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This paper describes a high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) method for the determination of serotonin (5-HT) and the related indoles tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid (IAA), and indole-3-proprionic acid (IPA) in whole blood. Ionization was achieved by atmospheric pressure chemical ionization (APCI). The method uses a solvent gradient to achieve baseline separation of all analytes. Linearity was established for all components from 10 to 500 ng/mL, with the exception of TRP, which was calibrated from 0.4 to 20 microg/mL. Limits of quantitation were established that were well below the normal endogenous concentrations of 5-HT and its related indoles. Recoveries of 5-HT, TRP, 5-HIAA, IAA, and IPA were 95%, 91%, 98%, 79%, and 65%, respectively, from whole blood matrix and varied less than 2%. Serotonin, TRP, and IAA results were verified by cross-validation with an external laboratory using a previously validated HPLC method with fluorometric detection. A regression analysis of the two data sets resulted in correlation coefficients of 0.981, 0.964, and 0.987 for 5-HT, TRP, and IAA, respectively.

A Liquid Chromatographic-Tandem Mass Spectrometric Method for the Analysis of Serotonin and Related Indoles in Human Whole Blood