To establish a highly efficient, sensitive, and accurate ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) method to determine the concentration of melatonin (MLT) in human plasma with heparin sodium.

The concentration of melatonin in plasma was measured using UHPLC-MS/MS. Plamsa sample preparation was done by protein precipitation using acetonitrile, its isotope melatonin-d4 (MLT-d4) was used as the internal standard (IS). Using UPLC BEH C₁₈ (2.1 mm×50.0 mm, 1.7 μm) reverse phase chromatography column, gradient elution was performed with a mobile phase consisting of acetonitrile-water (0.05 mM Ammonium fluoride) at a flow rate of 0.4 mL·min^-1 for 3 min. MLT was monitored using positive electrospray triple quadrupole mass spectrometer via multiple reaction monitoring (MRM) mode. The monitored transitions were set at m/z 233.0→174.0 for MLT. Moreover, the IS was set at m/z 237.2→178.2. This method was applied in a single-center, randomized, open-label, single oral dose pharmacokinetic trial, with 12 subjects in each group for fasting and after meals, administered a dose of 2 g (containing 4 mg of melatonin). Statistical analysis of pharmacokinetic parameters C_max (ΔC_max), AUC_0-t (ΔAUC_0-t), and AUC_0-∞ (ΔAUC_0-∞) using WinNonlin version 8.3 or above or SAS version 9.4 or above.

The achieved lower limit of quantification was 50.00 pg·mL^-1. The validated method had an excellent linearity in the range of 50.00-50 000.00 pg·mL^-1 for MLT y=2.00x+1.12×10^-4 (r²>0.999 4). The results of the intra-batch and inter-batch precision showed that the relative standard deviation (RSD) at different concentrations did not exceed 5.77%, and the accuracy was between 92.11% - 102.01% . The extraction recoveries of low, medium and high quality control samples of MLT were 103.35%, 113.75% and 131.21%, respectively. The coefficient of variation was 12.13%. No significant matrix effect was found. Through the pharmacokinetic study of 12 healthy humans, it was found for the first time that after taking the same dose of melatonin, the postprandial C_max was about 50% of that of fasting, but the AUC was very similar, indicating that food might affect the absorption and metabolism of melatonin in the body.

This method has strong specificity, good precision and high sensitivity, which can quickly and accurately determine the content of melatonin in human plasma with heparin sodium, and is used for the detection of human blood drug concentration and pharmacokinetics of melatonin.