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For the further study of [19F]‐alfatide, the development of highly sensitive analytical methods for its determination is an urgent issue. In this paper, a method for simultaneously determining [19F]‐alfatide and alfatide using liquid chromatography/tandem mass spectrometry was created and validated. The plasma samples were pretreated using the protein precipitation method. Poroshell 120 EC‐C18 (4.6 × 50 mm, 2.7 μm, Agilent) was used for the separation of the analytes to be measured. The mobile phase, consisting of acetonitrile and water (0.1% acetic acid, 50:50, v/v), was delivered at a flow rate of 0.60 ml/min for sample analysis. Positive electrospray ionization was performed using multiple‐reaction monitoring with transitions of m/z 715.2 → 636.6 for [19F]‐alfatide, the ion pairs 700.3 → 851.5 for alfatide and the ion pairs 237.1 → 194.2 for carbamazepine (internal standard). According to the results, the method had high specificity, precision and accuracy as well as an extended linear range. The matrix effect and extraction recovery were also acceptable. The method was successfully applied to the pharmacokinetic study of [19F]‐alfatide in rats.

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