Biomarkers of alcohol consumption in body fluids - possibilities and limitations of application in toxicological analysis

Ethyl alcohol is the most popular legal drug, but its excessive consumption causes social problems. Despite many public campaigns against alcohol use, car accidents, instances of aggressive behaviour, sexual assaults and deterioration in labor productivity caused by inebriated people is still commonplace. Fast and easy diagnosis of alcohol consumption is required in order to introduce proper and effective therapy, and is crucial in forensic toxicology analysis. The easiest method to prove alcohol intake is determination of ethanol in body fluids or in breath. However, since ethanol is rapidly metabolized in the human organism, only recent consumption can be detected using this method. Because of that, the determination of alcohol biomarkers was introduced for monitoring alcohol consumption over a wider range of time. The markers described in this article are ethanol, its non-oxidative metabolites (ethyl glucuronide, ethyl sulfate, phosphatidylethanol, ethyl phosphate, fatty acid ethyl esters) and oxidative metabolites (acetaldehyde and acetaldehyde adducts). The objective of this study is to review published studies focusing on the sample preparation methods and chromatographic or biochemical techniques for the determination of alcohol biomarkers in whole blood, plasma, serum and urine. Authors also described issues concerning the detection window of these biomarkers, and possibilities and limitations of their use in routine analytical toxicology for monitoring alcohol consumption or sobriety during alcohol therapy.

Bisphenols (A, S, and F) affect the basic hormonal activity determined for pharmaceuticals – Study of Saccharomyces cerevisiae

Pharmaceuticals and analogs of bisphenol A (BPA) are increasingly threatening environmental pollutants. In this study, mixtures of selected pharmaceuticals (diclofenac sodium salt, chloramphenicol, oxytetracycline hydrochloride, fluoxetine hydrochloride, estrone, ketoprofen, progesterone, gemfibrozil and androstenedione) were prepared with BPA and its two analogs (namely, bisphenols F and S) at such ratios to reflect environmentally detectable levels. Then, the mixture solutions were studied with a XenoScreen YES/YAS assay to determine the variations in the initial hormonal response of each pharmaceutical compound due to the presence of a bisphenol analog. The results obtained were modeled with the concentration addition (CA) and independent action (IA) approaches, the trueness of which was studied with model deviation ratios (MDR). The estrogenic agonistic activity of the drugs studied was most strongly affected by the presence of BPA in solution (twenty-one cases of synergy observed for CA models versus twelve cases of antagonism in the case of IA predictions). BPS shows a strong agonistic estrogenic impact on most of the drugs studied at medium and high concentration levels; androgenic agonistic activity was also impaired with elevated concentrations of BPS.

Ciecze jonowe jako medium sorpcyjne w mikroekstrakcji do pojedynczej kropli Cz. I. Podstawy teoretyczne zagadnienia

Technika mikroekstrakcji do pojedynczej kropli stanowi sztandarowy przykład rozwiązania technicznego spełniającego wymogi stawiane przez ideę zielonej chemii analitycznej.

Ciecze jonowe jako medium sorpcyjne w mikroekstrakcji do pojedynczej kropli. Cz.II. Praktyka analityczna

Pomimo rosnącego zainteresowania metodami analitycznymi opartymi na użyciu cieczy jonowych ich sprzężenie wraz z techniką mikroekstrakcji do pojedynczej kropli nie osiągnęło zadowalającej popularności oraz akceptacji ze strony środowiska naukowego.

Comparison of GC–MS and MEKC methods for caffeine determination in preworkout supplements

E. Waraksa , A. Pokrywka, M. Konop, J. Namieśnik , E. Kłodzińska – ELECTROPHORESIS – 2019
In this study, GC–MS- and MEKC-based methods for determination of caffeine (CAF) in preworkout supplements were developed and validated. The proposed protocols utilized minimal sample preparation (simple dilution and syringe filtration). The developed methods achieved satisfactory validation parameters, i.e. good linearity (R2 0.9988 and R2 0.9985 for GC–MS- and MEKC-based method, respectively), satisfactory intraand interaccuracy (within 92.6–100.7% for method utilizing GC–MS and 92.1–110.3% for protocol based on MEKC) and precision (CV 15.9% and CV 6.3% for GC– MS- and MEKC-based method, respectively) and recovery (within 100.1–100.8% for method utilizing GC–MS and 101.5–106.2% for protocol based on MEKC). The LOD was 0.03 and 3 g/mL formethod utilizing GC–MS andMEKC, respectively. The CAF concentrations determined by GC–MS- and MEKC-based methods were found to be in the range of 8.53–11.23 and 8.20–11.61 g/mL, respectively. Taking into consideration information on the labels, the investigated supplements were found to contain from 110.0 to 167.3% of the declared CAF content, which confirmed the literature reports on incompatibility of the declared product compositions with real ones. Nevertheless, the consumption of examined supplements as recommended by producers did not lead to exceeding the CAF safe limit of 400 mg per day. Additionally, the MEKC-based method allowed for detection and identification of vitamin B3 and B6 in all of the investigated supplement samples, which demonstrated that MEKC-based protocols may be an appropriate assays for simultaneous determination of CAF and vitamins.

