bims-metlip 2022-03-20 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2022–03–20
24 papers selected by
Sofia Costa, Icahn School of Medicine at Mount Sinai

An LC/MS/MS method for quantifying testosterone and dehydroepiandrosterone sulfate in four different serum samples during a single run.
Deep Learning-Assisted Peak Curation for Large-Scale LC-MS Metabolomics.
DIAMetAlyzer allows automated false-discovery rate-controlled analysis for data-independent acquisition in metabolomics.
Optimization and validation of direct gas chromatography-mass spectrometry method for simultaneous quantification of ten short-chain fatty acids in rat feces.
Profiling Urinary Sulfate Metabolites With Mass Spectrometry.
A concise review on lipidomics analysis in biological samples.
Integrative metabolic flux analysis reveals an indispensable dimension of phenotypes.
6-Glycosylaminoquinoline-assisted LDI MS for detection and imaging of small molecules with enhanced detection selectivity and sensitivity.
Mass Spectrometry Imaging Techniques Enabling Visualization of Lipid Isomers in Biological Tissues.
Rapid determination of hydrogen sulfide-related metabolites in human urine by UHPLC-ESI-MS/MS with fluorinated ion-pairing reagents.
Determination of 22 alternative plasticizers in wrap film by solid phase extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.
An Liquid Chromatography-Tandem Mass Spectrometry Method for the Simultaneous Determination of Afatinib, Alectinib, Ceritinib, Crizotinib, Dacomitinib, Erlotinib, Gefitinib, and Osimertinib in Human Serum: Erratum.
Mass Spectrometry Imaging.
Clinical Utility and Analytical Aspects of Direct Measurements of Free Hormones Using Mass Spectrometry-Based Methods.
Vitamin K (phylloquinone and menaquinones) in foods - Cost-effective quantification by LC-ESI-MS/MS.
Non-target analysis and stability assessment of reference materials using liquid chromatography‒high-resolution mass spectrometry.
LC-MS/MS method for quantifying aescinate A and B and assessing their relationship with phlebitis.
A novel, rapid and simple UHPLC-MS/MS method for quantification of warfarin in dried blood spots.
Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).
Simultaneous Quantification of 11 Antiseizure Medications and Metabolites in Serum for Therapeutic Drug Monitoring Using High-Performance Liquid Chromatography with Ultraviolet Detection: A Short Communication.
A Novel Approach for Monitoring the Volatile Metabolome in Biological Samples from Ruminants through Miniaturized Liquid-Liquid Extraction and Multiclass Gas Chromatography Analysis.
Ultra-High-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.
Simultaneous determination of the four key fluoroquinolones and two antipsychotics in fish and shrimp by LC-MS/MS.
Coupling paper spray ionization with the flexible microtube plasma for the determination of low polar biomarkers in mass spectrometry.

Anal Sci. 2022 Jan;38(1): 167-173

An LC/MS/MS method for quantifying testosterone and dehydroepiandrosterone sulfate in four different serum samples during a single run.

Shunsuke Fujimura, Takenori Ito, Shoujiro Ogawa, Takayuki Ishige, Shoichi Nishimoto-Kusunose, Tatsuya Higashi.

  Simultaneous measurements of the circulating testosterone (TS) and dehydroepiandrosterone sulfate (DHEAS) are deemed to be helpful for the assessment of men's health. Liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) is the most reliable methodology for this purpose; however, it has room for improvement in analysis throughput. In this study, a quadruplicate of the Girard reagents was used to develop an LC/ESI-MS/MS method capable of quantifying TS and DHEAS in four different serum samples in a single run. The four serum samples were separately pretreated, derivatized with one of four Girard reagents, and then combined. The LC/ESI-MS/MS analysis of the combined sample provided the androgen concentrations of four serum samples in parallel. The method had practical measuring ranges, in which good precision and accuracy, as well as negligible matrix effects were verified. The speed-up capability of the developed method was evaluated through the analysis of ten batches of serum samples (total 40 samples); the method saved a 60% post-pretreatment analysis time compared to the non-derivatization method for 40 samples.

Keywords:  Androgen; Derivatization; Girard reagent; High throughput; LC/ESI–MS/MS; Sample-multiplexing

DOI:  https://doi.org/10.2116/analsci.21P268
Anal Chem. 2022 Mar 15.

Deep Learning-Assisted Peak Curation for Large-Scale LC-MS Metabolomics.

Yoann Gloaguen, Jennifer A Kirwan, Dieter Beule.

