P173 Using Ion Mobility LC-MS and LC-MS/MS to resolve and identify isobaric glucuronide metabolites

Jayne Kirk , Waters Corporation, Wilmslow, United Kingdom
Rachel Sanig , Waters Corporation, Wilmslow, United Kingdom
Russell Mortishire-Smith , Waters Corporation, Wilmslow, United Kingdom
Sherri Naughton , Waters Corporation, Milford, MA
Yun Alelyunas , Waters Corporation, Milford, MA
Mark Wrona , Waters Corporation, Milford, MA
Common metabolic pathways often give rise to multiple instances of isobaric metabolites such as, (but not limited to), multiple mono-hydroxylations and di-hydroxylations with and without phase II conjugations, such as glucuronidation. LC-MS analysis of metabolic pathways for drugs requires accurate detection and identification of closely eluting species with similar RT’s and spectral profiles. This is further complicated by shifting abundance profiles at different time points, species differences and RT shifts due to biological matrix effects. Modern chromatography (and mass spectrometry) is sufficient in many cases, however isobaric species remain problematic. Ensuring closely eluting species have not shifted by RT requires standard addition or co-mixing of matrix and true isobaric coelutions may go undetected with conventional LC-MS approaches as they rely solely on discrimination using precursor mass. Furthermore, the MS/MS patterns from related compounds are often extremely similar and coelutions give rise to complex spectra which can hinder identification and structural elucidation. MS/MS of metabolites at low concentrations may also introduce noise from matrix at high concentrations.

Ion mobility is a property that can further resolve metabolites and yield additional structural information. Metabolism on different parts of the drug molecule yield slightly different molecular shapes which can then be measured through ion mobility, yielding a collision cross section (CCS) measurement. When ion mobility is coupled with LC-MS or LC-MS/MS, clean and fully resolved spectra can be generated even in the case of closely eluting metabolites, and the metabolites themselves can be differentiated using CCS.

A large number of closely eluting metabolites including mono/di-hydroxylations and various glucuronidated species were accurately resolved using IMS-MS. Data independent acquisition (HDMSE) was used to analyze the samples, which enabled detection by m/z and CCS with high accuracy and clean product ion spectra. Two commercially available drugs, nefazodone and buspirone were incubated in multiple hepatocyte species at 10 µM substrate concentration and samples prepared for analysis by protein precipitation. Aliquots of each sample set were spiked into human urine at a ratio of 1:10. HDMSE as well as Product Ion Confirmation (PICS) spectra were collected using a VION IMS QTof mass spectrometer.

Preliminary timecourse data for Monkey, Dog, Human and Rat metabolites was analyzed. For nefazodone, CCS values of between 213.4 and 214.4Å were measured for hydroxylated species, 216.1 and 217.9Å for dihydroxylated species, and 243.7 and 259.5Å for glucronidative species (Parent = 210.1Å). For Buspirone, CCS values of between 191.9 and 199.9Å were measured for hydroxylated species, 197.1 and 201.0Å for dihydroxylated species, and 245.2 and 247.2Å for glucronidative species (Parent = 194.9Å). Within isobaric classes, the spread in size varied from 0.5-6% change is shape which is indicativate of how much steric bulk a moeity adds to the molecule, thereby causing it to adopt a new (more extended or compact) orientiation. In most cases as expected, the metabolite occupies more space than parent, but in some cases the addition of oxygens actually could create a more compact structure as well. Furthermore, +O fragmentation patterns that caused larger size changes, tended to track with additional modifications (such as second oxidation, glucuronidation) and helped confirm their relatedness (in addition to fragmentation). This property enabled additional information which led to better characterization, structural confirmation and tracking of multiple isobaric metabolites across species and provided clues to the location of the biotransformation on the parent molecule beyond mass and fragments alone.