For analysis, HPLC grade solvents, e.g., methanol, acetonitrile, and formic acid (FA), were purchased from Thermo Scientific (Rockford, IL, United States). Water used for analysis was purified using Millipore Synergy® UV Ultrapure Water Purification System (EMD Millipore Corporation, MA, United States). Reference standards, primaquine diphosphate was purchased from Sigma (St Louis, MO, United States). Different metabolites of PQ and internal standards were synthesized at the National Center for Natural Products Research, University of Mississippi. Synthesis of reference standards have been described in the previous article; primaquine-5,6-orthoquinone (POQ) (Potter et al., 2015 (link)), carboxyprimaquine-5,7-orthoquinone (cPOQ) (Khan et al., 2021 (link)), PQ-N-CG (Fasinu et al., 2016 (link)), cPQ (McChesney and Sarangan, 1984 (link)), 4-methylprimaquine-5,6-orthoquinone (4-MePOQ), and deuterated primaquine (d3-PQ) (Khan et al., 2022b (link)). The racemic PQ, PQ enantiomers and placebo capsules used for the clinical study were prepared with complete characterization, purity, and stability, by ElSohly Laboratories, Inc (ELI), Oxford, MS. Each capsule contained the desired specific amount of PQ base as the diphosphate salt. Structure of all analytes are shown in Figure 1.
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