Available automated methods for peak detection in untargeted metabolomics suffer from poor precision. We present NeatMS, which uses machine learning based on a convoluted neural network to reduce the number and fraction of false peaks. NeatMS comes with a pre-trained model representing expert knowledge in the differentiation of true chemical signal from noise. Furthermore, it provides all necessary functions to easily train new models or improve existing ones by transfer learning. Thus, the tool improves peak curation and contributes to the robust and scalable analysis of large-scale experiments. We show how to integrate it into different liquid chromatography-mass spectrometry (LC-MS) analysis workflows, quantify its performance, and compare it to various other approaches. NeatMS software is available as open source on github under permissive MIT license and is also provided as easy-to-install PyPi and Bioconda packages.

DOI: https://doi.org/10.1021/acs.analchem.1c02220
Nat Commun. 2022 Mar 15. 13(1): 1347

DIAMetAlyzer allows automated false-discovery rate-controlled analysis for data-independent acquisition in metabolomics.

Oliver Alka, Premy Shanthamoorthy, Michael Witting, Karin Kleigrewe, Oliver Kohlbacher, Hannes L Röst.

The extraction of meaningful biological knowledge from high-throughput mass spectrometry data relies on limiting false discoveries to a manageable amount. For targeted approaches in metabolomics a main challenge is the detection of false positive metabolic features in the low signal-to-noise ranges of data-independent acquisition results and their filtering. Another factor is that the creation of assay libraries for data-independent acquisition analysis and the processing of extracted ion chromatograms have not been automated in metabolomics. Here we present a fully automated open-source workflow for high-throughput metabolomics that combines data-dependent and data-independent acquisition for library generation, analysis, and statistical validation, with rigorous control of the false-discovery rate while matching manual analysis regarding quantification accuracy. Using an experimentally specific data-dependent acquisition library based on reference substances allows for accurate identification of compounds and markers from data-independent acquisition data in low concentrations, facilitating biomarker quantification.

DOI: https://doi.org/10.1038/s41467-022-29006-z
J Chromatogr A. 2022 Mar 10. pii: S0021-9673(22)00156-X. [Epub ahead of print]1669 462958

Optimization and validation of direct gas chromatography-mass spectrometry method for simultaneous quantification of ten short-chain fatty acids in rat feces.

Jin-Hao Zhu, Qian Mao, Si-Yu Wang, Hui Liu, Shan-Shan Zhou, Wei Zhang, Ming Kong, He Zhu, Song-Lin Li.

  Short-chain fatty acids (SCFAs) play key roles in maintaining health and treating disease. Quantification of important fecal SCFAs is necessary to facilitate the clarification of their biological roles. However, the existing quantifying methods mainly depend on complicated precolumn derivatization, and/or are unable to determine formic acid, a SCFA commonly associated with toxicity. In this study, a direct gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of ten SCFAs including formic acid in rat feces was developed. The approach was optimized in terms of chromatographic and spectrometric conditions as well as sample preparation. DB-FFAP capillary column with temperature programming was used to get baseline separation and symmetrical peak shape of SCFAs without precolumn derivatization in a relatively short running time (8 min). Multiple reaction monitoring (MRM) scan mode was employed to enhance the sensitivity and selectivity of SCFAs. Acidification with 50% HCl and immediate extraction with diethyl ether were utilized to achieve sample preparation of ten SCFAs from feces. Furthermore, the developed method was validated with wide linear range, high sensitivity and precision, low matrix effect and acceptable accuracy. The established method was successfully applied to compare the contents of fecal SCFAs between normal and immunosuppressed animal models.

Keywords:  Direct analysis; Feces; Formic acid; Gas chromatography-mass spectrometry; Short-chain fatty acids

DOI:  https://doi.org/10.1016/j.chroma.2022.462958
Front Mol Biosci. 2022 ;9 829511

Profiling Urinary Sulfate Metabolites With Mass Spectrometry.

Christopher C J Fitzgerald, Rikard Hedman, Dimanthi R Uduwela, Bettina Paszerbovics, Adam J Carroll, Teresa Neeman, Adam Cawley, Lance Brooker, Malcolm D McLeod.

  The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through k-means clustering analysis of sulfate ester derived MS/MS fragmentation intensities. The utility of the method was highlighted in two applications. Firstly, the urinary metabolome of a thoroughbred horse was examined before and after administration of the anabolic androgenic steroid (AAS) testosterone propionate. The analysis detected elevated levels of ten sulfated steroid metabolites, three of which were identified and confirmed by comparison with synthesised reference materials. This included 5α-androstane-3β,17α-diol 3-sulfate, a previously unreported equine metabolite of testosterone propionate. Secondly, the hydrolytic activity of four sulfatase enzymes on pooled human urine was examined. This revealed that Pseudomonas aeruginosa arylsulfatases (PaS) enzymes possessed higher selectivity for the hydrolysis of sulfated metabolites than the commercially available Helix pomatia arylsulfatase (HpS). This novel method provides a rapid tool for the systematic, untargeted metabolic profiling of sulfated metabolites in a urinary matrix.

