Rapid UPLC-MS/MS method for the determination of ketoprofen in human dermal microdialysis samples

Dermal microdialysis (DMD) is a technique capable of determining the percutaneous penetration of drugs from topical formulations intended for local and/or regional activity. Typically, the concentrations of drug collected in dialysates are very low, generally in the ng/ml or even pg/ml range. An additional challenge is the very low volume of sample collected at each collection time and which can range from 1 to 30 microl only. Hence the objective was to develop and validate a rapid, accurate, precise, reproducible and highly sensitive LC-MS/MS method for the quantitative analysis of ketoprofen (KET) in dialystes following application of a topical gel product to the skin of human subjects. UPLC-MS/MS was used and KET was separated on an Acquity UPLC BEH C(18) column (100 mm x 2.1 mm i.d., 1.7 microm) and analysed in negative-ion (NI) electrospray ionisation (ESI) mode. The mobile phase (MP) consisted of acetonitrile:methanol:water (60:20:20, v/v/v) under isocratic conditions at a flow rate of 0.3 ml/min. Samples were extracted using ethyl acetate with ibuprofen (IBU) as internal standard (IS) and the organic solvent was then evaporated to dryness and the residue re-constituted in methanol. 5 microl samples were injected and analysis was performed at ambient temperature 22+/-0.5 degrees C. KET and IBU eluted at 1.07 and 1.49 min, respectively. KET and IBU responses were optimised at the transitions 253.00>209.00 and 205.00>161.00, respectively. Calibration curves were linear over the range 0.5-500 ng/ml with correlation coefficients>0.999. The accuracy and precision of the method were found to be between 99.97% and 104.67% (R.S.D.<2%) and the mean recovery of KET from normal saline was 88.03+/-0.3% (R.S.D.<2.20%). The LLOQ and LOD values were found to be 0.5 and 0.1 ng/ml respectively whereas the ULOD was set at 500 ng/ml. The method was successfully applied to determine the bioavailability of KET following application of topical KET gel, Fastum gel, to the skin of human volunteers.