Publications by authors named "Serge Rudaz"

232 Publications

Psychological Distress and Well-Being among Students of Health Disciplines: The Importance of Academic Satisfaction.

Int J Environ Res Public Health 2021 Feb 23;18(4). Epub 2021 Feb 23.

Faculty of Medicine, University of Geneva, Rue Michel-Servet 1, 1206 Geneva, Switzerland.

Background: Research on the mental health of students in health disciplines mainly focuses on psychological distress and nursing and medical students. This study aimed to investigate the psychological well-being and distress and related factors among undergraduate students training in eight different health-related tracks in Geneva, Switzerland.

Methods: This cross-sectional study used established self-filled scales for anxiety, depression, stress, psychological well-being, and study satisfaction. Descriptive statistics and hierarchical regression analyses were applied.

Results: In October 2019, out of 2835 invited students, 915 (32%) completed the survey. Lower academic satisfaction scores were strongly associated with depression (β = -0.26, < 0.001), anxiety (β = -0.27, < 0.001), and stress (β = -0.70, < 0.001), while higher scores were associated with psychological well-being (β = 0.70, < 0.001). Being female was strongly associated with anxiety and stress but not with depression or psychological well-being. Increased age was associated with enhanced psychological well-being. The nature of the academic training had a lesser impact on mental health and the academic year had none.

Conclusion: Academic satisfaction strongly predicts depression, anxiety, stress, and psychological well-being. Training institutions should address the underlying factors that can improve students' satisfaction with their studies while ensuring that they have access to psychosocial services that help them cope with mental distress and enhance their psychological well-being.
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http://dx.doi.org/10.3390/ijerph18042151DOI Listing
February 2021

Ion mobility-high resolution mass spectrometry in anti-doping analysis. Part I: Implementation of a screening method with the assessment of a library of substances prohibited in sports.

Anal Chim Acta 2021 Apr 31;1152:338257. Epub 2021 Jan 31.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis. In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, CCS values, and m/z ratios was constructed. Urine samples were analyzed using either a simple "dilute and shoot" procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and CCS values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses.
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http://dx.doi.org/10.1016/j.aca.2021.338257DOI Listing
April 2021

Evaluation of a nanoflow interface based on the triple-tube coaxial sheath-flow sprayer for capillary electrophoresis-mass spectrometry coupling in metabolomics.

J Chromatogr A 2021 Feb 9;1641:461982. Epub 2021 Feb 9.

School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland. Electronic address:

The performance of an original CE-MS interface that allows the in-axis positioning of the electrospray with respect to the MS inlet was evaluated. The variations in the geometrical alignment of this configuration in the absence of a nebulizing gas afforded a significant reduction in the sheath-liquid flow rate from 3 µL/min to as low as 300 nL/min. The sheath liquid and BGE were respectively composed of HO-iPrOHCHCOOH 50:50:1 (v/v/v) and 10% acetic acid (pH 2.2). A significant gain in sensitivity was obtained, and it was correlated to the effective mobility of the analytes. Compounds with low mobility values showed a greater sensitivity gain. Special attention was paid to the detection of proteinogenic amino acids. Linear response functions were obtained from 15 ng/mL to 500 ng/mL. The limits of quantification, as low as 34.3 ng/mL, were improved by a factor of up to six compared to the conventional configuration. The in-axis setup was ultimately applied to the absolute quantification of four important amino acids, alanine, tyrosine, methionine and valine, in standard reference material (NIST plasma). The accuracies ranged from 78 to 113%, thus demonstrating the potential of this configuration for metabolomics.
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http://dx.doi.org/10.1016/j.chroma.2021.461982DOI Listing
February 2021

Wipe-sampling procedure optimisation for the determination of 23 antineoplastic drugs used in the hospital pharmacy.

Eur J Hosp Pharm 2021 Mar 17;28(2):94-99. Epub 2019 Jul 17.

CYTOXLAB, Geneva University Hospitals, Geneva, Switzerland.

Purpose: Optimise a wipe sampling procedure to evaluate the surface contamination for 23 antineoplastic drugs used in the hospital pharmacy.

Methods: The influence of various parameters (ie, sampling device, sampling solution, desorption modes) was evaluated using a validated liquid chromatography-mass spectrometry (LC-MS/MS) method able to quantify 23 antineoplastic drugs widely used in the hospital pharmacy: 5-fluorouracil, busulfan, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, etoposide phosphate, fludarabine phosphate, ganciclovir, gemcitabine, idarubicin, ifosfamide, irinotecan, methotrexate, paclitaxel, pemetrexed, raltitrexed, topotecan and vincristine. Best conditions were tested with real samples from a hospital pharmacy chemotherapy compounding unit.

Results: Polyester swabs (TX714 and TX716) gave satisfactory results for the desorption step for all compounds with mean recoveries of 90% and 95%, respectively. For the wiping step, higher recoveries were obtained using TX716 and isopropanol 75% as wiping solution. As anticipated, most intense contaminations were found close to the chemotherapy production site, on surfaces the most frequently in contact with operators' hands.

Conclusion: Wipe sampling method was successfully developed and applied to real samples to determine surface contamination with 23 antineoplastic agents in trace amounts.
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http://dx.doi.org/10.1136/ejhpharm-2019-001983DOI Listing
March 2021

Approaches in metabolomics for regulatory toxicology applications.

