Development and validation of an analytical method for quantification of 15 non-tricyclic antidepressants in serum with UPLC-MS/MS

Abstract

Non-tricyclic antidepressants are often encountered in overdose cases, and for some of them therapeutic monitoring might be warranted. Therefore, we developed and validated an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of 15 common non-tricyclic antidepressants in serum: citalopram, desmethylcitalopram, desmethylfluoxetine, desmethylvenlafaxine, duloxetine, fluoxetine, fluvoxamine, melitracen, mianserin, mirtazapine, paroxetine, reboxetine, sertraline, trazodone and venlafaxine. The selected pharmaceuticals represent the most common non-tricyclic antidepressants available in Belgium. Serum samples (250 μL) were precipitated with methanol and acetonitrile containing 5 ng of the internal standards: mirtazapine-D 4 , paroxetine-D 5 , reboxetine-D 6 and venlafaxine-D 6 . The supernatant was evaporated at 56 °C under a flow of nitrogen and reconstituted in 100 μL of 50:50 methanol:water with 2 mM ammonium acetate. Five μL were injected and the target analytes were separated on a 100 × 2.1 mm ACQUITY BEH C18 column (Waters, Zellik, Belgium) using a Waters ACQUITY Ultra-Performance Liquid Chromatography system. The analytes were eluted within 7.5 min, using a gradient of 2 mM ammonium acetate and 0.1% formic acid in water and in methanol at a flow rate of 0.4 mL/min. Quantification was performed on a Waters triple-quadrupole ACQUITY TQD using multiple reaction monitoring (MRM) in positive mode (2 MRMs per analyte). A six-point calibration curve was used to cover a large concentration range from 10 ng/mL to 1000 ng/mL. The developed method was validated in terms of selectivity, linearity, precision, accuracy, matrix effect, recovery and stability. No interfering signals from matrix, internal standard or tricyclic antidepressant drugs were found. Limits of quantification and limits of detection ranged from 10 to 85 ng/mL and 2.0 to 28 ng/mL respectively. Ion suppression from matrix effects varied from 36 to 108% for the compounds and 47 to 102% for the internal standards. Process efficiency varied from 22 to 106%. Intra- and interassay precision varied from 87.7 to 96.5% and 87.7 to 96.9%, respectively. The bias of the assay was lower than 15% for all the compounds, except the lowest concentrations of desmethylcitalopram and trazodone (bias < 20%). Good validation performance was obtained thanks to the use of selected deuterated internal standards that adjusted for all of the compounds. This method is therefore suitable for quantification in suspected overdose cases and can be use in a clinical Toxicology lab in a daily basis.