Agilent Technologies has produced an application note demonstrating the use of high-resolution (HiRes) sampling 2D-LC for the separation and detection of impurities, exemplified for different isomers of chlorodifluorobenzoic acid at relative concentrations of 100 % and 0.05 %.
Introduction
Purity analysis of drug substances is of high importance regarding quality and patient safety. According to ICH Guideline Q3A(R2), an impurity in a new drug substance should be reported above a threshold of 0.05 % relative to the API. Separation and detection of impurities at these levels can be challenging, especially when the impurity is structurally related to the active pharmaceutical ingredient (API).
This application note demonstrates the separation of two coeluting compounds, one of which is present at very low concentration, and is hidden under the other more highly concentrated compound peak.
In HiRes sampling 2D-LC, target compounds can be determined by collecting several small fractions over a selected time range, covering the entire area of a peak from the 1D chromatogram. Each cut is parked in a sampling loop, and all cuts are analysed in the second dimension consecutively. In this mode, it is ensured that all of the selected compound is transferred to, and analysed in the second dimension. This way, selected coeluting compounds can be subjected to the HiRes sampling process for analysis in the second dimension.
Experimental and Software
For experimental and software details please refer to the application note by downloading it using the button below.
Conclusion
This application note demonstrates the detection of low-level impurities contained in a sample of a structurally related main compound using high‑resolution sampling 2D-LC. Using a mixture of chlorodifluorobenzoic acids, it is shown that compounds at relative concentrations of 0.05 %, 0.10 %, 0.15 % and 100 % according to the ICH Guideline about impurities in new drug substances, which cannot be detected separately in 1D analysis, are transferred to and separated on the 2D column by HiRes sampling 2D-LC.
