Intake of Artificial Sweeteners through Soft Drinks in the Preschool- and School-Aged Population.

One of the main public health issues that has recently been observed in a greater number of children is being overweight. The cause certainly lies in the decreasing physical activity of children, but mostly in their eating habits. Soft drinks are recognized as the most significant contributor to body overweight due to high sugar content; thus, as a result of numerous campaigns, part of the sugar is replaced by artificial sweeteners (ASs). Despite their advantage due to their low caloric value, WHO recommends that they should not be used to achieve weight control or as prevention for reducing the risk of non-communicable diseases, as there is no evidence of their effectiveness. Apart from beverages, artificial sweetener combinations are also added to a variety of "low fat" and "high protein" food products, which are highly favored especially among the young population. Therefore, it is necessary to take care of the cumulative intake. The conducted study included a survey of 323 parents of children aged 1-14 years, as well as an analysis of the AS content in the products most often consumed by the respondents. The results of the survey show that a large part of children (40%) aged 3-14 often consume soft drinks. Different products (soft drinks, juices/nectars, syrups) were sampled based on the respondents' responses, and an analysis showed that 54% of them contained one or more ASs. In addition, the survey indicated parents' lack of information about the presence of AS in products, as 51% of parents declared that they do not read the declarations of the products they buy. It is necessary to persist in consumer education and changes in dietary preferences and habits, especially among children.

Does Sunlight Affect the Quality for Purposes of DNA Analysis of Blood Stain Evidence Collected from Different Surfaces?

The aim of this study was to investigate the effect of sunlight on the degradation of DNA samples taken from blood stains from different types of surfaces. A blood sample obtained from a single male donor was placed on seven different surfaces (galvanized sheet, iron rod, newspaper, white printer paper, glass, soil, and ceramic panel). Samples were kept, during a 4-week summer period, in a room, but next to an open window. Every 7 days, 1 mm2 of blood sample was collected from each substrate and stored in labeled tube for later analysis. DNA was extracted with the Chelex method, amplified using AmpFISTRTM MinifilerTM Plus Amplification Kit, and quantified using a QuantifilerTM Human DNA Quantification kit. After 7 days of sun exposure, the highest DNA concentration was determined to be from the sample from a galvanized sheet stain, followed by, in order of decreasing concentration, the ceramic panel, glass, newspaper, iron rod, and white printer paper surface. As expected, the DNA concentration from all samples decreased as the sunlight exposure time progressed. The results obtained after the amplification in the MiniFilerTM system were in correlation with the DNA concentrations measured by the qPCR method for all samples, except for the glass, soil, and white printer paper samples. The obtained data show that DNA degradation is correlated to the length of sunlight exposure and to the type of surface the samples are collected from. A negative qPCR result does not mean negative PCR amplification in the STR system; therefore, both methods should be applied when analyzing forensic samples collected from trace evidence.

Real-Time PCR Method as Diagnostic Tool for Detection of Periodontal Pathogens in Patients with Periodontitis.

The most common type of periodontal disease is chronic periodontitis, an inflammatory condition caused by pathogenic bacteria in subgingival plaque. The aim of our study was the development of a real-time PCR test as a diagnostic tool for the detection and differentiation of five periodontopathogenic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Treponema denticola, in patients with periodontitis. We compared the results of our in-house method with the micro-IDent® semiquantitative commercially available test based on the PCR hybridization method. DNA was isolated from subgingival plaque samples taken from 50 patients and then analyzed by both methods. Comparing the results of the two methods, they show a specificity of 100% for all bacteria. The sensitivity for A. actinomycetemcomitans was 97.5%, for P. gingivalis 96.88%, and for P. intermedia 95.24%. The sensitivity for Tannerella forsythia and T. denticola was 100%. The Spearman correlation factor of two different measurements was 0.976 for A. actinomycetemcomitans, 0.967 for P. gingivalis, 0.949 for P. intermedia, 0.966 for Tannerella forsythia, and 0.917 for T. denticola. In conclusion, the in-house real-time PCR method developed in our laboratory can provide information about relative amount of five bacterial species present in subgingival plaque in patients with periodontitis. It is likely that such a test could be used in dental diagnostics in assessing the efficacy of any treatment to reduce the bacterial burden.

Differences in Plasma Cannabidiol Concentrations in Women and Men: A Randomized, Placebo-Controlled, Crossover Study.

