Determination of travoprost and travoprost free acid in human plasma by electrospray HPLC/MS/MS

A quantitative method for the analysis of AL-5848, the (+)-enantiomer of fluprostenol (FP), in human plasma is described. Plasma was spiked with a tetradeuterated analog of travoprost free acid (AL-5848X) as internal standard (IS) and acidified with 0.1 M formic acid. Sample clean up was performed using reversed phase solid-phase extraction. Following elution of the compounds of interest and evaporation to dryness, the residue was reconstituted in methanol:water (1:1) and chromatographed on an octadecylsilica (C18) column with negative ion electrospray ionization tandem mass spectrometry. The [M[bond]H](-) ions at m/z 457 and 461 for the analyte and IS, respectively, were subjected to collisional fragmentation with argon to yield the same intense 3-trifluoromethylphenolate (m/z 161) product ion. The validated concentration range was 0.010-3.00 ng/ml based on a 1.0 ml plasma aliquot. Fully adequate accuracy, precision, specificity, recovery and stability for routine use in clinical pharmacokinetic studies were demonstrated. Analysis of a second plasma aliquot following incubation with rabbit esterase allows the isopropyl ester pro-drug, travoprost (AL-6221), to be determined by difference.