Analyst 2021 Feb 19. Epub 2021 Feb 19.

School of Pharmaceutical Sciences, University of Geneva, Switzerland. and Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Switzerland and Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland.

Innovative methodological approaches are needed to conduct human health and environmental risk assessments on a growing number of marketed chemicals. Metabolomics is progressively proving its value as an efficient strategy to perform toxicological evaluations of new and existing substances, and it will likely become a key tool to accelerate chemical risk assessments. However, additional guidance with widely accepted and harmonized procedures is needed before metabolomics can be routinely incorporated in decision-making for regulatory purposes. The aim of this review is to provide an overview of metabolomic strategies that have been successfully employed in toxicity assessment as well as the most promising workflows in a regulatory context. First, we provide a general view of the different steps of regulatory toxicology-oriented metabolomics. Emphasis is put on three key elements: robustness of experimental design, choice of analytical platform, and use of adapted data treatment tools. Then, examples in which metabolomics supported regulatory toxicology outputs in different scenarios are reviewed, including chemical grouping, elucidation of mechanisms of toxicity, and determination of points of departure. The overall intention is to provide insights into why and how to plan and conduct metabolomic studies for regulatory toxicology purposes.
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http://dx.doi.org/10.1039/d0an02212hDOI Listing
February 2021

Respiratory tissue-associated commensal bacteria offer therapeutic potential against pneumococcal colonization.

Elife 2020 12 8;9. Epub 2020 Dec 8.

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

Under eubiotic conditions commensal microbes are known to provide a competitive barrier against invading bacterial pathogens in the intestinal tract, on the skin or on the vaginal mucosa. Here, we evaluate the role of lung microbiota in Pneumococcus colonization of the lungs. In eubiosis, the lungs of mice were dominantly colonized by . Differential analysis of 16S rRNA gene sequencing or specific qPCR of DNA from total organ homogenates broncho alveolar lavages implicated tight association of these bacteria with the host tissue. Pure conditioned culture medium inhibited growth and reduced the extension of pneumococcal chains. Growth inhibition in vitro was likely dependent on -produced lactic acid, since pH neutralization of the conditioned medium aborted the antibacterial effect. Finally, we demonstrate that provides a barrier against pneumococcal colonization in a respiratory dysbiosis model after an influenza A virus infection, when added therapeutically.
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http://dx.doi.org/10.7554/eLife.53581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723408PMC
December 2020

Determination of antiretroviral drugs for buyers' club in Switzerland using capillary electrophoresis methods.

Electrophoresis 2020 Dec 7. Epub 2020 Dec 7.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Switzerland.

Human immunodeficiency virus-acquired immunodeficiency syndrome continues to be a major global public health issue, having claimed almost 33 million lives to date. Due to the high cost of antiretroviral treatment, access to these drugs remains difficult for vulnerable populations, such as migrants and people living in prisons, who often do not have health insurance. These factors lead to poorer health outcomes and higher transmission rates. The personal importation scheme for unapproved generics from foreign countries is one option to access affordable human immunodeficiency virus treatment. However, the risk of importing falsified medicine remains high, and the quality control of unapproved drugs is lacking. In this context, three CE methods for the analysis of nine antiviral drugs found in commercial pharmaceutical formulations were evaluated. The selected compounds were emtricitabine, tenofovir disoproxil, tenofovir alafenamide, rilpivirine, efavirenz, raltegravir, dolutegravir, abacavir, and lamivudine. The developed methods were successfully applied to determine the active pharmaceutical ingredients of commercial formulations and unapproved generics. The quality control of unapproved generics by CE is an attractive approach due to its good standard of quality, low cost, ecofriendliness, and ease of implementation.
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http://dx.doi.org/10.1002/elps.202000216DOI Listing
December 2020

From a single steroid to the steroidome: Trends and analytical challenges.

J Steroid Biochem Mol Biol 2021 Feb 28;206:105797. Epub 2020 Nov 28.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Switzerland. Electronic address:

For several decades now, the analysis of steroids has been a key tool in the diagnosis and monitoring of numerous endocrine pathologies. Thus, the available methods used to analyze steroids in biological samples have dramatically evolved over time following the rapid pace of technology and scientific knowledge. This review aims to synthetize the advances in steroids' analysis, from classical approaches considering only a few steroids or a limited number of steroid ratios, up to the new steroid profiling strategies (steroidomics) monitoring large sets of steroids in biological matrices. In this context, the use of liquid chromatography coupled to mass spectrometry has emerged as the technique of choice for the simultaneous determination of a high number of steroids, including phase II metabolites, due to its sensitivity and robustness. However, the large dynamic range to be covered, the low natural abundance of some key steroids, the selectivity of the analytical methods, the extraction protocols, and the steroid ionization remain some of the current challenges in steroid analysis. This review provides an overview of the different analytical workflows available depending on the number of steroids under study. Special emphasis is given to sample treatment, acquisition strategy, data processing, steroid identification and quantification using LC-MS approaches. This work also outlines how the availability of steroid standards, the need for complementary analytical strategies and the improvement of calibration approaches are crucial for achieving complete steroidome quantification.
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http://dx.doi.org/10.1016/j.jsbmb.2020.105797DOI Listing
February 2021

[Buyers' club : an alternative for access to treatment ?]

Rev Med Suisse 2020 Nov;16(715):2228-2231

Direction médicale et qualité, HUG, 1211 Genève 14.

