Reconsidering mass spectrometric fragmentation in electron ionization mass spectrometry - new insights from recent instrumentation and isotopic labelling exemplified by ketoprofen and related compounds.

RATIONALE: In various fields of chemical analyses, structurally unknown analytes are considered. Proper structure confirmation may be challenged by the low amounts of analytes that are available, e.g. in early stage drug development, in metabolism studies, in toxicology or in environmental analyses. In these cases, mass spectrometric techniques are often used to build up structure proposals for these unknowns. Fragmentation reactions in mass spectrometry are known to follow definite pathways that may help to assign structural elements by fragment ion recognition. This work illustrates an investigation of fragmentation reactions for gas chromatography/electron ionization mass spectrometric characterization of benzophenone derivatives using the analgesic drug ketoprofen and seven of its related compounds as model compounds. METHODS: Deuteration and 18 O-labelling experiments along with high-resolution accurate mass and tandem mass spectrometry (MS/MS) were used to further elucidate fragmentation pathways and to substantiate rationales for structure assignments. Low-energy ionization was investigated to increase confidence in the identity of the molecular ion. RESULTS: The high-resolution mass analyses yielded unexpected differences that led to reconsideration of the proposals. Site-specific isotopic labelling helped to directly trace back fragment ions to their respective structural elements. The proposed fragmentation pathways were substantiated by MS/MS experiments. CONCLUSIONS: The described method may offer a perspective to increase the level of confidence in unknown analyses, where reference material is not (yet) available.

Photostability testing using online reactor HPLC hyphenation and mass spectrometric compound identification illustrated by ketoprofen as model compound.

Investigations on the photochemical stability of pharmaceutical substances are mandatory in drug development and licensing as photo-induced degradation of an active pharmaceutical ingredient (API) may not only lead to decreased API concentrations but also to toxic or reactive products. Thus, the US Food and Drug Administration (FDA) and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) issued Guidance for Industry Q1B "Photostability Testing of New Drug Substances and Products" for testing of pure but also packed drugs. However, photoproducts are also known to be generated in vivo under sunlight exposure of the skin and lead to considerable amounts of adverse drug effects. Herein we present an alternative system that may be used for photostability testing mimicking both situations. It combines a tailored photoreactor with an exchangeable pen light source and a modified HPLC system with online-SPE. Identification of photoproducts may be performed using mass spectrometry. The potential of accurate mass spectrometry as a tool for identification of photoproducts was demonstrated as well. A comparison of the online photoreactor system and the traditional photochamber irradiation was performed using ketoprofen for proof of concept. In both designs acetylbenzophenone and ethylbenzophenone were detected as main photoproducts. The new device allows for fast and easy photostability studies that may help to reduce time consuming in vitro experiments and animal trials. Using state of the art instruments kinetic studies could also easily be performed with qualitative and quantitative perspectives combined into one experimental design with only very low amounts of API needed. This may be useful in early drug development, where only small amounts of API are available. Scale-up may also be easily realized for the generation of reference material for quantification and quality control (QC) processes as well as for toxicity testing.