Evidence of the formation of direct covalent adducts of primaquine, 2-tert-butylprimaquine (NP-96) and monohydroxy metabolite of NP-96 with glutathione and N-acetylcysteine.
2-tert-Butylprimaquine (NP-96) is a novel quinoline anti-malarial compound with superior therapeutic profile than primaquine (PQ). Moreover, it is the first 8-aminoquinoline that is established to be devoid of methemoglobin toxicity. The purpose of the present study was to investigate covalent adduct formation tendency of PQ, NP-96 and their phase I metabolites with glutathione (GSH) and N-acetylcysteine (NAc). For the same, the two compounds were incubated in human and rat liver microsomes in the presence of trapping agents and NADPH. In a control set, NADPH was excluded, while a blank was also studied that was devoid of both NADPH and microsomes. The components in the reaction mixtures were initially separated on a C-18 column (250 mm×4.6mm, 5 μm) using a mobile phase composed of acetonitrile and 10 mM ammonium acetate in a gradient mode. The samples were then subjected to LC-MS(n) and LC-HR-MS analyses, and data were collected in full scan MS, data dependent MS/MS, targeted MS/MS, neutral loss scan (NLS) and accurate mass (MS/TOF) modes. In a significant finding, both PQ and NP-96 themselves showed potential to bind covalently with GSH and NAc, as adducts were observed even in the control and blank incubations. Intense peaks corresponding to covalent adduct of mono-hydroxy metabolite of NP-96 with GSH and NAc were also detected in NADPH supplemented reaction solution.
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar 160062, Punjab, India.
Amit
A
Garg
Bhagwat
B
Prasad
Hardik
H
Takwani
Meenakshi
M
Jain
Rahul
R
Jain
Saranjit
S
Singh
D000111:Acetylcysteine / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D011319:Primaquine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
C482216:2-tert-butylprimaquine; D005978:Glutathione; D011319:Primaquine; D000111:Acetylcysteine