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

Application de la chromatographie liquide à ultra-haute pression couplée à un spectromètre de masse quadripôle à temps de vol pour l'analyse antidopage

Pour la lutte antidopage, des outils analytiques toujours plus performants sont nécessaires afin de détecter des produits dopants en très faibles quantités dans des matrices biologiques. Dans le cadre de cette thèse, l'application de la chromatographie liquide à ultra-haute pression (UHPLC) couplée à la spectrométrie de masse quadripôle à temps de vol a été évaluée pour l'analyse antidopage. La première partie s'est consacrée à la mise au point d'une méthode d'analyse rapide de produits dopants dans l'urine. Une procédure de criblage, suivie d'une analyse de confirmation ont été développées pour permettre un gain en temps d'analyse, une procédure simplifiée de prétraitement de l'échantillon (dilution et injection) et la possibilité de retraiter les données ultérieurement. Dans la seconde partie, une approche de profilage de stéroïdes anabolisants dans l'urine a été mise au point, afin d'améliorer la fenêtre de détection d'un dopage à la testostérone, de par la découverte de nouveaux biomarqueurs

Fast chiral separation of drugs using columns packed with sub-2 mum particles and ultra-high pressure

The use of columns packed with sub-2 mum particles in liquid chromatography with very high pressure conditions (known as UHPLC) was investigated for the fast enantioseparation of drugs. Two different procedures were evaluated and compared using amphetamine derivatives and beta-blockers as model compounds. In one case, cyclodextrins (CD) were directly added to the mobile phase and chiral separations were carried out in less than 5 min. However, this strategy suffered from several drawbacks linked to column lifetime and low chromatographic efficiencies. In the other case, the analysis of enantiomers was carried out after a derivatization procedure using two different reagents, 2,3,4-tri-O-acetyl-alpha-D-arabinopyranosyl isothiocyanate (AITC) and N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent). Separation of several amphetamine derivatives contained within the same sample was achieved in 2-5 min with high efficiency and selectivity. The proposed approach was also successfully applied to the enantiomeric purity determination of (+)-(S)-amphetamine and (+)-(S)-methamphetamine. Similar results were obtained with beta-blockers, and the separation of 10 enantiomers was carried out in less than 3 min, whereas the individual separation of several beta-blocker enantiomers was performed in 1 min or less. Chirality, 2010. (c) 2009 Wiley-Liss, Inc

A distinct fatty acid profile underlies the reduced inflammatory state of metabolically healthy obese individuals.

Obesity is associated with numerous health complications; however, a subgroup of obese individuals (termed the metabolically healthy obese or MHO) appear to have lower risk for complications such as type 2 diabetes and cardiovascular disease. Emerging evidence suggests that MHO individuals have reduced inflammation compared to their metabolically unhealthy obese (MUO) counterparts. As it is recognized that fatty acids (FAs) have a strong relationship with inflammation, the current study aimed to uncover if the reduced inflammation observed in MHO individuals is mirrored by a more favourable FA profile.Fasted serum samples were collected from lean healthy (LH), MHO, and MUO participants (n = 10/group) recruited from the Diabetes Risk Assessment study. A panel of pro- and anti-inflammatory markers were measured by immunoassay. Total serum FA profiling, as well as the FA composition of circulating phospholipids (PL) and triglycerides (TG), was measured by gas chromatography. ANOVA and Mann-Whitney-Wilcoxon tests were used to assess statistical significance between the groups (P<0.05).MHO and MUO individuals had similar BMI and body fat %; however, lipid parameters in MHO individuals more closely resembled that of LH individuals. MHO individuals had circulating levels of high sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) similar to LH individuals, while levels of platelet derived growth factor-ββ (PDGF-ββ) were intermediate to that of LH and MUO individuals. FA profiling analysis combined with discriminant analysis modelling highlighted a panel of nine FAs (consisting of three saturated, three monounsaturated, and three polyunsaturated FAs) in PL and TG fractions that distinguished the three groups. Specifically, saturated FA (myristic and stearic acids) levels in MHO individuals resembled that of LH individuals.Our results suggest that the reduced inflammatory state of MHO individuals compared to MUO individuals may stem, in part, from a more favourable underlying FA profile

