Development of an LC-MS method to quantify coproporphyrin I and III as endogenous biomarkers for drug transporter-mediated drug-drug interactions.

Coproporphyrins are proposed as endogenous biomarkers of hepatic Organic Anion Transporting Polypeptide (OATP)1B functional activity. In this study, a new sample extraction method based on a mixed-mode anion exchange sorbent (SPE clean-up using Oasis 30mg Max 96 well plates) was developed for absolute quantification of coproporphyrin I and III (CP-I and CP-III) in human plasma. Chromatographic separation was performed with an Ace Excel 2 C18 PFP, 3µm, 2.1×150mm, maintained at 60°C. A 10mM ammonium formate containing 0.1% HCOOH and acetonitrile (100%) was used as mobile phase A and B, respectively. Mass transition, m/z 655.3→596.3 was selected to monitor CP-I and CP-III, while m/z 659.3→600.3 transition was used for the stable isotope labelled internal standard. Optimization of the liquid chromatography tandem mass spectrometry method ensured a lower limit of quantification (LLOQ) of 20pg/mL. Both CP-I and CP-III had extraction recoveries of 70%. The calibration range was 0.02-100ng/mL for both CP-I and CP-III, yielding calibration curves with correlation coefficients greater than 0.988. Inter day precision (CV<9%) and accuracy (84.3-103.9%) complied with the recommendation of the European Bioanalytical Forum. The optimized method was used to analyse plasma samples originating from three independent clinical studies. Obtained CP-I and CP-III plasma baseline levels in healthy volunteers were in good agreement with previously published data. Moreover, CP-I and CP-III plasma levels in human subjects dosed with a clinically confirmed OATP inhibitor were significantly increased compared to their baseline levels. These data demonstrate the potential of CP-I and CP-III as endogenous biomarkers to predict the drug-drug interaction (DDI) related to hepatic OATP1B inhibition. Stability of CP-I and CP-III in plasma and solvents under different processing and storage conditions was also evaluated.

The application of capillary microsampling in GLP toxicology studies.

AIM: Capillary microsampling (CMS) to collect microplasma volumes is gradually replacing traditional, larger volume sampling from rats in GLP toxicology studies. METHODOLOGY: About 32 µl of blood is collected with a capillary, processed to plasma and stored in a 10- or 4-µl capillary which is washed out further downstream in the laboratory. CMS has been standardized with respect to materials, assay validation experiments and application for sample analysis. CONCLUSION: The implementation of CMS has resulted in blood volume reductions in the rat from 300 to 32 µl per time point and the elimination of toxicokinetic satellite groups in the majority of the rat GLP toxicology studies. The technique has been successfully applied in 26 GLP studies for 12 different projects thus far.