Characterization of tenofovir, tenofovir disoproxil fumarate and emtricitabine in aqueous solutions containing sodium ions using ESI-MS, NMR and Ab initio calculations

Abstract

We have discerned some characteristics of protonated and sodiated forms of emtricitabine tenofovir and tenofovir disoproxil fumarate in aqueous solutions using mass spectrometry/mass spectrometry (MS/MS), nuclear magnetic resonance spectroscopy (NMR) and Ab initio calculations. Protonated species exhibited different mass spectral characteristics from sodiated species for the three antiretroviral drugs. Sodiation of the three compounds yields extremely stable conformers which require high excitation energies to detach the sodium cation using MS/MS. In the case of emtricitabine (FTC), collision induced dissociation (CID) of the protonated species with m/z 248 yielded m/z 130 as the major product ion by losing 2-ethyl-1,3-oxathiolane. MS/MS of the sodiated molecular ion [M+Na]+ with m/z 270 would be expected to yield [M+H]+ i.e. m/z 248 by losing the Na+ cation but instead lost 2-ethyl-1,3-oxathiolane and left the Na+ cation well sequestered within the ionic complex. Similar observations were made with tenofovir disoproxil fumarate (TDF) and tenofovir (TFV). Ab initio calculations showed in the case of sodiated TDF and TFV that, the sodium could only be cleaved when a nucleophilic species i.e. PO2 to which it was coordinated to, fragmented requiring activation energy of 1.5eV. The potential energy barrier to remove the Na+ cation alone was too high to allow the cleavage to proceed. NMR data showed that when TFV or TDF coordinates with Na+ ions at one of the nitrogens at position 1 in the purine moiety of the molecule, it also interacts with oxygens of the phospho group to form an ionic complex and perturbs the chemical environments of several atoms in the periphery. The abundance of sodium cations and their strong interactions with the core structures of FTC, TDF and TFV make sodiated species as the best candidates for their quantitation using MS/MS especially in biological fluids. Evidence for improved sensitivity when sodiated species of some of the antiretroviral (ARV) drugs were used for quantitation exists in the literature.