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

## TL;DR

This paper describes a new method to measure a specific impurity in oseltamivir phosphate using chiral HPLC after removing phosphate salt with solvent extraction.

## Contribution

A novel sample preparation method using solvent extraction and phosphate salt-out improves chiral HPLC analysis of oseltamivir phosphate impurities.

## Key findings

- The chiral HPLC method successfully separates and quantifies the (3S, 4S, 5R) enantiomeric impurity in oseltamivir phosphate.
- The method shows excellent linearity (0.035–0.300% w/w) and recovery rates (91–94%) for the impurity.
- Phosphate presence is confirmed in both organic and aqueous layers using ion-pair HPLC and 31P NMR.

## Abstract

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.

In this method, oseltamivir phosphate enantiomeric impurity is quantified through the chiral HPLC method by removal of phosphate salt through solvent extraction coupled with the salt‐out method. Chiral HPLC confirms the presence of drugs and impurities in the organic layer. Ion‐pair HPLC confirms the presence of phosphate in the aqueous layer.