bims-metlip 2024-02-04 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–02–04
twenty-one papers selected by
Sofia Costa, Matterworks

In-vivo tracking of deuterium metabolism in mouse organs using LC-MS/MS.
A LC-MS/MS method for the simultaneous quantitative determination of aldosterone, its precursor 18-hydroxycorticosterone and its metabolite tetrahydroaldosterone in human urine.
Analysis of tryptophan metabolites and related compounds in human and murine tissue: development and validation of a quantitative and semi-quantitative method using high resolution mass spectrometry.
Developing, validating, and comparing an analytical method to simultaneously detect z-drugs in urine samples using the QuEChERS approach with both liquid chromatography and gas chromatography-tandem mass spectrometry.
Quantification of prohibited substances and endogenous corticosteroids in saliva using traditional, alternative microextraction-based, and novel 3D printed sample-preparation methods coupled with LC-MS.
Pretreating and normalizing metabolomics data for statistical analysis.
Quantitative mass spectrometry imaging: therapeutics & biomolecules.
Development, Validation, and Clinical Application of an Ultra-High-Performance Liquid Chromatography Coupled With Tandem Mass Spectrometry Method for the Determination of 10 Antituberculosis Drugs in Human Serum.
Structural elucidation of derivatives of polyfunctional metabolites after methyl chloroformate derivatization by high-resolution mass spectrometry gas chromatography. Application to microbiota metabolites.
Screening and Confirmation of Psilocin, Mitragynine, Phencyclidine, Ketamine, and Ketamine Metabolites by Liquid Chromatography Tandem Mass Spectrometry.
Ultrahigh-Performance Supercritical Fluid Chromatography-Multimodal Ionization-Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts.
Untargeted metabolomics for lifestyle biomarker discovery in human hair.
Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.
Hollow-fiber liquid phase microextraction for determination of fluoxetine in human serum by nano-liquid chromatography coupled to high resolution mass spectrometry.
Method development for the quantification of nine nitazene analogs and brorphine in Dried Blood Spots utilizing liquid chromatography - tandem mass spectrometry.
PyMIDA: A Graphical User Interface for Mass Isotopomer Distribution Analysis.
Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.
Quantifying Gut Microbial Short-Chain Fatty Acids and Their Isotopomers in Mechanistic Studies Using a Rapid, Readily Expandable LC-MS Platform.
Development of chromatographic methods to determine multivitamins formulation depending on their solubility and polarity: comparative study using three greenness assessment tools.
Improved method robustness and ruggedness in liquid chromatography-mass spectrometry by increasing the acid content of the mobile phase.
Perspective on SPME-MS: Green and high-performance methods for rapid screening.

J Chromatogr A. 2024 Jan 28. pii: S0021-9673(24)00064-5. [Epub ahead of print]1717 464691

In-vivo tracking of deuterium metabolism in mouse organs using LC-MS/MS.

Siva Swapna Kasarla, Vera Flocke, Nay Min Thaw Saw, Antonia Fecke, Albert Sickmann, Matthias Gunzer, Ulrich Flögel, Prasad Phapale.

  Mass spectrometry-based metabolomics with stable isotope labeling (SIL) is an established tool for sensitive and precise analyses of tissue metabolism, its flux, and pathway activities in diverse models of physiology and disease. Despite the simplicity and broad applicability of deuterium (2H)-labeled precursors for tracing metabolic pathways with minimal biological perturbations, they are rarely employed in LC-MS/MS-guided metabolomics. In this study, we have developed a LC-MS/MS-guided workflow to trace deuterium metabolism in mouse organs following 2H7 -glucose infusion. The workflow includes isotopically labeled glucose infusion, mouse organ isolation and metabolite extraction, zwitterion-based hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry, targeted data acquisition for sensitive detection of deuterated metabolites, a spectral library of over 400 metabolite standards, and multivariate data analysis with pathway mapping. The optimized method was validated for matrix effects, normalization, and quantification to provide both tissue metabolomics and tracking the in-vivo metabolic fate of deuterated glucose through key metabolic pathways. We quantified more than 100 metabolites in five major mouse organ tissues (liver, kidney, brain, brown adipose tissue, and heart). Furthermore, we mapped isotopologues of deuterated metabolites from glycolysis, tricarboxylic acid (TCA) cycle, and amino acid pathways, which are significant for studying both health and various diseases. This study will open new avenues in LC-MS based analysis of 2H-labeled tissue metabolism research in animal models and clinical settings.

Keywords:  Deuterium tracing; LC-MS; Metabolic flux; Metabolomics; Tissue metabolism

DOI:  https://doi.org/10.1016/j.chroma.2024.464691
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jan 14. pii: S1570-0232(24)00020-5. [Epub ahead of print]1234 124012

A LC-MS/MS method for the simultaneous quantitative determination of aldosterone, its precursor 18-hydroxycorticosterone and its metabolite tetrahydroaldosterone in human urine.

