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Fentanyl, (N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylpropanamide) is a potent, synthetic opioid used as an anaesthetic, peri-operatively and for management of chronic pain. The delivery through transdermal patches (Durogesic®) is increasingly popular, with slow release of the drug into the bloodstream over 2-3 days using patches manufactured for different desorption rates (12-100 µg/h). For transdermal delivery, application of e.g. heat on the patch will increase plasma concentration. Analytical methods for pharmacokinetic studies of heating effects must be very sensitive, precise and accurate, as typical, steady-state plasma concentrations are below 1 ng/mL. Early research was based upon radioimmunoassay (RIA), however, these methods are no longer available and their performance suffered from cross-reactivity with metabolites causing overestimation at low concentrations.