![]() Nevertheless, the proposed method of estimating drug concentrations in bloodstains is characterized by many disadvantages, and the determined values should be treated with great caution, at best as estimates. The developed protocol has been extensively tested on authentic forensic samples and the obtained results were successfully compared with drug concentrations determined in whole blood. The conducted research indicates that estimation of the concentration of drugs in bloodstains from the blood of an unknown volume is possible. A special protocol was developed for this purpose. This is the first work in which quantitative analyses of drugs in bloodstains have been attempted. The Pearson correlation coefficients were: for plastic r(29) = 0.85 and for cotton r(25) = 0.91. The variables concentrations estimated from bloodstains and concentrations calculated from whole blood were found to be strongly correlated. The differences between amphetamine values estimated from bloodstains and determined from whole blood were analysed using the Bland-Altman difference plots that revealed significant agreement. However, for most of the cases, more accurate results were obtained. The accuracy was in the range from -87.7 to +471.0%. The results of bloodstains analyses were compared with the results of whole blood analyses. The developed protocol was tested on 32 authentic forensic samples (mostly cases of people driving under the influence of amphetamine). In order to isolate the analytes from bloodstains on different surfaces (plastic and cotton) the elution mixture consisted of an acetonitrile: water (60:40) and sodium chloride (0.9%) was used. In this work endogenous amino acids: valine and leucine were used to determine the unknown volume of blood from which the stain was formed. In this context, the aim of this work was to develop a simple procedure that would allow for the estimation of drug concentrations in forensic bloodstains of unknown volume. If the suspect was injured and bloodstains were revealed, they can become the best or sometimes, the only material to be used to prove driving under the influence of drugs. However, in some cases like hit-and-run, it may be collected after a long time since the incident, resulting in the complete elimination or a significant decrease in the concentrations of potentially present drugs. Blood is the primary material for quantitative toxicological analysis and interpretation of the results.
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