The potential therapeutic benefits of cannabidiol (CBD) require further study. Here, we report a triple-blind (participant, investigator, and outcome assessor) placebo-controlled crossover study in which 62 hypertensive volunteers were randomly assigned to receive the recently developed DehydraTECH2.0 CBD formulation or a placebo. This is the first study to have been conducted using the DehydraTECH2.0 CBD formulation over a 12-week study duration. The new formulation's long-term effects on CBD concentrations in plasma and urine, as well as its metabolites 7-hydroxy-CBD and 7-carboxy-CBD, were analyzed. The results of the plasma concentration ratio for CBD/7-OH-CBD in the third timepoint (after 5 weeks of use) were significantly higher than in the second timepoint (after 2.5 weeks of use; p = 0.043). In the same timepoints in the urine, a significantly higher concentration of 7-COOH-CBD was observed p < 0.001. Differences in CBD concentration were found between men and women. Plasma levels of CBD were still detectable 50 days after the last consumption of the CBD preparations. Significantly higher plasma CBD concentrations occurred in females compared to males, which was potentially related to greater adipose tissue. More research is needed to optimize CBD doses to consider the differential therapeutic benefits in men and women.

Trial of a Novel Oral Cannabinoid Formulation in Patients with Hypertension: A Double-Blind, Placebo-Controlled Pharmacogenetic Study.

Cannabidiol (CBD) is a non-psychoactive cannabinoid, and available evidence suggests potential efficacy in the treatment of many disorders. DehydraTECH™2.0 CBD is a patented capsule formulation that improves the bioabsorption of CBD. We sought to compare the effects of CBD and DehydraTECH™2.0 CBD based on polymorphisms in CYP P450 genes and investigate the effects of a single CBD dose on blood pressure. In a randomized and double-blinded order, 12 females and 12 males with reported hypertension were given either placebo capsules or DehydraTECH™2.0 CBD (300 mg of CBD, each). Blood pressure and heart rate were measured during 3 h, and blood and urine samples were collected. In the first 20 min following the dose, there was a greater reduction in diastolic blood pressure (p = 0.025) and mean arterial pressure MAP (p = 0.056) with DehydraTECH™2.0 CBD, which was probably due to its greater CBD bioavailability. In the CYP2C9*2*3 enzyme, subjects with the poor metabolizer (PM) phenotype had higher plasma CBD concentrations. Both CYP2C19*2 (p = 0.037) and CYP2C19*17 (p = 0.022) were negatively associated with urinary CBD levels (beta = -0.489 for CYP2C19*2 and beta = -0.494 for CYP2C19*17). Further research is required to establish the impact of CYP P450 enzymes and the identification of metabolizer phenotype for the optimization of CBD formulations.

Leafy Vegetable Nitrite and Nitrate Content: Potential Health Effects.

The aim of this research was to determine the concentrations of nitrates and nitrites in different types of vegetables that are commonly represented in the diet of the inhabitants of Split and Dalmatian County. Therefore, using the method of random selection, there were 96 samples of different vegetables. The determination of the nitrate and nitrite concentrations was carried out by high-pressure liquid chromatography (HPLC) with a diode array detector (DAD). The nitrate concentrations in the range 2.1-4526.3 mg kg-1 were found in 92.7% of the analyzed samples. The highest nitrate values were found in rucola (Eruca sativa L.) followed by Swiss chard (Beta vulgaris L.). In 36.5% of the leafy vegetables intended for consumption without prior heat treatment, nitrite was found in the range of 3.3-537.9 mg kg-1. The high levels of nitrite in the vegetables intended for fresh consumption and the high nitrate values in Swiss chard indicate the need to establish maximum nitrite limits in vegetables, as well as the broadening of legal nitrate limits to wide varieties of vegetables.

Knowledge and Attitudes of Cannabidiol in Croatia among Students, Physicians, and Pharmacists.

