Development and Validation of a Method for the Analysis of Bisoprolol and Atenolol in Human Bone

A method based on gas chromatography–mass spectrometry (GC–MS) is described for the determination of bisoprolol and atenolol in human bone. After the addition of lobivolol as internal standard, pulverized samples were incubated in acetonitrile for 1 h under ultrasounds. After adjusting the pH of the samples to 6, they were centrifuged, and the supernatants were subjected to solid phase extraction. Elution was achieved by using 3 mL of 2% ammonium hydroxide in 80:20 dichloromethane:isopropanol solution. Eluted samples were evaporated and derivatized. Chromatography was performed on a fused silica capillary column and analytes were determined in the selected-ion-monitoring (SIM) mode. The assay was validated in the range 0.1–0.3 ng/mg (depending on the drug) to 150 ng/mg, the mean absolute recoveries were 60% for bisoprolol and 106% for atenolol, the matrix effect was 69% for bisoprolol and 70% for atenolol and process efficiency was 41% for bisoprolol and 80% for atenolol. The intra- and inter-assay accuracy values were always better than 12%. The validated method was then applied to bone samples from two real forensic cases in which toxicological analysis in blood were positive for atenolol in the first case (0.65 µg/mL) and bisoprolol in the second case (0.06 µg/mL). Atenolol was found in bone samples from the corresponding case at the approximate concentration of 148 ng/mg and bisoprolol was found at 8 ng/mg.