Keywords:  anti-doping; mass spectrometry; metabolomics; steroid; sulfatase; sulfate ester; sulfation

DOI:  https://doi.org/10.3389/fmolb.2022.829511
ADMET DMPK. 2021 ;9(1): 1-22

A concise review on lipidomics analysis in biological samples.

Ramani Venkata Addepalli, Ramesh Mullangi.

  Lipids are a complex and critical heterogeneous molecular entity, playing an intricate and key role in understanding biological activities and disease processes. Lipidomics aims to quantitatively define the lipid classes, including their molecular species. The analysis of the biological tissues and fluids are challenging due to the extreme sample complexity and occurrence of the molecular species as isomers or isobars. This review documents the overview of lipidomics workflow, beginning from the approaches of sample preparation, various analytical techniques and emphasizing the state-of-the-art mass spectrometry either by shotgun or coupled with liquid chromatography. We have considered the latest ion mobility spectroscopy technologies to deal with the vast number of structural isomers, different imaging techniques. All these techniques have their pitfalls and we have discussed how to circumvent them after reviewing the power of each technique with examples..

Keywords:  LC-MS/MS; Lipids; bioanalysis; samples processing

DOI:  https://doi.org/10.5599/admet.913
Curr Opin Biotechnol. 2022 Mar 09. pii: S0958-1669(22)00028-3. [Epub ahead of print]75 102701

Integrative metabolic flux analysis reveals an indispensable dimension of phenotypes.

Richard C Law, Aliya Lakhani, Samantha O'Keeffe, Sevcan Erşan, Junyoung O Park.

Complete understanding of a biological system requires quantitation of metabolic fluxes that reflect its dynamic state. Various analytical chemistry tools, enzyme-based probes, and microscopy enable flux measurement. However, any method alone falls short of comprehensive flux quantitation. Here we show that integrating these techniques results in a systems-level quantitative map of absolute metabolic fluxes that constitute an indispensable dimension of characterizing phenotypes. Stable isotopes, mass spectrometry, and NMR spectroscopy reveal relative pathway fluxes. Biochemical probes reveal the physical rate of environmental changes. FRET-based and SRS-based microscopy reveal targeted metabolite and chemical bond formation. These techniques are complementary and can be computationally integrated to reveal actionable information on metabolism. Integrative metabolic flux analysis using various quantitative techniques advances biotechnology and medicine.

DOI: https://doi.org/10.1016/j.copbio.2022.102701
Anal Chim Acta. 2022 Apr 08. pii: S0003-2670(22)00191-X. [Epub ahead of print]1201 339620

6-Glycosylaminoquinoline-assisted LDI MS for detection and imaging of small molecules with enhanced detection selectivity and sensitivity.

Ge Ma, Xiaoyong Zhao, Mengzhe Guo, Yaqin Liu, Keqiang Shi, Cheng Guo, Yuanjiang Pan.

  Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a powerful tool in the analysis and imaging of small molecules. However, MALDI MS analysis is easily subjected to poor signal reproducibility and selectivity, especially for complex samples. In this study, a matrix glycosylation strategy was proposed to synthesize glycosylated matrices with excellent performances by enhancing the interaction of the matrix with small molecules. A series of glycosylated matrices including 3-glycosylaminoquinoline (3-GAQ), 6-glycosylaminoquinoline (6-GAQ), and 1-amino-5-glycosylaminoquinoline (GDAN) were synthesized by connecting glucose with the existing amine matrices. Compared with their parent matrices and the existing matrix (1,5-diaminonaphathelene, 1,5-DAN), the glycosylated matrices exhibited remarkably-improved sensitivity, higher signal reproducibility (RSD < 9%) in detecting metabolites, demonstrating the effectiveness of the glycosylation strategy. Among them, 6-GAQ exhibited the best performance. Using 6-GAQ, the detection limit of citric acid reached the low fmol range, and the calibration curve of citric acid had ideal linearity (R2 > 0.99), proving that 6-GAQ was capable of accurate quantitative analysis of metabolites. Furthermore, 6-GAQ was used for the imaging of metabolites in the mouse kidney section, showing higher sensitivity and lower background noise than the commonly-used matrices, 9-aminoquinoline (9-AA), and 1,5-DAN. More importantly, 6-GAQ can selectively detect the hydrophilic metabolites, especially the hydrophilic lipids in the mouse kidney. Overall, 6-GAQ is an ideal matrix potentially applied in the imaging and quantitative analysis of hydrophilic small molecules in complex samples.

Keywords:  Glycosylated matrix; Mass spectrometry imaging (MSI); Matrix-assisted laser desorption/ionization (MALDI); Metabolite detection; Quantitative analysis

DOI:  https://doi.org/10.1016/j.aca.2022.339620
Anal Chem. 2022 Mar 18.

