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n automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: The aim of this work was to develop a trypsin-based micro-immobilized enzyme reactor prepared on a monolithic ethylenediamine BIA Separations CIM (convective interaction media) minidisk. The micro-immobilized enzyme reactor (IMER) was integrated in a liquid chromatography system hyphenated to electrospray ionization tandem mass spectrometry to carry out on-line protein digestion and identification. The performance of this IMER was compared with that obtained using a previously developed bioreactor prepared on a conventional CIM ethylenediamine disk and with that of the commercially available Poroszyme immobilized trypsin cartridge. In this work, we showed how different proteins were identified with good recoveries using a digestion time of 10min only. [Copyright &y& Elsevier]

Abstract: Two cytochrome P450 (CYP)-based immobilized enzyme reactors (IMERs) were developed to perform automated on-line phase I drug metabolism studies. For this purpose, biotinylated recombinant CYP2D6 or CYP3A4 reconstituted systems were anchored to the surface of two monolithic mini-columns (2mm×6mm I.D.), which had been covalently grafted with NeutrAvidin. After optimization of immobilization conditions, the obtained IMERs were integrated on-line into a LC hyphenated to an electrospray ionization MS/MS system. Studies with probe substrates and a known competitive inhibitor were performed, showing the potential of CYP-based IMERs in drug metabolism. In the optimized conditions, ca. 15 experiments were carried out with each bioreactor. [Copyright &y& Elsevier]

Abstract: The preparation and characterization of three trypsin-based monolithic immobilized enzyme reactors (IMERs) developed to perform rapid on-line protein digestion and peptide mass fingerprinting (PMF) are described. Trypsin (EC 3.4.21.4) was covalently immobilized on epoxy, carbonyldiimidazole (CDI) and ethylenediamine (EDA) Convective Interaction Media® (CIM) monolithic disks. The amount of immobilized enzyme, determined by spectrophotometric measurements at 280nm, was comprised between 0.9 and 1.5mg per disk. Apparent kinetic parameters and , as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-α-benzoyl-l-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, α1-acid glycoprotein, ovalbumin and albumin) was evaluated by inserting the IMERs into a liquid chromatography system coupled to an electrospray ionization ion-trap mass spectrometer (LC-ESI–MS/MS) through a switching valve. Results were compared to the in-solution digestion in terms of obtained scores, number of matched queries and sequence coverages. The most efficient IMER was obtained by immobilizing trypsin on a CIM® EDA disk previously derivatized with glutaraldehyde, as a spacer moiety. The proteins were recognized by the database with satisfactory sequence coverage using a digestion time of only 5min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12μM) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections. [Copyright &y& Elsevier]

Analysis of human insulin and its synthetic analogues is increasingly requested for clinical monitoring, for anti-doping purposes, but also for forensic cases. Indeed, insulin analogues may be abused for suicide or homicide – whence their forensic interest. Collection and storage conditions, as well as the phenomenon of degradation make post-mortem serum samples analytically challenging and consequently, the rate of exogenous insulin administration as cause of death is undoubtedly underestimated. However, with recent technological advances and the development of new extraction techniques particularly for anti-doping analyses, detection of insulins in post-mortem samples seems to be achievable. This study describes the first validated quantitative method for analysis human insulin and its six analogues (lispro, aspart, glulisine, glargine, detemir and degludec) in plasma and post-mortem sera. Various extraction processes, namely precipitation + solid phase extraction (SPE), filtration + SPE, precipitation + SPE + immunopurification, and filtration + immunopurification, were assessed to evaluate the lowest limit of detection for all target analogues. The selected sample preparation consists of filtration step followed by immunopurification extraction with an anti-body precoated ELISA plate for plasma. For post-mortem sera, the first step of precipitation was added to remove matrix interferences. The extracts were analyzed by ultra-high-performance liquid chromatography-high resolution mass spectrometry (LC-HRMS), interfaced by electrospray (ESI). The method was validated with respect linearity, precision, accuracy, recovery, matrix effect, dilution and carryover. The limit of quantification (LOQ) in plasma was 0.5 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. Two types of post-mortem sera were studied based on the post-mortem interval (PMI): inferior or superior to 48 h. The obtained LOQ were the same for each analogue, independent from the PMI: 1.0 ng/mL for human insulin and rapid-acting insulins, 1.0 ng/mL for glargine, 2.5 ng/mL for degludec and 10 ng/mL for detemir. At the LOQ level, for all insulins and all samples, accuracy was between 70 and 130% and precision inferior to 30%. The validated method was applied to five subjects participating in therapeutic monitoring of insulin and to seven post-mortem cases. Image 1 • Optimization of sample preparation for insulins analysis in plasma & post-mortem sera. • Five different workflows were compared to reach the lowest LOD for all insulins. • Validation of the LC-MS/HRMS assay from plasma and post-mortem sera. • Application from authentic clinical and post-mortem cases. [ABSTRACT FROM AUTHOR]

