Investigations on drug related compounds using online photoreactor HPLC hyphenation and complementary mass spectrometric characterization illustrated by ketoprofen and related benzophenone compounds

Abstract

Here a comprehensive strategy for both photostability testing of drug substances and a complementary structural characterisation approach for drug related compounds, including photoproducts was established and tested. The photostability studies were performed using a tailored online photoreactor-SPE-LC system and its potential was demonstrated by irradiation of Ketoprofen as a model compound. The new devise allows fast and easy photostability testing that may help to reduce time and animal trials. Using this state of the art setup, kinetic studies can easily be performed with qualitative and quantitative perspectives combined into one experimental design with only very low amounts of pharmaceutical substance needed. The system allows online coupling using ESIMS or offline EI-mass spectrometric characterization of photoproducts after fractionation. Moreover, scale-up process can be realized for structure confirmation and can be used for further toxicity tests and as reference material for product quantification. Deuterated photoproducts were derived via photolysis experiments and used for further inspections on structural proposals. In order to increase the confidence level of MS-characterization of unknown compounds, a tiered mass spectrometric approach was suggested through this work for EI-mass spectrometric characterization of ketoprofen and its related benzophenone derivatives including those identified as photoproducts. In addition to high resolution accurate mass measurements derived from recent mass spectrometric instrumentation, the established approach comprises isotopic labelling together with tandem mass spectrometry. The isotopic labelling was performed by both deuteration and 18O labelling using different methods in order to produce compounds that contain deuterium or oxygen-18O at definite positions. The suggested approach demonstrated its potential as a powerful tool for mass spectrometric characterization of benzophenone derivatives and fragment ion recognition. Upon these promising results, it can be used to increase the level of confidence in the identification of unknown analytes, where reference material is not (yet) available. The new photostability setup testing together with the established mass spectrometric approach can be beneficial for the simulation of photoreactions in vivo. This could be achieved by irradiation of mixtures to mimic the biological situation and identify the resulted compounds.. A similar strategy may also be used for other stability testing utilizing a slightly different reactor design, in which various factors affecting drug substances such as temperature and environmental conditions can be investigated. Structure elucidation as well as generation of reference materials upon these stability testing can then be realized.