Determination of N-desethylamodiaquine by hydrophilic interaction liquid chromatography with tandem mass spectrometry: Application to in vitro drug metabolism studies

The antimalarial drug amodiaquine is extensively metabolized to N-desethylamodiaquine (DEAQ) by cytochrome P450 2C8 (CYP2C8). DEAQ formation is an enzyme specific reaction that is used to quantify in vitro CYP2C8 activity. A rapid and sensitive method for the determination of DEAQ in human liver microsomes was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC–MS/MS). Microsomal incubation samples were processed by protein precipitation with acetonitrile. The analytes were separated on a BETASIL Silica-100 (50 mm × 2.1 mm, 5 μm) column by isocratic elution at a flow rate of 220 μl/min with a mobile phase consisting of 85% acetonitrile containing 5 mM ammonium acetate and 0.1% formic acid. Detection was by positive electrospray ionization on a TSQ Quantum Discovery triple quadrupole mass spectrometer operated in the selective reaction monitoring mode. The precursor–product ion pair was m/z 328 → 283 for DEAQ and m/z 331 → 283 for DEAQ-d3. The lower limit of quantification was 10 nM for DEAQ and linearity was observed over the concentration range of 10–1500 nM. Intra- and inter-day accuracy and precision were within 3.4 and 7.0%, respectively. The method was successfully applied to CYP2C8 drug metabolism studies in pooled human liver microsomes.