Final ProgramCPSA 2011
Science and Technology Coming Together to Make a Difference
October 3 - 6, 2011
Bucks County Sheraton Hotel
Langhorne, PA
Poster Abstract #12
Quantitation of metabolites in plasma samples by UV-MS correction using a dual-cell-linear ion trap mass spectrometer
Thermo Fisher Scientific, San Jose, California, 95134 USA
The quantitative assessment of metabolites in samples from development in vivo studies has become an area of increasing study with the release of guidances from regulatory bodies (MIST and M3 R2) and their subsequent interpretation and implementation. We have applied a UV correlation to assign concentrations to metabolites from in vitro generated samples and used the results to quantify plasma samples on the same instrument.
Initially, metabolites of dextromethorphan were generated in vitro through incubations for 2 hours at 100 uM Dextromethorphan for 2 hours to assure the creation of significant concentration of metabolites. Samples were analyzed by UHPLC-UV along with a standard curve of dextromethorphan prepared in microsomal matrix (50 µM to 1 µM) to assign absolute metabolite levels. The molar absorptivity of dextromethorphan and its metabolites was assumed to be the same. The in vitro sample was diluted to create a standard curve in plasma at known concentrations for UHPLCMS analysis on a Velos Pro dual pressure linear ion trap. The significant difference in formation rates in vitro required two separate dilutions be prepared to prepare standard curves of the appropriate expected range for each analyte. Plasma samples for UHPLCMS analysis were prepared by spiking known amounts of dextrorphan and methoxymorphinan (between 750 and 5 ng/mL) into plasma extraction matrix followed by mixing with in vitro microsomal matrix. A total of 12 spiked concentration values were prepared for each analyte. Quantification of the spiked plasma samples against the UV-generated metabolite standard curve was compared to the known spike values and also confirmed for two metabolites by standard curves created with synthetic standards. For all the samples analyzed, the calculated concentration was within 2X the known value when quantifying based on the in vitro UV-MS correlation standard curve. Quantitation was performed by targeted selective reaction monitoring (SRM) as well as full scan MS2 on dextromethorphan, dextrorphan, and methoxymorphinan without the use of timed segments.