Simultaneous determination of selective serotonin reuptake inhibitors and their main metabolites in human breast milk by liquid chromatography-electrospray mass spectrometry

• Quantification of all SSRI antidepressants in human breast milk by HPLC–MS.
• Method validation over a large concentration range including major metabolites.
• Method application to patient samples from a clinical pharmacokinetic study.

A bioanalytical method by high performance liquid chromatography coupled to electrospray mass spectrometry (HPLC-ESI–MS), adapted from a previously published method in plasma, was validated in breast milk for the simultaneous quantification of all antidepressants belonging to the class of selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their major metabolites (desmethylcitalopram and norfluoxetine). Milk samples (250 μl) first underwent protein precipitation followed by solid-phase extraction on a reversed phase/cation exchange sorbent. Analytes were thereafter separated on a XBridge C18 column (2.1 mm × 100 mm; 3.5 μm) using a mobile phase composed of ammonium acetate buffer (pH 8.1; 50 mM) and acetonitrile in gradient mode. Detection was performed by a single quadrupole mass spectrometer running in selected ion monitoring in positive ionization mode. Method validation covered a wide concentration range of 2–500 ng/ml for citalopram, desmethylcitalopram and paroxetine, 5–500 ng/ml for sertraline, and 2–1000 ng/ml for fluoxetine, norfluoxetine and fluvoxamine. Validation performances such as trueness (90.3–111.6%), repeatability (0.8–9.3%) and intermediate precision (0.9-9.5%) were in agreement with criteria from international guidelines and matrix effects for the analyte/internal standard ratios ranged from 92% to 110% (relative standard deviation <15%). Accuracy profiles (total error of trueness and precision) were lying within the limits of ±30% accepted in bioanalysis. Finally, the method was successfully applied to patient samples collected in a clinical pharmacokinetic study of nursing mothers taking an antidepressant treatment.

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