• Methadone enantiomers separation and quantitation using SFC-MS/MS in DBS and DMS. • Methadone enantiomers separation and quantitation using LC-MS/MS in DBS and DMS. • SFC-MS/MS and LC-MS/MS comparison between method validation parameters. • Quantification and ratio of R- and S-methadone in real post mortem samples fluids. This study describes two bioanalytical methods for the quantitation of the two methadone enantiomers in dried matrix spots using high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) and high performance supercritical chromatography tandem mass spectrometry (HPSFC-MS/MS). Dried matrix spots were obtained by spotting 10 µL of each sample fluid on a Whatman paper. Methadone and its main metabolite, EDDP, were extracted with 100 µL methanol and subsequently injected into the LC-MS/MS and SFC-MS/MS systems. Enantiomeric separation was achieved with AGP-column for the LC conditions and with Chiralpak IH-3 in SFC. The two methods were fully validated and 93 post-mortem samples were analysed with both analytical methods. Results from validation parameters and results obtained for all post-mortem samples were compared with a significant spearman correlation of r s = 0.9978 for R-methadone and r s = 0.9981 for S-methadone. The LC method provided better results in terms of uncertainty, retention factor and resolution, whereas SFC provides better sensitivity, with lower LOD. Median R-/S-methadone ratio in peripheral blood was found equal to 1.60 (N = 32), varying from 0.79 to 4.23. The reported values were in good agreement with previously published results. Based on the results obtained here, SFC-MS/MS can be considered a reliable alternative to the widely used LC-MS/MS for the quantitation of methadone enantiomers in bioanalysis and should be evaluated for other bioanalytical methods. Both methods can be easily and quickly used in toxicological routine analysis for the methadone quantitation in human fluids matrices, even if considering that the polysaccharide coated column IH-3 used in SFC does not allow the enantiomeric EDDP separation. [ABSTRACT FROM AUTHOR]

Music therapy has long been used as a non-pharmacological intervention to improve cognitive function and mood in humans. Mounting rodent evidence also supports beneficial impact of music exposure on animal cognitive performance. The zebrafish (Danio rerio) is an important emerging aquatic animal model in translational biomedical and neuroscience research. Here, we evaluate the effects of intermittent (2-h or 6-h twice daily) and continuous (24-h) solfeggio-frequency music exposure on behavioral, cognitive and endocrine parameters in adult zebrafish whose circadian rhythm was disturbed by a 24-h light exposure. Overall, a 24-h light exposure stress evokes overt cognitive deficits in the inhibitory avoidance test and elevates zebrafish whole-body cortisol levels. However, these effects were reversed by solfeggio-frequency music exposure for 2 or 6 h twice daily, and by continuous 24-h exposure. Collectively, these findings suggest a positive modulation of cognitive and endocrine responses in adult zebrafish by environmental enrichment via the long-term exposure to music, and reinforces zebrafish as a robust, sensitive model organism for neurocognitive and neuroendocrine research. [ABSTRACT FROM AUTHOR]