Detection of cocaine and its major metabolites in bone following outdoor decomposition after chronic cocaine administration using 2D-LC/MS/MS
Mella, Malorie Ann
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In the field of forensic toxicology, several challenges exist with quantification analysis of cocaine and metabolites in post mortem samples. Cocaine can prove difficult to detect and quantify in blood, urine, and soft tissues following extensive decomposition. Alternative matrices, such as hair, nails, and bone could prove useful in detecting chronic drug use in post-mortem toxicology cases. Detection and quantification of drugs in complex matrices is difficult to accomplish due to time-consuming extraction processes, and inability to detect an analyte at trace levels. Further, analysis of drugs in hard tissues, such as hair and bone, has only been attempted in recent years. Even fewer studies have investigated detection of drugs following decomposition of remains, specifically outdoor decomposition. The objective of this study was to develop a robust extraction and clean up methodology, in which a homogenization step precedes, to efficiently extract drugs from complex matrices, reach a target limit of detection (LOD) and to maintain instrument performance using multidimensional chromatography. Multi-dimension chromatography platform such as two dimensional liquid chromatography tandem mass spectrometry ( 2D-LC/MS/MS,) offers options not compatible with single dimension v units. With large volume injection capabilities of aqueous and organic extracts, the analytical process be reduced from multiple hours to minutes. All rat specimens used for this study fell under an Institutional Animal Care and Use Committee (IACUC) protocol. The rodents underwent a 10-12 weeks chronic intravenous self-administration of cocaine. This was followed by a six-week period of abstinence, followed again by a three-week period of cocaine self-administration before being euthanized. Average daily dosages for each rat fell within a range of 13-19 mg/kg. A total of 14 cocaine positive rats were placed outside and above ground in the Boston University Forensic Anthropology Outdoor Research Facility (Holliston, MA, U.S.A) for a period of 12 months. All recoverable skeletal samples were collected for testing. Drug free control rat bones were also acquired by placing drug-free rats outdoors, above ground, until full decomposition occurred. In this study, a method analyzing cocaine and its major metabolites benzoylecgonine and ecgonine methyl ester was developed. After homogenization of whole bones, the extraction process was performed using a mixed mode reversed-phase/ion exchange sorbent. The use of a 2D LC/MS/MS technology eliminates the need for a lengthy evaporation step in the extraction method. The chosen 2D LC/MS/MS used in this application was identified using a 6x6 automated method development protocol. The manual extraction of the bone samples was completed in less than an hour. The analysis was performed using 100μL of the final organic solvent (MeOH) extracts. vi The limit of quantitation (LOQ) for cocaine and benzoylecgonine was measured at 0.05ng/g (0.05ng/mL or 50pg/g) of sample material and the LOQ for ecgonine methyl ester was measured at 0.1ng/g (0.1 ng/mL or 100pg/g). The extraction method for cocaine proved to give a linear dynamic range of 2.5 orders of magnitude (0.05 ng/g to 10ng/g with an R2 = 0.998. The micro extraction protocol combined with a multi-dimension chromatography used in this study decreased sample preparation time without sacrificing the quality seen with current single dimension chromatography techniques. The procedure developed in this study can be utilized on bone and completed in less than an hour before injection into the 2D-LC/MS/MS system.