One of the standard tests carried out when a corpse is discovered is a tox screen to look for the presence of any drugs or poisons that could indicate foul play or suicide.
SourcBody fluids and tissues are removed for analysis by a set of standard procedures and results are generally available in a reasonable time frame. In cases of severe decomposition, when the corpse is essentially a skeleton, the bones are the only source of toxicological information and the tox screen, while still possible, becomes more difficult.
Various classes of drug such as benzodiazepines and opiates have been analysed in bone tissue but the trickiest stage could be regarded as drug recovery. The extraction methods published to date involve passive methanolic extraction, Soxhlet extraction and acid digestions. However, they are very time-consuming, taking up to 72 hours for completion. A team of forensic science researchers from Canada decided to try and speed up this process by evaluating microwave-assisted extraction (MAE).
MAE is a popular technique used in environmental analysis to extract pollutants from a range of matrices including soil, sediments and animal and
Splant tissues. Several samples can be extracted at the same time in some commercial instruments and the parameters can be closely controlled. Its use in forensic toxicology is extremely rare, so James Watterson, Nathalie Desrosiers and Caroline Betit from the Laurentian University at Sudbury, Ontario, applied it to the extraction of drugs from the bones of rats as a test case.
They selected three drugs, ketamine, diazepam and pentobarbital, on the basis of their forensic relevance and diverse chemical properties and gave them to rats. The team confirmed that the drugs themselves were resistant to breakdown under microwave irradiation. After 20 minutes, the rats were killed then left in secure cages in the open air to decompose naturally. It took three weeks for nearly complete skeletonisation, by which time only the fur, skin and bones remained. The different bones were collected and taken back to the lab.
Skeletonisation actually simplifies the collection of some bones, such as the spinal and pelvic bone, which are difficult to remove from fleshy animals. For this project, it also created a more relevant application since skeletal tissues are more likely to be required for toxicological analysis when there are no soft tissues or body fluids available.
The bones from the animals exposed to ketamine and pentobarbital were ultrasonicated in sodium hydroxide-sodium chloride solution for cleaning and marrow removal until no soft tissue remained. The diazepam-exposed bones were ultrasonicated in phosphate buffer since the alkaline solution reduced the subsequent response during drug detection.
The cleaned bones were pooled according to drug type because different bone types have been reported to store varied levels of drugs. After grinding, the bone tissue was extracted by MAE in a domestic microwave oven with phosphate buffer (pH 6), pure methanol or 50% aqueous methanol and the data were compared with the results from passive extraction in methanol.
The extracts were reconstituted in phosphate buffer (pH 6), apart from the diazepam samples, for analysis by ELISA. The results were expressed as the percentage decrease in absorbance compared with that from a drug-free control sample. Detection limits were 0.25, 2.5 and 2.5 ng/mL for diazepam, pentobarbital and ketamine, respectively, and the corresponding precision CVs for replicate analysis were 2-11, 0.5-5 and 0.9-6%.
The semi-quantitative nature of ELISA allowed the extraction variables to be studied. Each solvent brought about significant extraction but the methanolic solvents extracted diazepam and ketamine more quickly than the buffer. However, the buffer gave the greatest yield for diazepam. The solvent effect was not as marked for pentobarbital. Larger sample sizes required longer extraction times for maximal extraction.
Effectively, the optimum extraction procedure appeared to be drug-dependent, which was not unexpected. However, MAE provided a notable improvement over passive extraction, with times as low as 5 minutes possible depending on the drug, its original dose, the bone mass and the solvent.
In addition, the use of phosphate buffer, while not necessarily giving maximal extraction, yields a solution directly compatible with ELISA, so sufficient drug could be extracted to give a positive response in an initial screen. Subsequent reanalysis could be carried out for positive samples.
This preliminary examination has illustrated the value of MAE for the extraction of drugs from bone tissue in the forensic context. It could easily be extended to human bones and, possibly, to other types of solid forensic sample such as hair or nails. The researchers recommended that future work should examine the relationship between levels of a drug and its metabolites, which could provide an indication of the pattern of drug use.
Source - http://www.separationsnow.com/coi/cda/detail.cda?id=21649&type=Feature&chId=1&page=1
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