Detection and quantitation of cocaine and benzoylecgonine in skeletal tissue using laminar flow triple quadrupole mass spectrometry
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Abstract
Toxicological analysis can be a critical factor in determining the cause of an individual's death especially when no other physical signs of trauma are present. However, there are cases where human remains are discovered several years after a person's death, leaving toxicologists with very few biological matrices to choose from for analysis. In the last 20 years, developments in the field of forensic toxicology have led to several promising methods for the use of human bone as a matrix for drug analysis. This presents a unique challenge for toxicologists due to the fact that bone is a hard tissue, requiring extensive homogenization and extraction procedures. Further, there is very little reference material available on the topic of skeletal remains as an alternative drug matrix. The objective of this study was to develop a quick and efficient method of extraction as well as subsequent analytical detection and quantitation of cocaine and benzoylecgonine in the complex matrix of skeletal remains following a 3-month period of chronic drug administration. All rodent specimens utilized in this research were in accordance with the Institutional Animal Care and Use Committee (IACUC) protocol at Boston University. The 16 rats used for this study underwent a 12-week chronic self-administration of cocaine, followed by a 5-week period of abstinence, and then a 3-week period of resumed consumption before euthanasia. Twelve of the sixteen rat specimens received a pericardial infusion of paraformaldehyde and phosphate buffered saline at the time of death before being frozen. The carcasses were allowed to return to room temperature before undergoing dissection to remove the left humerus, left femur, and 13th thoracic vertebrae from each rat for toxicological analysis.
Segments of each bone were taken, and all internal bone marrow was removed. Fragments of each bone in the weight of 0.25g were then homogenized using a bead mill, methanol, and deionized water. Solid phase extraction was conducted on the homogenates using a mixed mode, reverse phase/ion exchange column. The resulting product was evaporated and reconstituted in 180 µL 10mM ammonium formate in DI water and 20 µL methanol prior to LC-MS/MS analysis employing the use of gradient elution, and electrospray ionization. The MS was operated in positive ion mode, with MRM ion detection.
The linear dynamic range for cocaine and benzoylecgonine was found to be 0.25 ng/mL – 200 ng/mL with a limit of quantitation (LOQ) of 0.25 ng/mL. Calibration curves were generated using 8 spiked human urine calibrators providing R² values greater than 0.98 for both compounds. This procedure utilized bead mill homogenization, mixed mode solid phase extraction, and LC-MS/MS analysis utilizing a laminar flow, triple quadrupole mass spectrometer to obtain drug concentrations in skeletal tissue in as short as 1 hour.