bims-metlip 2023-12-10 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–12–10
fiveteen papers selected by
Sofia Costa, Matterworks

Development of targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry method for acyl-Coenzyme A covering short- to long-chain species in a single analytical run.
SFC-MS/MS for orthogonal separation of hydroxylated 17α-methyltestosterone isomers.
Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.
Rapid profiling of cytokinins using supercritical fluid chromatography coupled with tandem mass spectrometry.
Establishment of a modified QuEChERS extraction and liquid chromatography-tandem mass spectrometry method for multiple pesticide residues followed by determination of the residue levels and exposure assessment in livestock urine.
Development of a second-tier method for C4, C5 and C2 acylcarnitine analysis in plasma.
Deep Learning-Enabled MS/MS Spectrum Prediction Facilitates Automated Identification Of Novel Psychoactive Substances.
Comparison of calibration strategies for accurate quantitation by isotope dilution mass spectrometry: a case study of ochratoxin A in flour.
On-tissue chemical derivatization-enhanced spatially resolved lipidomics reveals abnormal metabolism in type 2 diabetic rat brain.
Reverse metabolomics for the discovery of chemical structures from humans.
Combination of a solid phase extraction and a two-dimensional LC-UV method for the analysis of vitamin D3 and its isomers in olive oil.
Application of Skyline software for detecting prohibited substances in doping control analysis.
Automated magnetic-bead-assisted sequential extraction technology for simultaneous detection of Aβ1-42 and Aβ1-40 in cerebrospinal fluid: An advance toward fully automated liquid chromatography-tandem mass spectrometry method.
Pyoverdine Analysis - From High-resolution MS/MS Fragmentation to Ion Mobility Measurements.
Development and validation of equilibrium dialysis UHPLC-MS/MS measurement procedures for total and unbound concentrations of bictegravir, dolutegravir, darunavir and doravirine in human plasma. Application to patients with HIV.

J Chromatogr A. 2023 Nov 27. pii: S0021-9673(23)00749-5. [Epub ahead of print]1714 464524

Development of targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry method for acyl-Coenzyme A covering short- to long-chain species in a single analytical run.

Madhulika Singh, Ligia Akemi Kiyuna, Christoff Odendaal, Barbara M Bakker, Amy C Harms, Thomas Hankemeier.

  Acyl-CoAs play a significant role in numerous physiological and metabolic processes making it important to assess their concentration levels for evaluating metabolic health. Considering the important role of acyl-CoAs, it is crucial to develop an analytical method that can analyze these compounds. Due to the structural variations of acyl-CoAs, multiple analytical methods are often required for comprehensive analysis of these compounds, which increases complexity and the analysis time. In this study, we have developed a method using a zwitterionic HILIC column that enables the coverage of free CoA and short- to long-chain acyl-CoA species in one analytical run. Initially, we developed the method using an LC-QTOF instrument for the identification of acyl-CoA species and optimizing their chromatography. Later, a targeted HILIC-MS/MS method was created in scheduled multiple reaction monitoring mode using a QTRAP MS detector. The performance of the method was evaluated based on various parameters such as linearity, precision, recovery and matrix effect. This method was applied to identify the difference in acyl-CoA profiles in HepG2 cells cultured in different conditions. Our findings revealed an increase in levels of acetyl-CoA, medium- and long-chain acyl-CoA while a decrease in the profiles of free CoA in the starved state, indicating a clear alteration in the fatty acid oxidation process.

Keywords:  Acyl-CoA; Biomarker; HILIC; HepG2; LC-MS

DOI:  https://doi.org/10.1016/j.chroma.2023.464524
Drug Test Anal. 2023 Dec 04.

SFC-MS/MS for orthogonal separation of hydroxylated 17α-methyltestosterone isomers.

Felix Bredendiek, Maria Kristina Parr.

