Development and validation of a method for the determination of nine benzodiazepines and metabolites in dried blood spots (DBS) using UPLC-QTOF-MS

Abstract

In forensic toxicology benzodiazepines are common compounds implicated in drug impaired driving cases. Detection of these compounds is ideally determined from whole blood collected from the individual after impairment has been established and qualified personnel are available to draw blood. This study focused on the development and validation of a screening method that could also be applied to quantification of a set of nine benzodiazepines and metabolites extracted from dried blood spots and analyzed using UPLC-QTOF-MS: diazepam (DZP), temazepam (TMZ), oxazepam (OXZ), nordiazepam (NOZ), lorazepam (LRZ), clonazepam(CLZ), 7-aminoclonazepam (7CLZ), alprazolam (APZ), and α-hydroxyalprazolam (aHAM). Advantages of DBS over whole blood sampling are less invasive sampling, potential for increased stability of analytes in the card matrix, small sample volumes, simplified extraction and ease of storage and transport. All dried blood spots were 20 μL of sheep blood spiked with a 2.86μg/mL mixture of the compounds included in this study and left to try overnight at ambient temperature. The optimized method immersed a ½” diameter punch of card containing the entire blood spot in an extraction solvent of 1:1 (v/v) methanol:acetonitrile and sonicated for 30 minutes. The extraction solvent was separated from the spot, 1 mL of acetonitrile was added followed by protein filtration. Filtrate was evaporated down to dryness and analytes were then reconstituted in 1:1 (v/v) acetonitrile:water before analysis. Each step of extraction and sample preparation was optimized for this study. All analytes were stable within the card matrix for 14 days under refrigeration at 4°C. All analytes were stable up to 12 hours in the autosampler of the instrument. Hematocrit over a range of 20 - 70% did not affect interpretation of results. Validation produced calibration curves over a range of 7.8 – 500 ng/mL that had R2 values ranging from 0.998-1.00 with a quadratic line of best fit. Bias was <20% for high blind samples and ≤25.6% for low blind samples. A screening method using UPLC-QTOF-MS to analyze DBS extracts able to identify all benzodiazepines of interest was developed and validated. Calibration curves representing impairment ranges of the analytes of interest predicted concentrations within a reliable range that allowed for quantification of the analytes.