Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MSE with a single-gradient elution of 36min (including re-equilibration time) in the negative electrospray ionization mode. MSE analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids. Figure UHPLC-QTOF-MSE acquisition mode for DHEAG in urine sampl

Etude métabolomique de la réponse à la blessure chez "Arabidopsis thaliana" par chromatographie liquide à ultra-haute pression (UHPLC) couplée à la spectrométrie de masse à temps de vol (TOF-MS)

Un concept d'analyse métabolomique séquentielle basée sur la chromatographie liquide à ultra-haute pression couplée à la spectrométrie de masse à temps de vol (UHPLC-TOF-MS) a été développé pour évaluer de la manière la plus complète possible la réponse à la blessure chez "Arabidopsis thaliana". Lors de la première étape, une méthode rapide de criblage a permis l'évaluation des modifications métaboliques chez un grand nombre d'individus stressés. Cette étape a fourni des renseignements sur les composés potentiellement induits lors du phénomène de stress et a permis de former des assemblages d'individus représentatifs. Dans une seconde étape, des analyses confirmatoires ont été effectuées avec une haute résolution chromatographique sur des assemblages où la variabilité biologique est réduite. Cette deuxième étape a ainsi permis de confirmer les résultats obtenus en criblage et de sélectionner les biomarqueurs les plus pertinents en vue d'une caractérisation structurale complète

Oral Liquid Formulation of Levothyroxine Is Stable in Breakfast Beverages and May Improve Thyroid Patient Compliance

Patients on treatment with levothyroxine (T4) are informed to take this drug in the morning, at least 30 min before having breakfast. A significant decrease of T4 absorption was reported, in fact, when T4 solid formulations are taken with food or coffee. According to preliminary clinical study reports, administration of T4 oral solution appears to be less sensitive to the effect of breakfast beverages on oral bioavailability. In the present study, stability of T4 oral solution added to breakfast beverages was investigated. A 1 mL ampoule of single-dose Tirosint® oral solution (IBSA Farmaceutici Italia, Lodi, Italy) was poured into defined volumes of milk, tea, coffee, and coffee with milk warmed at 50 °C, as well as in orange juice at room temperature. Samples were sequentially collected up to 20 min and analyzed by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. The results of the study demonstrated that T4 is stable in all beverages after 20 min incubation. Demonstration of T4 stability is a prerequisite for a thorough evaluation of the effect of breakfast beverages on the bioavailability of T4 given as oral solution and for a better understanding of the reasons underlying a decreased T4 bioavailability administered as solid formulations

Synergy at the 'Ecole de Pharmacie Geneve-Lausannel': Methodology developments for the treatment of complex metabolomic datasets with data mining

With the advances in analytical techniques and data mining, the chances to elucidate a plant metabolome and understand the metabolite variation in response to external stimuli such as stress are gradually becoming feasible in a global manner. This approach represents a very important research challenge because of the structural diversity of the metabolites and the convolute nature of biological matrices. Based on a new collaborative framework emerging from the recent creation of the EPGL, a project aimed at the development of methodology for the treatment of complex metabolomic datasets with data mining has been initiated with the expertise of different EPGL laboratories. In this paper the strategies used for the study of the metabolic variations in a biological model (the effect of mechanical wounding on Arabidopsis thaliana) are described. Metabolite profiling based on micro-extraction and LC/MS analysis with various detection methods has been used. Data were parsed in the form of ion maps and treatment of this complex set of MS information was performed with dedicated bioinformatic tools. This approach should enable the evaluation of metabolome variations in a comprehensive manner for a better understanding of complex biological mechanisms and for the detection of novel bioactive molecule

Highlights of Analytical Chemistry in Switzerland: Plant Metabolomics – Strategies for Biomarker Detection, Isolation, and Identification

Recent developments in analytical methods and data mining have permitted metabolomics to evolve from an ambitious concept to a valuable technology which provides a global picture of molecular organisation at the metabolite level

Detection of cocaine on euro banknotes; Development of a practical approach for the interpretation of suspect cases.

