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 (50mmx2.1mm, 5microm) column by isocratic elution at a flow rate of 220microl/min with a mobile phase consisting of 85% acetonitrile containing 5mM 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-d(3). The lower limit of quantification was 10nM for DEAQ and linearity was observed over the concentration range of 10-1500nM. 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.
Dravid
Prajakta V
PV
Department of Pharmacy Practice, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA.
Frye
Reginald F
RF
D000655:Amodiaquine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D000962:Antimalarials / Q000032:analysis* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003627:Data Interpretation, Statistical; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D012997:Solvents; D053719:Tandem Mass Spectrometry
D000962:Antimalarials; D007202:Indicators and Reagents; D012997:Solvents; D000655:Amodiaquine; C047386:desethylamodiaquine