Rapid medication management for patients infected with HIV, HCV or HBV is key in optimizing a more favourable clinical response, in terms of morbidity, mortality, quality-of-life and reduced risk of transmission. If a drug is expensive, access to treatment for an uninsured patient with limited resources can be a hurdle that leads to forgoing healthcare for economic reasons. The buyers' club's objective is to provide logistics and/or financial assistance to a patient aiming to import qualitative generics for his personal use at an affordable price oversea. The drug is purchased on the internet.
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November 2020

Evaluation of Different Tandem MS Acquisition Modes to Support Metabolite Annotation in Human Plasma Using Ultra High-Performance Liquid Chromatography High-Resolution Mass Spectrometry for Untargeted Metabolomics.

Metabolites 2020 Nov 15;10(11). Epub 2020 Nov 15.

School of Pharmaceutical Sciences, University of Geneva, Rue Michel-Servet 1, 1211 Geneva 4, Switzerland.

Ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) is a powerful and essential technique for metabolite annotation in untargeted metabolomic applications. The aim of this study was to evaluate the performance of diverse tandem MS (MS/MS) acquisition modes, i.e., all ion fragmentation (AIF) and data-dependent analysis (DDA), with and without ion mobility spectrometry (IM), to annotate metabolites in human plasma. The influence of the LC separation was also evaluated by comparing the performance of MS/MS acquisition in combination with three complementary chromatographic separation modes: reversed-phase chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) with either an amide (aHILIC) or a zwitterionic (zHILIC) stationary phase. RPLC conditions were first chosen to investigate all the tandem MS modes, and we found out that DDA did not provide a significant additional amount of chemical coverage and that cleaner MS/MS spectra can be obtained by performing AIF acquisitions in combination with IM. Finally, we were able to annotate 338 unique metabolites and demonstrated that zHILIC was a powerful complementary approach to both the RPLC and aHILIC chromatographic modes. Moreover, a better analytical throughput was reached for an almost negligible loss of metabolite coverage when IM-AIF and AIF using ramped instead of fixed collision energies were used.
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http://dx.doi.org/10.3390/metabo10110464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697060PMC
November 2020

Exploring blood alterations in chronic kidney disease and haemodialysis using metabolomics.

Sci Rep 2020 11 11;10(1):19502. Epub 2020 Nov 11.

Service of Nephrology and Hypertension, Department of Medicine, Geneva University Hospitals, Rue Gabrielle-Perret-Gentil 4, 1205, Geneva, Switzerland.

Chronic kidney disease (CKD) is characterized by retention of uremic solutes. Compared to patients with non-dialysis dependent CKD, those requiring haemodialysis (HD) have increased morbidity and mortality. We wished to characterise metabolic patterns in CKD compared to HD patients using metabolomics. Prevalent non-HD CKD KDIGO stage 3b-4 and stage 5 HD outpatients were screened at a single tertiary hospital. Various liquid chromatography approaches hyphenated with mass spectrometry were used to identify 278 metabolites. Unsupervised and supervised data analyses were conducted to characterize metabolic patterns. 69 patients were included in the CKD group and 35 in the HD group. Unsupervised data analysis showed clear clustering of CKD, pre-dialysis (preHD) and post-dialysis (postHD) patients. Supervised data analysis revealed qualitative as well as quantitative differences in individual metabolites profiles between CKD, preHD and postHD states. An original metabolomics framework could discriminate between CKD stages and highlight HD effect based on 278 identified metabolites. Significant differences in metabolic patterns between CKD and HD patients were found overall as well as for specific metabolites. Those findings could explain clinical discrepancies between patients requiring HD and those with earlier stage of CKD.
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http://dx.doi.org/10.1038/s41598-020-76524-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658362PMC
November 2020

Network principal component analysis: a versatile tool for the investigation of multigroup and multiblock datasets.

Bioinformatics 2020 Nov 9. Epub 2020 Nov 9.

School of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland.

Motivation: Complex data structures composed of different groups of observations and blocks of variables are increasingly collected in many domains, including metabolomics. Analysing these high-dimensional data constitutes a challenge, and the objective of this article is to present an original multivariate method capable of explicitly taking into account links between data tables when they involve the same observations and/or variables. For that purpose, an extension of standard principal component analysis called NetPCA was developed.

Results: The proposed algorithm was illustrated as an efficient solution for addressing complex multigroup and multiblock datasets. A case study involving the analysis of metabolomic data with different annotation levels and originating from a chronic kidney disease (CKD) study was used to highlight the different aspects and the additional outputs of the method compared to standard PCA. On the one hand, the model parameters allowed an efficient evaluation of each group's influence to be performed. On the other hand, the relative relevance of each block of variables to the model provided decisive information for an objective interpretation of the different metabolic annotation levels.

Availability: NetPCA is available as a Python package with NumPy dependencies.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa954DOI Listing
November 2020

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Anal Chem 2020 10 1;92(20):14103-14112. Epub 2020 Oct 1.

Division of Systems Biomedicine and Pharmacology, Leiden Academic Centre for Drug Research, Leiden University, 2311 G Leiden, The Netherlands.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (μ) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The μ was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the μ for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.
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http://dx.doi.org/10.1021/acs.analchem.0c03129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581015PMC
October 2020

Is pain temporary and glory forever? Detection of tramadol using dried blood spot in cycling competitions.