The presence of traces of narcotics, particularly cocaine, on banknotes in circulation is a known and widespread fact in all countries. While linked to consumption and trafficking (primary contamination), their spread is due to direct contact with other banknotes during machine counting and cash financial transactions. The mere detection of traces of cocaine on a sample of banknotes is therefore not sufficient evidence to establish the banknote's illegal origin. Increasing levels of contamination are recorded close to (in terms of both place and time) the first direct contact with the substance. The analysis must thus be able to demonstrate that the concentration of narcotics on the banknotes is significantly higher (statistically) in terms of value and frequency than would be expected from background noise alone. Even in that event, however, this evidence has to be substantiated with additional confirmations linking banknotes to the person and this latter to drug trafficking and/or dealing. In general, an in-depth and systematic analysis of all seized banknotes to search for traces of narcotics is not only prohibitive in terms of cost, but also unnecessary. If the sampling procedure is respected, the Swiss Federal Supreme Court actually recognizes IMS (ion mobility spectrometry) as a lawful method for checking the degree of banknote contamination, as well as all the statistical conclusions that can be drawn from it. In special cases, the prosecutor may require confirmation of IMS results by a laboratory test (liquid/gas chromatography-mass spectrometry). Using a non-destructive sampling procedure (suction on swabs) we determined the presence of cocaine on 978 circulating euro banknotes, randomly collected at 5 swiss customs offices, with IMS and LC-MS/MS in order to establish a normal (background) contamination level. A significant proportion (46.4%) of the euro banknotes analysed by LC-MS/MS had cocaine concentrations above the quantification limit (1 ng/swab). However, the extent of contamination is a determining factor: 94.6% of the banknotes in circulation have cocaine concentrations equal to or less than 10 ng/swab and only 3.4% have cocaine concentrations above 20 ng/swab. By comparison, only 27.3% and 13.4% respectively of the seized banknotes (2 real cases) had cocaine concentrations equal to or less than 10 ng/swab, but 63.5% and 86.7% respectively had cocaine concentrations above 20 ng/swab. We also describe a Komolgorov-Smirnov test model used to determine the presence of an "abnormal" level of contamination relative to the reference banknotes (banknotes in circulation or background noise) effectively and within realistic practical and theoretical frameworks. This model provides a quantifiable and statistically significant result that not only simplifies data interpretation, but also facilitates admissibility as forensic evidence in proceedings. When applied to the sized banknotes using both IMS and LC-MS/MS data, we obtain fully consistent and sounding conclusions

Multivariate data analysis of rapid LC-TOF/MS experiments from Arabidopsis thaliana stressed by wounding

A metabolomic strategy based on a rapid high performance liquid chromatography (LC) method coupled with a high resolution time-of-flight (TOF) mass spectrometer (MS) has been developed to detect metabolomic modifications occurring in Arabidopsis thaliana upon stress induction. The method was evaluated for its potential of fast discrimination between stressed (wounding by forceps) versus control Arabidopsis specimens, based on a metabolomic fingerprinting survey. Multivariate analysis was applied to handle the large amount of data generated and extract relevant information. Signal variations were filtered with an ANOVA test to select discriminant detected analytes between plant sets. Selected ions were then processed through a data reduction procedure applied to the chromatographic information generating Total Mass Spectra (TMS) and further investigated by multivariate analysis. Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) on principal coordinates were combined for data treatment. PCA and HCA demonstrate a clear clusterisation of plant specimens selecting the highest discriminating ions given by the complete data analysis and leading to the specific identification of discrete induced metabolites or spiked compounds. Putative stress induced compounds issued from this screening procedure were analysed using a conventional chromatographic gradient. This sequential strategy (screening-confirmation) was developed for the investigation of new low molecular mass regulators involved in plant defence signalling

Metabolite profiling of plant extracts by ultra-high-pressure liquid chromatography at elevated temperature coupled to time-of-flight mass spectrometry

Detailed metabolite profiling of crude plant extracts, mandatory for both quality control and metabolomics purposes, requires high-resolution separation and sensitive detection with a reasonable sample throughput. In this respect, the use of ultra-high-pressure liquid chromatography (UHPLC) working at high temperature (HT) and coupled to time-of-flight mass spectrometry (TOF-MS) was evaluated in the present study in terms of achievable peak capacities for given analysis times. Prior to the analysis of complex mixtures, the effects of TOF-MS detection on peak capacity were evaluated, and a loss of 15-30% compared to UV was observed due to the additional band broadening generated by this detector. Extracts from a model plant Arabidopsis thaliana and from a widely used phytochemical preparation Ginkgo biloba, as well as a standard mixture of representative natural products (NPs), have been analyzed. As expected from the theory, the increase in mobile phase temperature of up to 90 degrees C for the profiling of extracts containing metabolites spread over a large polarity range (e.g., Arabidopsis thaliana) generated similar peak capacities to those obtained at room temperature, but with a 2- to 3-fold reduction in analysis time, demonstrating the power of this approach for such applications. On the other hand, for the analysis of more polar extracts (e.g., Ginkgo biloba), the use of higher temperature was not beneficial, as it induced a significant decrease in retention, and thus resolving power, because of the increase in elution strength. The use of HT-UHPLC-TOF-MS raised the question of NP stability under high temperature conditions. This work demonstrated that no apparent degradation was evidenced at high temperature for a representative mixture of NPs and also for the different metabolites detected in the selected plant extracts

Optimized liquid chromatography-mass spectrometry approach for the isolation of minor stress biomarkers in plant extracts and their identification by capillary nuclear magnetic resonance

A LC-MS approach is presented for the isolation of minor key plant biomarkers, in view of their characterization by NMR at the microgram scale. Due to the complexity of plant extracts, the purification of metabolites present in low concentrations is critical. The strategy used relies on the optimization of the chromatographic analysis using ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOF-MS), thanks to modelling software. The optimized method is then transferred to semi-preparative LC conditions with MS detection. The approach is illustrated by the isolation of wound-induced jasmonate derivatives revealed by a metabolomic study in Arabidopsis thaliana leaves and their subsequent characterization by capillary NMR (CapNMR)