  Because of their performance-enhancing effect, anabolic androgenic steroids (AAS) are often misused in sports. Nearly half of the adverse analytical findings (AAF) in 2022 doping controls are correlated to AAS misuse. Metabolites play a crucial role in the bioanalysis of endogenous and exogenous steroids. Therefore, one important field in antidoping research is the investigation on drug metabolizing and steroidogenic enzymes. The introduction of a hydroxy group is the most common reaction, which is catalyzed by cytochrome P450 (CYP) enzymes in phase-I metabolism. Analysis of AAS metabolites is commonly performed using gas chromatography mass spectrometry (GC-MS) systems. Laborious sample preparation and extended run times compared to liquid chromatography (tandem) mass spectrometry (LC-MS/MS) methods are usually correlated with this type of analysis. On the other hand, liquid chromatography (tandem) mass spectrometry (LC-MS[/MS]) methods have a lower separation efficiency than GC-MS methods. Both techniques lack selectivity for hydroxylated 17α-methyltestosterone metabolites. Therefore, as an orthogonal analytical approach, a supercritical fluid chromatography tandem mass spectrometry method was developed to separate four hydroxy metabolites of 17α-methyltestosterone (2α-/2β-/4-/6β-hydroxy-17α-methyltestosterone). This project aimed to get a more in-depth look at the metabolization and analysis of 17α-methyltestosterone and its hydroxylated metabolites. The developed method revealed lower limits of quantitation between 0.6 and 6 ng/ml at an accuracy of 85-115% using a matrix matched calibration. An in vitro study with human liver microsomes shows 6β-hydroxy-17α-methyltestosterone as main metabolite (15.9%) as well as the metabolite 2β-hydroxy-17α-methyltestosterone (0.5%). The results show that the developed method is sensitive and robust. In addition, the method allows a previously missing discrimination of the hydroxylated metabolites in a short analysis time without prior, complex derivatizations.

Keywords:  SFC; metabolism; methyltestosterone; tandem mass spectrometry

DOI:  https://doi.org/10.1002/dta.3620
Anal Chem. 2023 Dec 06.

Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Corey D Broeckling, Richard D Beger, Leo L Cheng, Raquel Cumeras, Daniel J Cuthbertson, Surendra Dasari, W Clay Davis, Warwick B Dunn, Anne Marie Evans, Alvaro Fernández-Ochoa, Helen Gika, Royston Goodacre, Kelli D Goodman, Goncalo J Gouveia, Ping-Ching Hsu, Jennifer A Kirwan, Dritan Kodra, Julia Kuligowski, Renny Shang-Lun Lan, María Eugenia Monge, Laura W Moussa, Sindhu G Nair, Nichole Reisdorph, Stacy D Sherrod, Candice Ulmer Holland, Dajana Vuckovic, Li-Rong Yu, Bo Zhang, Georgios Theodoridis, Jonathan D Mosley.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.3c02924
Anal Chim Acta. 2024 Jan 02. pii: S0003-2670(23)01231-X. [Epub ahead of print]1285 342010

Rapid profiling of cytokinins using supercritical fluid chromatography coupled with tandem mass spectrometry.

Ivan Petřík, Aleš Pěnčík, Jakub Stýskala, Lenka Tranová, Petra Amakorová, Miroslav Strnad, Ondřej Novák.

   BACKGROUND: The determination of plant hormones is still a very challenging analytical discipline, mainly due to their low concentration in complex plant matrices. Therefore, the involvement of very sensitive high-throughput techniques is required. Cytokinins (CKs) are semi-polar basic plant hormones regulating plant growth and development. Modern methods for CK determination are currently based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), which enables the separation of CK isomeric forms occurring endogenously in plants. Here, ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry (UHPSFC-MS/MS) was used for the simultaneous determination of 37 CK metabolites.
RESULTS: The chromatographic conditions were tested on three different columns with various retention mechanisms. Hybrid silica modified with 2-picolylamine was selected as the stationary phase. Several parameters such as column temperature, back pressure regulation, mobile phase composition and make-up solvent were investigated to achieve efficient separation of CK isomers and reasonable sensitivity. Compared to UHPLC-MS/MS, a 9-min chromatographic analysis using a mobile phase of supercritical CO2 and 5 mM ammonia in methanol represents a three-fold acceleration of total run time. The quantification limit of UHPSFC-MS/MS method was in the range of 0.03-0.19 fmol per injection and the method validation showed high accuracy and precision (below 15 % for most analytes). The method was finally applied to the complex plant matrix of the model plant Arabidopsis thaliana and the obtained profiles of CK metabolites were compared with the results from the conventional UHPLC-MS/MS method.
SIGNIFICANCE: The presented work offers a novel approach for quantification of endogenous CKs in plants. Compared to the conventional UHPLC-MS/MS, the total run time is shorter and the matrix effect lower for the key CK metabolites. This approach opens the opportunity to utilize UHPSFC-MS/MS instrumentation for targeted plant hormonomics including other plant hormone families.

