Dave van Staveren (Presenter)
Tecan Schweiz AG
Authorship: Marianne Hädener (1), Wolfgang Weinmann (1), Stefan König (1), Dave R. van Staveren (2)
(1) Institute of Forensic Medicine, Forensic Toxicology and Chemistry, University of Bern, Bühlstrasse 20, 3012 Bern, Switzerland (2) Tecan Schweiz AG, Seestrassse 103, 8708 Männedorf, Switzerland
| Short Abstract Antidepressants are widely prescribed medications for the treatment of depression and other mental illnesses. Although in general having good safety profiles, they still carry some risk of side effects and fatal intoxication due to suicidal/accidental overdose. Therefore, the analysis of antidepressant drugs in blood samples is important in clinical and forensic toxicology. This poster describes the determination of different classes of antidepressants in human whole blood employing a straightforward sample preparation procedure based on the AC Extraction Plate and subsequent LC-MS/MS analysis. |
Long Abstract
Introduction:
Antidepressants are widely prescribed medications for the treatment of depression and other mental illnesses such as anxiety disorder or obsessive compulsive disorder. Although the newer generation antidepressants have improved tolerability and safety profiles, they still carry some risk of side effects and fatal intoxication due to suicidal/accidental overdose. Therefore, in recent years, the analysis of antidepressant drugs in blood samples has become increasingly important in clinical and forensic toxicology (e.g. therapeutic drug monitoring, driving under the influence of drugs, cases of violent crime, and cases of unknown cause of death). This poster describes the determination of different classes of antidepressants in human whole blood employing a straightforward sample preparation procedure based on the AC Extraction Plate and subsequent LC-MS/MS analysis.
Methods:
Sample preparation was achieved with Tecan AC Extraction PlatesTM. The inner surface of each well of these 96 well plates is partly coated with a sorptive material which acts as an extraction phase for relatively non-polar small molecules from aqueous solution. Sample processing was performed directly with whole blood samples according to the workflow illustrated in Figure 1 using the following solutions:
• modifier buffer: H2O/MeCN 95/05 (v/v)
• wash solution: H2O/MeCN 95/05 (v/v)
• elution solvent: H2O/MeCN 10/90 (v/v)
The extraction mix was generated in situ in the AC Extraction Plate wells by adding an internal standard solution (5 µg/ml in acetonitrile) to the modifier buffer. The wash step was performed a total of three times which resulted in full decoloration of the wells.
Figure 1. Sample preparation workflow with the Tecan AC Extraction PlateTM.
5 μL of the processed sample was injected onto the analytical column (Phenomenex Synergi Polar RP, 2.5 µm, 50 x 2.0 mm) and eluted by a gradient using water + 0.1% formic acid and acetonitrile + 0.1% formic acid as the mobile phases. Detection of the analytes was accomplished by a triple stage quadrupole mass spectrometer (AB Sciex, 3200 QTrap), operated in positive electrospray ionization and selected reaction monitoring mode.
Results:
In a first step, we used spiked whole blood calibration samples with 19 different antidepressants. Linearity of each analyte was assessed with seven calibration levels (10, 25, 50, 100, 250, 500 and 1000 ng/ml), covering the forensically relevant range. For trazodone, levels ranging from 50 - 5000 ng/ml were chosen, the reason being that therapeutic blood concentrations of trazodone are higher than those of the other antidepressants. Linear regression analysis with a weighting factor of 1/x demonstrated good linearity for all analytes (r ≥ 0.9935), except for venlafaxine and desvenlafaxine. For these two analytes the lowest calibration sample showed an insufficient signal-to-noise ratio and could therefore not be reliably quantified.
The presented method was applied to the analysis of eight authentic blood samples, collected from impaired drivers during road traffic controls or after accidents. The results were compared to those obtained by using our validated method, employing the same LC-MS/MS method but protein precipitation for sample preparation. Reported results for the protein precipitation method are mean values of two determinations, whereas with the AC Extraction Plate only a single determination was made. The agreement between the two methods was acceptable (within ± 26%) for all of the detected antidepressants, except for mirtazapine and desvenlafaxine.
Conclusions:
This work represents the first investigation on the suitability of the AC Extraction Place for the preparation of whole blood samples. All of the nineteen antidepressants could be extracted from blood using the AC Extraction Plate. Extraction efficiencies were found to depend on the logP value of the analytes. Sample extracts were subjected to LC-MS/MS for quantitative analysis. Calibration curves were linear over the tested range for all analytes, except for the two antidepressants showing the lowest extraction efficiencies. The presented AC Extraction Plate method was successfully applied to authentic whole blood samples from forensic toxicology cases and yielded results comparable to those obtained with a protein precipitation method.
The presented method has not been validated and is not ready for routine use. However, the preliminary results are very encouraging, showing that the AC Extraction Plate is also compatible with whole blood which is a commonly encountered matrix in clinical and forensic toxicology.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Tecan Schweiz AG |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Tecan Schweiz AG |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |