Last Month's Most Accessed Feature: Filter paper prop

Multiple tissue and fluid analysis

Filter paper has been used as a support in 96-well plates for ten types of biological fluids and tissues in the post-mortem LC/MS analysis of the alkaloid colchicine, in which the calibration curve for dried blood spots can be applied to most of the other sample types. In 2012, a team of scientists in Switzerland had successfully used filter paper in this way to analyse drugs in dried blood spots and they wanted to see how far the technique could be extended to other biological samples.

Julien Déglon and colleagues from the Forensic Medicine Unit and the Forensic Toxicology and Chemistry Unit at the University Center of Legal Medicine, Lausanne-Geneva, described their method in Drug Testing and Analysis. Successful application would effectively map the distribution of a drug throughout the body in a post-mortem examination.

The particular drug they chose to study was colchicine, an alkaloid which is prepared from seeds and corns of the meadow saffron plant and is prescribed for the treatment of gout, rheumatoid arthritis and familial Mediterranean fever. It is one of those drugs that must be taken with extra care because the plasma concentration at which toxic effects begin to appear is close to the therapeutic concentration. It can kill within 7-10 days of overexposure.

Liquid chromatography-tandem mass spectrometry is one of the most popular methods of drug analysis in the forensic lab and the researchers wanted to develop an automated procedure for analysing different types of post-mortem samples to increase throughput. They succeeded by generalising the sample preparation protocol for different tissues and fluids to give a rapid analysis time.

Filter paper support

To begin with, the research team developed their methodology on biological media that were spiked with colchicine. Whole blood, urine, bile, vitreous humour, pericardial fluid and cerebrospinal fluid were spotted onto a filter paper card. Frozen samples of brain, liver, kidney and heart tissue were prepared and 60-µm sections were cut out and applied to the filter paper.

Small sections were cut out manually and placed in a well on the 96-well plate and an autosampler was set in motion to carry out the extraction procedure. Methanol containing deuterated 7-aminoclonazepam as a chromatographic standard was dispensed into each well. The contents were mixed by drawing air into the well followed by drawing and dispensing half of the methanolic solvent. The automated extraction from the filter paper took just two minutes.

After mixing, the syringe withdrew a small volume to inject into the liquid chromatograph for LC/MS/MS analysis. After passing through a C18 column to separate it from any other compounds extracted from the biological fluids and tissues, colchicine was detected and measured by electrospray ionisation in selected reaction monitoring mode. Two transitions each were monitored for colchicine and the standard.

Colchicine in dried blood spots on filter paper was also analysed at different concentrations under full validation to produce a calibration curve over 0.5-5 ng/mL, which corresponds to the toxic range for colchicine. There were no interferences from any other compounds present in blood and the analytical data, including precision and repeatability, were all acceptable.

Drug distribution

When the calibration curve for dried blood spots was applied to the other fluids and tissues that had been spiked with known concentrations of colchicine, the values were found to be accurate. For tissues, the results also matched those determined following liquid-liquid extraction (LLE). The only exception was bile, for which the mass spectrometric signal was almost fully suppressed by another component. In this case, LLE or a longer chromatographic separation is required to remove this interference.

For the remaining biological matrices, successful separation from matrix components was achieved and the chromatographic step and mass spectrometric detection were completed within five minutes, making a total of just seven minutes for extraction and analysis.

The role of the filter paper is important. Without it, the tissue sections float to the surface once methanol has been added to the well, preventing the removal of solvent for direct injection into the liquid chromatographic inlet. However, when the tissues are added to filter paper first, they remain adhered and do not interfere with the subsequent extraction.

Forensic case study

Having established the ability of dried blood spots to calibrate for the other fluids and tissues, the method was applied to a real case, in which a man died after deliberately ingesting a mixture of drugs, including colchicine, acetaminophen, allopurinol and acemetacin, as well as two litres of wine.

On admission to the intensive care unit, the patient admitted to taking 10-20 times the recommended daily dose of colchicine. This was confirmed by analysis of ante-mortem and post-mortem blood, in which the levels of colchicine were found to be sufficient to cause death.

The distribution of colchicine was measured across the various fluids and tissues. The concentrations in liver and heart were very similar, being about half the level in kidney. The very low concentrations in brain, cerebrospinal fluid and vitreous humour were consistent with a weak ability to cross the blood-brain barrier.

The automated LC/MS/MS method using filter paper to support the samples in 96-well plates should provide a useful platform for forensic and clinical investigations into the distribution of drugs and toxic compounds throughout the body.