UPLC-MS/MS method for quantifying non-stimulant ADHD medications in human breast milk and plasma: Application in a...

Non-stimulant medications, such as atomoxetine (ATX) and guanfacine (GFC), are increasingly prescribed for attention-deficit/hyperactivity disorder (ADHD), including among women of childbearing age. However, experimental data on the transfer of these drugs into human breast milk remain extremely limited. In this study, we developed and validated a highly sensitive and precise ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantification of ATX, its major metabolites (4-hydroxy ATX: 4-OH ATX and N-desmethyl ATX: N-DAT), and GFC in human plasma and breast milk. Stable isotope-labeled compounds were used as internal standards (IS). Plasma samples were prepared using liquid-liquid extraction with ethyl acetate, and breast milk samples were treated by protein precipitation with acetonitrile containing 0.5% formic acid. The method required only 100 µL of sample volume and showed good linearity, accuracy, and precision over the tested ranges (plasma: 0.5-50 ng/mL; breast milk: 0.8-50 ng/mL). Matrix effects were evaluated using six individual lots of plasma and breast milk, and acceptable variability was achieved. The validated method was applied to plasma and breast milk samples from an ATX-treated lactating woman, yielding milk-to-plasma ratios (M/P) of 0.089 for ATX and 1.17 for 4-OH ATX. The relative infant dose (RID) was estimated at 0.086% for ATX and 0.059% for 4-OH ATX, and the combined RID was estimated to be 0.145%. This study provides a robust and reliable tool for assessing the transfer of non-stimulant ADHD medications into human breast milk and strengthens the evidence base for safe pharmacotherapy during lactation.