Mass Spectrometry Imaging Techniques Enabling Visualization of Lipid Isomers in Biological Tissues.

Antonín Bednařík, Vadym Prysiazhnyi, Dominika Bezdeková, Jens Soltwisch, Klaus Dreisewerd, Jan Preisler.

This Feature focuses on a review of recent developments in mass spectrometry imaging (MSI) of lipid isomers in biological tissues. The tandem MS techniques utilizing online and offline chemical derivatization procedures, ion activation techniques such as ozone-induced dissociation (OzID), ultraviolet photodissociation (UVPD), or electron-induced dissociation (EID), and other techniques such as coupling of ion mobility with MSI are discussed. The importance of resolving lipid isomers in diseases is highlighted.

DOI: https://doi.org/10.1021/acs.analchem.1c05108
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Mar 07. pii: S1570-0232(22)00102-7. [Epub ahead of print]1196 123198

Rapid determination of hydrogen sulfide-related metabolites in human urine by UHPLC-ESI-MS/MS with fluorinated ion-pairing reagents.

Bassam Lajin, Barbara Obermayer-Pietsch, Walter Goessler.

  Although primarily considered as a toxic gas, hydrogen sulfide is naturally produced in humans from the amino acid cysteine in low quantities and is commonly referred to as the "third gaseous signaling molecule". There is a need to establish reliable biomarkers for exposure to hydrogen sulfide in human matrices not only at toxic levels but also at trace levels. Herein, we report optimized analytical methods for the direct determination of three compounds related to hydrogen sulfide metabolism in human urine without sample preparation; namely, trimethylsulfonium, thiosulfate, and cystine. The methods were based on ion-pair ultra-high performance liquid chromatography (UHPLC) with an anionic fluorinated acid or a cationic fluoroalkylamine, a member of a new generation of ion-pairing reagents, and detection by the electrospray ionization tandem mass spectrometry (ESI-MS/MS). Rapid chromatographic separation was achieved in 2-3 min with limits of detection of 0.1, 2.5, and 50 nmol L-1 (nM) for trimethylsulfonium, cystine, and thiosulfate, respectively. Isotopically labeled internal standards were used for each analyte to account for matrix effects and qualifier ions were employed to ensure selectivity. The method was validated for recovery (generally within 80-120%) and repeatability (RSD% 1.0-10%), and applied to investigate the normal concentrations of the three analytes in forty morning first-pass urine samples of healthy volunteers.

Keywords:  (U)HPLC; Hydrogen sulfide metabolism; Mass spectrometry; Microbiome; Third gaseous signaling molecule

DOI:  https://doi.org/10.1016/j.jchromb.2022.123198
J Chromatogr A. 2022 Feb 21. pii: S0021-9673(22)00114-5. [Epub ahead of print]1669 462916

Determination of 22 alternative plasticizers in wrap film by solid phase extraction and ultra-high performance supercritical fluid chromatography-tandem mass spectrometry.

Pei-Xia He, Yun Ling, Wei Yong, Mei-Yi Yao, Yu-Jia Zhang, Xue-Song Feng, Yuan Zhang, Feng Zhang.

  A rapid, accurate and novel analytical method based on ultra-high performance supercritical fluid chromatography-tandem mass spectrometry for determination of 22 alternative plasticizers in wrap film was developed. Instrumental analysis and sample preparation procedures were systematically optimized. The targets were separated on Torus 1-AA column (100 mm × 3 mm, 1.7 µm). Mobile phase A was supercritical carbon dioxide, and mobile phase B was ethanol/methanol (7:3, v/v) containing 0.1% formic acid and 0.5 mM ammonium acetate. Gradient elution was performed. The analytes were extracted by 10 mL n-hexane/dichloromethane (1:1, v/v), and further purified on silica solid phase extraction cartridges. The analytes were quantified by ultra-high performance supercritical fluid chromatography-tandem mass spectrometry with electrospray ionization source, and detection was performed on multiple reaction monitoring mode. Two commercially available isotopically-labelled internal standards were used for quantification calibration, and analytes were divided into two groups according to the more appropriate internal standards (chemistry similarity, closeness of retention time). Method validation was performed in terms of recovery, repeatability, linearity, sensitivity and matrix effect. Linearity was assessed using matrix-matched standard calibration. Satisfactory linearity (r2 ≥ 0.995), intra-day precision (RSDs ≤ 9.6%), inter-day precision (RSDs ≤ 10.9%), recovery (75.6-124.5%) as well as good selectivity was observed. The limits of detection were 0.04-10 µg/kg, while the limits of quantification were 1.0-50 µg/kg. Most targets did not show significant matrix effect. Validation results verified that the proposed method was efficient, rapid and sensitive. Eventually it was successfully applied to food wrap film analysis, and results indicated that DEHA, ATBC, DBA and TnBP were the most frequently detected plasticizers in wrap film samples,which was worthy of attention.

