Determination of Atropine Enantiomers in Ophthalmic Solutions by Liquid Chromatography Using a Chiral AGP Column

Many therapeutic agents are commercialized under their racemic form. The enantiomers can show differences in the pharmacokinetic and pharmacodynamic profile. The use of a pure enantiomer in pharmaceutical formulations may result in a better therapeutic index and fewer adverse effects. Atropine, an alkaloid of Atropa belladonna, is a racemic mixture of l-hyoscyamine and d-hyoscyamine. It is widely used to dilate the pupil. To quantify these enantiomers in ophthalmic solutions, an HPLC method was developed and validated using a Chiral AGP column at 20C. The mobile phase consisted of a buffered phosphate solution (containing 10 mM 1-octanesulfonic acid sodium salt and 7.5 mM triethylamine, adjusted to pH 7.0 with orthophosphoric acid) and acetonitrile (99 + 1, v/v). The flow rate was 0.6 mL/min, with UV detection at 205 nm. In the concentration range of 14.026.0 g/mL, the method was found to be linear (r > 0.9999), accurate (with recovery of 100.1100.5), and precise (RSD system 0.6; RSD intraday 1.1; RSD interday 0.9). The method was specific, and the standard and sample solutions were stable for up to 72 h. The factorial design assures robustness with a variation of 10 in the mobile phase components and 2C of column temperature. The complete validation, including stress testing and factorial design, was studied and is presented in this research.