Identification of the carboxylic acid functionality in protonated drug metabolite model compounds by using tandem mass spectrometry based on ion-molecule reactions coupled with high performance liquid chromatography

Abstract

The carboxylic acid functionality is among the many functionalities that drug metabolites, degradation products and impurities may contain. Therefore, analytical techniques that can be used to rapidly identify the carboxylic acid functionality are important for pharmaceutical industry. In this study, a tandem mass spectrometric approach based on diagnostic gas-phase ion-molecule reactions with (isopropenoxy)trimethylsilane (ITS) was developed for the identification of protonated compounds containing the carboxylic acid functionality in a modified linear quadrupole ion trap mass spectrometer. All protonated carboxylic acid, N-oxide, sulfoxide, carboxamide, ketone, amine, hydroxylamine, sulfone, sulfonamide, amide, ether, ester and aldehyde model compounds studied react with ITS to form protonated ITS and an adduct that has lost an acetone molecule, [adduct – acetone]+. Protonated compounds containing the carboxylic acid functionality can be differentiated from most other types of protonated compounds based on the formation of a secondary product, 2° [adduct – acetone]+, upon reactions of the [adduct – acetone]+ primary product with a second ITS molecule via addition and elimination of an acetone molecule. Quantum chemical calculations suggest that both addition/acetone elimination reactions are initiated by proton transfer to ITS. Nucleophilic substitution of acetone at the silicon atom by the carbonyl oxygen atom of the carboxylic acid or the primary reaction product leads to elimination of acetone. An equimolar mixture of 4-aminobenzoic acid, clozapine N-oxide, oxcarbamazepine, diphenylsulfoxide, naproxen and celecoxib was subjected to analogous HPLC/tandem mass spectrometry experiments to demonstrate the utility of this method for high-throughput drug metabolite characterization. A limitation of this method is that compounds that contain a phenol group and an additional basic functionality can show a false positive result.