Effect of anionic additive type on ion pair formation constants of basic pharmaceuticals

Due to their beneficial effect on selectivity, peak shape, and sample loading, the use of mobile phase anionic additives, such as formate (HCOO−), chloride (Cl−), and trifluoroacetate (CF3COO−), is increasing in both reversed-phase chromatography (RPLC) and liquid chromatography-mass spectrometry (LC/MS). Similarly, perchlorate is a common “ion pair” agent in reversed-phase separation of peptides. Although many studies have suggested that anions effect in chromatography is due to the formation of ion pairs in the mobile phase between the anions and cationic analytes, there has been no independent verification that ion pairs are, in fact, responsible for these observations. In order to understand the mechanisms by which anionic additives influence retention in chromatography and ionization efficiency in electrospray mass spectrometry, we studied the formation of ion pairs between a number of prototypical basic drugs and various additives by measuring the effect of anionic additives on the electrophoretic mobility of the probe drugs under solvent conditions commonly used in chromatography. For the first time, ion pair formation between basic drugs and anionic additives under conditions commonly used in reversed-phase liquid chromatography has been confirmed independently with all anions (i.e. hexafluorophosphate, perchlorate, trifluoroacetate, and chloride) used in this study. We measured ion pair formation constants (Kip) for different anionic additives using capillary electrophoresis (CE) and obtained quantitative estimates for the extent of ion pairing in buffered acetonitrile–water. The data clearly indicate that different anionic additives ion pair with cationic drugs to quite different extents. The ion pair formation constants show a clear trend with the order being: PF6− > ClO4− > CF3COO− > Cl−. However, the extent of ion pairing is not large. At a typical RPLC mobile phase additive concentration of 20 mM, the percentages of the analytes that are present as ion pairs are about 15%, 6%, and 3% for hexafluorophosphate, perchlorate, and trifluoroacetate, respectively. The fraction of the analytes present as a chloride pair is even smaller.