Keywords:  Arabidopsis; Cytokinin; Phytohormone; Quantification; Separation; UHPSFC-MS/MS

DOI:  https://doi.org/10.1016/j.aca.2023.342010
J Chromatogr A. 2023 Dec 01. pii: S0021-9673(23)00772-0. [Epub ahead of print]1714 464547

Establishment of a modified QuEChERS extraction and liquid chromatography-tandem mass spectrometry method for multiple pesticide residues followed by determination of the residue levels and exposure assessment in livestock urine.

Youzhi Su, Jianjiang Lu, Fang Li, Jun Liu.

  The establishment of an analytical method for pesticide residues in livestock urine can realize the real-time monitoring of pesticide pollution in livestock breeding. In this study, a novel method was developed for the determination of 106 pesticide residues in livestock urine based on a modified QuEChERS extraction and liquid chromatography-tandem mass spectrometry. Acetonitrile was used to extract target analytes through acidic and alkaline switching of the sample environment. The purification effect of captiva EMR-Lipid on samples was investigated. Three kinds of materials, C18, polar enhanced polymer (PEP), N-propylethylenediamine (PSA), were selected from 20 kinds of materials as adsorbents for QuEChERS. A mass analysis was carried out using simultaneous scanning in both positive and negative ion mode and multiple reaction monitoring mode. All analytes showed good linearity, with correlation coefficients (R2) greater than 0.9923; their limits of quantification were 0.02-1.95 ng/mL. The average recoveries at low, medium, and high spiked levels were in the range of 70.1 %-117.3 %, with intra-day precision ranging from 3.4 % to 16.9 % and inter-day precision ranging from 4.0 % to 19.3 %. The established analytical method was used to analyze the pesticide residue in swine urine and bovine urine collected from farms in Yining, Xinjiang, China. A total of 8 pesticides were detected, and the residue ranged from less than the limit of quantitation to 22.4 ng/mL. The top three pesticides with the highest detection frequency were clothianidin, thiamethoxam, and dinotefuran. The exposure assessment based on the monitored pesticide residue concentration levels showed that the detected pesticides could pose little risk to cattle and pigs.

Keywords:  Exposure assessment; Liquid chromatography–tandem mass spectrometry; Livestock urine; Pesticide residues; QuECHERS

DOI:  https://doi.org/10.1016/j.chroma.2023.464547
Clin Biochem. 2023 Dec 02. pii: S0009-9120(23)00226-6. [Epub ahead of print] 110698

Development of a second-tier method for C4, C5 and C2 acylcarnitine analysis in plasma.

Josko Ivica, Faisal Adam, Lyse Wortel, Teresa Kalika, Heather Pelly, Jeannette Gauthier, Murray Potter.

   INTRODUCTION: Acylcarnitines are typically analyzed using either a flow injection analysis (FIA) method or liquid chromatography-mass spectrometry (LC-MS/MS) methods. The FIA method is a fast, efficient method, however it does not have the capability to separate compounds with the same molecular weight. These isobaric interferences can be removed by chromatographic separation with LC-MS/MS. In this study, we aimed to develop and optimize a qualitative LC-MS/MS method to separate the isobaric interferences for two-, four- and five-carbon acylcarnitines.
METHODS: The samples were first prepared by acylcarnitine derivatization with butanolic HCl. The developed LC-MS/MS method is a combination of isocratic and gradient elution used to separate acylcarnitines. Multiple reaction monitoring was used for determination of precursor and product ions for each acylcarnitine species as well as known interferences used in our study. We used this method to analyze quality assurance and patient samples with elevated two-, four- and five-carbon acylcarnitines.
RESULTS: Butyryl- and isobutyrylcarnitines as well as valeryl- and isovalerylcarnitines were successfully separated using the developed method. This method was able also to separate and distinguish acetylcarnitine from glutamate interference that has been causing overestimation of acetylcarnitine. In patients, the dominant five-carbon acylcarnitine was found to be isovalerylcarnitine. We confirmed that the majority of analyzed patient samples had additional carnitine adducts present but not valerylcarnitine. Butyryl- and isobutyrylcarnitines, in variable ratios, were present in every patient sample.
CONCLUSION: We developed a qualitative LC-MS/MS method for butyl-ester derivatized acylcarnitines, which can be used as a second-tier method for diagnosis and monitoring of various inborn errors of metabolism in our hospital network.

