Continuous sampling/transfer strategy to overcome sampling-volume mismatch in heart-cutting two-dimensional liquid...

Heart-cutting two-dimensional liquid chromatography (2D-LC) is widely used for pharmaceutical impurity profiling but suffers from sampling‑volume mismatch between the fixed loop volume and the elution volume of the target peak. Under‑sampling with small loops causes loss of sensitivity, whereas over‑sampling using an oversized loop leads to severe band broadening. Here we introduce a Continuous Sampling/Transfer (CST) strategy that overcomes this mismatch using remarkably simple hardware: only two identical loops and a standard 2‑position/4‑port duo valve. This system enables quantitative transfer of entire peaks irrespective of width by slicing the target peak into discrete fractions and transferring each immediately to the second dimension. Transfer efficiency exceeds 98%, with excellent linearity (r > 0.9999) and repeatability (RSD = 0.2%, n = 6). The utility of CST was demonstrated by impurity profiling of ertapenem for injection using size‑exclusion - reversed‑phase 2D‑LC. The CST interface precisely transferred eight peaks of varying widths from the first dimension and resolved 15 impurities in the second dimension, including two co‑eluting pairs undetectable by one‑dimensional RP method. High‑resolution mass spectrometry identified five previously unreported impurities. The CST strategy offers a hardware‑simple, robust solution to sampling‑volume mismatch, enabling in‑depth impurity profiling and separation performance assessment.