Development and validation of a rapid LC-MS/MS method for methylphenidate, atomoxetine, and their metabolites with...

Therapeutic drug monitoring of attention-deficit/hyperactivity disorder (ADHD) pharmacotherapy is challenged by substantial interindividual variability and the lack of rapid, multiplex analytical platforms. We developed and validated a fast, integrated 5-minute LC-MS/MS assay for the simultaneous quantification of stimulant (methylphenidate, ritalinic acid) and non-stimulant (atomoxetine, 4-hydroxy-atomoxetine) ADHD medications in human serum. The method employs a single-step protein precipitation and requires only 200 µL of serum. Validation demonstrated excellent linearity, precision, accuracy, minimal matrix effects, and robust analyte stability in accordance with FDA and CLSI guidelines. Application to pediatric ADHD patients revealed formulation- and dose-dependent concentration differences for methylphenidate and atomoxetine, along with strong parent-metabolite correlations. No significant associations were observed with age, sex, or body mass index. This rapid and reliable LC-MS/MS platform supports clinically actionable therapeutic drug monitoring and provides a practical framework for personalized ADHD treatment.