Drug Test Anal 2020 Nov 6;12(11-12):1649-1657. Epub 2020 Sep 6.

School of Pharmaceutical Sciences, University of Geneva, University Medical Centre, Geneva 4, Switzerland.

Tramadol is a synthetic opioid drug used in the treatment of chronic and acute pain. An abnormal prevalence of its misuse in elite sport to overcome pain resulting from prolonged physical effort was recently reported. However, besides its antinociceptive effects, tramadol consumption is associated with negative effects such as numbness, confusion, and reduced alertness. This fact prompted the Union Cycliste Internationale to ban the use of tramadol in cycling competitions. Herein, we present the development of a dried blood spot (DBS) sample collection and preparation method followed by a liquid-chromatography mass spectrometry (LC-MS) analysis to rapidly determine the presence of tramadol and its two main metabolites in blood samples. The detection window of each analyte was evaluated and the analysis of performance on various MS platforms (HRMS and MS/MS) was assessed. Tramadol and its two main metabolites were detected up to 12 h after the intake of a single dose of 50 mg of tramadol in positive controls. In professional cycling competitions, 711 DBS samples collected from 361 different riders were analysed using the developed methodology, but all returned negative results (absence of parent and both metabolite compounds). In the context of professional cycling, we illustrate a valid method bringing together the easiness of collection and minimal sample preparation required by DBS, yet affording the performance standards of MS determination. The proposed method to detect tramadol and its metabolites was successfully implemented in cycling races with a probable strong deterrent effect.
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http://dx.doi.org/10.1002/dta.2923DOI Listing
November 2020

Evaluation of ion mobility in capillary electrophoresis coupled to mass spectrometry for the identification in metabolomics.

Electrophoresis 2021 Feb 6;42(4):342-349. Epub 2020 Sep 6.

Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, Geneva, Switzerland.

Currently, feature annotation remains one of the main challenges in untargeted metabolomics. In this context, the information provided by high-resolution mass spectrometry (HRMS) in addition to accurate mass can improve the quality of metabolite annotation, and MS/MS fragmentation patterns are widely used. Accurate mass and a separation index, such as retention time or effective mobility (μ ), in chromatographic and electrophoretic approaches, respectively, must be used for unequivocal metabolite identification. The possibility of measuring collision cross-section (CCS) values by using ion mobility (IM) is becoming increasingly popular in metabolomic studies thanks to the new generation of IM mass spectrometers. Based on their similar separation mechanisms involving electric field and the size of the compounds, the complementarity of CCS and μ needs to be evaluated. In this study, a comparison of CCS and μ was achieved in the context of feature identification ability in untargeted metabolomics by capillary zone electrophoresis (CZE) coupled with HRMS. This study confirms the high correlation of CCS with the mass of the studied metabolites as well as the orthogonality between accurate mass and μ , making this combination particularly interesting for the identification of several endogenous metabolites. The use of IM-MS remains of great interest for facilitating the annotation of neutral metabolites present in the electroosmotic flow (EOF) that are poorly or not separated by CZE.
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http://dx.doi.org/10.1002/elps.202000120DOI Listing
February 2021

Development and validation of an UHPLC-MS/MS method for extended serum steroid profiling in female populations.

Bioanalysis 2020 Jun 1;12(11):753-768. Epub 2020 Jun 1.

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne & Geneva, Lausanne University Hospital & University of Lausanne, Switzerland.

Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC-MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20%). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. The UHPLC-MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.
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http://dx.doi.org/10.4155/bio-2020-0046DOI Listing
June 2020

Comprehensive Examination of the Mouse Lung Metabolome Following Infection Using a Multiplatform Mass Spectrometry Approach.

J Proteome Res 2020 05 23;19(5):2053-2070. Epub 2020 Apr 23.

Centro de Metabolómica y Bioanálisis (CEMBIO), Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, Boadilla del Monte 28660, Spain.

The mechanisms whereby () rewires the host metabolism in vivo are surprisingly unexplored. Here, we used three high-resolution mass spectrometry platforms to track altered lung metabolic changes associated with infection of mice. The multiplatform data sets were merged using consensus orthogonal partial least squares-discriminant analysis (cOPLS-DA), an algorithm that allows for the joint interpretation of the results from a single multivariate analysis. We show that infection triggers a temporal and progressive catabolic state to satisfy the continuously changing energy demand to control infection. This causes dysregulation of metabolic and oxido-reductive pathways culminating in -associated wasting. Notably, high abundances of trimethylamine--oxide (TMAO), produced by the host from the bacterial metabolite trimethylamine upon infection, suggest that could exploit TMAO as an electron acceptor under anaerobic conditions. Overall, these new pathway alterations advance our understanding of the link between pathogenesis and metabolic dysregulation and could serve as a foundation for new therapeutic intervention strategies. Mass spectrometry data has been deposited in the Metabolomics Workbench repository (data-set identifier: ST001328).
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http://dx.doi.org/10.1021/acs.jproteome.9b00868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199213PMC
May 2020

Applicability of Supercritical fluid chromatography-Mass spectrometry to metabolomics. II-Assessment of a comprehensive library of metabolites and evaluation of biological matrices.