Keywords:  acylcarnitines; chromatography; inborn errors of metabolism; mass spectrometry

DOI:  https://doi.org/10.1016/j.clinbiochem.2023.110698
Anal Chem. 2023 Dec 04.

Deep Learning-Enabled MS/MS Spectrum Prediction Facilitates Automated Identification Of Novel Psychoactive Substances.

Fei Wang, Daniel Pasin, Michael A Skinnider, Jaanus Liigand, Jan-Niklas Kleis, David Brown, Eponine Oler, Tanvir Sajed, Vasuk Gautam, Stephen Harrison, Russell Greiner, Leonard J Foster, Petur Weihe Dalsgaard, David S Wishart.

The market for illicit drugs has been reshaped by the emergence of more than 1100 new psychoactive substances (NPS) over the past decade, posing a major challenge to the forensic and toxicological laboratories tasked with detecting and identifying them. Tandem mass spectrometry (MS/MS) is the primary method used to screen for NPS within seized materials or biological samples. The most contemporary workflows necessitate labor-intensive and expensive MS/MS reference standards, which may not be available for recently emerged NPS on the illicit market. Here, we present NPS-MS, a deep learning method capable of accurately predicting the MS/MS spectra of known and hypothesized NPS from their chemical structures alone. NPS-MS is trained by transfer learning from a generic MS/MS prediction model on a large data set of MS/MS spectra. We show that this approach enables a more accurate identification of NPS from experimentally acquired MS/MS spectra than any existing method. We demonstrate the application of NPS-MS to identify a novel derivative of phencyclidine (PCP) within an unknown powder seized in Denmark without the use of any reference standards. We anticipate that NPS-MS will allow forensic laboratories to identify more rapidly both known and newly emerging NPS. NPS-MS is available as a web server at https://nps-ms.ca/, which provides MS/MS spectra prediction capabilities for given NPS compounds. Additionally, it offers MS/MS spectra identification against a vast database comprising approximately 8.7 million predicted NPS compounds from DarkNPS and 24.5 million predicted ESI-QToF-MS/MS spectra for these compounds.

DOI: https://doi.org/10.1021/acs.analchem.3c02413
Anal Bioanal Chem. 2023 Dec 04.

Comparison of calibration strategies for accurate quantitation by isotope dilution mass spectrometry: a case study of ochratoxin A in flour.

Jennifer Bates, Adilah Bahadoor, Sheryl A Tittlemier, Jeremy E Melanson.

  Analysis of low-level organic contaminants in complex matrices is essential for monitoring global food safety. However, balancing sample throughput with complex experimental designs and/or sample clean-up to best reduce matrix effects is a constant challenge. Multiple strategies exist to mitigate these effects, with internal standard-based methods such as isotope dilution mass spectrometry (IDMS) being the most advantageous. Here, multiple internal calibration strategies were investigated for the quantification of ochratoxin A (OTA) in wheat samples by liquid chromatography-mass spectrometry (LC-MS). Internal standard-based quantitation methods such as single (ID1MS), double (ID2MS), and quintuple (ID5MS) isotope dilution mass spectrometry, as well as external standard calibration, were explored and compared. A certified reference material (CRM) of OTA in flour, MYCO-1, was used to evaluate the accuracy of each method. External calibration generated results 18-38% lower than the certified value for MYCO-1, largely due to matrix suppression effects. Concurrently, consistently lower OTA mass fractions were obtained for the wheat samples upon quantitation by external calibration as opposed to ID1MS, ID2MS, and ID5MS. All isotope dilution methods produced results that fell within the expected range for MYCO-1 (3.17-4.93 µg/kg), validating their accuracy. However, an average 6% decrease in the OTA mass fraction was observed from results obtained by ID1MS compared to those by ID2MS and ID5MS. Upon scrutiny, these differences were attributed to an isotopic enrichment bias in the isotopically labelled internal standard [13C6]-OTA that was used for ID1MS, the OTAL-1 CRM. The advantages and limitations of each isotopic method are illustrated.

Keywords:  Internal standard; Liquid chromatography-mass spectrometry; Mycotoxins; Trace contaminants in food

DOI:  https://doi.org/10.1007/s00216-023-05053-3
Talanta. 2023 Nov 30. pii: S0039-9140(23)01242-0. [Epub ahead of print]269 125491

On-tissue chemical derivatization-enhanced spatially resolved lipidomics reveals abnormal metabolism in type 2 diabetic rat brain.

