Desarrollo y validación de un método para el análisis de opiáceos y cocaína en tejido óseo humano

Abstract

In recent years, a great advance has occurred in the study of bones as toxicological matrix, resulting in a wide range of substances analysed in this tissue. Some of these studies have been developed in humans, but most of the researchers use experimental animals under controlled conditions of dose, route of administration, delay between administration and death, degree of decomposition, etc. These studies have examined several conditions that may influence on toxicological measurements in bone such as the patterns of drug exposure or the delay between the last exposure and death. Furthermore, a variety of different skeletal tissues have been analysed, and a number of different methods for tissue treatment (microwave-assisted extraction or SPE) and analyte detection (ELISA, ELISA-LC-MS, UPLC-MS, GC-MS) have been tested. Despite the advances made in the study of bone as toxicological matrix in recent years, there are no standardized protocols for the extraction and quantification of drugs from this matrix in human. Opioids and cocaine are drugs of great medical-legal interest, since both are usually involved in acute drug-related damage. Cocaine is widely consumed and deaths involving it have increased, although many of these may be due to heroin overdoses among people who also used cocaine since opioids frequently generate fatal poisonings. Therefore, this study aims to develop and to validate a fast method for simultaneous analysis of the following substances in human bone: 6-monoacetylmorphine, morphine, cocaine, benzoylecgonine, tramadol and methadone. We applied the methodology to real forensic cases whose toxicological analysis for these drugs in blood was positive to check the reliability and robustness of the method and to investigate preliminarly the relationship between bone and blood concentrations. Bone fragments were taken from 100 cadavers autopsied in the Institute of Legal Medicine of Murcia. Samples were dried, pulverized, and incubated in acetonitrile under ultrasounds. Then, samples were subjected to a solid phase extraction and the analysis was performed by gas chromatography-mass spectrometry. The assay was validated in the range 0.3-1 ng/mg (depending on the drug) to 150 ng/mg, the mean absolute recoveries ranging from 66% to 110%, the matrix effect from 62% to 121% and process efficiency from 61% to 89% depending on the analyte. The intra- and inter-assay accuracy values were always better than 20%. The validated method was then successfully applied to real bone samples from six forensic cases in which toxicological analysis for these drugs in blood was positive. Drugs were detected in bone in 12 of the 15 blood positive results. The approximate concentration range was 3-5 ng/g for 6-monoacetylmorphine, 3-7 ng/g for morphine, 14-28 ng/g for methadone and 6 ng/g and 11 ng/g for tramadol and benzoylecgonine.