J Chromatogr A 2020 Jun 7;1620:461021. Epub 2020 Mar 7.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

In this work, the impact of biological matrices, such as plasma and urine, was evaluated under SFCHRMS in the field of metabolomics. For this purpose, a representative set of 49 metabolites were selected. The assessment of the matrix effects (ME), the impact of biological fluids on the quality of MS/MS spectra and the robustness of the SFCHRMS method were each taken into consideration. The results have highlighted a limited presence of ME in both plasma and urine, with 30% of the metabolites suffering from ME in plasma and 25% in urine, demonstrating a limited sensitivity loss in the presence of matrices. Subsequently, the MS/MS spectra evaluation was performed for further peak annotation. Their analyses have highlighted three different scenarios: 63% of the tested metabolites did not suffer from any interference regardless of the matrix; 21% were negatively impacted in only one matrix and the remaining 16% showed the presence of matrix-belonging compounds interfering in both urine and plasma. Finally, the assessment of retention times stability in the biological samples, has brought into evidence a remarkable robustness of the SFCHRMS method. Average RSD (%) values of retention times for spiked metabolites were equal or below 0.5%, in the two biological fluids over a period of three weeks. In the second part of the work, the evaluation of the Sigma Mass Spectrometry Metabolite Library of Standards containing 597 metabolites, under SFCHRMS conditions was performed. A total detectability of the commercial library up to 66% was reached. Among the families of detected metabolites, large percentages were met for some of them. Highly polar metabolites such as amino acids (87%), nucleosides (85%) and carbohydrates (71%) have demonstrated important success rates, equally for hydrophobic analytes such as steroids (78%) and lipids (71%). On the negative side, very poor performance was found for phosphorylated metabolites, namely phosphate-containing compounds (14%) and nucleotides (31%).
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http://dx.doi.org/10.1016/j.chroma.2020.461021DOI Listing
June 2020

Interlaboratory and Interplatform Study of Steroids Collision Cross Section by Traveling Wave Ion Mobility Spectrometry.

Anal Chem 2020 04 24;92(7):5013-5022. Epub 2020 Mar 24.

LABERCA, Oniris, INRAE, F-44307 Nantes, France.

Collision cross section (CCS) databases based on single-laboratory measurements must be cross-validated to extend their use in peak annotation. This work addresses the validation of the first comprehensive CCS database for steroids. First, its long-term robustness was evaluated (i.e., a year and a half after database generation; Synapt G2-S instrument; bias within ±1.0% for 157 ions, 95.7% of the total ions). It was further cross-validated by three external laboratories, including two different TWIMS platforms (i.e., Synapt G2-Si and two Vion IMS QToF; bias within the threshold of ±2.0% for 98.8, 79.9, and 94.0% of the total ions detected by each instrument, respectively). Finally, a cross-laboratory CCS database was built for 87 steroids (142 ions). The cross-laboratory database consists of average CCS values obtained by the four TWIMS instruments in triplicate measurements. In general, lower deviations were observed between CCS measurements and reference values when the cross-laboratory database was applied as a reference instead of the single-laboratory database. Relative standard deviations below 1.5% were observed for interlaboratory measurements (<1.0% for 85.2% of ions) and bias between average values and CCS measurements was within the range of ±1.5% for 96.8% of all cases. In the context of this interlaboratory study, this threshold was also suitable for CCS measurements of steroid metabolites in calf urine. Greater deviations were observed for steroid sulfates in complex urine samples of adult bovines, showing a slight matrix effect. The implementation of a scoring system for the application of the CCS descriptor in peak annotation is also discussed.
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http://dx.doi.org/10.1021/acs.analchem.9b05247DOI Listing
April 2020

Semen endocannabinoids are correlated to sperm quality in a cohort of 200 young Swiss men.

Andrology 2020 09 6;8(5):1126-1135. Epub 2020 Apr 6.

Service of Clinical Chemistry & Toxicology, Central Institute of Hospitals, Hospital of Valais, Sion, Switzerland.

Background: A role for endocannabinoids in the male and female reproductive systems has been highlighted during the recent decades. Some of these compounds bind the cannabinoid CB1 receptor, which is abundantly expressed in the central nervous system but also present in the reproductive system, while others act as 'entourage compounds' modulators.

Objectives: The present study aimed at evaluating the relationship between sperm quality and endocannabinoid profiles in a cohort of 200 young Swiss men and whether the presence of specific xenobiotics could influence these profiles.

Materials And Methods: Semen analysis was performed according to WHO guidelines. Endocannabinoid profiles in blood and semen, as well as bisphenol A and S in urine, were determined by LC-MSMS methods. The presence of selected drugs was tested in urine by immunological screening, and urinary tetrahydrocannabinol (THC) metabolites were quantified by GC-MS.

Results: Anandamide concentrations in seminal fluid and oleoylethanolamide (OEA) concentrations in blood serum appeared inversely correlated with sperm motility, while semen palmytoylethanolamide (PEA) was positively linked to sperm concentration. Moreover, OEA and PEA in seminal fluid were associated with better sperm morphology. Interestingly, the concentrations of the same endocannabinoids measured in both blood and semen were not correlated, and the presence of THC metabolites in some individuals was linked to lower concentrations of endocannabinoids.

Conclusions: In the context of the general decline of the sperm count observed within the male population, endocannabinoids in semen constitute a class of promising biochemical markers that open new perspectives as a complement for the usual evaluation of semen quality or for the toxicological screening of individuals' exposure to putative endocrine disruptors.
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http://dx.doi.org/10.1111/andr.12785DOI Listing
September 2020

Bacterial cell cycle control by citrate synthase independent of enzymatic activity.

