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

• Main Authors: Lucia Fernandez-Lopez, Manuela Pellegrini, Maria Concetta Rotolo, Aurelio Luna, Maria Falcon, Rosanna Mancini
• Format: Article
• Language: English
• Published: MDPI AG 2019-06-01
• Series: Molecules
• Subjects: beta-blockers; human bone; gas chromatography–mass spectrometry
• Online Access: https://www.mdpi.com/1420-3049/24/13/2400

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.