The effect of analyte acidity on signal suppression and the implications to peak purity determinations using atmospheric pressure ionization mass spectrometry

The effect of a co-eluting halogenated phenol, spiked at 1% of the main analyte level, has been examined for a series of halogenated phenols using LC–MS techniques. Similarly, the effect of co-eluting anilines has been investigated. The purpose of the work presented here was to evaluate the degree of signal suppression for structurally similar halogenated phenols and for similar anilines utilizing atmospheric pressure chemical ionization (APCI) in the negative mode and electrospray (ESI) in positive mode, respectively. A correlation between the effects of analyte ionization efficiency resulting from co-eluting compounds (signal suppression) and pKa has been made for these compounds. It was found that minimal signal suppression occurs when the spiked impurity has a similar (ΔpKa < 1.5) acidity when compared to the main peak it is co-eluting with. The degree of signal suppression sharply increases when the difference in pKa's between the main peak and the spiked impurity was greater than 1.5 units. Thus, when the main peak is much less acidic (more than 1.5 pKa difference) than the co-eluting impurity, signal suppression of the latter would not occur in negative mode APCI. Similarly, when the main peak is much less basic than the co-eluting peak, signal suppression of the impurity will also not be found for aniline compounds in positive mode ESI. Furthermore, the degree of signal suppression decreases as a function of sample load such that injections of 3 μg or less show no discernible impact on the spiked impurity peak. Ultimately, these results indicate that the use of mass spectrometry (MS) in peak purity determinations requires numerous considerations prior to assessing main peak purity. The optimization of sample load during an impurities assay will maximize co-eluting impurity signal as purity determinations by mass spectrometry made at sample loads above the 3 μg (sample load) threshold increase the risk for false negative assessment of impurities.