Quantitation of Parent Drug and Its Unstable Metabolites by in Situ Coulometric Oxidation and Liquid Chromatography−Tandem Mass Spectrometry

摘要

Recent FDA and ICH guidances on safety testing of drugnmetabolites have challenged the way we traditionally thinknabout quantitative bioanalytical methods. Such assays, inngeneral, require a reference standard for each analyte tonconstruct calibration curves and prepare quality controlnsamples. However, early in the drug development process,nmetabolite standards may not be readily available, and ifnthey are inherently unstable, they are difficult to synthesizenor purify. In this paper, we describe a novel in-linenmethod for producing and then quantifying a very unstablenmetabolite which is based upon the in situ postcolumnncoulometric oxidation of the parent drug. Lacking anynmetabolite standards, the feasibility of simultaneouslynquantifying a development drug (compound A) and itsnunstable hydroxylated metabolites (metabolite B) wasninvestigated. Reference standards for these ostensiblynmajor human metabolites could not be reliably obtainedndue to rapid degradation upon purification and/or subsequentnstorage. Following high-performance liquid chromatographyn(HPLC) separation, parent drug and itsn[13C3-15N] isotopically labeled internal standard werenquantitatively converted to equal amounts of a diastereomericnpair of hydroxylated metabolites using a postcolumnncoulometric electrochemical cell before reachingnthe mass spectrometer. The concentration of theninjected parent (which is equal to the total concentrationnof the in-line generated metabolites since thenconversion to metabolite is quantitative) and the tandemnmass spectrometry (MS/MS) signals of the electrochemicallyngenerated metabolites were used tonconstruct a calibration curve for quantifying both thenparent drug and its hydroxylated metabolites. Plasmanextracts from humans dosed with compound A containednchromatographically distinct liquid chromatography-nmass spectrometry (LC-MS) signals (m/zn538) for in vivo formed hydroxylated metabolites andnthe electrochemically oxidized parent drug which hadnbeen converted in-line into its chemically identicalntwin. Both peaks in this study sample could be quantifiednusing a single calibration curve obtained under thensame coulometric conditions using known amounts ofnthe parent drug. Although no attempt was made to fullynvalidate a bioanalytical method, the practicality of thisnin situ quantification approach was further confirmednby the preliminary bioanalytical analysis of a selectionnof plasma samples collected following oral administrationn(50 mg) of compound A in a clinical study.