Determination of serotonin and its precursors in chocolate samples by capillary liquid chromatography with mass spectrometry detection

A method for the analysis of serotonin (5-HT) and its precursors, 5-hydroxytryptophan (5-HTP) and l-tryptophan (TP) in chocolate samples by capillary liquid chromatography–mass spectrometry (cLC–MS) has been developed. Optimum chromatographic conditions were established by using a personalized multifactorial experimental design. Finally the cLC separation was achieved through a mixture of acetonitrile and 5 mM ammonium formate at pH 4 (3:97, v/v) as mobile phase in gradient elution, setting the injection volume at 10 μL and using pure water as injection solvent for focusing purposes on the head of the capillary column. For extraction of targets in chocolate samples a new, fast and simple procedure based on the use of acidic extraction medium and sonication was developed. Working in selected ion mode (m/z 177 for 5-HT, m/z 205 for l-tryptophan and m/z 221 for 5-HTP) detection limits were between 0.01 and 0.11 μg g−1 and linearity was in the concentration range of 0.5–25 μg g−1. Recoveries higher than 76% with RSDs lower than 8% were obtained from spiked samples for all analytes, showing the effectiveness of the proposed method. Serotonin and its precursors were determined in 5 kinds of commonly consumed chocolates with different cocoa contents (70–100%). The highest serotonin content was found in chocolate with a cocoa content of 85% (2.93 μg g−1). Regarding l-tryptophan, the highest content of this amino acid (13.27–13.34 μg g−1) was found in chocolate samples with the lowest cocoa content (70–85%). 5-Hydroxytryptophan was not detected in any chocolate samples.