Simultaneous analysis of several antiretroviral nucleosides in rat-plasma by high-performance liquid chromatography with UV using acetic acid/hydroxylamine buffer

Zalcitabine (ddC), lamivudine (3TC), didanosine (ddI), stavudine (d4T), carbovir (CBV), zidovudine (AZT), tenofovir (PMPA) and its administrated form (tenofovir diisoproxyl fumarate, TDF), are nucleosides currently approved in HIV therapy. To facilitate pharmacokinetics studies, a specific reversed-phase high-performance liquid chromatography (HPLC) method was developed for their analysis in rat plasma. The method involved a quantitative recovery of these drugs from rat plasma by solid-phase extraction on Oasis® HLB Waters cartridges followed by optimised HPLC separation on an Atlantis™ dC18 column with acetic acid-hydroxylamine buffer (ionic strength 5 mM, pH 7)-acetonitrile elution gradient. Quantitation was performed by HPLC/UV at 260 nm. Linear calibration curves were obtained within a 30-10,000 ng/mL plasma concentration range. Correlation coefficients (r2) greater than 0.992 were obtained by least-squares regression and limits of quantification were in 30-90 ng/mL concentration range. Quantitative parameters (accuracy, intra-day repeatability and inter-day reproducibility) yielded satisfactory results. Finally, a new buffer, obtained with acetic acid and hydroxylamine, has been tested in HPLC/ESI-MS/MS and appears to be an efficient volatile buffer in the medium 5-7 pH range. Indeed, at pH 7 and low ionic strength (5 mM), its buffer capacity is one hundred times higher to that obtained for the usual acetic acid/ammonia buffer.