Analyse métabolomique dans l'urine par UHPLC-QTOF-MSE : profilage des stéroïdes appliqué au dépistage antidopage

Une approche métabolomique est proposée pour l'analyse non-ciblée des métabolites de stéroïdes anabolisants androgéniques présents dans l'urine. Une méthode analytique, associant la chromatographie liquide à ultra-haute pression et un spectromètre de masse hybride quadripôle à temps de vol, a été utilisée pour l'analyse d'échantillons d'une étude clinique sur l'effet de la prise de testostérone. L'ensemble de données obtenu est caractérisé par une structure à trois entrées (volontaires x temps x stéroïdes mesurés) a été utilisée pour exploiter un profil de métabolites étendu permettant d'améliorer la sensibilité des tests antidopage relatifs à l'administration de testostérone

Users of ‘diet’ drinks who think that sweetness is calories

We present the first experiment that was based on a novel analysis of the mental processes of choice. Sensed material characteristics such as the sweetness of a drink and symbolic attributes such as the source of sweetness stated on the label are put into the same units of influence on the response. Most users of low-calorie drinks thought about the energy in a drink quite differently from the way they decided how sweet and how low in calories they liked the drink to be. Also the female diet drink users thought about energy content differently from most of the male users of sugar drinks. In both groups’ ratings of likelihood of choice and in sugar drink users’ estimates of energy content, sweetness and labelled calories were usually treated as separate stimuli or ideas. In contrast, some female diet drink users treated sweetness and perceived calories as the same, whereas no male sugar drink user did. Such findings illustrate how this approach spans the gap between sensory perception and conceptualised knowledge

Associations between amino acids and derivatives, and fatty acids, with fasting and postprandial indices of insulin sensitivity.

<p>Significance was set at p ≤ 0.01 using Bonferroni correction for multiple testing. Linear regressions were adjusted for age, sex, and BMI.</p><p>Associations between amino acids and derivatives, and fatty acids, with fasting and postprandial indices of insulin sensitivity.</p

Mean %PP of plasma amino acid and derivatives measured by CE-MS for lean healthy (LH, n = 10, white bars), metabolically healthy obese (MHO, n = 10, grey bars) and metabolically unhealthy obese (MUO, n = 10, black bars).

<p>Significant %PP amino acids between the three groups were identified using a non-parametric ANOVA Kruskal-Wallis (p < 0.05) test followed by a post-hoc Mann-Whitney test (p < 0.05). Data is presented as mean %PP ± SEM.</p

Mean circulating concentration of inflammatory markers.

<p>Data represented as mean±SEM. LH, lean healthy; MHO, metabolically healthy obese; MUO, metabolically unhealthy obese; hsCRP, high sensitivity C-reactive protein; IL-6, interleukin-6, IFN-γ, interferon γ; IP-10, interferon-γ inducible protein 10; PDGF-ββ, platelet-derived growth factor ββ; RANTES, regulated upon activation normal T-cell expressed and secreted; HMW adiponectin, high molecular weight adiponectin; IL-1Ra, interleukin-1 receptor antagonist. A non-parametric ANOVA Kruskal-Wallis test followed by a post-hoc Mann-Whitney-Wilcoxon test was used to determine significance between groups (P<0.05).</p

Mean relative percentage values of total fatty acids in serum.

<p>Total fatty acids (FAs) are reported as relative % values in lean healthy (LH), metabolically healthy obese (MHO), and metabolically unhealthy obese (MUO) groups. A non-parametric ANOVA Kruskal-Wallis test followed by a post-hoc Mann-Whitney-Wilcoxon test was used to determine significance between groups. FAs in bold font were significant in the ANOVA test (P<0.05).</p