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Pharmacokinetic studies of topical ketoprofen formulations using continuous sampling techniques such as microdialysis (MD) or open-flow microperfusion (OFM) require sensitive assays due to small sample volumes. A simple and easy online-SPE-MS/MS method for ketoprofen analysis was developed for both MD and OFM samples obtained from rat dermal tissue. The quantification range is 25-5000 ng/ml with a limit of detection of 3 ng/ml using only 10 mu l sample volume. The method is characterized by a simple setup using a short polymeric SPE column (OASIS HLB) for desalting with 1.5 min run times in combination with a sensitive MS detection in negative ESI MRM mode. An easy sample workup procedure was used which enables high throughput analysis of a large number of samples for pharmacokinetic studies. In addition, a commercial available (fenoprofen) as well as an isotopically labelled (deuterated ketoprofen) standard were investigated as potential internal standards. The method was validated according to FDA guidelines for bioanalytical validation in terms of accuracy, intra-batch and inter-batch precision, linearity, matrix effect, recovery and stability for both internal standards. Accuracies were 98-113% (fenoprofen) and 95-108% (deuterated ketoprofen), intra-batch precision was 2-3% R.S.D. (fenoprofen) and 2-6% R.S.D. (deuterated ketoprofen), and inter-batch precision was 2-6% R.S.D. (fenoprofen) and 3-6% R.S.D. (deuterated ketoprofen) over the entire quantification range. The presented method was applied to dermal interstitial fluid samples obtained in a topical administration study of ketoprofen in rats. (c) 2007 Elsevier B.V. All rights reserved.