Keywords:  Alternative plasticizers; Solid phase extraction; Ultra-high performance supercritical fluid chromatography-tandem mass spectrometry; Wrap film

DOI:  https://doi.org/10.1016/j.chroma.2022.462916
Ther Drug Monit. 2022 Apr 01. 44(2): 357

An Liquid Chromatography-Tandem Mass Spectrometry Method for the Simultaneous Determination of Afatinib, Alectinib, Ceritinib, Crizotinib, Dacomitinib, Erlotinib, Gefitinib, and Osimertinib in Human Serum: Erratum.

DOI: https://doi.org/10.1097/FTD.0000000000000958
Mass Spectrom (Tokyo). 2022 ;11(1): A0102

Mass Spectrometry Imaging.

Shuichi Shimma.

  Mass spectrometry imaging (MSI) is a technique for obtaining information on the distribution of various molecules by performing mass spectrometry directly on the sample surface. The applications range from small molecules such as lipids to large molecules such as proteins. It is also possible to detect pharmaceuticals and elemental isotopes in interstellar matter. This review will introduce various applications of MSI with examples.

Keywords:  instruments; isotopes; lipids; mass spectrometry imaging; pharmaceuticals; proteins

DOI:  https://doi.org/10.5702/massspectrometry.A0102
J Appl Lab Med. 2022 Mar 17. pii: jfac010. [Epub ahead of print]

Clinical Utility and Analytical Aspects of Direct Measurements of Free Hormones Using Mass Spectrometry-Based Methods.

Mark M Kushnir, Heather A Nelson, Kelly Doyle.

   BACKGROUND: The free hormone (FH) hypothesis states that hormone action and the corresponding biological effects are mediated by the unbound (free) fraction of hormone in circulation. The in vivo relationship between protein-bound and FH is complex and dynamic. In most individuals, measurement of total hormone (TH) is usually adequate to reflect the hormone status; however, certain physiological conditions and/or medications can affect protein binding and alter FH concentration. In these cases, measurement of FH will provide a better measure of the bioactive hormone status than measurement of the TH. Measurement of FH presents many challenges, as the concentrations are very low and there are number of pitfalls, which may affect the measured concentrations.
CONTENT: In this review, we discuss techniques used in the separation and direct quantitation of FH concentrations in biological samples using mass spectrometry for analysis. We also highlight clinical situations in which FH analysis is warranted and when mass spectrometry should be the preferred methodology over immunoassays.
SUMMARY: Equilibrium dialysis, ultrafiltration, or size-exclusion separation coupled with liquid chromatography-tandem mass spectrometry provides a sensitive and specific method to measure FH concentrations. These direct methods are useful in iatrogenic or physiological states that alter hormone binding or metabolism.

Keywords:  laboratory methods and tools; mass spectrometry; steroids and steroid hormones; thyroid hormones

DOI:  https://doi.org/10.1093/jalm/jfac010
Food Chem. 2022 Mar 09. pii: S0308-8146(22)00634-3. [Epub ahead of print]385 132672

Vitamin K (phylloquinone and menaquinones) in foods - Cost-effective quantification by LC-ESI-MS/MS.

Marie Bagge Jensen, Kari Elin Rød, Petra Ložnjak Švarc, Eystein Oveland, Jette Jakobsen.

  Further research on vitamin K is necessary as growing evidence of vitamin K's importance in human health beyond blood coagulation and bone health is emerging. We present a cost-effective LC-ESI-MS/MS method for quantification of phylloquinone (PK), and menaquinones (MK) 4-10 in food using deuterium labelled (d7) compounds (d7-PK, d7-MK-4, d7-MK-7 and d7-MK-9) as internal standards. The validation of the method included assessment of matrix effect, limit of quantification (LOQ), precision, and trueness. The LC-ESI-MS/MS method runtime is 9 min. The method was compared to a validated LC-FLD method (CEN 14148), for quantification of vitamin K in broccoli, cheese, natto, liver, and microalgae. LOQs of the LC-ESI-MS/MS method were ≤4 µg/100 g food. The intra- and inter-assay precision was <15% for PK, MK-4, MK-7 and MK-9; <20% for MK-5, MK-8, and MK-10, and ≤25% for MK-6. No significant differences between the quantified content by the LC-ESI-MS/MS and LC-FLD methods were observed.

Keywords:  Internal standards; LC-ESI-MS/MS; Matrix effect; Method comparison; Trueness; Vitamin K

DOI:  https://doi.org/10.1016/j.foodchem.2022.132672
Anal Chim Acta. 2022 Apr 08. pii: S0003-2670(22)00193-3. [Epub ahead of print]1201 339622

Non-target analysis and stability assessment of reference materials using liquid chromatography‒high-resolution mass spectrometry.