Haohang Dong, Bin Hu, Wei Chen, Liping Wang, Huiying Sun, Jianfeng Zhou, Jianchun Chen, Baorong Chen, Zhenxia Du.

  Aldosterone (ALD), its precursor 18-hydroxycorticosterone (18-OHB) and its metabolite tetrahydroaldosterone (TH-ALD) are important biomarkers for the diagnosis of primary aldosteronism (PA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized in the detection of small molecules of hormones because it has advantages in terms of specificity and sensitivity. The objective of this study is to develop a new LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB, and TH-ALD in human urine and attempt to diagnose primary aldosteronism using different indicators. The urine samples were treated with a solid-phase extraction pretreatment technique and the three analytes were separated on a reversed-phase column and detected on a triple quadrupole mass spectrometer. The established method was validated according to CLSI C62-A standard guidelines. The calibration ranges from 25 pg/mL to 5000 pg/mL for aldosterone (free), 18-hydroxycorticosterone and tetrahydroaldosterone, and the lower limit of quantification for these three analytes was 25 pg/mL. The matrix effects and recoveries of these three analytes ranged from 85.1 % to 115 % and from 86.3 % to 114 %, respectively. The intra-day and inter-day precision ranged from 1.29 % to 6.78 % and from 1.77 % to 8.64 %, respectively. The performance of the method met the requirements of the guidelines. 40 clinical urine samples including 22 PA patients and 18 non-PA patients were detected, and the ROC curves of three diagnostic indicators were established. The area under the curve (AUC) of ALD (free) is the biggest, so ALD (free) was the best compound to be used as a diagnostic indicator in this study. When the cut-off point was taken as 141 ng/24-h, the sensitivity was 72.7 % and the specificity was 88.9 %. We developed and validated an LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB and TH-ALD in human urine. Our study provides a reference for the use of new biomarkers for the diagnosis of primary aldosteronism.

Keywords:  18-hydroxycorticosterone; Aldosterone (free); Liquid chromatography-tandem mass spectrometry; Primary aldosteronism; Tetrahydroaldosterone

DOI:  https://doi.org/10.1016/j.jchromb.2024.124012
Anal Methods. 2024 Jan 29.

Analysis of tryptophan metabolites and related compounds in human and murine tissue: development and validation of a quantitative and semi-quantitative method using high resolution mass spectrometry.

Sandy Abujrais, S J Kumari A Ubhayasekera, Jonas Bergquist.

This study explores the metabolic differences between human and murine plasma in addition to differences between murine subcutaneous and visceral white adipose tissue. A quantitative and semi-quantitative targeted method was developed and validated for this purpose. The quantitative method includes tryptophan and its metabolites in addition to tyrosine, phenylalanine, taurine, B vitamins, neopterin, cystathionine and hypoxanthine. While the semi-quantitative method includes; 3-indoleacetic acid, 5-hydroxyindoleacetic acid, acetylcholine, asymmetric dimethylarginine, citrulline and methionine. Sample preparation was based on protein precipitation, while quantification was conducted using ultrahigh-performance liquid chromatography coupled to a quadrupole Orbitrap tandem mass spectrometer with electrospray ionization in the parallel reaction monitoring (PRM) mode. The low limit of quantification for all metabolites ranged from 1 to 200 ng mL-1. Matrix effects and recoveries for stable isotope labelled internal standards were evaluated, with most having a coefficient of variation (CV) of less than 15%. Results showed that a majority of the analytes passed both the intra- and interday precision and accuracy criteria. The comparative analysis of human and murine plasma metabolites reveals species-specific variations within the tryptophan metabolic pathway. Notably, murine plasma generally exhibits elevated concentrations of most compounds in this pathway, with the exceptions of kynurenine and quinolinic acid. Moreover, the investigation uncovers noteworthy metabolic disparities between murine visceral and subcutaneous white adipose tissues, with the subcutaneous tissue demonstrating significantly higher concentrations of tryptophan, phenylalanine, tyrosine, and serotonin. The findings also show that even a semi-quantitative method can provide comparable results to quantitative methods from other studies and be effective for assessing metabolites in a complex sample. Overall, this study provides a robust platform to compare human and murine metabolism, providing a valuable insight to future investigations.

DOI: https://doi.org/10.1039/d3ay01959d
Saudi Pharm J. 2024 Feb;32(2): 101950

Developing, validating, and comparing an analytical method to simultaneously detect z-drugs in urine samples using the QuEChERS approach with both liquid chromatography and gas chromatography-tandem mass spectrometry.

Varsha Chauhan, Manu Sharma, Abhishek Tiwari, Varsha Tiwari, Manish Kumar, Ajay Sharma, Arya Lakshmi Marisetti, Ashok Kumar, Abdulsalam Alhalmi, Omar M Noman, Ahmad Alahdab.