Lingzhi Wang, Qingce Zang, Xinyi Tu, Xinzhu Li, Ting Li, Shiyu Zhu, Ruiping Zhang, Zeper Abliz.

  Neurologic disorders are often accompanied by alterations in lipids and oxylipins in the brain. However, the complexity of the lipidome in the brain and its changes during brain damage caused by diabetes remain poorly understood. Herein, we developed an enhanced spatially resolved lipidomics approach with the assistance of on-tissue chemical derivatization to study lipid metabolism in the rat brain. This method enabled the spatially resolved analysis of 560 lipids and oxylipins in 19 brain microregions in coronal and sagittal sections and remarkably improved the coverage of lipidome detection. We applied this method to lipidomic studies of the diabetic rat brain and found that lipid dysregulation followed a microregion-specific pattern. Carnitines and glycerolipids were mainly elevated in the corpus callosum (midbrain) and pineal gland regions, respectively. In addition, most oxylipins, including fatty aldehydes and oxo fatty acids, were significantly upregulated in nine brain microregions. We produced a spatially resolved analysis of lipids and oxylipins, providing a novel analytical tool for brain metabolism research.

Keywords:  Brain; Diabetes; Mass spectrometry imaging; On-tissue chemical derivatization; Spatially resolved lipidomics

DOI:  https://doi.org/10.1016/j.talanta.2023.125491
Nature. 2023 Dec 05.

Reverse metabolomics for the discovery of chemical structures from humans.

Emily C Gentry, Stephanie L Collins, Morgan Panitchpakdi, Pedro Belda-Ferre, Allison K Stewart, Marvic Carrillo Terrazas, Hsueh-Han Lu, Simone Zuffa, Tingting Yan, Julian Avila-Pacheco, Damian R Plichta, Allegra T Aron, Mingxun Wang, Alan K Jarmusch, Fuhua Hao, Mashette Syrkin-Nikolau, Hera Vlamakis, Ashwin N Ananthakrishnan, Brigid Boland, Amy Hemperly, Niels Vande Casteele, Frank J Gonzalez, Clary B Clish, Ramnik J Xavier, Hiutung Chu, Erin S Baker, Andrew D Patterson, Rob Knight, Dionicio Siegel, Pieter C Dorrestein.

Determining structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here, we present reverse metabolomics as a discovery strategy, where MS/MS spectra are acquired from newly synthesized compounds and searched for in public metabolomics data to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans - N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository scale analysis,15,16 we discovered that some conjugated bile acids were associated with inflammatory bowel disease (IBD). Using four distinct human IBD cohorts for validation, we found that Glu, Ile/Leu, Phe, Thr, Trp and Tyr conjugated cholic acids were elevated in Crohn's disease. Several of these compounds and related structures were shown to affect pathways associated with inflammatory bowel disease, such as interferon-gamma production in CD4+ T cells40 and PXR agonism.41 Bacteria belonging to the Bifidobacterium, Clostridium, and Enterococcus genera were able to produce these bile amidates when cultured. Because searching repositories with MS/MS spectra has only recently become possible, reverse metabolomics approach can now be employed as a general strategy to discover other molecules from human and animal ecosystems.

DOI: https://doi.org/10.1038/s41586-023-06906-8
Talanta. 2023 Nov 25. pii: S0039-9140(23)01237-7. [Epub ahead of print]269 125486

Combination of a solid phase extraction and a two-dimensional LC-UV method for the analysis of vitamin D3 and its isomers in olive oil.

Francesca Rinaldi, Sara Tengattini, Erika Amore, Filippo Scarabelli, Gabriella Massolini, Enrica Calleri, Caterina Temporini.

  The current HPLC methods for the quantification of vitamin D3 (VitD3) and its two isomers previtamin D3 (PreVitD3) and trans-vitamin D3 (trans-VitD3) in olive oil preparations present some limitations mainly due to peak overlapping of the oily matrix components with the compounds of interest. The use of two-dimensional liquid chromatography (2D-LC) with different retention mechanism can reach higher resolving power thus allowing the analysis of complex samples. The present paper proposes a new alternative method including a solid phase extraction sample preparation step and a two-dimensional liquid chromatographic analysis using routine instrumentation, fitting the needs of quality assurance and quality control laboratories of pharmaceutical companies. The extraction protocol was demonstrated to provide a clean-up of the sample and a quantitative recovery of the species of interest. The 2D method proved its suitability in the isolation of vitamins from oil components in the first dimension and the separation and quantification of the analytes in the second dimension thanks to the orthogonal selectivities of phenyl and porous graphitic carbon (PGC) stationary phases. The method was validated following ICH guidelines and possesses an adequate sensitivity to quantify the impurity trans-VitD3 in pharmaceuticals considering the limits imposed by regulatory agencies. The applicability of the phenyl x PGC 2D-LC-UV method to quality control of medicinal products based on VitD3 in olive oil was confirmed by the successful quantification of vitamins in olive oil formulations.