Elife 2020 03 9;9. Epub 2020 Mar 9.

Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

Proliferating cells must coordinate central metabolism with the cell cycle. How central energy metabolism regulates bacterial cell cycle functions is not well understood. Our forward genetic selection unearthed the Krebs cycle enzyme citrate synthase (CitA) as a checkpoint regulator controlling the G→S transition in the polarized alpha-proteobacterium , a model for cell cycle regulation and asymmetric cell division. We find that loss of CitA promotes the accumulation of active CtrA, an essential cell cycle transcriptional regulator that maintains cells in G-phase, provided that the (p)ppGpp alarmone is present. The enzymatic activity of CitA is dispensable for CtrA control, and functional citrate synthase paralogs cannot replace CitA in promoting S-phase entry. Our evidence suggests that CitA was appropriated specifically to function as a moonlighting enzyme to link central energy metabolism with S-phase entry. Control of the G-phase by a central metabolic enzyme may be a common mechanism of cellular regulation.
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http://dx.doi.org/10.7554/eLife.52272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083601PMC
March 2020

Implementation of liquid chromatography-high resolution mass spectrometry methods for untargeted metabolomic analyses of biological samples: A tutorial.

Anal Chim Acta 2020 Apr 2;1105:28-44. Epub 2020 Jan 2.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Rue Michel-Servet 1, 1211, Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland. Electronic address:

Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. By its nature, this is a highly interdisciplinary field of research, and mastering all of the steps comprised in the pipeline can be a challenging task, especially for those researchers new to the topic. In this tutorial, we aim to provide an overview of the most widely adopted methods of performing LC-HRMS-based untargeted metabolomics of biological samples. A detailed protocol is provided in the Supplementary Information for rapidly implementing a basic screening workflow in a laboratory setting. This tutorial covers experimental design, sample preparation and analysis, signal processing and data treatment, and, finally, data analysis and its biological interpretation. Each section is accompanied by up-to-date literature to guide readers through the preparation and optimization of such a workflow, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls.
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http://dx.doi.org/10.1016/j.aca.2019.12.062DOI Listing
April 2020

Combining the advantages of multilevel and orthogonal partial least squares data analysis for longitudinal metabolomics: Application to kidney transplantation.

Anal Chim Acta 2020 Feb 23;1099:26-38. Epub 2019 Nov 23.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland. Electronic address:

Kidney transplantation is one of the renal replacement options in patients suffering from end-stage renal disease (ESRD). After a transplant, patient follow-up is essential and is mostly based on immunosuppressive drug levels control, creatinine measurement and kidney biopsy in case of a rejection suspicion. The extensive analysis of metabolite levels offered by metabolomics might improve patient monitoring, help in the surveillance of the restoration of a "normal" renal function and possibly also predict rejection. The longitudinal follow-up of those patients with repeated measurements is useful to understand changes and decide whether an intervention is necessary. The time modality, therefore, constitutes a specific dimension in the data structure, requiring dedicated consideration for proper statistical analysis. The handling of specific data structures in metabolomics has received strong interest in recent years. In this work, we demonstrated the recently developed ANOVA multiblock OPLS (AMOPLS) to efficiently analyse longitudinal metabolomic data by considering the intrinsic experimental design. Indeed, AMOPLS combines the advantages of multilevel approaches and OPLS by separating between and within individual variations using dedicated predictive components, while removing most uncorrelated variations in the orthogonal component(s), thus facilitating interpretation. This modelling approach was applied to a clinical cohort study aiming to evaluate the impact of kidney transplantation over time on the plasma metabolic profile of graft patients and donor volunteers. A dataset of 266 plasma metabolites was identified using an LC-MS multiplatform analytical setup. Two separate AMOPLS models were computed: one for the recipient group and one for the donor group. The results highlighted the benefits of transplantation for recipients and the relatively low impacts on blood metabolites of donor volunteers.
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http://dx.doi.org/10.1016/j.aca.2019.11.050DOI Listing
February 2020

Optimized one-pot derivatization and enantioseparation of cysteine: Application to the study of a dietary supplement.

J Pharm Biomed Anal 2020 Feb 23;180:113066. Epub 2019 Dec 23.

University of Perugia, Department of Pharmaceutical Sciences, Via Fabretti, 48, 06123 Perugia, Italy. Electronic address:

Cysteine is a sulfur-containing amino acid which plays an outstanding role in many biological pathways in mammals. The analysis and quantification of native cysteine remains a critical issue due to its highly reactive thiol group evolving to the disulfide cystine derivative through oxidation reaction. Aimed at improving the derivative stability, cysteine was labelled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), which reacts with both amino and thiol groups. The derivatization was optimized and the chemical identity of the reaction product was assessed via high-resolution mass spectrometry. The NBD-cysteine derivative resulted stable for 10 days. This derivative was enantioresolved (α and R equal to 1.25 and 2.70, respectively) thanks to a (R,R)-Whelk-O1 phase with the following chromatographic setting: eluent, MeOH/water-90/10 (v/v) with 15 mM ammonium formate (pwsH 6.0); column temperature, 35 °C; flow rate, 1.0 mL/min. The developed method was validated following the ICH guidelines and applied for the quality control of a L-cysteine containing dietary supplement.
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http://dx.doi.org/10.1016/j.jpba.2019.113066DOI Listing
February 2020

Profiling of anabolic androgenic steroids and selective androgen receptor modulators for interference with adrenal steroidogenesis.