  Detecting z-drugs, a sedative-hypnotic medication, is also misused for criminal activities. Therefore, the analysis of urine samples is crucial for clinical and forensic purposes. We conducted a study where we developed, validated, and compared an analytical method for simultaneously detecting z-drugs in urine samples. Our approach uses the QuEChERS method for sample preparation, combined with liquid chromatography (LC) and gas chromatography (GC) coupled with tandem mass spectrometry (MS/MS). We optimized the QuEChERS method to effectively extract z-drugs from urine samples while minimizing matrix effects and achieving high recovery rates. After extraction, we split the samples into two parts for analysis using LC-MS/MS and GC-MS/MS. We validated our methods, and the results showed good linearity over a broad concentration range (1-200 ng/mL) for each z-drug. The limits of detection and quantification were within clinically relevant ranges, ensuring sensitivity for detecting z-drugs in urine samples. We compared the two chromatographic techniques by analyzing a set of urine samples spiked with known concentrations of z-drugs using both LC-MS/MS and GC-MS/MS methods and then applied to the real samples. The results were statistically analyzed to assess any significant differences in accuracy and precision above 95 %, and both methods offered reliable and consistent results with the samples as well. In conclusion, our analytical method coupled with both LC-MS/MS and GC-MS/MS using the QuEChERS approach provides a comprehensive and robust solution for the simultaneous detection of z-drugs in urine samples. The choice between the two chromatographic techniques can be based on the specific z-drugs of interest and the required analytical performance. This method holds promise for applications in clinical toxicology, forensic analysis, and monitoring z-drug usage.

Keywords:  Eszopiclone; GC–MS/MS; LC−MS/MS; QuEChERS; Zolpidem; Zopiclone

DOI:  https://doi.org/10.1016/j.jsps.2023.101950
Anal Chim Acta. 2024 Feb 22. pii: S0003-2670(24)00037-0. [Epub ahead of print]1291 342236

Quantification of prohibited substances and endogenous corticosteroids in saliva using traditional, alternative microextraction-based, and novel 3D printed sample-preparation methods coupled with LC-MS.

Krzysztof Goryński, Łukasz Sobczak.

  Oral fluid has gained significant interest as an alternative matrix for drug testing due to its easy and non-invasive collection. Despite these advantages, achieving suitably low limits of detection remains a clear challenge in the use of oral fluids for drug screening. In this study, we demonstrate that the application of commercially available SPME fibers followed by liquid chromatography tandem mass spectrometry can enable the comprehensive detection and confirmation of drugs in oral fluid samples. To this end, we develop and test a sample-preparation protocol for a panel of 46 drugs covering the most popular drugs of abuse and doping agents available worldwide. Human saliva samples were collected using a Salivette® device (CE IVD certified) and sampled using SPME devices coated with a C18 extraction phase. The proposed protocol was validated with respect to its lower limits of quantification (LLOQ), linearity, matrix effects, precision, and extraction recovery. Linearity was confirmed for all compounds (R2 > 0.97), except for testosterone (R2 = 0.953) and metandrostenolon (R2 = 0.958). Furthermore, 4 compounds suffered from matrix effects, with less than 10 % deviation from acceptance criteria. After analytical validation, saliva samples from volunteers were analyzed to determine free concentrations of cortisol at different times after awaking. Finally, a 3D-printed prototype device was designed and successfully applied to extract small molecules, thus demonstrating a new modern low-cost approach for bioanalysis.

Keywords:  3D printing; Doping substances; Drugs; Polyamide non-coated device for adsorption-based microextraction; Saliva; Solid-phase microextraction

DOI:  https://doi.org/10.1016/j.aca.2024.342236
Genes Dis. 2024 May;11(3): 100979

Pretreating and normalizing metabolomics data for statistical analysis.

Jun Sun, Yinglin Xia.

  Metabolomics as a research field and a set of techniques is to study the entire small molecules in biological samples. Metabolomics is emerging as a powerful tool generally for precision medicine. Particularly, integration of microbiome and metabolome has revealed the mechanism and functionality of microbiome in human health and disease. However, metabolomics data are very complicated. Preprocessing/pretreating and normalizing procedures on metabolomics data are usually required before statistical analysis. In this review article, we comprehensively review various methods that are used to preprocess and pretreat metabolomics data, including MS-based data and NMR -based data preprocessing, dealing with zero and/or missing values and detecting outliers, data normalization, data centering and scaling, data transformation. We discuss the advantages and limitations of each method. The choice for a suitable preprocessing method is determined by the biological hypothesis, the characteristics of the data set, and the selected statistical data analysis method. We then provide the perspective of their applications in the microbiome and metabolome research.

Keywords:  Data centering and scaling; Data normalization; Data transformation; MS-Based data preprocessing; Missing values; NMR Data preprocessing; Outliers; Preprocessing/pretreatment

DOI:  https://doi.org/10.1016/j.gendis.2023.04.018
Chem Commun (Camb). 2024 Jan 29.

Quantitative mass spectrometry imaging: therapeutics & biomolecules.