Due to cannabidiol's health benefits and absence of serious side effects, its use is constantly growing. This is a survey-based cross-sectional study that was conducted to determine Croatian pharmacists', physicians', and students' knowledge and attitudes about cannabidiol (CBD). Two questionnaires were created, one for students and the other for physicians and pharmacists. Our participants (in total 874: 473 students and 401 physicians and pharmacists) generally had positive attitudes towards CBD therapy as approximately 60% of them believe that CBD treatment is generally efficacious. Participants had positive attitudes toward the therapeutic value of CBD, especially pharmacists and pharmacy students (63.8% and 72.2%, respectively). Pharmacists were significantly more convinced that CBD could reduce the use of opioids prescribed for chronic pain (p < 0.05). Only 17.5% of students had read scientific papers about CBD, compared to a significantly higher percentage of physicians and pharmacists (43.0% and 47.8%, respectively) (p < 0.05). This study revealed a gap in knowledge regarding CBD, since 89.3% of pharmacists and physicians, as well as 84.8% of students, believe they need more education about CBD. We conclude that it is important to improve the educational curricula so that medical professionals can recommend CBD use to their patients when needed.

New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia.

We believe that analysing pooled urine samples for recreational drugs used at mass events can provide useful information about trends in drug use. An opportunity arose with the Ultra Europe music festival, which is attended by more than 150,000 people from over 150 countries every year. We analysed 30 pooled urine samples collected from portable chemical toilets located at or close to the Ultra Europe music festival venue in Split, Croatia in 2016-2018 to detect the presence of classic and new psychoactive substances (NPS). Four urine samples collected in 2016 were from a toilet without added chemicals (otherwise used to kill the smell) while the remaining samples were collected from toilets with added chemicals. Samples were qualitatively analysed with gas chromatography-mass spectrometry (GC/MS) using the full-scan mode. Data were compared with the Wiley mass spectral library of designer drugs and our in-house library containing about 1000 compounds and metabolites. We identified forty-six different substances and metabolites, 26 of which were classic substances/metabolites, mostly from the stimulants group, while 20 were NPS. In the NPS group, most of them were phenethylamines and cathinones. The variety of substances was the highest on the first day of the festival regardless of the year, but 2018 showed a significant drop compared to the previous two years. The results of our study revealed a stable trend of classic drug consumption, while NPS trend changed from one year to another.

NUTRITION AND MINERAL CONTENT IN HUMAN TEETH THROUGH THE CENTURIES.

OBJECTIVES: The objectives of the study were: (i) to compare mineral values of calcium, phosphorus, magnesium, zinc and their ratios, in teeth from Croatian populations that lived in different historical periods, namely the Medieval period, the early 20th century, and the 21 st century; and (ii) to elucidate the influence of diet and lifestyle on tooth quality in these groups. DESIGN: Determination of mineral content (Ca, Mg, P and Zn) in 294 teeth from three different periods of history (Medieval Period [G1], Second World War period [G2] and the contemporary period [G3]) using atomic absorption spectrometer and UV/VIS spectrometer methods. RESULTS: Our results showed that the mineral content in teeth has changed over the centuries. Between analysed samples from all groups, there were significant differences in the elemental content as well as in the interrelationship of the elements. The largest differences were observed in the mean values of magnesium and zinc concentrations while smaller differences were observed for changes in calcium and phosphorus. CONCLUSIONS: From the obtained results, it is evident that there are changes in the tooth mineral composition in specimens centuries apart that could be a reflection of changes in eating habits, food quality and the environment. In addition to differences in individual elements, differences in their mutual ratios were also observed.

ABCB1, CYP2B6, and CYP3A4 genetic polymorphisms do not affect methadone maintenance treatment in HCV-positive patients.

The aim of this study was to determine the influence of ABCB1, CYP2B6, and CYP3A4 genetic polymorphisms on methadone metabolism in patients with hepatitis C virus (HCV) undergoing methadone maintenance treatment (MMT). The study included 35 participants undergoing MMT, who were divided in three groups: HCV-positive (N=12), HCV-negative (N=16), and HCV clinical remission (CR) (N=7). The concentrations of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) were determined with gas chromatography-mass spectrometry. The patients were genotyped for ABCB1 rs1045642, CYP2B6 rs3745274, CYP3A4 rs2242480, and CYP3A4 rs2740574 polymorphisms. Differences between single nucleotide polymorphism (SNP) genotypes and methadone-to-EDDP ratio were analysed with one-way ANOVA, which showed no significant difference between the genes (p=0.3772 for ABCB1 rs1045642, p=0.6909 for CYP2B6 rs3745274, and p=0.6533 for CYP3A4 rs2242480). None of the four analysed SNP genotypes correlated with methadone-to-EDDP concentration ratio. A major influence on it in hepatitis C-positive patients turned out to be the stage of liver damage.

