A LC-MS/MS Assay for Quantification of Amodiaquine and Desethylamodiaquine in Dried Blood Spots on Filter Paper

Artesunate-amodiaquine (ARS-AQ) is a first-line antimalarial treatment recommended by the World Health Organization. AQ is the long acting partner drug in this combination, and therapeutic success is correlated with the terminal exposure to AQ. Dried blood spot (DBS) sampling for AQ is a convenient and minimally invasive technique, especially suitable for clinical studies in resource limited settings and pediatric studies. Our primary aim was to develop and validate a bioanalytical method for quantification of AQ and its active metabolite in capillary blood applied onto filter paper as a DBS sample. The separation was achieved using a reverse phase column (Zorbax SB-CN 50 × 4.6 mm, I.D. 3.5 μm) and a mobile phase consisting of acetonitrile:ammonium formate 20 mM with 0.5% formic acid (15:85, v/v). A 50 μL DBS was punctured with five 3.2 mm punches from the filter paper, and the punches collected correspond to approximately 15 μL of dried blood. The blood was then extracted using a mixture of 0.5% formic acid in water:acetonitrile (50:50, v/v), along with stable isotope-labeled internal standards (AQ-D10 and desethylamodiaquine [DAQ]-D5). Mass spectrometry was used for quantification over the range of 2.03-459 ng/mL for AQ and 3.13-1570 ng/mL for DAQ. The validation of the method was carried out in compliance with regulatory requirements. The intra- and interbatch precisions were below 15% and passed all validation acceptance criteria. No carryover and no matrix effects were detected. Normalized matrix factors (analyte/internal standard) ranged from 0.96 to 1.03 for all analytes, hence no matrix effects. AQ and DAQ were stable in all conditions evaluated. Long-term stability in DBS samples was demonstrated for up to 10 years when stored at -80°C and for 15 months when stored at room temperature. The developed method was demonstrated to be reliable and accurate. This assay may be particularly useful in the context of resource limited settings and in pediatric field studies.