Joseph H Holbrook, Gabrielle E Kemper, Amanda B Hummon.

Mass spectrometry imaging (MSI) has become increasingly utilized in the analysis of biological molecules. MSI grants the ability to spatially map thousands of molecules within one experimental run in a label-free manner. While MSI is considered by most to be a qualitative method, recent advancements in instrumentation, sample preparation, and development of standards has made quantitative MSI (qMSI) more common. In this feature article, we present a tailored review of recent advancements in qMSI of therapeutics and biomolecules such as lipids and peptides/proteins. We also provide detailed experimental considerations for conducting qMSI studies on biological samples, aiming to advance the methodology.

DOI: https://doi.org/10.1039/d3cc05988j
Ther Drug Monit. 2024 Jan 24.

Development, Validation, and Clinical Application of an Ultra-High-Performance Liquid Chromatography Coupled With Tandem Mass Spectrometry Method for the Determination of 10 Antituberculosis Drugs in Human Serum.

Xudong Fan, Suhang Guo, Ruoying Zhang, Qingshan Cai, Yazhen Lang, Jinpeng Huang, Yuanyuan Chen, Ying Zhang, Yingying Xu, Meng Chen, Gaoyi Yang, Xinjun Cai.

INTRODUCTION: Linezolid, moxifloxacin, rifapentine, rifabutin, cycloserine, clofazimine, bedaquiline, levofloxacin, prothionamide, and ethionamide are commonly used second-line antituberculosis (anti-TB) drugs. To support therapeutic drug monitoring in regular clinical practice, the authors sought to develop a method based on ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) that would allow for the simultaneous quantification of multiple second-line anti-TB drugs in human serum.
METHODS: Analytes were extracted from human serum by protein precipitation. UHPLC-MS/MS was performed using a gradient at a flow rate of 0.3 mL/min, and each sample was taken for 7.5 minutes. The mass spectrometry scanning mode used was electrospray ionization with multiple reaction monitoring in the positive mode.
RESULTS: Validation showed that endogenous substances in the sample did not interfere with the assay, and the relationship between X and Y was highly linear, with a coefficient of determination (R2) >0.9954 for each curve. The accuracy (85.0%-114.7%) and precision (intraday: 0.27%-9.32%; interday: 0.20%-7.66%) were less than 15.0%, and the internal standard-normalized matrix effects were consistent (coefficient of variation ≤4.40%). The analytes were stable in the final extract and human serum under various storage conditions (recovery: 87.0%-115.0%). The clinical applicability of the method was demonstrated by quantitative determination of analytes in serum samples obtained from patients with TB. Reproducibility of the drug concentrations measured in clinical samples was confirmed by incurred sample reanalysis.
CONCLUSIONS: A simple and reliable analytical method was developed and validated for the simultaneous determination of 10 anti-TB drugs in human serum using UHPLC-MS/MS. Quantitation of anti-TB drugs in clinical samples confirmed that the assay is suitable for therapeutic drug monitoring in regular clinical practice.

DOI: https://doi.org/10.1097/FTD.0000000000001170
J Chromatogr A. 2024 Jan 17. pii: S0021-9673(24)00029-3. [Epub ahead of print]1717 464656

Structural elucidation of derivatives of polyfunctional metabolites after methyl chloroformate derivatization by high-resolution mass spectrometry gas chromatography. Application to microbiota metabolites.

M Bajo-Fernández, G Montero, V Alonso-Herranz, C Barbas, F Rey-Stolle, A García.

  Metabolomics has become an essential discipline in the study of microbiome, emerging gas chromatography coupled to mass spectrometry as the most mature, robust, and reproducible analytical technique. Silylation is the most widely used chemical derivatization strategy, although it has some limitations. In this regard, alkylation by alkyl chloroformate offers some advantages, such as a rapid reaction, milder conditions, better reproducibility, and the generation of more stable derivatives. However, commercial spectral libraries do not include many of the alkyl derivatives, mainly for polyfunctional metabolites, which can form multiple derivatives. That introduces a huge bias in untargeted metabolomics leading to common errors such as duplicates, unknowns, misidentifications, wrong assignations, and incomplete results from which non-reliable findings and conclusions will be retrieved. For this reason, the purpose of this study is to overcome these shortcomings and to expand the knowledge of metabolites in general and especially those closely related to the gut microbiota through the thorough study of the reactivity of the different functional groups in real matrix derivatized by methyl chloroformate, a common representative alkylation reagent. To this end, a systematic workflow has been developed based on exhaustive structural elucidation, along with computational simulation, and taking advantage of the high sensitivity and high-resolution gas chromatography-mass spectrometry. Several empirical rules have been established according to chemically different entities (free fatty acids, amino acids, polyols, sugars, amines, and polyfunctional groups, etc.) to predict the number of derivatives formed from a single metabolite, as well as their elution order and structure. In this work, some methyl chloroformate derivatives not previously reported as well as the mechanisms to explain them are given. Extremely important is the interconversion of E- and Z- geometric isomers of unsaturated dicarboxylic acids (case of fumaric-maleic and case of citraconic-mesaconic acids), or the formation of cycled derivatives for amino acids, as well as common metabolites, as in the case of serine and cysteine, and many others.

