Trapping mode two-dimensional liquid chromatography for quantitative low-level impurity enrichment in pharmaceutical development.

ABSTRACT

Trapping mode two-dimensional liquid chromatography (2D-LC) has recently found applications in pharmaceutical analysis to clean, refocus, and enrich analytes. Given its enrichment capability, 2D-LC with multiple trappings is appealing for low-level impurity monitoring that cannot be solved by single dimensional LC (1D-LC) or unenriched 2D-LC analysis. However, the quantitative features of multi-trapping 2D-LC remain largely unknown at impurity levels from parts-per-million (ppm) to 0.15% (w/w). We present a simple heart-cutting trapping mode 2D-LC workflow using only common components and software found in typical off-the-shelf 1D-LC instruments. This robust, turn-key system's quantitative capabilities were evaluated using a variety of standard markers, demonstrating linear enrichment for up to 20 trapping cycles and achieving a recovery of over 97.0%. Next, the trapping system was applied to several real-world low-level impurity pharmaceutical case studies including (1) the identification of two unknown impurities at sub-ppm levels resulting in material discoloration, (2) the discovery of a new impurity at 0.05% (w/w) co-eluted with a known impurity, making the undesired summation above the target specification, and (3) the quantification of a potential mutagenic impurity at 10-ppm level in a poorly soluble substrate. The recovery in all studies was better than 97.0% with RSD lower than 3.0%, demonstrating accuracy and precision of the 2D-LC trapping workflow. As no specialized equipment or software is required, we envision that the system could be used to develop low-impurity monitoring methods suitable for validation and potential execution in quality-control laboratories.