Identifying Drug Metabolites
A lab within the department of chemistry at Purdue University is developing methods for metabolite identification. “We first started developing these as a compliment to established methods of identifying functional groups in an unknown analyte,” explains Steven Habicht, a scientist working in the lab. He says there are certain situations where identifying specific impurities is not possible by MS/MS alone, and the more additional compounds, the longer it takes for analysis and the more expensive it is.
This HPLC-MS/MS method is based on ion-molecule reactions for identifying tertiary N-oxide functional groups in unknown analytes. “This is applied early in the drug discovery phase, but it could also be used for stability-indicating assays to see if any compounds will be formed when exposed to high heat, humidity, or from storage in plastic containers. If extra products are formed from the compound, you need to identify what those are and whether they are toxic.”
In order to introduce neutral molecules into a commercial quadrupole ion trap MS, his group developed an external reagent mixing manifold. This allows tri (dimethyl amino) borane (TDMAB) to be mixed with the helium buffer gas used in the trap. The analyte of interest is isolated in the trap and reacts with TDMAB for a specific time. The reaction delivers only protonated N-oxide analytes as they elute from the HPLC column. This process was demonstrated using Clozapine N-oxide and Olanzapine-N oxide.
“We’re creating our own niche in metabolite identification with these methods,” states Habicht. “There are situations where you can’t distinguish two different types of metabolites where these ion-molecule reactions can fill the void.” He adds that the main focus is to continue to develop a library of different reagents to make functional group identification a straight-forward process.
There is little doubt that many more potential applications for LC/MS will continue to be developed as more targeted drugs become available through the efforts of genomics, proteomics, and metabolomics. LC/MS may become standard for clinicians searching to identify sources of side effects or potential toxins. Who knows where these technologies will lead medicine over the next 100 years?