Effect of Alternative Mobile Phase Additives on the Peak Shape of Basic Analytes in RP-HPLC

Sammanfattning

Introduction: Reversed phase liquid chromatography (RPLC) is a central analytical technique in pharmaceutical and biopharmaceutical science, where it is essential to reliably detect, separate, quantify, and purify drugs, of which a majority contain basic groups. Background: Basic analytes often exhibit poor peak shape, for example tailing, due to mixed-mode interactions with the stationary phase. Acidic additives are commonly used to lower pH and act as ion pairing agents, improving chromatographic performance. Unfortunately, strong ion pair agents often suppress the signal in mass spectrometry detection. Followingly, it is desirable to find alternative additives that fit the silver lining between good chromatographic performance and MS compatibility. Aim(s): This study aims to evaluate additive effects on peak shape, mainly tailing factor (Tf), of amines and peptides in analytical and overload conditions on two columns. Methods: Different additives of varying concentrations were used to analyse Tf and w50% for amines and peptides in both analytical and overload conditions on HPLC-UV and -MS. Tf was calculated with the help of an Excel template and statistical analyses such as ANOVAs were conducted in RStudio. Results: PrA and GA generally yielded worse tailing for the amines, than all the other additives. Peptides exhibited charge-dependant tailing and increased additive concentrations mitigated the extent of that tailing in both analytical and overload conditions. TCA was found not to be a suitable alternative to TFA with regard to MS compatibility, as indicated by previous studies. Conclusion: No universally best additive was found, and the importance of MS compatibility was highlighted. Additive effects are highly dependent on analyte type and column.