Method transfer from ion pair chromatography to charged aerosol detector-compatible mixed-mode chromatography: A case study using carbocisteine

Abstract

To take advantage of the charged aerosol detector (CAD) for sensitive LC analysis of analytes with weak chromophore, the volatility of the entire mobile phase is a prerequisite. Therefore, it is not feasible to use common non-volatile ion-pairing reagents such as tetrabutylammonium hydroxide for the analysis of polar, acidic amino acids.This case study presents a possible procedure for transferring an ion pair chromatography to an HPLCCAD method using the example of carbocisteine and its two main degradation products, carbocisteine sulfoxide and carbocisteine lactam. In screening experiments, substitution by various volatile alkylamines resulted in inferior chromatographic separation. As an alternative, mixed-mode chromatography using a column with strong anion exchange functionalities and a mobile phase containing 12 mM formic acid and 2 mM trifluoroacetic acid, led to a selectivity equivalent to ion pair chromatography with tetrabutylammonium hydroxide. A change in the level of background noise generated by non-volatile impurities eluting from the column during measurements – often called column bleeding – invalidated the settings predicted from response surface methodology experiments in terms of modelling chromatographic selectivity and CAD sensitivity, and a reduction of the acetonitrile ratio to 22% (v/v) was required. At a flow rate of 1.2 mL/min and an evaporation temperature of 65 °C, the experimentally determined LOQs of both degradation products were below 50 ng on column. The combination of mixed-mode chromatography with CAD offers a viable alternative to ion pair chromatography with UV detection in the analysis of acidic analytes with a weak chromophore such as amino acids and impurities thereof.