Sub Topic | Secondary Topic: Analytical Methodology - Non-biological Matrix | HPLC (Small Molecule)
Authors: Yue Hu, Bristol-Myers Squibb Company (Main Author, Presenting Author)
Presenting Author: Yue Hu
Purpose: Determination of trace level polymeric impurities in small-molecule pharmaceuticals presents significant challenge to separation scientists. Polymeric impurities are a mixture of molecules with same repeating unit but a distribution in the number of repeat units. They can be retained by multiple retention mechanisms such size-exclusion, precipitation/re-dissolution, and adsorption/desorption on analytical columns and typically elute as a broad band which often co-elute with their parent compounds and other small molecule impurities. Polymeric impurities may differ significantly in their solubility and retention mechanisms from their small-molecule parent compounds, further complicating sample preparation and chromatographic separation. This poster presents a systematic approach in developing a quantitative method for the determination of trace level (0.05%) of a polymeric impurity in a marketed small-molecule pharmaceutical compound. The general application of the finally adopted methodology is also discussed.
Methods: Different types of chromatographic separations were explored step-by-step from size-exclusion chromatography (SEC), normal-phase chromatography (NPC), to reversed-phase chromatography (RPLC). In order to apply RPLC, the polymeric impurity is derivatized (hydrolysis) to improve its solubility in diluent of acetonitrile/water mixture. The un-derivatized parent compound is evaluated as external standard for the quantification of the polymeric impurity. Method validation is performed after the final chromatographic conditions are selected.
Results: In SEC analysis, the polymeric impurity elutes as an extremely broad band making this separation mode unsuitable for trace analysis due to lack of method sensitivity. NPC encounters similar challenge in sensitivity. Derivatization (hydrolysis) of the polymeric impurity greatly improves its solubility in acetonitrile/water mixture making analysis under RPLC conditions feasible. However, when the derivaitzed polymeric impurity is retained under typical RPLC conditions, the peak is very broad and co-elutes with other small-molecule analytes and its retention behavior follows gradient polymer elution chromatography. A size-exclusion chromatography is finally developed under RPLC conditions, where the derivatized polymeric impurity is excluded from the pores of the column stationary phase and elutes as a sharp peak before solvent front while other small-molecule analytes including the parent compound elute around or after the solvent front. The use of a small-molecule external standard instead of a polymer standard for the quantification of the polymeric impurity is proven acceptable. A relative response factor, RRF, is required for calculation due to two facts: 1) the polymeric impurity is quantified against a small-molecule standard and 2) the polymeric impurity itself is derivatized. The final method is successfully validated with a recovery of 80.0, 90.0% and%96.6% at 0.05%, 0.3% and 0.6% levels, respectively. The Minimum Quantitation Limit of the method is 0.02%.
Conclusion: A size-exclusion chromatography under reversed-phase liquid chromatography conditions was developed for the determination of a polymeric impurity in a small-molecule pharmaceutical. The approach utilizes the large molecular mass of the polymeric impurity to separate it from all other small-molecule analytes including its parent compound by size exclusion. The approach reaps the benefit of high efficiency of RPLC which is unrivaled by SEC or NPC. The use of the un-derivatized parent compound as external standard for quantitation purpose proves to be a very practical choice considering the significant efforts would be required for the characterization, qualification and inventory management of a polymer standard. The methodology presented here can be applied to other polymeric impurities present in small-molecule pharmaceuticals as long as the solubility of those polymeric impurities can be manipulated to make their analysis feasible under RPLC conditions.
See attached abstract pdf for images.