LC-MS/MS METHOD FOR THE DETERMINATION OF ANTIDEPRESSANTS AND BENZODIAZEPINES IN MECONIUM.

An LC-MS/MS method for the determination of 14 benzodiazepines (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25 ± 0.02 g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed-phase, with a gradient of 0.1% formic acid in 2 mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of antidepressants and another for benzodiazepines. Analytes were monitored by tandem mass spectrometry employing ESI+ in MRM mode (2 transitions per compound). Method validation included: linearity (n = 5, LOQ to 400 ng/g), limits of detection (n = 6, 1-20 ng/g), limits of quantification (n = 9, 5-20 ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n = 15, 90.6-111.5%), imprecision (n = 15, 0-14.6%), matrix effect (n = 10, -73% to 194.9%), extraction efficiency (n = 6, 35.9-91.2%), process efficiency (n = 6, 20.1-188.2%), stability 72 h in the autosampler (n = 3, -8.5% to 9%) and freeze/thaw stability (n = 3, -1.2 to -47%). The method was applied to 4 meconium specimens, which were analysed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC-MS/MS method allows a high throughput determination of the most common benzodiazepines and antidepressants in meconium, which could be useful in clinical and forensic settings.