Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies

An 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. Injection setups used for in-capillary CYP450 assay

Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach

Abstract.: A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4min. Method performances were evaluated. The method showed good linearity in the range of 25-1000ngmL−1 with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25ngmL−1 for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma sample

Innovative methodology to transfer conventional GC-MS heroin profiling to UHPLC-MS/MS

Nowadays, in forensic laboratories, heroin profiling is frequently carried out by gas chromatography coupled with mass spectrometry (GC-MS). This analytical technique is well established, provides good sensitivity and reproducibility, and allows the use of large databases. Despite those benefits, recently introduced analytical techniques, such as ultra-high-pressure liquid chromatography (UHPLC), could offer better chromatographic performance, which needs to be considered to increase the analysis throughput for heroin profiling. With the latter, chromatographic conditions were optimized through commercial modeling software and two atmospheric pressure ionization sources were evaluated. Data obtained from UHPLC-MS/MS were thus transferred, thanks to mathematical models to mimic GC-MS data. A calibration and a validation set of representative heroin samples were selected among the database to establish a transfer methodology and assess the models' abilities to transfer using principal component analysis and hierarchical classification analysis. These abilities were evaluated by computing the frequency of successful classification of UHPLC-MS/MS data among GC-MS database. Seven mathematical models were tested to adjust UHPLC-MS/MS data to GC-MS data. A simplified mathematical model was finally selected and offered a frequency of successful transfer equal to 95%. Figur

Single-run separation of closely related cationic and anionic compounds by CE-ESI-MS: application to the simultaneous analysis of melamine and its analogs in milk

In recent years, two adulteration incidents concerning the addition of melamine, a nitrogen-rich industrial small polar compound, to pet food and infant formula products have occurred in China. These issues prompted laboratories to develop methods for the analysis of melamine and related compounds in a wide variety of food products and ingredients. In this context, a CE-ESI-MS method was developed to simultaneously analyze melamine and its related products (ammeline, ammelide and cyanuric acid) that possess close physico-chemical properties. This method allows the simultaneous analysis of both cations and anions in a single run, using CE to divide the run into two time segments in normal polarity mode. For this purpose, ESI polarity was switched once during the run, increasing sensitivity and data quality. The method was applied to spiked powdered milk and melamine-contaminated powdered milk, with two sample preparation procedures

Single-run separation of closely related cationic and anionic compounds by CE-ESI-MS: application to the simultaneous analysis of melamine and its analogs in milk

In recent years, two adulteration incidents concerning the addition of melamine, a nitrogen-rich industrial small polar compound, to pet food and infant formula products have occurred in China. These issues prompted laboratories to develop methods for the analysis of melamine and related compounds in a wide variety of food products and ingredients. In this context, a CE-ESI-MS method was developed to simultaneously analyze melamine and its related products (ammeline, ammelide and cyanuric acid) that possess close physico-chemical properties. This method allows the simultaneous analysis of both cations and anions in a single run, using CE to divide the run into two time segments in normal polarity mode. For this purpose, ESI polarity was switched once during the run, increasing sensitivity and data quality. The method was applied to spiked powdered milk and melamine-contaminated powdered milk, with two sample preparation procedures

Evaluation of Solid-Phase Microextraction Desorption Parameters for Fast GC Analysis of Cocaine in Coca Leaves

By its simplicity and rapidity, solid-phase microextraction (SPME) appears as an interesting alternative for sample introduction in fast gas chromatography (fast GC). This combination depends on numerous parameters affecting the desorption step (i.e., the release of compounds from the SPME fiber coating to the GC column). In this study, different liner diameters, injection temperatures, and gas flow rates are evaluated to accelerate the thermal desorption process in the injection port. This process is followed with real-time direct coupling a split/splitless injector to a mass spectrometer by means of a short capillary. It is shown that an effective, quantitative, and rapid transfer of cocaine (COC) and cocaethylene (CE) is performed with a 0.75-mm i.d. liner, at 280 degrees C and 4 mL/min gas flow rate. The 7-microm polydimethylsiloxane (PDMS) coating is selected for combination with fast GC because the 100-microm PDMS fiber presents some limitations caused by fiber bleeding. Finally, the developed SPME-fast GC method is applied to perform in less than 5 min, the quantitation of COC extracted from coca leaves by focused microwave-assisted extraction. An amount of 7.6 +/- 0.5 mg of COC per gram of dry mass is found, which is in good agreement with previously published results

BioCreative III interactive task: an overview

The BioCreative challenge evaluation is a community-wide effort for evaluating text mining and information extraction systems applied to the biological domain. The biocurator community, as an active user of biomedical literature, provides a diverse and engaged end user group for text mining tools. Earlier BioCreative challenges involved many text mining teams in developing basic capabilities relevant to biological curation, but they did not address the issues of system usage, insertion into the workflow and adoption by curators. Thus in BioCreative III (BC-III), the InterActive Task (IAT) was introduced to address the utility and usability of text mining tools for real-life biocuration tasks. To support the aims of the IAT in BC-III, involvement of both developers and end users was solicited, and the development of a user interface to address the tasks interactively was requested