A Pharmaceutical Company was finding higher than expected plasma drug concentrations during a Phase I study for a chemotherapeutic prodrug and suspected that in vitro conversion of the prodrug to the drug might be the cause.
The assay employed by the Client was for the analysis of all three substances (the prodrug, drug, and a metabolite) simultaneously. This process was leading to the conversion of the prodrug to the drug during the extraction process and elevating the measured drug concentrations.
Our scientists carefully studied the solubility of each substance and developed a new assay which first selectively extracted the drug (formed in vivo) from plasma as soon as it was thawed leaving the prodrug and metabolite behind. The drug was then quantified by an LC/MS/MS system, while the prodrug and the metabolite were extracted separately from the plasma samples and analyzed by a HPLC-fluorescence system. This selective extraction process enabled the accurate determination of the in vivo drug concentration.