Keywords:  Alkyl derivatization; Chloroformates; Gas chromatography; High-resolution mass spectrometry; Microbiome

DOI:  https://doi.org/10.1016/j.chroma.2024.464656
J Anal Toxicol. 2024 Jan 27. pii: bkae002. [Epub ahead of print]

Screening and Confirmation of Psilocin, Mitragynine, Phencyclidine, Ketamine, and Ketamine Metabolites by Liquid Chromatography Tandem Mass Spectrometry.

Madeleine E Wood, Glenna J Brown, Erin L Karschner, Joshua Z Seither, Jordan T Brown, Jessica L Knittel, Jeffrey P Walterscheid.

  A safe and productive workplace requires a sober workforce, free from substances that impair judgment and concentration. Although drug monitoring programs already exist, the scope and loopholes of standard workplace testing panels are well-known, allowing other substances to remain a source of risk. Therefore, a high-throughput urine screening method for psilocin, mitragynine, phencyclidine, ketamine, norketamine, and dehydronorketamine was developed and validated in conjunction with a urine and blood confirmation method. There are analytical challenges to overcome with psilocin and mitragynine, particularly when it comes to drug stability and unambiguous identification in authentic specimens. Screening and confirmation methods were validated according to ANSI/ASB Standard 036, Standard Practices for Method Validation in Forensic Toxicology. An automated liquid handling system equipped with dispersive pipette extraction tips was utilized for preparing screening samples, whereas an offline SPE method was used for confirmation sample preparation. Both methods utilized liquid chromatography tandem mass spectrometry to achieve limits of detection between 1-5 ng/mL for the screening method and 1 ng/mL for the confirmation method. Automation allows for faster throughput and enhanced quality assurance, which improves turnaround time. Compared to previous in-house methods, specimen volumes were substantially decreased for both blood and urine, which is an advantage when volume is limited. This screening technique is well-suited for evaluating large numbers of specimens from those employed in safety-sensitive workforce positions. This method can be utilized by workplace drug testing, human performance, and postmortem laboratories seeking robust qualitative screening and confirmation methods for analytes that have traditionally been too challenging to routinely analyze.

Keywords:  LC-MS/MS; hallucinogens; high-throughput; workplace drug testing

DOI:  https://doi.org/10.1093/jat/bkae002
Anal Chem. 2024 Feb 01.

Ultrahigh-Performance Supercritical Fluid Chromatography-Multimodal Ionization-Tandem Mass Spectrometry as a Universal Tool for the Analysis of Small Molecules in Complex Plant Extracts.

Kateřina Plachká, Veronika Pilařová, Štefan Kosturko, Jan Škop, Frantisek Svec, Lucie Nováková.

Complex analysis of plant extracts usually requires a combination of several analytical approaches. Therefore, in this study, we developed a holistic two-injection approach for plant extract analysis, which is carried out within one instrument without the need for any manual intervention during the analysis. Ultrahigh-performance supercritical fluid chromatography (UHPSFC) was employed for the analysis of 17 volatile terpenes on a porous graphitic carbon column within 7.5 min, followed by analysis on short diol column where flavonoids, phenolic acids, and terpenoic acids were analyzed within 15.5 min. A multimodal ionization source combining electrospray and atmospheric pressure chemical ionization (ESCi) was selected for mass spectrometry detection as a simultaneous ionization of both lipophilic and polar compounds was required. The quantitative aspects of the final UHPSFC-ESI/ESCi-MS/MS two-injection approach were determined, and it was applied to the analysis of Eucalyptus sp. extracts prepared by supercritical fluid extraction. Current methods reported in the literature typically require a labor-intensive combination of liquid and gas chromatography for the complex analysis of plant extracts. We present for the first time a new UHPSFC approach requiring only a single instrument that provides an alternative approach to the analysis of complex plant extracts.

DOI: https://doi.org/10.1021/acs.analchem.3c03599
Forensic Sci Int. 2024 Jan 18. pii: S0379-0738(24)00019-7. [Epub ahead of print]356 111938

Untargeted metabolomics for lifestyle biomarker discovery in human hair.

Ana M F Pego, Edward J Knaven, Anke P C van de Plas, Jos F Brouwers, Eva Cuypers, Bryn Flinders, Ron M A Heeren, Arian C van Asten, Ben M de Rooij.