Elliott J Wright, Daniel G Beach, Pearse McCarron.

  Development and characterization of biological and environmental matrix certified reference materials (CRMs) for organic analytes typically relies heavily on targeted analytical methods, such as liquid chromatography (LC) with triple-quadrupole mass spectrometry detection. LC with high-resolution mass spectrometry (LC‒HRMS) can also provide high quality data for both targeted and non-targeted analytes, with the potential for retrospective data analysis. Here, we demonstrate the utility of non-target analysis (NTA) using LC‒HRMS for profiling and stability assessment of a mussel tissue matrix CRM certified for several classes of marine algal toxins (CRM-FDMT1). First, the NTA method was developed using data-dependent MS/MS acquisition and commercial metabolomics software for data processing. Of 128 toxin analogues previously reported in CRM-FDMT1, 125 were detected by LC-HRMS, with 97 triggered for MS/MS by data dependant acquisition. Automated data processing detected 119 of these compounds and 109 were retained after automated filtering of results for putative toxin analogues. Those analogues not detected were low abundance ions, or poorly resolved isomers. The method was then used to demonstrate new strategies for CRM stability assessment considering the stability of certified analytes, related toxin analogues, and unrelated matrix compounds. Several analogues from each toxin class in CRM-FDMT1 as well as other unrelated matrix compounds were observed to be significantly less stable than the certified toxins. Using this method, no instability was measured for any compounds at conditions ≤4 °C, providing a greater degree of confidence in CRM stability than could be achieved using conventional approaches to stability assessment targeting only the certified analytes. The NTA method and stability assessment approach presented are applicable to future CRM development with other matrices and organic analyte classes.

Keywords:  Algal toxin; Compound Discoverer; Data-dependent analysis; Metrology; Phycotoxin; Stability

DOI:  https://doi.org/10.1016/j.aca.2022.339622
Anal Biochem. 2022 Mar 10. pii: S0003-2697(22)00092-6. [Epub ahead of print] 114636

LC-MS/MS method for quantifying aescinate A and B and assessing their relationship with phlebitis.

Cailian Fei, Yuehao Han, Zhipeng Wang, Dongyuan Lu, Hanglin Li, Wansheng Chen, Shouhong Gao, You Yin.

  The purpose of this study is to establish and validate a sensitive, robust and rapid liquid chromatography-tandem mass spectrometry method for quantifying the aescinate A and aescinate B in human plasma and assessing the association of phlebitis and aescinate A and aescinate B in vivo exposure. The chromatographic separation was completed on Agilent ZORBAX SB-C18 (2.1 mm × 100 mm, 3.5 μm, Agilent, USA) column with isocratic elution. The flow rate was 0.3 mL/min and the total run time was optimized within 5 min. The protein precipitation was applied to pretreat plasma sample using methanol as precipitant. The data acquisition was achieved with positive electrospray ionization in multi-reaction monitoring mode for both aescinate A and aescinate B. The calibration range of aescinate A and aescinate B are constructed in 100-2000 ng/mL, and their correlation coefficients are both >0.990. The intra-day and inter-day precision and accuracy of this method are less than 9.04% and within -13.75% and -0.93%. This analytical method has been successfully applied for the determination of plasma aescinate A and aescinate B concentrations in patients with cerebral infarction, and the results showed that the incidence and grade of phlebitis were not associated with the in vivo exposure of aescinate A and aescinate B.

Keywords:  Aescinate A; Aescinate B; Liquid chromatography-tandem mass spectrometry; Phlebitis; Plasma

DOI:  https://doi.org/10.1016/j.ab.2022.114636
Anal Biochem. 2022 Mar 14. pii: S0003-2697(22)00120-8. [Epub ahead of print] 114664

A novel, rapid and simple UHPLC-MS/MS method for quantification of warfarin in dried blood spots.

Wenhao Qu, Xianglian Li, Guidi Tian, Linsheng Liu, Liyan Miao.

  Warfarin is a common first line anticoagulant with a narrow therapeutic window. Because of the large blood volume needed, previous warfarin determination methods were not applicable to small animals, such as mice. To reduce the number of small animals used needed, we developed and validated a sensitive rapid assay for the simultaneous detection of warfarin enantiomers in mouse dried blood spot (DBS) samples. Analytes were extracted by tert-butyl methyl ether and then separated by a chiral Cellulose-1 column with a mobile phase of 75% acetonitrile (containing 0.1% formic acid). The total chromatographic run time was 3 min. Negative mode electrospray ionization was used for MS/MS detection, where the monitored ion transitions were m/z 307.1 → 161.0 and 341.1 → 284.0 for warfarin and coumachlor (internal standard) respectively. The calibration curves were linear with a correlation coefficient of ≥0.994 for both enantiomers over a concentration range of 10-1000 ng/mL. The satisfactory accuracy and adequate reproducibility of both warfarin enantiomers were validated in terms of intra- and interday precision with mouse DBS cards. The samples were stable at room temperature for at least 14 days. The validated method was applied to a pharmacokinetic study in mice.

