Quantification of drug metabolising enzymes and transporter proteins in the paediatric duodenum via LC-MS/MS proteomics using a QconCAT technique.

Characterising the small intestine absorptive membrane is essential to enable prediction of the systemic exposure of oral formulations. In particular, the ontogeny of key intestinal Drug Metabolising Enzymes and Transporter (DMET) proteins involved in drug disposition needs to be elucidated to allow for accurate prediction of the PK profile of drugs in the paediatric cohort. Using pinch biopsies from the paediatric duodenum (n = 36; aged 11 months to 15 years), the abundance of 21 DMET proteins and two enterocyte markers were quantified via LC-MS/MS. An established LCMS nanoflow method was translated to enable analysis on a microflow LC system, and a new stable-isotope-labelled QconCAT standard developed to enable quantification of these proteins. Villin-1 was used to standardise abundancy values. The observed abundancies and ontogeny profiles, agreed with adult LC-MS/MS-based data, and historic paediatric data obtained via western blotting. A linear trend with age was observed for duodenal CYP3A4 and CES2 only. As this work quantified peptides on a pinch biopsy coupled with a microflow method, future studies using a wider population range are very feasible. Furthermore, this DMET ontogeny data can be used to inform paediatric PBPK modelling and to enhance the understanding of oral drug absorption and gut bioavailability in paediatric populations.

Electrochemiluminescent detection of methamphetamine and amphetamine.

Direct detection of amphetamine type stimulants (ATSs) including methylamphetamine (MA) in street samples and biological matrices without the need for pretreatment or extraction is a great challenge for forensic drug analysis. Electrochemical techniques, such as electrochemiluminescence (ECL), are promising tools for this area of analysis. This contribution focuses on the electrochemical and photochemical properties of [Ru(bpy)3](2+) Nafion composite films and their subsequent use for the detection of ATS in particular MA. Under optimised conditions, the response linearly increased with the concentration over the concentration range 50pM≤[MA]≤1mM while an equivalent dynamic range was obtained for amphetamine with a correlation coefficient of 0.9903 and 0.9948 respectively. The ECL signal was monitored at ∼620nm, representing the λmax for the [Ru(bpy)3](2+) Nafion composite films. This wavelength is shifted by approximately 15nm compared to the photoexcited λmax for the same system. The modified films were formed by direct interaction with the electrode surface without the need for surface modification or chain linkers. This is a major advantage for the fabrication of any sensor as it reduces the synthesis times resulting in more economically and cheaper production costs. This technique is simple, rapid, selective and sensitive, and shows potential for the high-throughput quantitation of ATS as well as possibilities for adaptation with other techniques such as FIA or LC systems.