Development and application of a dual isotopic labeling method for enhanced detection and quantification of stimulants in urine samples using high-resolution mass spectrometry

Given the critical nature of anti-doping efforts, the detection of stimulant substances is shifting from accurate qualitative analysis to precise quantitative analysis. Additionally, the use of liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in detecting stimulants is becoming more widespread. However, the lack of isotope-labeled internal standards is causing increasing issues with quantitative accuracy. Furthermore, challenges such as the mass spectrometric response of small molecules and the separation of isomers present additional difficulties. We have developed a quantitative method for stimulant substances containing amine or phenol hydroxyl groups, using a dual-label derivatization system. This method offers a new perspective for analyzing and detecting low molecular weight substances, isomers, or those with poor LC-MS response, and proposes a solution to the problem of missing isotope-labeled internal standards. Methodological validation has shown that this approach has promising application potential.