The investigation of the effect of bacteria on drug concentration in dried blood stain

The dried blood spot method is an alternative to traditional toxicological analysis methods because it is minimal and more practical in terms of collection, storage and analysis. With the dried blood spot method, it is possible to conduct newborn screening, heavy metal detection, monitoring of therapeutic drugs, detection of psychoactive substances and their metabolites, toxicology, microbiology and epidemiological studies. Cocaine is an illegal, addictive and stimulant substance used as a recreational drug. Detection of cocaine can be made from various body fluids such as blood, urine and sweat, as well as from various biological materials such as hair, nails and dried blood spots. However, in the literature review, it was seen that the effect of environmental conditions and microorganisms on the substance concentrations detected in dried blood samples has not yet been investigated. In this thesis study, it was aimed to examine the effect of bacteria on the determination of cocaine and its metabolites from dried blood spots. In the study, a certified reference blood sample containing cocaine, benzoylecgonine and cocaethylene was purchased, turned into a dried blood spot and contaminated with different bacterial mixtures, in three different environments (indoor and outdoor environment, bags), at 5 different time intervals (1, 7, 14, 21, 28 days) and then toxicological analyzes were performed. In the study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) system was used for the quantification of cocaine, benzoylecgonine and cocaethylene using the liquid-liquid extraction method. In order to understand the effect of bacteria on the substance concentration, samples were studied separately, without bacteria, with bacterial mixture, and with E. coli and Enterococcus spp.. In the studies conducted, a 9-point calibration graph in the range of 0,5-50 ng/mL was obtained for cocaine, benzoylecgonine and cocaethylene in methanol solvent (r>0,99). The limit of detection (LOQ) values obtained in methanol for cocaine, benzoylecgonine and cocaethylene were found to be 0.1 ng/mL. In repeatability studies, percent relative standard deviation (RSD%) was obtained as <20 in all indoor and outdoor samples. Although the RSD% value of cocaethylene on the 14th day was 23.20 in samples containing bacteria mixtures stored in the bag environment, and the RSD% values of cocaine and cocaethylene on the 7th day were 58,16 and 35,45 respectively, in the samples containing E. coli kept in the bag environment, the other RSD% values obtained on days are <20. The results of percentage recovery studies of cocaine, benzoylecgonine and cocaethylene have values in the range of 66,33-158,32% in indoor samples, 88,99-249,26% in samples stored in bags, and 30,27-275,76% in outdoor samples has been obtained. When the effects of factors such as different environments, different times and bacterial exposure on the substance concentration were evaluated in the analysis of illegal substances from dried blood spots, it was observed that all three substances lost their stability over time. While the substance with the highest stability among these three substances is cocaine, the substance that degrades most rapidly is cocaethylene. When the indoor, bag and outdoor samples were compared, it was observed that the environment in which the substances were most stable was the indoor environment, while the environment in which they degraded the fastest was the outdoor environment. In all three environments, it was observed that the substance concentrations and % recovery values in the samples containing the bacterial mixture were lower than in the samples containing bacteria. In all three environments, it was observed that the substance concentrations and % recovery values in samples containing only E. coli were higher than in samples containing only Enterococcus spp.. It is thought that this study has unique value as it is the first study showing that the substance concentrations in dried blood spots may vary depending on the bacterial type, storage environment and time, and will guide future research. Keywords: dried blood spot, bacteria, cocaine, benzoilecgonine, cocaethylene, forensic toxicology, forensic microbiology