successful imaging mass spectrometry by exhibiting an organ-specific distribution of endogenous ions as well as a localized distribution of exogenously applied ions with an ultra-high spatial resolution image in the size of 18.5 megapixels.

DOI: https://doi.org/10.1063/5.0037370