Objective To evaluate the sources of uncertainty in determination of melatonin in dietary supplements using ultra-high performance liquid chromatography-mass spectrometry (LC-MS) technology, to explore the effects of each component, and to improve the accuracy of the determination method.

Methods The sources of uncertainty in establishment of a method for determining melatonin in dietary supplements using liquid chromatography-mass spectrometry were analyzed. These sources mainly included non-uniformity, balance weighing, repeatability of sample testing, solution preparation, standard curve fitting, and instrument tolerance error, etc, and the synthesis of these uncertainties was also discussed.

Results The factors that contributed significantly to uncertainty were mainly the process of sample preparation and curve fitting. The expanded uncertainty for 500 mg melatonin tablets in content determination was 15.624 ng·mL^-1 (P=95%, k=2).

Conclusion The uncertainty of measuring melatonin content by liquid chromatography-mass spectrometry is mainly introduced in the context of standard curve fitting, solution preparation, and sample pretreatment. The experimental process should be strictly standardized, steps should be simplified, and the accuracy of experimental measurement results should be improved.