Comparison of dental caries in Croats from the early medieval period and the 20th century.

OBJECTIVE: To compare dental caries frequency in the Croatian population exhumed from two archeological periods and compare two methods: International Caries Detection and Assessment System (ICDAS) and DMFT (Decayed-Missing-Filled-Tooth) index. MATERIALS AND METHODS: The study included 279 teeth from 69 human remains: Set I of 30 remains and 126 teeth dated from 9th to10th centuries A.D. and Set II of 39 remains and 153 teeth from the recent 20th century. Methods used for caries prevalence were ICDAS and DMFT. Tooth wear was recorded according to the Brabant index. RESULTS: ICDAS scoring system showed significantly higher caries frequency in Set I of 64.34% and in Set II 59.47%, compared to DMFT method with 16.52% for Set I and 28.75% for Set II. Dental wear in Set I showed 73.91% and in Set II 73.15%, so no significant difference was observed. CONCLUSIONS: Depending on the ICDAS or DMFT method used for caries detection, different results have been obtained whereby the ICDAS system has a more precise and advanced approach for caries lesions.

Examination of a New Delivery Approach for Oral Cannabidiol in Healthy Subjects: A Randomized, Double-Blinded, Placebo-Controlled Pharmacokinetics Study.

INTRODUCTION: Therapeutic effects of cannabidiol (CBD) in specialized populations continue to emerge. Despite supra-physiological dosing being shown to be tolerable in various pathologies, optimization of CBD absorption has obvious benefits for general health and recreational usage. Our objectives were to: (1) to investigate a joint pharmacokinetic-physiological time course of multiple recreational-equivalent (< 100 mg) dosages of oral CBD in young healthy adults and (2) evaluate a newly developed technology (TurboCBD™) for the enhanced delivery of CBD. METHODS: In a double-blinded, placebo-controlled, cross-over design, 12 participants received placebo, generic 45 or 90 mg of CBD, or TurboCBD™ delivery technology capsules on five separate occasions. RESULTS: Although there were no differences in the 45 mg conditions, circulating CBD levels were higher with the TurboCBD™ 90 mg group at both 90 (+ 86%) and 120 (+ 65%) min compared with the 90 mg control (p < 0.05). Total area under the curve tended to be higher with TurboCBD™ 90 mg compared with 90 mg (10,865 ± 6322 ng ml-1 vs. 7114 ± 2978 ng ml-1; p = 0.088). Only the TurboCBD™ 90 mg dose was elevated greater than placebo at 30 min (p = 0.017) and remained elevated at 4 h (p = 0.002). CONCLUSION: Consistent with higher bioavailability, TurboCBD™ 90 mg at the peak CBD concentration was associated with an increase in cerebral perfusion and slight reduction in blood pressure compared with baseline and the 90 mg control. Further studies are needed to establish the mechanisms of action of this technology and to explore the therapeutic potential of acute and chronic dosing on more at-risk populations. FUNDING: Lexaria Bioscience Corp. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT03295903.

A case of synthetic cannabinoid poisoning in Croatia.

The number of new psychoactive substances (NPS), synthetic cannabinoids (SCs) in particular, is growing constantly. Because of the insufficiently explored effects on consumer health, they have become a major problem in the emergency departments. They are difficult to identify, and there are no antidotes that could reverse their detrimental effects. We report a case of poisoning of a young man who used SCs. The patient was admitted to the emergency department of the Clinical Hospital Merkur, Zagreb (Croatia) after sniffing and smoking a herbal product bought on the street. He presented with severe cognitive difficulties and visible eye redness. Other symptoms included somnolence, disorientation, loss of coordination, unsteady gait, hyporeflexia, stiffness, cramps and cold limbs, blurred vision, teeth grinding, dry mouth, tinnitus, fear, suicidal thoughts, impaired focus, memory, and speech, sedation, fatigue, depression, thought blocking, and autistic behaviour. His skin was dry, and his mucosa dry and irritated. Herbal products "Rainbow Special" and "Luminated Aroma" used by the patient were qualitatively analysed with gas chromatography / mass spectrometry (GC/MS) after direct extraction with an organic solvent. Solid-phase extraction method was used to analyse serum and urine samples. Despite the negative findings of biological samples, mostly due to the limitations of GC/MS, the clinical picture infallibly pointed to the poisoning with SCs. This was confirmed by the findings of 5-fluoro AMB (methyl 2-(1-(5-fluoropentyl)-1H-indazole-3-carboxamido)-3-methylbutanoate) in the herbal products.