Keywords:  Isomer separation; Olive oil matrix; Phenyl column; Porous graphitic carbon; Solid phase extraction; Two-dimensional liquid chromatography; Vitamin D(3); trans-VitD(3)

DOI:  https://doi.org/10.1016/j.talanta.2023.125486
PLoS One. 2023 ;18(12): e0295065

Application of Skyline software for detecting prohibited substances in doping control analysis.

Hyeon-Jeong Lee, Mijin Jeon, Yoondam Seo, Inseon Kang, Wooyeon Jeong, Junghyun Son, Eugene C Yi, Hophil Min.

As the number of prohibited drugs has been progressively increasing and analytical methods for detecting such substances are renewed continuously for doping control, the need for more sensitive and accurate doping analysis has increased. To address the urgent need for high throughput and accurate analysis, liquid chromatography with tandem mass spectrometry is actively utilized in case of most of the newly designated prohibited substances. However, because all mass spectrometer vendors provide data processing software that is incapable of handling other instrumental data, it is difficult to cover all doping analysis procedures, from method development to result reporting, on one platform. Skyline is an open-source and vendor-neutral software program invented for the method development and data processing of targeted proteomics. Recently, the utilization of Skyline has been expanding for the quantitative analysis of small molecules and lipids. Herein, we demonstrated Skyline as a simple platform for unifying overall doping control, including the optimization of analytical methods, monitoring of data quality, discovery of suspected doping samples, and validation of analytical methods for detecting newly prohibited substances. For method optimization, we selected the optimal collision energies for 339 prohibited substances. Notably, 195 substances exhibited a signal intensity increase of >110% compared with the signal intensity of the original collision energy. All data related to method validation and quantitative analysis were efficiently visualized, extracted, or calculated using Skyline. Moreover, a comparison of the time consumed and the number of suspicious samples screened in the initial test procedure highlighted the advantages of using Skyline over the commercially available software TraceFinder in doping control.

DOI: https://doi.org/10.1371/journal.pone.0295065
J Chromatogr A. 2023 Nov 23. pii: S0021-9673(23)00756-2. [Epub ahead of print]1713 464531

Automated magnetic-bead-assisted sequential extraction technology for simultaneous detection of Aβ1-42 and Aβ1-40 in cerebrospinal fluid: An advance toward fully automated liquid chromatography-tandem mass spectrometry method.

Yutong Zou, Xiaoli Ma, Chenhui Mao, Jian Zhong, Yifei Wang, Danchen Wang, Songlin Yu, Jing Gao, Ling Qiu.

  Traditional solid-phase extraction (SPE) LC-MS/MS is limited by high costs, turnaround times, and procedural complexity, which limited the usage in clinical practice. This study aimed to establish a robust UPLC-MS/MS method with automated magnetic-bead-assisted sequential extraction (MBASE) technology to simultaneously measure Aβ1-42 and Aβ1-40 in cerebrospinal fluid (CSF). A Waters TQ-XS triple quadrupole mass spectrometer and Acquity UPLC Protein BEH C4 column were used. The targeted analytes were extracted and concentrated using the automated MBASE technology with chemically modified magnetic MCX beads. Analytical performance was verified referring to the CLSI C62-A and EP-15-A3 guidelines. A total of 68 CSF samples were collected and analyzed using the MBASE UPLC-MS/MS method, traditional SPE UPLC-MS/MS method, and Lumipulse G fully automated chemiluminescence detection system, and method comparison analysis is conducted. The MBASE UHPLC-MS/MS method showed an analytical performance equivalent to that of traditional SPE technology, with a higher sample throughput and smaller amount of materials ($34.98 vs. $493.96) and labor cost (101 min vs. 140 min) for 96 samples. The limit of quantification (LOQ) of Aβ1-42 and Aβ1-40 was 0.10 ng/mL and 0.05 ng/mL; recovery was 88.35-107.07 % and 95.72-96.60 %; and total imprecision was 3.69-6.83 % and 3.02-3.61 %, respectively. The measurements were faithfully reproduced within the allowable levels of uncertainty using certified reference materials. The correlations between this MBASE UPLC-MS/MS method, the SPE UPLC-MS/MS method, and Lumipulse G fully automated biochemical analysis method are all deemed good (r = 0.869-0.936), and the MBASE- and SPE-UPLC-MS/MS methods showed comparable measurements. To our knowledge, our study firstly verified the robust performance of the MBASE UPLC-MS/MS method to simultaneously determine Aβ1-42 and Aβ1-40 in CSF. With further introduce of automation, the assay with high accuracy and low material and labor costs will become a promising clinical technology.

