Increasing bioanalytical throughput using pcSFC-MS/MS: 10 minutes per 96-well plate.

ABSTRACT

The utility of packed-column supercritical, subcritical, and enhanced fluidity liquid chromatographies (pcSFC) for high-throughput applications has increased during the past few years. In contrast to traditional reversed-phase liquid chromatography, the addition of a volatile component to the mobile phase, such as CO2, produces a lower mobile-phase viscosity. This allows the use of higher flow rates which can translate into faster analysis times. In addition, the resulting mobile phase is considerably more volatile than the aqueous-based mobile phases that are typically used with LC-MS, allowing the entire effluent to be directed into the MS interface. High-throughput bioanalytical quantitation using pcSFC-MS/MS for pharmacokinetics applications is demonstrated in this report using dextromethorphan as a model compound. Plasma samples were prepared by automated liquid/liquid extraction in the 96-well format prior to pcSFC-MS/MS analysis. Three days of validation data are provided along with study sample data from a patient dosed with commercially available Vicks 44. Using pcSFC and MS/MS, dextromethorphan was quantified in 96-well plates at a rate of approximately 10 min/plate with average intraday accuracy of 9% or better. Daily relative standard deviations (RSDs) were less than 10% for the 2.21 and 14.8 ng/mL quality control (QC) samples, while the RSDs were less than 15% at the 0.554 ng/mL QC level.