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Etoricoxib is a potent and novel selective inhibitor of cyclooxygenase-2 (COX-2) which has been developed for the treatment of osteoarthritis, rheumatoid arthritis and several other inflammatory conditions. To support clinical pharmacokinetics studies, a method for the determination of etoricoxib in human plasma was developed. During the development of the method it was found that highly fluorescent products were formed when etoricoxib was exposed to UV light (254 nm). The formation of highly fluorescent products was the basis for the development of a highly sensitive HPLC/fluorescent assay for the indirect determination of etoricoxib in human plasma; the limit of quantification (LOQ) was 1 ng/mL. To unequivocally determine the chemical structures of the photolysis products of etoricoxib, a series of studies was conducted. When etoricoxib was irradiated online in a photochemical reactor, three products were detected in an HPLC-UV system. These products were characterized by HPLC-UV-fluorescence and HPLC-MS/MS. Possible structures of these products were proposed based on these data. The major photolysis products of etoricoxib were further isolated and their structures were elucidated using NMR and HPLC-NMR. The results of these experiments indicate that etoricoxib undergoes a photocyclization reaction when irradiated with UV light (254 nm), leading to the formation of two major isomeric photocyclization products.

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6'-methyl-[2,3']bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard (II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C8 SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C8/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP guard column (20 x 4.6 mm) connected to a Prism-RP analytical column (150 x 4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH = 4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (lambda(ex) = 260 nm, lambda(em) =375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5-500 ng/ml for human plasma and human urine yielded a linear response (R2>0.99) when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n = 5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.