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United States Department of Agriculture

Agricultural Research Service

Research Project: DISCOVERY AND DEVELOPMENT OF NATURAL PRODUCT FOR PHARMACEUTICAL AND AGRICHEMICAL APPLICATIONS

Location: Natural Products Utilization Research

Title: Profiling primaquine metabolites in primary human hepatocytes by UPLC-QTOF-MS with 13c stable isotope labeling

Authors
item Bharathi, Avula -
item Tekwani, Babu -
item Chauarasiya, Narayan -
item Nanayakkara, Dhammika -
item Wang, Yan-Hong -
item Khan, Shabana -
item Adelli, Vijender -
item Sahu, Rajnish -
item Elsohly, Mahmoud -
item Mcchesney, James -
item Khan, Ikhlas -
item Walker, Larry -

Submitted to: Journal of Mass Spectrometry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 20, 2012
Publication Date: February 1, 2013
Citation: Bharathi, A., Tekwani, B.L., Chauarasiya, N.D., Nanayakkara, D.N., Wang, Y., Khan, S.I., Adelli, V.R., Sahu, R.K., Elsohly, M.A., Mcchesney, J.D., Khan, I., Walker, L.A. 2013. Profiling primaquine metabolites in primary human hepatocytes by UPLC-QTOF-MS with 13c stable isotope labeling. Journal of Mass Spectrometry. 48(2):276-285.

Interpretive Summary: Primaquine (PQ), an 8-aminoquinoline is the clinical drug of choice for the radical cure. Its antimalarial activity and toxicity of primaquine, has been attributed to one, or more, of its metabolites. The major plasma metabolite identified in humans and animals, carboxyprimaquine (cPQ), appears not to be responsible for this toxicity. Identification of minor metabolites in biological matrices poses a major challenge. A simple and sensitive LC-MS-QTOF method is developed for the qualitative and quantitative analysis of PQ and other metabolites. Acquity UPLC was integrated with QTOF-MS to combine the efficiency of separation with high sensitivity, selectivity of detection, and accurate mass. Fourteen (14) compounds showing twin peaks with mass difference of 6, structure and fragmentation pathways were proposed and characterized. The MS/MS fragmentation pattern presented for the individual metabolites provided enough information for prediction of the plausible structures for these metabolites.

Technical Abstract: Primaquine (PQ) is an important antimalarial agent because of its activity against exoerythrocytic forms of Plasmodium spp. However, hemolytic anemia is a dose-limiting side effect of primaquine therapy that limits its widespread use. The major plasma metabolite identified in humans and animals, carboxyprimaquine (cPQ), appears not to be responsible for this toxicity. Identification of minor metabolites in biological matrices poses a major challenge. Drug candidates labeled with stable isotopes in combination with LC/MS can be used to significantly lessen this challenge. This study was undertaken to identify the metabolites of PQ from an in vitro incubation of a 1:1 w/w mixture of 13C6-PQ/PQ with primary human hepatocytes using UPLC-QTOF-MS. An Acquity UPLCTM BEH Shield RP18 column (100 mm × 2.1 mm I.D., 1.7 µm) was used. The mobile phase consisted of water and acetonitrile both containing formic acid at a flow rate of 0.25 mL/min with gradient elution. Acquity UPLC was integrated with QTOF-MS to combine the efficiency of separation with high sensitivity, selectivity of detection, and accurate mass. The qualitative metabolite identification was performed using MetaboLynx XS software.

Last Modified: 4/23/2014
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