Methadone Maintenance Treatment: A 15-year Retrospective Study in Split-Dalmatia County, Croatia.

BACKGROUND: The aim of this retrospective study is to use existing epidemiological data on patients in methadone maintenance treatment in Split-Dalmatia County from 2001 to 2015 to evaluate the substitution treatment system and policy of opiate addicts treatment, the epidemiological trend, and risk of overdose mortality. In addition, we would like to highlight the problem of poor control of methadone therapy and thus the possibility of selling methadone. The result is a greater number of deaths caused by methadone in people who were not in methadone therapy. METHODS: The research included data collected from 3189 patients who had been in the drug abuse treatment program in Split-Dalmatia County during 2001-2015 and data gathered from autopsy examinations of patients in Split-Dalmatia County with emphasis on data for 186 overdosed patients during research period. RESULTS: The total number of patients in the methadone treatment program in Split-Dalmatia County during the research period, except in 2005 and 2006, has been stable, while the number of new opiate patients, being in first-time treatment, decreased by 62.5%. The number of addicts who were in a long-term maintenance program has increased by 198%, whereas the number of addicts who were in a short-term detoxification treatment has decreased by 96.4%. According to results obtained from performed autopsies, 186 death cases were determined as overdoses. Methadone was found in 56 of those cases and was declared as the cause of death in 39 cases (70%). Of the total number of autopsied patients with diagnosed methadone overdose, only 23 (59%) had been recorded to receive methadone therapy in Public Health Institute of Split-Dalmatia County database. CONCLUSIONS: The results of this study show the favorable epidemiological trend because of the decreasing number of new opiate patients in treatment. The retention of opiate patients in substitution therapy indicates the effectiveness of methadone maintenance programs. Our research did not determine any influence of methadone substitution therapy on an increasing risk of specific (overdose) mortality.

Liver damage indices as a tool for modifying methadone maintenance treatment: a cross-sectional study.

AIM: To assess the effect of liver damage on methadone metabolism in opiate addicts undergoing methadone maintenance treatment (MMT). METHODS: This cross-sectional study recruited 74 patients treated at the outpatient clinic of Public Health Institute of Split-Dalmatia County from 2013-2016. Concentrations of methadone and its main inactive metabolite were measured in participants' biological samples on regular check-ups. Urine samples obtained before oral methadone intake, and blood and urine samples obtained 90 minutes after methadone intake were analyzed using gas chromatography/mass spectrometry. Participants were divided into groups according to liver damage criteria: hepatitis C virus status (positive, negative, or clinical remission); aspartate aminotransferase to platelet ratio (APRI) index (<0.7 and ≥0.7); and fibrosis-4 score (<1.45, 1.45-3.25, >3.25). RESULTS: Metabolic ratio and methadone metabolite concentration in plasma decreased linearly with HCV infection status by the factor of 1.67 (P=0.001) and 2.2 (P=0.043), respectively. Metabolic ratio in plasma decreased in patients with APRI index ≥0.7 by the average factor of 2.12 (P=0.01) and methadone metabolite concentration in plasma decreased by the factor of 6.16 (P=0.009). Metabolic ratio in urine decreased with the severity of fibrosis-4 score by the average factor of 1.63 (P=0.008), whereas methadone metabolite concentration decreased by the factor of 3.53 (P=0.007). CONCLUSION: Liver damage decreases methadone metabolism. Indices of liver function should be calculated regularly during MMT for methadone dose titration.

Comparison of the solid phase and liquid-liquid extraction methods for methadone determination in human serum and whole blood samples using gas chromatography/mass spectrometry.

The aim of this study was to determine the optimal biological sample and the optimal extraction technique for monitoring methadone concentrations in biological samples. We analysed methadone in serum and whole blood samples using gas chromatography/mass spectrometry (GC/MS). Before analysis, we compared five solid-phase extraction (SPE) and two liquid-liquid extraction (LLE) methods and determined that SPE with Supelco LC-18 in serum yielded the best extraction efficiency. The limit of detection was 10 ng mL-1 and the limit of quantification 25 ng mL-1. Correlation coefficient was over 0.999 for the methadone calibration curve in linear range from 50 to 2000 ng mL-1. Intra and inter-day accuracy and precision of the method was satisfactory. The method was successfully applied for determining serum methadone in patients on maintenance therapy.

