Pharmaceuticals

While tandem mass spectrometry experiments based on collision-activated dissociation often cannot be used to differentiate between isomeric ions, diagnostic gas-phase ion-molecule reactions have been demonstrated to provide a powerful tool for the identification of specific functional groups in ionized analytes. This approach involves isolation of protonated analytes in a tandem mass spectrometer and allowing them to react with reagents that yield diagnostic product ions only for protonated analytes containing specific functionalities. It has proven to be especially beneficial for the characterization of drug metabolites and impurities. Most drugs contain undesirable impurities that are formed during the synthesis process. Regulatory authorities limit the levels of impurities that contain potentially mutagenic functional groups. The purpose of this research is to develop automated methodology based on HPLC/tandem mass spectrometry experiments utilizing diagnostic gas-phase ion-molecule reactions for the structural characterization of unknown drug impurities containing potentially mutagenic functional groups. In order to completely automate these experiments, they are being coupled to machine learning in collaboration with Professor Chopra’s group (Chemistry, Purdue). A nine pulsed-valve interface is used to introduce nine reagents into the mass spectrometer for ion-molecule reactions while the analytes elute from the HPLC in order to monitor nine different reactions for each analyte in one HPLC run.

Figure 4. The diagnostic utility of employing reagents, such as 2-methoxypropene (MOP), to identify functional groups via diagnostic gas-phase ion-molecule reactions in protonated metabolites of a drug. After the metabolites were (a) protonated and isolated in the tandem mass spectrometer, (b) they were allowed to react with MOP and (c) the formation of a diagnostic addition product (DP) as opposed to proton transfer (PT) or no reaction was monitored. Only the protonated sulfoxide metabolites generated the diagnostic addition product ion (DP) with MOP.

Figure 5. Pulsed-valve assembly for the introduction of nine different reagents into an ion trap mass spectrometer that consists of nine pulsed-valve “stems” that plug into a manifold wheel that is mounted to a modified back plate of the mass spectrometer.

Figure 6. (Left) A selected ion chromatogram showing reactions of MOP, DMDS, DEMB, TMB, TMMS, HSiCl₃, ACE, TDMAB, and DMA (structures shown on top of the figure) with protonated albendazole sulfoxide, 4-hydroxy-3,5-dimethoxybenzoic acid, omeprazole N-oxide, dibenzothiophene sulfone, and clozapine N-oxide in the ion trap mass spectrometer as the compounds eluted from the HPLC, were protonated in an APCI source, and then were transferred into the ion trap. Signals for MS/MS ion-molecule reaction product ions specific to each reagent are represented by different colored circles and lines (each line corresponds to one pulsed introduction of a reagent into the mass spectrometer). (Right) Typical pulse sequence for the introduction of nine different reagents or reagent systems into the mass spectrometer for gas-phase ion-molecule reactions. Letters A − I refer to the nine different pulsed valves. The open time for each valve was 150 μs. A 1 s delay was used between the different reagent pulses to enable pumping of the reagents out of the ion trap before the next reagent was introduced.

References:

1. Fine, J; Liu, J.K-Y.; Beck, A.; Alzarieni, K.Z.; Ma, X.; Boulos, V.M.; Kenttämaa, H.I.; Chopra, G. Graph-Based Machine Learning Interprets and Predicts Diagnostic Isomer-Selective Ion–Molecule Reactions in Tandem Mass Spectrometry. Sci. 2020, 11, 11849−11858.
2. Kong, J.Y.; Hilger, R.T.; Jin, C.; Yerabolu, R.; Zimmerman, J.R.; Replogle, R.W.; Jarrell, T.M.; Easterling, L.; Kumar, R.; Kenttämaa, H.I. Integration of a Multichannel Pulsed-Valve Inlet System to a Linear Quadrupole Ion Trap Mass Spectrometer for the Rapid Consecutive Introduction of Nine Reagents for Diagnostic Ion/Molecule Reactions. Chem. 2019, 91, 15652−15660.