  There is a risk of crimes remaining unsolved when no matching DNA profiles or fingermarks are found. If this is the case, forensic investigations are faced with a significant shortage of evidence and information regarding the unknown perpetrator and/or victim as well as any missing persons. However, a rather commonly found biological trace encountered at crime scenes is human hair. As hair acts as a biochemical reservoir, it may contain valuable information regarding one's characteristics and habits. This study aimed to build an analytical method capable of determining a marker set of relevant metabolites in hair, eventually building up a profile of its donor. To find potential markers, an untargeted metabolomics approach was developed to select and identify statistically significant features. For that purpose, a total of 68 hair samples were collected at several hairdresser shops in varying neighbourhoods. Compound extraction was achieved via methanolic incubation overnight and analysis performed using a high-resolution mass spectrometry (HRMS) Orbitrap Q Exactive Focus. The acquired data was uploaded and statistically evaluated using two free online software/libraries, where a total of eight compounds have given a match on both tools. Their presumptive identity was confirmed using reference standards and consequently added to a dynamic target donor profiling list. These results show the potential of using untargeted metabolomics for the search for lifestyle biomarkers capable of differentiating individuals. Such tools are of paramount importance in a forensic setting with little or no evidence available and no clear tactical leads.

Keywords:  Donor profiling; Forensic science; Hair; Lifestyle markers; Metabolomics; Untargeted screening

DOI:  https://doi.org/10.1016/j.forsciint.2024.111938
Anal Chem. 2024 Jan 31.

Three-Minute Enantioselective Amino Acid Analysis by Ultra-High-Performance Liquid Chromatography Drift Tube Ion Mobility-Mass Spectrometry Using a Chiral Core-Shell Tandem Column Approach.

Simon Jonas Jaag, Younes Valadbeigi, Tim Causon, Harald Gross, Michael Lämmerhofer.

Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core-shell particle tandem column with weak anion exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (Rs > 1.5 except for glutamic acid with Rs = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section (DTCCSN2) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.

DOI: https://doi.org/10.1021/acs.analchem.3c05426
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Jan 17. pii: S1570-0232(24)00026-6. [Epub ahead of print]1234 124018

Hollow-fiber liquid phase microextraction for determination of fluoxetine in human serum by nano-liquid chromatography coupled to high resolution mass spectrometry.

Beatriz Isabella Cestaro, Kelly Cavalcanti Machado, Michel Batista, Bruno José Gonçalves da Silva.

  Therapeutic drug monitoring (TDM) is a personalized care tool based on the determination of a target drug concentration in human serum. An antidepressant drug of interest for such investigations is fluoxetine (FXT), due to a severe impact of genetic polymorphisms on its metabolism. A bioanalytical method employed for TDM purposes must exhibit satisfactory selectivity and detectability, which becomes more difficult due to highly complex biological matrices. In this study, a highly selective bioanalytical method for the determination of FXT in human serum is proposed, which provides excellent clean-up efficiency based on a low cost hollow fiber liquid-phase microextraction (HF-LPME) sample preparation step and nano-liquid chromatography coupled to high-resolution mass spectrometry (nano-LC-HRMS). HF-LPME was performed using a two-phase "U" configuration, with 6 cm fiber, 20 µL of 1-octanol acting as supported liquid membrane, and ammonium hydroxide (pH 10) as the donor phase with NaCl (10 % m/v) and methanol (5 % v/v) as additives, requiring only 250 µL of the sample. The procedure was conducted for 30 min under a 750 rpm stirring rate. Gradient elution was carried out employing an acetonitrile-water as mobile phase, the composition of 30:70 to 100:00 (v/v) for 15 min, using formic acid 0.1 % (v/v) as an additive. MS1 was acquired in an Orbitrap mass analyzer, while MS2 was acquired in a linear trap quadrupole. Satisfactory linearity (Pearson's r = 0.99709) was obtained for a concentration range of 0.02 to 2.5 µg mL-1, which is compatible with the therapeutic and toxic range for FXT. The developed method presents adequate precision (1.61 to 7.45 %) and accuracy (95 to 114 %) and allows the dilution of high concentration samples in a 1:4 ratio (v/v), enabling its application for forensic serum samples. To our knowledge, this is the first study reporting a method based on HF-LPME and nano-LC-HRMS with any analytical purpose, especially with a TDM focus.

Keywords:  Fluoxetine; HF-LPME; High resolution mass spectrometry; Human serum; nano-LC-MS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124018
J Pharm Biomed Anal. 2024 Jan 12. pii: S0731-7085(24)00015-3. [Epub ahead of print]241 115975

Method development for the quantification of nine nitazene analogs and brorphine in Dried Blood Spots utilizing liquid chromatography - tandem mass spectrometry.

Christina Ververi, Martina Galletto, Marta Massano, Eugenio Alladio, Marco Vincenti, Alberto Salomone.

