Postmortem analysis of human bone marrow aspirate - Quantitative determination of SSRI and SNRI drugs

For the first time the MAE/UHPLC-TOFMS method was developed and used in order to determine the antidepressant drugs within the human bone marrow aspirate in real forensic cases. The following drugs, belonging to the group of selective serotonin (or serotonin-norepinephrine) reuptake inhibitors, were tested in this study: venlafaxine, citalopram, fluoxetine, sertraline and paroxetine. The sample preparation proposed in the present article included several steps: homogenization in ultrasound bath, liquid-liquid extraction, fat removal and evaporation under nitrogen. The extraction involved microwave-assisted extraction, performed for 15 min at 55 °C, with hexane-isoamyl alcohol (99:1, v/v) mixture, as an extraction solvent. Time and temperature of extraction were optimized using the simplex method. The fat removal step was introduced because of the fatty nature of the bone marrow that resulted in insufficiently purified samples, impossible to analyze using HPLC. It was achieved by adding a mixture of ethanol, water and formic acid to samples consisting of hexane and isoamyl alcohol, so that the analytes could diffuse to polar phase and fat could stay in non-polar phase. The method was validated and parameters such as: LOD, LOQ, linearity, matrix effect, recovery were determined. The validation parameters obtained allowed to recognize the method as quantitative. Due to lack of the data on therapeutic and toxic levels of considered drugs in bone marrow, data regarding serum has been used for reference. Under this assumption, the developed method allows for quantification of all mentioned drugs at therapeutic levels. Moreover this method has been used in real cases. Finally four analytes: venlafaxine (104 ng/mL), fluoxetine (84 ng/mL), paroxetine (about 3.6 μg/mL) and citalopram (68 ng/mL) were found in three case samples.