Development and Validation of a Method for Alcohol Analysis in Brain Tissue by Headspace Gas Chromatography with Flame Ionization Detector

Abstract Ethanol is the most widely used and abused drug. While blood is the preferred specimen for analysis, tissue specimens such as brain serve as alternative specimens for alcohol analysis in post-mortem cases where blood is unavailable or contaminated. A method was developed using headspace gas chromatography with flame ionization detection (HS-GC-FID) for the detection and quantification of ethanol, acetone, isopropanol, methanol and n-propanol in brain tissue specimens. Unfixed volatile-free brain tissue specimens were obtained from the Department of Pathology at Virginia Commonwealth University. Calibrators and controls were prepared from 4-fold diluted homogenates of these brain tissue specimens, and were analyzed using t-butanol as the internal standard. The chromatographic separation was performed with a Restek BAC2 column. A linear calibration was generated for all analytes (mean r2 > 0.9992) with the limits of detection and quantification of 100–110 mg/kg. Matrix effect from the brain tissue was determined by comparing the slopes of matrix prepared calibration curves with those of aqueous calibration curves; no significant differences were observed for ethanol, acetone, isopropanol, methanol and n-propanol. The bias and the CVs for all volatile controls were ≤10%. The method was also evaluated for carryover, selectivity, interferences, bench-top stability and freeze-thaw stability. The HS-GC-FID method was determined to be reliable and robust for the analysis of ethanol, acetone, isopropanol, methanol and n-propanol concentrations in brain tissue, effectively expanding the specimen options for post-mortem alcohol analysis. Introduction Ethanol is one of the most commonly found psychoactive substances in clinical and forensic toxicology, particularly in post-mortem analysis, as over-consumption of alcohol induces impairments that often lead to fatal accidents, violent crimes and suicides. A myriad of factors are important to the analysis and interpretation of post-mortem alcohol concentrations including ante-mortem trauma and life-saving treatment, interval between death and analysis, environmental factors, condition or decomposition of the body and types and location of the specimen collected (1). These factors can contribute to post-mortem production of ethanol and other volatile organic compounds (e.g. methanol, n-propanol, n-butanol, acetaldehyde) from microbial fermentation of natural substrates like glucose (2). Since the human body does not store glucose evenly within various fluids and tissues, post-mortem alcohol production is largely site-dependent and the choice of specimen for analysis is crucial to distinguish between post-mortem production and ante-mortem ingestion. As with the analysis of other drugs of abuse, blood is the preferred specimen for alcohol analysis. Blood concentrations are used as indicators of intoxication in human performance testing such as driving under the influence or in post-mortem cases. Blood alcohol concentration (BAC) is used as the standard to which the alcohol concentrations in other biological specimens are compared (3–12). Alcohol analysis is generally performed either using direct injection gas chromatography (GC) or headspace gas chromatography (HS-GC) with flame ionization detection (FID). Of the two, headspace is preferred over direct injection, because HS-GC takes advantage of the volatility of alcohol and an extraction procedure is not necessary. Injection of the headspace gas also protects the column from non-volatile components in the matrix that would be present using direct injection. Determination of BAC has been extensively studied using both direct injection GC (13–15) and HS-GC (15, 16). Although blood is the preferred specimen, there are occasions when blood is unavailable or contaminated due to traumatic injuries or there is suspected post-mortem alcohol production. In such instances, alternative specimens are utilized. Urine is commonly submitted for post-mortem alcohol analysis as it generally has low risk of microbial contamination and low glucose concentration, reducing the likelihood of post-mortem alcohol production (1). Similarly, vitreous humor is used by virtue of its low glucose concentration and anatomic location, isolated from the spread of bacteria in the gut during decomposition (1). Both direct injection GC and HS-GC have been applied to urine and vitreous humor alcohol analysis (3, 4, 14, 17, 18). Tissue specimens, though less often, have also been analyzed for alcohols using both GC techniques. One study investigated post-mortem ethanol formation in unadulterated kidney and muscle tissue specimens using HS-GC (19). Skeletal muscle alcohol concentration has been determined from 1:4 diluted homogenate samples using direct injection GC; the muscle to blood alcohol ratio found was on average 0.94–1.48 (5). Liver tissue as a matrix has been analyzed using HS-GC, and the liver to blood alcohol ratio found was on average 0.47–0.85 (6). Another alternative specimen for alcohol analysis is brain tissue. Where blood is easily lost due to ante-mortem trauma, the brain may be preserved due to its encasement in the protective skull. The isolated location and lack of glucose storage also make the brain an attractive specimen for analysis as it is less susceptible to post-mortem alcohol diffusion, such as from the stomach to heart blood (20) and post-mortem alcohol formation (2). Whereas there is concern over differences in alcohol concentration in post-mortem blood collected from various sites (2), the regional distribution of alcohol in the brain does not differ significantly (7). As a highly vascularized tissue with a rich blood supply, the brain displays rapid alcohol equilibrium with blood (8) and thus may be a good indicator of ante-mortem BAC. In cases where there is an acute subdural hemorrhage from head trauma and the victim survives hours before death, peripheral blood may become unreliable due to ongoing metabolism, yet ethanol concentration in brain tissues underneath the subdural hematoma may still reflect the concentration at the time of injury (9). Though the value of brain as an alternative specimen for post-mortem alcohol analysis cannot be overstated, unlike other specimens, there have been few studies performed to analyze alcohol concentrations in brain. Human brain alcohol concentrations, from aliquots of steam-distilled brain, have been found to correlate strongly with BAC (brain–blood ratio of 0.64–1.20) using direct injection GC (10). A brain–blood ratio of 0.80–1.50 (from 82% of the values within the study) was observed with homogenization, extraction and analysis via direct injection GC (11). Thus it seems that different extraction techniques for brain alcohol determination can substantially affect the measured ratios. There is only one published study that examined the reliability of the HS-GC method for the analysis of ethanol in brain tissue specimen. Disti (...truncated)