Quantification of Melatonin, Caffeine, and Paraxanthine in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

Melatonin (MEL) and caffeine (CA) are mediated by cytochrome P450 1A2 (CYP1A2), and the plasma concentration of MEL is reportedly affected by CA intake and CYP1A2 activity. Because the plasma concentrations of MEL and CA or paraxanthine (PX) differ by approximately 10⁶, MEL, CA, and PX (a metabolite of CYP1A2) have not been quantified in a single-sample preparation. This study aimed to evaluate a liquid chromatography-tandem mass spectrometry method for quantification of MEL, CA, and PX in the same sample preparation. Initially, an injection volume of 10 µL produced a sharp peak for MEL, but the peaks for CA and PX were not suitable for quantification due to their asymmetric peaks. Therefore, CA and PX were separately quantified using 0.1 μL sample, which enabled measurement of both compounds with good peak symmetry. Under these conditions, the relative error for MEL, CA, and PX ranged from  − 9.62% to 1.01%, 0.96% to 9.39%, and − 2.77% to 5.67%, with relative standard deviations of 7.23%, 4.95%, and 8.54%, respectively. In conclusion, the developed method is suitable for use in future studies on the relationship among MEL, CA, and PX.