Biochem Pharmacol 2020 02 27;172:113781. Epub 2019 Dec 27.

Swiss Centre for Applied Human Toxicology (SCAHT), University of Basel, Basel, Switzerland; Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland. Electronic address:

Anabolic-androgenic steroids (AAS) are testosterone derivatives developed for steroid-replacement and treatment of debilitating conditions. They are widely used by athletes in elite sports and bodybuilding due to their muscle-building and performance-enhancing properties. Excessive AAS use is associated with cardiovascular diseases, mood changes, endocrine and metabolic disorders; however, the underlying mechanisms remain unknown. Selective androgen receptor modulators (SARMs) aim to reduce adverse androgenic effects, while maximizing anabolic effects. This study assessed potential steroidogenic disturbances of 19 AAS and 3 SARMs in human adrenocortical carcinoma H295R cells, comparing basal and forskolin-activated states by mass spectrometry-based quantification of nine major adrenal steroids. Mesterolone, mestanolone and methenolone increased mineralocorticoid but decreased adrenal androgen production, indicating CYP17A1 dysfunction. Cell-free activity assays failed to detect direct CYP17A1 inhibition, supported by molecular modeling. The mRNA expression levels of 3β-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2 were unaffected, suggesting indirect inhibition involving post-translational modification and/or impaired protein stability. Clostebol and oxymetholone decreased corticosteroid but increased dehydroepiandrosterone biosynthesis in H295R cells, suggesting CYP21A2 inhibition, sustained by molecular modeling. These AAS did not affect the expression of key steroidogenic genes. None of the SARMs tested interfered with steroidogenesis. The chosen approach allowed the grouping of AAS according to their steroidogenic-disrupting effects and provided initial mechanistic information. Mesterolone, mestanolone and methenolone potentially promote hypertension and cardiovascular diseases via excessive mineralocorticoid biosynthesis. Clostebol and oxymetholone might cause metabolic disturbances by suppressing corticosteroid production, resulting in adrenal hyperplasia. The non-steroidal SARMs exhibit an improved safety profile and represent a preferred therapeutic option.
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http://dx.doi.org/10.1016/j.bcp.2019.113781DOI Listing
February 2020

Supercritical fluid chromatography-mass spectrometry in routine anti-doping analyses: Estimation of retention time variability under reproducible conditions.

J Chromatogr A 2020 Apr 12;1616:460780. Epub 2019 Dec 12.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland. Electronic address:

The aim of this study was to estimate the retention time variability under reproducible conditions of an SFC-MS analytical method for routine anti-doping analyses. For this purpose, a set of 51 doping agents, as neat standards and spiked in diluted urine, was used to assess their retention times variability over a period of four months, as well as the column inter-batch reproducibility. Three UHPSFC stationary phases have been employed, the Acquity UPC Torus 2-Picolylamine (2-PIC), UPC Viridis BEH and Acquity UPLC HSS C18 SB. Four columns, per column chemistry, have been purchased to represent three different production lots, with a total of twelve columns employed in this study. The two columns from the same lot were applied to the first part of the study (repeatability), whereas the representative of three different lots were employed in the second part (robustness). In terms of organic modifier, a mixture of 98% MeOH and 2% water containing 20 mM ammonium formate was selected in order to limit the formation of methyl-silyl ethers on the surface of the silica particles, thus potentially improving the repeatability of retention times. A comparison with an UHPLC reference analytical method was made with the same set of analytes. The average relative standard deviations (RSD%), represented in split violin plots, illustrate how two of the UHPSFC columns assessed in this study were able to generate an excellent repeatability of retention times, with results that are in a similar range of those generated by UHPLC. Moreover, the Torus 2-PIC has proven to be the best of the three stationary phases, with an impressive RSD% of 0.5% in diluted urine relative to the inter-month variability. Finally, the inter-batch reproducibility assessment has highlighted a good reproducibility of the same stationary phase belonging to different production lots for all three column chemistries assessed, with the Viridis BEH silica generating an RSD% of 0.7% in diluted urine. Higher values of RSD (%) were found for Torus 2-PIC and HSS C18 SB, respectively of 1.0% and 1.6%.
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http://dx.doi.org/10.1016/j.chroma.2019.460780DOI Listing
April 2020

Electromembrane Extraction of Highly Polar Compounds: Analysis of Cardiovascular Biomarkers in Plasma.

Metabolites 2019 Dec 18;10(1). Epub 2019 Dec 18.

Division of Systems Biomedicine and Pharmacology, Leiden Academic Center for Drug Research, Leiden University, Einsteinweg 55, 2333 CC Leiden, The Netherlands.

