The STEP method (statistical test of equivalent pathways): application to pharmaceuticals

The STEP method (Statistical Test of Equivalent Pathways), recently developed to determine primary and secondary fragmentation in the MS/MS of peptides and carbohydrates, is applied in the current study to common pharmaceutical antibiotics. The classification of product ions as primary or secondary is then utilized to construct genealogy diagrams that aid in the structural characterization of the product ions. Four compounds were subjected to the MS/MS conditions used for the STEP method, and the method was used to correctly identify primary and secondary ions in three of the four pharmaceuticals. Calculated STEP values for erythromycin did not match previously characterized fragmentation assignments. This provided an opportunity to explore potential limitations of STEP analysis. It was determined that inaccurate STEP assignments could result, if the starting compound is classified as “fragile”, because fragile ions, such as erythromycin can produce abnormally low STEP ratios. While this finding represents a limitation of using the STEP method to determine whether product ions are due to primary or secondary fragmentation for fragile ions, it suggests the possibility of identifying the presence of “fragile ions” by STEP analysis.