  The detection of nitazenes in biological fluids is increasingly needed as they are repeatedly reported in intoxication and overdose cases. A simple method for the quantification of low levels of nine nitazene analogs and brorphine in Dried Blood Spots (DBS) was developed and validated. 10 μL of spiked whole blood is deposited on a Capitainer®B card and allowed to dry. The spot is punched out, and extracted with 500 μL methanol:acetonitrile (3:1 v/v) added with 1.5 μL of fentanyl-D5 as the internal standard. After stirring, sonication, and centrifugation of the vial, the solvent is dried under nitrogen, the extract is reconstituted in 30 μL methanol, and 1 μL is injected into a UHPLC-MS/MS instrument. The method validation showed linear calibration in the 1-50 ng/mL range, LOD values ranging between 0.3 ng/mL (isotonitazene) and 0.5 ng/mL (brorphine), average CV% and bias% within 15 % and 10 % for all compounds, respectively. The matrix effect due to blood and filter paper components was within 85-115 % while recovery was between 15-20 %. Stability tests against time and temperature showed no significant variations for storage periods up to 28 days. Room temperature proved to represent the best samples storage conditions. UHPLC-MS/MS proved capable to reliably identify all target analytes at low concentration even in small specimen volumes, as those obtained from DBS cards, which in turn confirmed to be effective and sustainable micro-sampling devices. This procedure improves the efficiency of toxicological testing and provides an innovative approach for the identification of the nitazene class of illicit compounds.

Keywords:  DBS; Dried blood spots; HPLC–MS/MS; Isotonitazene; Nitazenes

DOI:  https://doi.org/10.1016/j.jpba.2024.115975
Anal Chem. 2024 Feb 02.

PyMIDA: A Graphical User Interface for Mass Isotopomer Distribution Analysis.

Naveed Ziari, Marc K Hellerstein.

Mass isotopomer distribution analysis (MIDA) is an analytical technique that measures the synthesis rate of biological polymers using combinatorial probabilities and stable isotope labeling. Over the past few decades, this method has been developed and applied to a wide range of uses that have increased our understanding of metabolism and the etiology and monitoring of disease. There is currently no publicly available piece of software for performing MIDA calculations in a targeted manner without its functionality being limited to a specific use case. We present a cross-platform Python graphical user interface implementation for research to obtain kinetic parameters easily from stable-isotope labeling studies and provide the code and user manual on GitHub.

DOI: https://doi.org/10.1021/acs.analchem.3c02211
ISME Commun. 2024 Jan;4(1): ycad006

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Yuto Fukuyama, Shigeru Shimamura, Sanae Sakai, Yuta Michimori, Tomomi Sumida, Yoshito Chikaraishi, Haruyuki Atomi, Takuro Nunoura.

  Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1 μl of 100 nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

Keywords:  13C tracer; capillary electrophoresis-tandem mass spectrometry (CE-MS); metabolomics; methanogen

DOI:  https://doi.org/10.1093/ismeco/ycad006
Anal Chem. 2024 Jan 30.

Quantifying Gut Microbial Short-Chain Fatty Acids and Their Isotopomers in Mechanistic Studies Using a Rapid, Readily Expandable LC-MS Platform.

Cheng-Yu Charlie Weng, Christopher Suarez, Shawn Ehlers Cheang, Garret Couture, Michael L Goodson, Mariana Barboza, Karen M Kalanetra, Chad F Masarweh, David A Mills, Helen E Raybould, Carlito B Lebrilla.

Short-chain fatty acids (SCFAs) comprise the largest group of gut microbial fermentation products. While absorption of most nutrients occurs in the small intestine, indigestible dietary components, such as fiber, reach the colon and are processed by the gut microbiome to produce a wide array of metabolites that influence host physiology. Numerous studies have implicated SCFAs as key modulators of host health, such as in regulating irritable bowel syndrome (IBS). However, robust methods are still required for their detection and quantitation to meet the demands of biological studies probing the complex interplay of the gut-host-health paradigm. In this study, a sensitive, rapid-throughput, and readily expandible UHPLC-QqQ-MS platform using 2-PA derivatization was developed for the quantitation of gut-microbially derived SCFAs, related metabolites, and isotopically labeled homologues. The utility of this platform was then demonstrated by investigating the production of SCFAs in cecal contents from mice feeding studies, human fecal bioreactors, and fecal/bacterial fermentations of isotopically labeled dietary carbohydrates. Overall, the workflow proposed in this study serves as an invaluable tool for the rapidly expanding gut-microbiome and precision nutrition research field.

DOI: https://doi.org/10.1021/acs.analchem.3c04352
BMC Chem. 2024 Jan 27. 18(1): 20

Development of chromatographic methods to determine multivitamins formulation depending on their solubility and polarity: comparative study using three greenness assessment tools.

Sara Abdel Basset Galal, Eman Saad Elzanfaly, Emad Mohamed Hussien, Enas Abdel Hakim Amer, Hala Elsayed Zaazaa.