Keywords:  Dried blood spot; LC-MS/MS; Pharmacokinetics; Warfarin

DOI:  https://doi.org/10.1016/j.ab.2022.114664
Appl Environ Microbiol. 2022 Mar 14. e0241621

Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

Mamoru Oshiki, Komei Nagai, Satoshi Ishii, Yoshiyuki Suzuki, Nobuo Saito, Takashi Yamaguchi, Nobuo Araki, Satoshi Okabe.

  In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15N atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO2-, indophenol for NH4+, and p-aminobenzaldehyde for N2H4), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH2OH and NO3- were chemically converted to NO2- by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15N-labeled compound, and a linear relationship was found between the determined 15N/14N peak ratio and 15N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO2- oxidation and NO3- reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE 15N/14N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N atom% of NO2- can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.

Keywords:  15N atom%; MALDI-TOF MS; colorimetric reagent; mass spectrometry; nitrogen cycle; nitrogen isotope

DOI:  https://doi.org/10.1128/aem.02416-21
Ther Drug Monit. 2022 Apr 01. 44(2): 345-350

Simultaneous Quantification of 11 Antiseizure Medications and Metabolites in Serum for Therapeutic Drug Monitoring Using High-Performance Liquid Chromatography with Ultraviolet Detection: A Short Communication.

Yannick Lipecki, Ekkehard Haen.

BACKGROUND: Therapeutic drug monitoring (TDM) of antiseizure medications (ASMs) is widely used to guide therapy, avoid toxicity, and assess patient compliance. Commercial immunologic quantification methods are common practice; however, as they are only applicable to one specific drug and prone to cross-reacting metabolites, their practical applicability is limited. In this article, the authors proposed a high-performance liquid chromatography method using ultraviolet detection (HPLC-UV) for simultaneous quantification of 11 ASMs and active metabolites (carbamazepine, felbamate, lacosamide, lamotrigine, levetiracetam, phenobarbital, phenytoin, primidone, zonisamide, carbamazepine-10,11-epoxide, and licarbazepine) in serum.
METHODS: Chromatographic separation was performed on a Phenomenex Luna PFP(2) (3-µm particle size; 150 × 4.6 mm i.d.) analytical column. The mobile phase comprised phosphate buffer (20 mM; pH 3), acetonitrile (ACN), and methanol using gradient elution. Analyses were conducted at 35°C and a 1.3-mL/min flow rate. The detection wavelength for all analytes was 210 nm. The samples were prepared by protein precipitation using ACN.
RESULTS: The HPLC-UV method was validated according to the FDA guidelines and applied to measure patient samples in TDM. Calibration curves showed excellent linearity (r2 > 0.99) and covered the entire reference range for each analyte. Intraday and interday imprecisions and inaccuracies were <10% for all samples. Extensive stability testing showed no significant degradation (<15%), and interference measurements additionally ensured clinical applicability. Furthermore, the sensitivity was comparable with that of previously published HPLC methods using mass spectrometry.
CONCLUSIONS: The authors developed an HPLC-UV method for the simultaneous quantification of 11 ASMs in the human serum and demonstrated its practical applicability in TDM. The method requires only standard laboratory equipment and simple sample preparation, making TDM available in less specialized laboratories.

DOI: https://doi.org/10.1097/FTD.0000000000000908
J Agric Food Chem. 2022 Mar 17.

A Novel Approach for Monitoring the Volatile Metabolome in Biological Samples from Ruminants through Miniaturized Liquid-Liquid Extraction and Multiclass Gas Chromatography Analysis.

Liliana Cordeiro, Ana R J Cabrita, Hugo M Oliveira, Margarida R G Maia, José A Rodrigues, António J M Fonseca, Inês M Valente.

  A straightforward and versatile methodology for the extraction of volatile metabolites in biological samples from ruminants for gas chromatography analysis is proposed. The methodology was applied in the determination of multiclass metabolites (short-chain fatty acids, aldehydes, alcohols, ketones, esters, phenols, and sulfides) in different analytical matrices (rumen fluid, urine, and feces) collected from Holstein cows. The 24 multiclass volatile metabolites reported in the different biological samples and their respective concentrations were critically discussed in the context of digestive physiology. Most detected compounds are derived from the rumen and lower gut fermentation of carbohydrates, proteins, and lipids or their metabolism, being consistent with the prior state of the art. The proposed method also takes advantage of the already existing tools in animal nutrition laboratories, providing a novel methodological ground that can generate relevant bioanalytical information with a significant impact on ruminant's nutritional studies.

