Microwave-assisted extraction technique for preparation of bone tissue

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  • Published: Sep 15, 2015
  • Author: Ryan De Vooght-Johnson
  • Channels: Sample Preparation
thumbnail image: Microwave-assisted extraction technique for preparation of bone tissue

Toxicological analysis of bone

During a post-mortem examination, determining whether the deceased has ingested a drug – and if so, when and how much – can be critical to establishing the cause of death.

During a post-mortem examination, determining whether the deceased has ingested a drug – and if so, when and how much – can be critical to establishing the cause of death. Usually, this involves analysis of blood, urine, hair, and perhaps tissue samples from the liver or kidneys.

In circumstances where the body has lain undiscovered for long periods, however, these conventional samples may not be available due to decomposition. In situations where the body has skeletonised, bone tissue becomes a valuable specimen.

Although bone has received less attention than other sample types due to difficulties with its use, a number of studies have shown that it is possible to detect of a wide range of drugs in bone, which can aid investigation of deaths that would otherwise be challenging.

However, there remains room for improvement. Drug levels measured in bone often correlate poorly to those measured in blood, leading to concerns about their analytical value. In addition, the process of decomposition can affect the measurable levels of a drug in bone, further complicating the interpretation of results.

Microwave-assisted drug extraction

Work is ongoing to enhance the use of this unusual biological matrix for toxicological analysis. A group based at Laurentian University in Canada has conducted experiments in animals that indicate that it may be possible to discriminate between patterns of drug exposure using bone. Their method relies on analysis of the quantitative relationship between the levels of a drug and its metabolite(s) in decomposed bone. “The variability in drug levels in the different bones in the body makes interpretation of any single measurement nearly impossible, but it appears that quantitative relationships between drug and metabolite levels may have discrimination power between different exposure patterns,” explained lab leader Professor James Watterson.

To build on these initial findings, and ultimately improve the accuracy of bone toxicological analysis, they need to test a large number of samples and a wide range of exposure conditions. In their recent Drug Testing and Analysis paper, the Canadian team describes a technique that makes this possible.

The lab developed a method to analyse dextromethorphan (DXM) – a cough suppressant – and its primary metabolite, dextorphan (DXT), in bone. They administered 100 mg/kg of dextromethorphan to three rats, which were sacrificed approximately 20 minutes after receiving the dose, and their remains allowed to decompose outdoors. Their vertebral bones were then recovered, cleaned and ground up.

The first step in preparing the samples was drug extraction, which was performed using a novel, microwave-assisted extraction (MAE) technique. Solutions were prepared in methanol then irradiated using a microwave oven. This was followed by clean up via microplate solid-phase extraction (MP-SPE) and analysis by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode. After 1 hour of MAE, MPSPE-GCMS analysis successfully detected DXM and its metabolite in all samples.

The total ion chromatograms of the bone extracts taken from DXM-exposed rats revealed clear peaks that were not present in the drug-free control, which likely correspond to other metabolites of DXM. The group say their procedure could also be used to identify these metabolites.

Delineating exposure patterns

The MAE-MPSPE method developed in this research is a rapid means of preparing bone tissue for toxicological analysis using GC-MS. In future work, the authors aim to use the protocol to investigate the impact of different patterns of exposure on levels of DXM and its metabolites in bone. By analysing the relationship between the levels of a drug and its metabolites, it may be possible to differentiate between patterns of exposure. For instance, to determine whether a single high dose or many smaller doses were taken, and how long after exposure death occurred.

This requires studies using a large number of samples, as Watterson explains: “Since there are so many factors that influence measured drug and metabolite levels in skeletal remains, we must analyse a large number of samples to maximise statistical power.” This makes the time it takes to prepare a sample a major obstacle to throughput. In some cases, solvent extractions have required incubation times of over 18 hours.

This microwave-based method could help to reduce the obstacle of analytical throughput. It can process 40 bone samples in parallel with extraction times substantially lower than those in previously published work.

This study therefore not only contributes to the understanding of the importance of drug and metabolite levels in the toxicological analysis of bone but will also support the use of MAE-MPSPE-GC-MS to investigate drug exposure patterns. Looking ahead, Watterson indicates that future studies will utilise LC-MS to expand the range of measurable metabolites and increase analytical throughput even further.

Related Links

Drug Testing and Analysis, 2015, 7(8), 708-713, Analysis of dextromethorphan and dextrorphan in decomposed skeletal tissues by microwave assisted extraction, microplate solid-phase extraction and gas chromatography-mass spectrometry (MAE-MPSPE-GCMS).

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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