LC–MS/MS determination in rabbit plasma of the main photoproduct of RLP068/Cl, a cationic sensitizer proposed for photodynamic therapy (PDT) of microbial infections

The clinical development of a sensitizer for photodynamic therapy (PDT) requires the structural identification of the photoproducts and their quantification in biological fluids and tissues. We describe the LC–MS identification of the most important photoproducts of a cationic phthalocyanine sensitizer (RLP068/Cl) and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of the main photoproduct (the cationic phthalimide derivative 3-[(1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl)oxy]-N,N,N-trimethylbenzenaminium chloride) in rabbit plasma. The tri-deuterated product was used as co-eluting internal standard. The cationic photoproduct was isolated from plasma samples by protein precipitation with perchloric acid in methanol (7%, v/v). HPLC step was performed on a Phenomenex Synergi Hydro-RP column (20 mm × 2.0 mm, 2 μm particles) with a mobile phase of 0.5% (v/v) aqueous TFA/methanol (85:15, v/v). Flow rate was 0.2 mL/min and 40 μL injection were performed. Run time was 10 min. Detection was achieved by means of a Bruker Esquire 3000+ ion trap mass spectrometer equipped with an ESI source working in positive mode. A multiple reaction monitoring method following the transitions 297.1 → 282.1 for the analyte and 300.1 → 282.1 + 285.1 for the internal standard was used. The analytical method was validated over the concentration range 0.46–91.2 ng/mL and lower limits of detection (LLOD) and quantification (LLOQ) respectively of 0.2 and 0.5 ng/mL were found.