Bone mineral density of skeletal remains: Discordant results between chemical analysis and DXA method.

Dual-energy X-ray absorptiometry (DXA) scanning is a gold standard for bone mineral density measurement and diagnosis of primary and secondary osteoporosis in living persons. DXA is becoming widespread when analysing archaeological material, and is considered to provide an accurate diagnosis of osteoporosis in skeletal samples. The aim of this study was to explain the differences in results between bone mineral density (obtained with DXA) and chemical determination of calcium and phosphorus concentrations in skeletal remains. We examined bone mineral density (BMD) and mineral content of femoral bone samples exhumed from mass graves of the Second World War. BMD was determined by Hologic QDR 4500 C (S/N 48034) Bone Densitometer. Concentrations of calcium and phosphorus were determined with AAS (Atomic absorption spectroscopy) and UV/VIS (Ultraviolet-visible) spectroscopy. The results obtained in this study do not support the hypothesis according to which BMD measured by DXA scan has positive correlation with chemically determined concentrations of calcium and phosphorus in bones, especially in acidic soils where there was significant impact of diagenesis observed.

Identification process of skeletal remains from mass graves: Our experience and proposal guidelines.

Aim of this paper is to present our experience and proposal guidelines for reducing the number of samples for DNA analysis of skeletal remains from mass graves, whether for scientific purpose or for the identification of mass graves victims. Therefore, the analysis of 94 bone fragments included the following measurements: femur length and the femoral head diameter, the diameter of the upper, wider portion and lower wider portion of the bone fragment, densitometry of the fragments and measurement of mass and volume of fragments. Bone density was determined on the basis of measured values of mass and volume. The results of fragment matching by physical analyses were compared with the pairing results obtained by previously conducted DNA analysis. Deviation in measured values of matching bone fragments that made a pair was calculated for all successfully matched fragments. By the results of DNA analysis 36 femoral pairs were successfully formed. Measured values were added to the DNA analysis. Out of 36 pairs, positively ascertained by the DNA analysis, 29 pairs were formed after adding the results of physical measurements and removing the data where femur samples were damaged. Total correspondence in measurements of the femoral length was noted in 25.9% pairs, while the correspondence within the 5% error was 100%. Density of the tested femurs was significantly different for the same person (DNA match), both for the left and the right femoral fragment. It would be optimal to choose only the whole-length left or right femur and thus reduce the number of samples by 50%. With regard to the results of our research and the observations deriving from them, as well as to the guidelines we used in the study, we suggested these guidelines be used both for scientific researches and to identify mass graves victims.

Skeletal remains from late Roman period: "as old as Diocletian's Palace".

In 2000, human skeletal remains were discovered in Split (Croatia). As archaeologists confirmed, it was an ancient skeleton accompanied by ceramics and bracelet characteristic for late Roman period whose possible violent death was excluded. The bone sample was radiocarbon dated by AMS to 1750 years. DNA was successfully extracted from the bone sample and subsequently typed using mt DNA and STR systems. The metal content was determined by atomic absorption spectrometry (AAS) in flame mode. Mercury concentration was determined by direct consecutive measures taken with a mercury analyzer. According to our results, we consider that the bones could belong to the one of the last citizens of the Diocletian's Palace.

Blood alcohol stability in postmortem blood samples.

The effect of long-term storage on alcohol stability in postmortem blood samples was investigated. Seventy-nine blood samples were used in this study. For the first time, blood alcohol concentrations were measured within 1 to 4 days after being taken at the Laboratory of Forensic Toxicology. After storage (ranging from 191 to 468 days), each sample was reanalyzed for blood alcohol concentration, and results were compared with the original analysis. Results showed good agreement between the experimental measurements. About 90% of the results lie within 95% limits, and 10% were outside. Despite these positive correlations, the results of some blood samples showed a high variation in ethanol concentration. Deviation in alcohol concentration between replicates of up to 10% (observed in 39% samples) is not acceptable as precise forensic evidence.