Identification of unstable metabolites of Lonafarnib using liquid chromatography-quadrupole time-of-flight mass spectrometry, stable isotope incorporation and ion source temperature alteration.

Structural characterization of unstable metabolites and other drug-derived entities poses a serious challenge to the analytical chemist using instrumentation such as LC-MS and LC-MS/MS, and may lead to inaccurate identification of metabolite structures. The task of structural elucidation becomes even more difficult when an analyte is unstable in the ion source of the mass spectrometer. However, a judicious selection of the experimental conditions and the advanced features of new generation mass spectrometers can often overcome these difficulties. We describe here the identification of three drug-derived peaks (A, B and C) that were detected from a Schering-Plough developmental compound (Lonafarnib) following incubation with cDNA-expressed human CYP3A4. Definitive characterization was achieved using (1) accurate mass measurement, (2) stable isotope incorporation, (3) reduced ion source temperature, (4) alkali ion attachment and (5) MS/MS fragmentation studies. The protonated ions of compounds A and B fragmented almost completely in the source, yielding ions of the same mass-to-charge ratio (m/z) as that of protonated C (CH+). Fortunately, the presence of Na+ and K+ adducts of A and B provided information crucial to distinguishing AH+ and BH+ from their fragment ions. Metabolite A was shown to be an unstable hydroxylated metabolite of Lonafarnib. The metabolite C was shown to be a dehydrogenated metabolite of Lonafarnib (Lonafarnib-2H), unstable in the presence of protic solvents. Finally, B was artifactually formed most likely from C by the solvolytic addition of methanol during sample preparation. MS/MS fragmentation experiments assisted in identifying the site of metabolism in A and chemical modification in B. A and C readily interconvert through hydration/dehydration, and B and C through addition/elimination of methanol present in the sample-processing solvents. Finally, NMR experiments were performed to confirm the structures of A and C.