Development and validation of a LC-MS/MS method for the determination of sleep quality-related biomarkers in human urine for clinical testing

• A dual-strategy LC-MS/MS method was developed for 7 sleep-related biomarkers in urine. • Innovative precipitation-purification approach improved recovery of polar analytes. • Establishment of reference intervals for these biomarkers in morning urine from a healthy Chinese population. A sensitive and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of seven sleep quality-related biomarkers (SQBs) in human urine: cortisol, testosterone, 17α-hydroxyprogesterone, dehydroepiandrosterone sulfate, melatonin, 6-sulfatoxymelatonin, and serotonin. Due to the wide polarity range of the analytes, two distinct sample pretreatment approaches, liquid-liquid extraction for steroid hormones and a novel precipitation-purification strategy for polar neurotransmitters. The new approaches were optimized, along with dual LC-MS/MS analytical methods. The method demonstrated excellent linearity ( R ² > 0.99), precision (RSD < 15%), recovery (85.85–111.57%), and minimal matrix effects (88.47–97.03%). It was successfully applied to morning urine samples from 70 healthy volunteers to establish preliminary reference intervals. This study provides a robust analytical tool for sleep biomarker analysis and supports future clinical and epidemiological research on sleep health.