Development and validation of a method for the determination of nine benzodiazepines and metabolites in dried blood spots (DBS) using UPLC-QTOF-MS
Year: 2017
Type: dissertation
Abstract: In forensic toxicology benzodiazepines are common compounds implicated in \ndrug impaired driving cases. Detection of these compounds is ideally determined from \nwhole blood collected from the individual after impairment has been established and \nqualified personnel are available to draw blood. This study focused on the development \nand validation of a screening method that could also be applied to quantification of a set \nof nine benzodiazepines and metabolites extracted from dried blood spots and analyzed \nusing UPLC-QTOF-MS: diazepam (DZP), temazepam (TMZ), oxazepam (OXZ), \nnordiazepam (NOZ), lorazepam (LRZ), clonazepam(CLZ), 7-aminoclonazepam (7CLZ), \nalprazolam (APZ), and α-hydroxyalprazolam (aHAM). Advantages of DBS over whole \nblood sampling are less invasive sampling, potential for increased stability of analytes in \nthe card matrix, small sample volumes, simplified extraction and ease of storage and \ntransport. All dried blood spots were 20 μL of sheep blood spiked with a 2.86μg/mL \nmixture of the compounds included in this study and left to try overnight at ambient \ntemperature. The optimized method immersed a ½” diameter punch of card containing \nthe entire blood spot in an extraction solvent of 1:1 (v/v) methanol:acetonitrile and \nsonicated for 30 minutes. The extraction solvent was separated from the spot, 1 mL of \nacetonitrile was added followed by protein filtration. Filtrate was evaporated down to \ndryness and analytes were then reconstituted in 1:1 (v/v) acetonitrile:water before \nanalysis. Each step of extraction and sample preparation was optimized for this study. All \nanalytes 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 \na range of 20 - 70% did not affect interpretation of results. Validation produced \ncalibration curves over a range of 7.8 – 500 ng/mL that had R2 values ranging from \n0.998-1.00 with a quadratic line of best fit. Bias was <20% for high blind samples and \n≤25.6% for low blind samples. \nA screening method using UPLC-QTOF-MS to analyze DBS extracts able to \nidentify all benzodiazepines of interest was developed and validated. Calibration curves \nrepresenting impairment ranges of the analytes of interest predicted concentrations within \na reliable range that allowed for quantification of the analytes.
Author: Seana L. Hill
Language: English
Subfield: Toxicology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2747804464