Quantitative Determination and Stability of Trace Levels of Oxytocin in Human and Rhesus Macaque Urine by LC–MS/MS

Oxytocin (OT) quantification in biological samples is vital for understanding its role in social behavior, emotion, and brain development in humans and animals. Urinary OT analysis has gained attention but is challenging due to variability in matrix effects, background interference, low concentrations, and stability concerns. This study developed an efficient LC-MS/MS method using mixed-mode solid-phase extraction (SPE) for sample preparation and serially coupled columns for separation. It achieved a lower limit of quantification (LLOQ) of 1 pg/mL, surpassing the sensitivity of previous methods. The method was fully validated per FDA guidance. The extraction recovery was > 80% in human urine. The precision was < 9%, and accuracy was between 101% and 109%. Partial validation for rhesus macaque urine showed accuracy ranged between 93% and 109%, with precision under 11%. Stability studies confirmed that OT in urine remains stable at room temperature for 4 h and at -80°C for 30 days. SPE-extracted samples were stable at 4°C for 24 h, and OT was unaffected by up to three freeze-thaw cycles. These results indicate that the LC-MS/MS method is accurate, consistent, and reliable for measuring OT in human and rhesus macaque urine and provide guidance for proper sample handling and storage.