Cardiovascular diseases (CVDs) represent a major concern in today's society, with more than 17.5 million deaths reported annually worldwide. Recently, five metabolites related to the gut metabolism of phospholipids were identified as promising predictive biomarker candidates for CVD. Validation of those biomarker candidates is crucial for applications to the clinic, showing the need for high-throughput analysis of large numbers of samples. These five compounds, trimethylamine N-oxide (TMAO), choline, betaine, l-carnitine, and deoxy-l-carnitine (4-trimethylammoniobutanoic acid), are highly polar compounds and show poor retention on conventional reversed phase chromatography, which can lead to strong matrix effects when using mass spectrometry detection, especially when high-throughput analysis approaches are used with limited separation of analytes from interferences. In order to reduce the potential matrix effects, we propose a novel fast parallel electromembrane extraction (Pa-EME) method for the analysis of these metabolites in plasma samples. The evaluation of Pa-EME parameters was performed using multi segment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Recoveries up to 100% were achieved, with variability as low as 2%. Overall, this study highlights the necessity of protein precipitation prior to EME for the extraction of highly polar compounds. The developed Pa-EME method was evaluated in terms of concentration range and response function, as well as matrix effects using fast-LC-MS/MS. Finally, the developed workflow was compared to conventional sample pre-treatment, i.e., protein precipitation using methanol, and fast-LC-MS/MS. Data show very strong correlations between both workflows, highlighting the great potential of Pa-EME for high-throughput biological applications.
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http://dx.doi.org/10.3390/metabo10010004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022788PMC
December 2019

Steroid profile analysis by LC-HRMS in human seminal fluid.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Jan 9;1136:121929. Epub 2019 Dec 9.

Analytical Sciences, Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Rue Michel-Servet 1, 1206 Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland. Electronic address:

Steroids are essential hormones that play a crucial role in homeostasis of many biological processes including sexual development, spermatogenesis, sperm physiology and fertility. Although steroids have been largely studied in many biological matrices (such as urine and plasma), there is very limited information of the steroid content and their study as potential indicators of the quality of the seminal fluid. In this study, a LC-HRMS (liquid chromatography-high resolution mass spectrometry) strategy has been developed in order to obtain the extended steroid profile of human seminal fluid. A comparison between supported liquid extraction (SLE) and solid liquid extraction (SPE) was carried out and the chosen SPE method was further optimized to evidence the largest possible number of compounds. Steroids were automatically annotated by using DynaStI, a publicly available retention time prediction tool developed in our lab, to match the experimental data (i.e. accurate mass and t). Altogether, these resources allowed us to develop a post-targeted approach able to consistently detect 41 steroids in seminal fluid (with half of them being androgens). Such steroid pattern was found to be stable across different extraction times and injection days. In addition to accurate mass and retention time, the identity of 70% of the steroids contained in such steroid profile was confirmed by comparing their fragmentation patterns in real samples to those of pure commercial standards. Finally, the workflow was applied to compare and distinguish the steroid profile in seminal fluid from healthy volunteers (n = 7, with one of them being a vasectomized subject). In all, the developed steroidomics strategy allows to reliably monitor an extended panel of 41 steroids in human seminal fluid.
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http://dx.doi.org/10.1016/j.jchromb.2019.121929DOI Listing
January 2020

Analytical strategies for the determination of amino acids: Past, present and future trends.

J Chromatogr B Analyt Technol Biomed Life Sci 2019 Nov 22;1132:121819. Epub 2019 Oct 22.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Switzerland.

This review describes the analytical methods that have been developed over the years to tackle the high polarity and non-chromophoric nature of amino acids (AAs). First, the historical methods are briefly presented, with a strong focus on the use of derivatization reagents to make AAs detectable with spectroscopic techniques (ultraviolet and fluorescence) and/or sufficiently retained in reversed phase liquid chromatography. Then, an overview of the current analytical strategies for achiral separation of AAs is provided, in which mass spectrometry (MS) becomes the most widely used detection mode in combination with innovative liquid chromatography or capillary electrophoresis conditions to detect AAs at very low concentration in complex matrixes. Finally, some future trends of AA analysis are provided in the last section of the review, including the use of supercritical fluid chromatography (SFC), multidimensional liquid chromatography and electrophoretic separations, hyphenation of ion exchange chromatography to mass spectrometry, and use of ion mobility spectrometry mass spectrometry (IM-MS). Various application examples will also be presented throughout the review to highlight the benefits and limitations of these different analytical approaches for AAs determination.
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http://dx.doi.org/10.1016/j.jchromb.2019.121819DOI Listing
November 2019

Choosing an Optimal Sample Preparation in for Untargeted Metabolomics Approaches.

Metabolites 2019 Sep 20;9(10). Epub 2019 Sep 20.

Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University Medical Centre, 1206 Geneva, Switzerland.

Untargeted metabolomics aims to provide a global picture of the metabolites present in the system under study. To this end, making a careful choice of sample preparation is mandatory to obtain reliable and reproducible biological information. In this study, eight different sample preparation techniques were evaluated using as a model for Gram-negative bacteria. Two cell retrieval systems, two quenching and extraction solvents, and two cell disruption procedures were combined in a full factorial experimental design. To fully exploit the multivariate structure of the generated data, the ANOVA multiblock orthogonal partial least squares (AMOPLS) algorithm was employed to decompose the contribution of each factor studied and their potential interactions for a set of annotated metabolites. All main effects of the factors studied were found to have a significant contribution on the total observed variability. Cell retrieval, quenching and extraction solvent, and cell disrupting mechanism accounted respectively for 27.6%, 8.4%, and 7.0% of the total variability. The reproducibility and metabolome coverage of the sample preparation procedures were then compared and evaluated in terms of relative standard deviation (RSD) on the area for the detected metabolites. The protocol showing the best performance in terms of recovery, versatility, and variability was centrifugation for cell retrieval, using MeOH:HO (8:2) as quenching and extraction solvent, and freeze-thaw cycles as the cell disrupting mechanism.
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http://dx.doi.org/10.3390/metabo9100193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836107PMC
September 2019