  High performance liquid chromatography is one of the techniques of choice for the separation and quantitative determination of drugs in mixture form. Ipriflavone, ascorbic acid, pyridoxine, vitamin D3, and lysine are formulated together as an adjuvant combination in osteoporosis. In this work, we developed and validated two complementary high performance liquid chromatographic methods to determine the five compounds in their pharmaceutical dosage form. The first method (method A) was capable of determining ipriflavone, ascorbic acid, pyridoxine, and vitamin D3 in their bulk and combined pharmaceutical formulation. The method is based on Liquid Chromatographic separation with UV detection at 254 nm using Agilent Eclipse XDB-C18 column with a mobile phase consisting of 25 mM ammonium acetate buffer (pH 4.2): methanol in gradient mode. Due to the high polarity of lysine, it was difficult to achieve satisfactory retention on reversed phase columns. So, we separated it on a strong cation exchange column (Exsil 100 SCX) without derivatization with a mobile phase consisting of 10 mM sodium dihydrogen phosphate and 200 mM sodium chloride (pH 6) with UV detection at 210 nm (method B). Validation of the proposed methods was performed according to ICH guidelines Q2(R1). The proposed methods proved to be valid for selective analysis of the stated drugs in their bulk and combined pharmaceutical formulation. Greenness assessment of the developed methods was evaluated using three assessment tools: ESA, GAPI and the most recently developed tool AGREE, showing a satisfactory comprehensive guide of the greenness of the developed methods.

Keywords:  Ascorbic acid; Greenness Assessment; Ipriflavone; Lysine HCl; Pyridoxine HCl; RP-HPLC method; Vitamin D3

DOI:  https://doi.org/10.1186/s13065-024-01118-1
J Chromatogr A. 2024 Jan 29. pii: S0021-9673(24)00067-0. [Epub ahead of print]1717 464694

Improved method robustness and ruggedness in liquid chromatography-mass spectrometry by increasing the acid content of the mobile phase.

A Kaufmann, P Butcher, K Maden, S Walker, M Widmer, R Kaempf.

  A routine multiresidue method developed for the detection and quantification of veterinary drug residues in animal-based food was used to analyze sheep (ovine) liver. Unlike when working with previously validated matrices (e.g., bovine liver), some of the analytes of interest chromatographed in the form of split- or even fully baseline separated peaks. In other cases a significantly longer retention times (tR) was observed. A detailed investigation led to the elucidation of taurocholic acid as the causative agent. This compound is present in sheep liver at significantly higher concentrations than in most other animal tissues. Taurocholic acid is a zwitterionic compound and likely acts as an ion pairing agent, which modifies the selectivity of the stationary phase in a highly spatial and dynamic way. Injecting smaller volumes of matrix extract or the use of a significantly higher formic acid concentration in the mobile phase reduced or even completely eliminated the peak splitting. A more detailed examination led to the observation that the problem is not restricted to this particular matrix and extraction procedure or the used stationary phase. In fact, a higher formic acid concentration (e.g., 1.0 % versus 0.1 %) significantly improves the peak shape of many analytes present in fortified matrix samples as well as in pure standard solutions. In addition, analytical column aging was observed as being slower with a higher formic acid concentration. Finally the peak shape of analytes interacting with the metallic parts along the flow path of the liquid chromatograph was also significantly improved. Use of 0.1 % acid in mobile phases is often taken for granted in LC-MS. Regardless of the stationary phase, a higher ionic strength better stabilizes the pH and reduces unwanted interactions, which ultimately improves the method robustness. Flow injection experiments often show that 0.1 % acid concentrations produce the highest analyte signals. Yet, the use of 1 % acid in the mobile phase often leads to narrower and therefore taller chromatographic peaks, which may lead to lower detection limits for many analytes and to an improved separation efficiency.

Keywords:  Bioanalysis; Calibration; LC–MS; Mass spectrometry; Signal suppression

DOI:  https://doi.org/10.1016/j.chroma.2024.464694
Anal Chim Acta. 2024 Feb 22. pii: S0003-2670(24)00045-X. [Epub ahead of print]1291 342244

Perspective on SPME-MS: Green and high-performance methods for rapid screening.

Wei Zhou, Janusz Pawliszyn.

  The direct coupling of solid-phase microextraction (SPME) with mass spectrometry (MS) offers rapid analysis with high sensitivity and low matrix effects by benefiting from the integration of sampling, high enrichment, and clean-up functions of SPME. Eliminating chromatographic separation reduces the amount of gas/solvent needed for analysis, while direct desorption in SPME-MS consumes none or few microliters of organic solvents per sample, further enhancing the greenness of the SPME technology. Over the past two decades, the rapid evolution of SPME-MS has given rise to numerous novel technologies that employ diverse ionization techniques and interfaces, several of which have already been commercialized. Drawing from an extensive review published earlier this year and our research experience, we provide perspectives on three aspects of these technologies: interface design and automation, integration with state-of-art MS instrumentation, and anticipated future developments.

Keywords:  Direct coupling; Green analytical chemistry; Mass spectrometry; Rapid analysis; Solid-phase microextraction

DOI:  https://doi.org/10.1016/j.aca.2024.342244