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

Abstract

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.

CASMI 2013: Identification of Small Molecules by Tandem Mass Spectrometry Combined with Database and Literature Mining

“…e literature was searched for published tandem mass spectra of these uoroquinolones and compared with the provided data with the best match to o oxacin. [13][14][15] e spectral match further supported the proposed molecular formula. O oxacin was submitted as the sole structure candidate with good con dence (Score= 0.85).…”

Application of the statistical test of equivalent pathways (STEP) method to the triple quadrupole mass spectrometer

“…Two MS/MS spectra were acquired at high and low dissociation energy conditions at each of these collision gas pressures. For each experiment, data was acquired for 2 min with a scan time of 10 s and interscan time of 0.1 s. For initial method development studies, both high and low dissociation spectra were acquired at conditions similar to those previously used on an ion trap mass spectrometer 4, 18. Briefly, the high‐energy CID spectrum was obtained when the precursor ion was depleted to 0–2% relative abundance, as described previously.…”

“…The study of the nature of characteristic fragmentations of gaseous ions by mass spectrometry is important because understanding basic fragmentation information is helpful in deducing precursor ion structures for unknown compounds 1–3. Fragmentation information can be used to establish genealogical relationships, which are important in discerning information about the connectivity of groups that fragment during tandem mass spectrometric (MS/MS) experiments 4. Since first‐generation product ions (primary product ions) directly originate from the precursor ion while the second generation of ions (secondary product ions) originate from other product ions in the MS/MS experiment, genealogy information can be useful in piecing together the structure of a precursor ion, based on the product ions detected in the MS/MS data 5–7…”

“…The STEP (statistical test of equivalent pathways) analysis, which was originally developed on the ion trap mass spectrometer, is another tool that differentiates primary product ions from secondary product ions 4, 18. Applicability of this method was previously tested with peptides and carbohydrates 18.…”

Tunable transition metal-ligand complexation for enhanced elucidation of flavonoid diglycosides by electrospray ionization mass spectrometry

“…Tandem mass spectrometry has been used extensively for structural elucidation of isomers [1][2][3][4][5][6][7][8][9][10][11][12] and there continues to be interest in applying existing tandem mass spectrometric methods, such as MS n [9,13,14], and developing new ones, such as STEP [15,16], a statistically based method that relies on comparison of abundances of fragment ions obtained for different collision activation conditions, to differentiate isomers. Our group has focused on the development of metal complexation strategies to enhance the differentiation of isomers [17][18][19][20][21][22][23] because metal complexation often alters the dissociation pathways of ions, thus giving additional diagnostic fragment ions.…”