Cocaine compromised by formalin

Cocaine use is on the rise but the number of deaths related to cocaine alone, in the absence of opiates such as heroin, remains relatively small. In general, autopsies will involve the removal of body organs for histological examination before storage, but toxicological analyses for drugs of abuse would not be called upon.

Later on, if evidence is received that the deceased might have been a cocaine user, the organs can be recovered and tested. In many instances, this might be the only way forward, since the body may have been buried or cremated in the meantime. The delayed forensic analysis of stored tissue depends on the integrity of the specimens but is that a reliable practice?

Autopsied organs and tissue are stored in formalin, a solution of formaldehyde in water, which is a preservative and "fixes" the tissue, making it firmer. However, there might be problems with cocaine. It has already been reported that the primary cocaine metabolite, benzoylecgonine, is leached from tissues by formalin where it remains in the solution. So, measuring it in tissue would give a false reading.

It stands to reason that cocaine itself might behave in a similar fashion. Indeed, there is also the possibility that cocaine reacts with the formalin solution to make detection even more difficult.

The consequences of formalin fixation on cocaine have been examined by Santo Davide Ferrara and colleagues from the Section of Legal Medicine in the Department of Environmental Medicine and Public Health at the University of Padua, Italy. In the first instance, they studied the stability of cocaine in formalin solutions at pH 3.5 and 7.4. Portions of the solution were withdrawn over 4 weeks of storage at room temperature and extracted by SPE for GC/MS analysis.

In unbuffered formalin at pH 3.5, cocaine was stable over the whole period but in buffered formalin at pH 7.4, cocaine was hydrolysed rapidly to benzoylecgonine, undergoing 80% depletion in about 15 days. The reaction followed pseudo first order kinetics with a half life of 7 days. The drug reacts with water and not formaldehyde.

Postmortem brain and liver tissue from a cocaine-related death were also stored in both formalin solutions for up to 30 days and the tissues and solution were both analysed. Both cocaine and benzoylecgonine were rapidly leached from the tissues into the formalin solution.

After 30 days, about 80% of both compounds were lost from liver and about 60% from brain. The difference was attributed to the greater lipophilicity of brain tissue which would hold back penetration of the solution to the inner portions of the samples. The degree of loss in pH 7.4 solutions was slightly lower than at pH 3.5.

This behaviour following formalin fixation is important in the pathology lab because buffered formalin at pH 7.0 is normally used to fix tissues. So, if cocaine were present within the stored samples, most of it would be leached into solution within 15 days, where it would be largely transformed into benzoylecgonine. However, it is not all so simple because a size effect also comes into play. At a recent court case in

Italy, evidence was presented that drugs might have played a role in the death of a young man. Two of his liver sections had been fixed in formalin, so they were tested for drugs by the researchers.

Benzoylecgonine was found in the formalin and benzoylecgonine and cocaine were both detectable in the core of the larger liver piece (ca. 60 cm³). The smaller piece, at about 5 cm³, showed no signs of cocaine but did retain some benzoylecgonine. Quite simply, formalin took longer to penetrate and leach the drug from the larger sample.

The results of this study illustrate the difficulty in quantifying cocaine in formalin-fixed tissues. The tissue type and size, the formalin pH, and the rate of hydrolysis to benzoylecgonine all come into play. The most reliable time for analysis is within days of formalin fixation before the conversion and extraction have inflicted too much damage.

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