Keywords:  Amyloid; Cerebrospinal fluid; Magnetic-bead-assisted sequential extraction technology; UPLC–MS/MS

DOI:  https://doi.org/10.1016/j.chroma.2023.464531
Chimia (Aarau). 2023 Apr 26. 77(4): 250-253

Pyoverdine Analysis - From High-resolution MS/MS Fragmentation to Ion Mobility Measurements.

Karoline Rehm, Vera Vollenweider, Rolf Kümmerli, Laurent Bigler.

  Microorganisms produce iron chelators called siderophores that are a rich source for drug discovery or plant protective agents. Pyoverdines are a class of siderophores from fluorescent Pseudomonas members and consist of different peptide chains specific to each bacterial species. The structural elucidation and characterization of pyoverdines require comprehensive analytical methods as bacterial extracts are complex mixtures. Here, we present a high-throughput UHPLC-MS/MS pipeline and the application of ion mobility spectrometry to facilitate research in the field of medicine and agriculture.

Keywords:  Mass Spectrometry; Pseudomonas; Siderophore; Structural Elucidation

DOI:  https://doi.org/10.2533/chimia.2023.250
Clin Chim Acta. 2023 Nov 30. pii: S0009-8981(23)00480-1. [Epub ahead of print]552 117678

Development and validation of equilibrium dialysis UHPLC-MS/MS measurement procedures for total and unbound concentrations of bictegravir, dolutegravir, darunavir and doravirine in human plasma. Application to patients with HIV.

Raúl Rigo-Bonnin, Laura García-Tejada, Virgínia Mas-Bosch, Arkaitz Imaz, Juan Manuel Tiraboschi, Sofía Scévola, Jordi Niubó, Antonio Navarro-Alcaraz, Daniel Podzamczer.

   BACKGROUND: Fixed-dose combinations of antiretroviral drugs are commonly used to treat HIV infection and therapeutic monitoring is not part of routine clinical practice. However, drug concentrations monitoring might have role in different clinical scenarios as well as for research purposes. This study aimed to develop and validate UHPLC-MS/MS procedures for measuring total and unbound concentrations of bictegravir, dolutegravir, darunavir and doravirine in human plasma.
MATERIAL AND METHODS: Equilibrium dialysis preceded sample preparation (based on protein precipitation) for measuring unbound antiretroviral concentrations. Chromatographic separations were achieved on an Acquity®-UPLC® HSS™-T3 column (50 mm × 2.1 mm; 1.8 µm) using a non-linear water/acetonitrile gradient containing 0.1 % formic acid at a 0.5 mL/min flow rate. Antiretrovirals were detected by tandem mass spectrometry in positive electrospray ionisation and multiple reaction monitoring modes.
RESULTS: No significant interferences or carry-over were observed. Imprecisions, absolute relative biases, normalised matrix effects and recoveries were ≤15.0 %, ≤11.1 %, (94.7-104.1)% and (96.7-105.5)%, respectively. Non-linear measuring intervals were observed between (25-10,000) µg/L for total/plasma dialysate concentrations and linearity schemes (1.00-100) µg/L for buffer dialysate concentrations.
CONCLUSIONS: The UHPLC-MS/MS procedures developed could be used for research purposes and therapeutic drug monitoring of antiretrovirals in routine clinical practice.

Keywords:  Bictegravir; Darunavir; Dolutegravir; Doravirine; Rapid equilibrium dialysis; UHPLC-MS/MS; Unbound (free)

DOI:  https://doi.org/10.1016/j.cca.2023.117678