Quantification of Oseltamivir Phosphate Enantiomeric Impurity by Chiral HPLC Method With the Aid of Solvent Extraction and Phosphate Salt-Out Method.

A robust chiral high-performance liquid chromatography (HPLC) method was established to separate and quantify the enantiomeric impurity (3S, 4S, 5R) in oseltamivir phosphate. A new sample preparation approach was used, involving the solvent extraction method to remove phosphate salt from the drug, thereby preventing the column's clogging and confirming method repeatability. Thin-layer chromatography (TLC) was employed to identify oseltamivir in the organic layer, while 31P nuclear magnetic resonance (NMR) spectroscopy and the molybdenum blue method were used to confirm the presence of phosphate in the aqueous layer. An ion-pair reversed-phase HPLC method with indirect UV detection was utilized to quantify the phosphate in both the organic and aqueous phases. Chromatographic separation of enantiomeric impurity (3S, 4S, 5R) from oseltamivir phosphate drug substances (3R, 4R, 5S) was accomplished using a Chiralpak IC-3 column with a mobile phase consisting of n-hexane, methanol, isopropyl alcohol, and diethyl amine (85:10:5:0.2, v/v/v/v) at a flow rate of 0.6 mL/min and a detection wavelength of 225 nm. The selectivity of method is clearly proved by separating the impurity from oseltamivir phosphate drug substance, with a resolution of more than 3.0. The method displayed exceptional linearity over a range of 0.035-0.300%w/w, with limits of detection of 0.005%w/w and quantification of 0.035%w/w. Consistent recovery rates were obtained between 91% and 94%, and the analytical solution remains stable for up to 72 h at 2°C-8°C.