Keywords:  ruminant animals; salt-assisted liquid−liquid extraction; volatile compounds

DOI:  https://doi.org/10.1021/acs.jafc.1c06662
Anal Chem. 2022 Mar 15.

Ultra-High-Throughput Ambient MS: Direct Analysis at 22 Samples per Second by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry.

Andrew J Radosevich, Fan Pu, David Chang-Yen, James W Sawicki, Nari N Talaty, Nathaniel L Elsen, Jon D Williams, Jeffrey Y Pan.

Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry is an ambient-direct sampling method that is being developed for high-throughput, label-free, biochemical screening of large-scale compound libraries. Here, we report the development of an ultra-high-throughput continuous motion IR-MALDESI sampling approach capable of acquiring data at rates up to 22.7 samples per second in a 384-well microtiter plate. At top speed, less than 1% analyte carryover is observed from well-to-well, and signal intensity relative standard deviations (RSD) of 11.5% and 20.9% for 3 μM 1-hydroxymidazolam and 12 μM dextrorphan, respectively, are achieved. The ability to perform parallel kinetics studies on 384 samples with a ∼30 s time resolution using an isocitrate dehydrogenase 1 (IDH1) enzyme assay is shown. Finally, we demonstrate the repeatability and throughput of our approach by measuring 115200 samples from 300 microtiter plate reads consecutively over 5.54 h with RSDs under 8.14% for each freshly introduced plate. Taken together, these results demonstrate the use of IR-MALDESI at sample acquisition rates that surpass other currently reported direct sampling mass spectrometry approaches used for high-throughput compound screening.

DOI: https://doi.org/10.1021/acs.analchem.1c04605
Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2022 Mar 18. 1-9

Simultaneous determination of the four key fluoroquinolones and two antipsychotics in fish and shrimp by LC-MS/MS.

Keyi Shen, Xiaoshuang Zou, Jun Wang.

  In this study, we developed and validated a liquid chromatography triple quadrupole tandem mass spectrometry (LC-MS/MS) method used to simultaneously determine levels of four fluoroquinolones (ofloxacin, norfloxacin, lomefloxacin, and pefloxacin) and two antipsychotics (diazepam and methaqualone) in fish and shrimp. The samples were extracted with a mixture of anhydrous sodium sulfate and acetonitrile, and purified by C18 cartridge solid-phase extraction with an optimized eluent. The MS2 method was applied to recognize the molecular structure of these compounds according to a main fragmentation scheme. The key ions of identification and quantification were deduced from chemical structures. Multiple reaction monitoring was used to quantitatively analyse the compounds of interest. Satisfactory linearities were obtained (R2 ≥0.99) with the limits of quantitation (LOQs) ranging between 0.03 and 1.96 μg kg-1. The recoveries were 74-122%, with a relative standard deviation (RSD) below 4.9% for these compounds at the spiking level of three, five, and ten times the LODs, respectively. The LC-MS/MS method allows precise and sensitive determination of residues of six important banned veterinary drugs in fish and shrimp tissue. This methodological approach solved the problem imposed by the need for two or more analysis methods to analyse the compounds of interest described in this study.

Keywords:  Veterinary drug residues; aquatic products; fragmentation pathways; multiple reaction monitoring; quantitative analysis

DOI:  https://doi.org/10.1080/19440049.2022.2032381
Anal Chim Acta. 2022 Apr 08. pii: S0003-2670(22)00190-8. [Epub ahead of print]1201 339619

Coupling paper spray ionization with the flexible microtube plasma for the determination of low polar biomarkers in mass spectrometry.

Daniel Foest, Alexander Knodel, Sebastian Brandt, Joachim Franzke.

  A fast and precise analysis of complex biological samples is one of the most important challenges in clinical- and life-sciences. In this field, the paper spray ionization (PSI) becomes a more and more successful ambient ionization technique for mass spectrometry. The PSI is based on the electrospray mechanism and is limited to polar target analytes. In this work, a transition from the paper spray ionization to a corona discharge under standard PSI conditions is observed and evaluated by using a complex liver sample. This evaluation leads to an advancement of the PSI by adding a flexible microtube plasma (FμTP) that is more efficient in respect to non- and low polar molecules. The combination of the PSI and the FμTP in a sequential way allows the determination of polar lipids as well as non-polar compounds like cholesterol and possible lung cancer biomarkers. As add-on for PSI, this approach enhances the number of detectable species in one single measurement and seems to be a powerful tool for the rapid analysis of complex biological samples in clinical- and life-sciences.

Keywords:  Biomarker; Cholesterol; Flexible microtube plasma; Paper spray chemical ionization; Paper spray ionization

DOI:  https://doi.org/10.1016/j.aca.2022.339619