Comparison of Redox Properties of Flavonoid Aglycones and Corresponding Glycosides and Their Mixtures in the Cellular Model

Flavonoids are polyphenolic compounds commonly found in plants. As dietary components, they have been shown to exhibit numerous pro-health properties that are believed to be associated with their antioxidant effects. In this study, the antioxidant activity of four flavonoid compounds was determined by cellular antioxidant activity assay using HT29 cells as a model of the alimentary tract. The strongest impact on cellular redox status was observed for aglycones which acted as both antioxidants (quercetin) and prooxidants (naringenin). Interestingly, mixtures of tested compounds displayed only antioxidative properties.

Determination of Antioxidant Activity of Vitamin C by Voltammetric Methods

Voltammetric methods—cyclic (CV) and differential pulse voltammetry (DPV) are considered the most appropriate way to evaluate antioxidant activity of redox active compounds. They provide information about both mechanism and kinetics of electrochemical oxidation of antioxidants as well as their physical and chemical properties such as the redox potential or the number of electrons transferred. These methods are helpful for understanding the mechanisms of oxidation or reduction processes of antioxidant compounds. This work presents the electrochemical properties of vitamin C obtained by both CV and DPV methods.

Determination of free tyrosine in equestrian supplements by LC–MS/MS and comparison of its quantity with total free amino acids content in view of doping control

E. Waraksa , K. Kowalski , R. Rola, E. Kłodzińska, T. Bieńkowski, J. Namieśnik MICROCHEMICAL JOURNAL – 2019
The reports on the probable beneficial impact of tyrosine (TYR) supplementation on performance enhancement have contributed to the growth of interest in TYR in equestrian sports field and related industries, such as the manufacture of dietary and nutritional supplements. In this study, the first attempt to the assessment of horses exposure to TYR during nutritional supplementation was demonstrated by quantification of unbound TYR and a total free amino acids (AAs), and subsequent determination of the ratio of TYR to a total free AAs content in equestrian supplement samples. Within the scope of this study, a rapid and sensitive LC–MS/MS-based method for the determination of unbound TYR and a simple spectrophotometric protocol for the quantification of a total free AAs have been developed and validated according to the international guidelines for bioanalytical methods. For sample preparation, an ultrasound-assisted solid-liquid extraction (USLE; for solid samples) and ultrasound-assisted liquid-liquid extraction (ULLE; for liquid samples) have been optimized. The comprehensive approach on the simultaneous assessment of matrix effect, recovery, process efficiency, accuracy, precision, curve factor and internal standard association demonstrated satisfactory validation parameters for LC–MS/MS-based procedure, such as global matrix effect in the range of 89.9–91.9%, good linearity (R2 > 0.9929), limit of detection (0.6 ng/mL) and recovery within 89.2–108.0%. For spectrophotometric method, limit of detection was 1100 ng/mL, recovery ranged from 103.2 to 108.8%, both, intra- and inter-day accuracy was within 89.5–108.7%, and both, intra- and inter-assay precision was below 8.5%. In view of the satisfactory validation parameters obtained for both methods, the procedures can be utilized for routine analysis of dietary and nutritional supplements.

Development and validation of a GC–MS/MS method for the determination of 11 amphetamines and 34 synthetic cathinones in whole blood

M. Woźniak , L. Banaszkiewicz , M. Wiergowski, E. Tomczak, M. Kata, B. Szpiech, J. Namieśnik , M. Biziuk Forensic Toxicology – 2019
Purpose Psychoactive compounds that contain a phenylethylamine structure (such as amphetamine-type stimulants and synthetic cathinones) are one of the major classes of stimulants on the recreational drug market. Approximately 670 new psychoactive substances (NPS) are monitored only in Europe; however, new psychoactive compounds are being developed for illicit trade each year. In this context, the development of new analytical procedures for the determination of such compounds in biological specimens for forensic toxicology is of great importance. Methods Gas chromatography–tandem mass spectrometry (GC–MS/MS) technique was applied for analysis of amphetamines and synthetic cathinones. The volumes of 200 µL of each whole blood sample and 1 mL of liquid-liquid extraction solvent were used for extraction, followed by pentafuoropropionyl derivatization. Results A high-throughput, robust, rapid, and sensitive procedure involving a simple liquid-liquid extraction for the simultaneous determination of 45 amphetamine-type stimulants and synthetic cathinones in whole blood was developed. The assay was validated based on its recovery (83.2–106%), interday accuracy (89.0–108%), and interday precision (≤8.1%). In view of the low limits of detection (ranged between 0.02 and 0.72 ng/mL) and limits of quantifcation (1 and 2.5 ng/mL), the developed method can serve as a less expensive and more ecologically friendly alternative to the liquid chromatography–tandem mass spectrometric methods. Conclusions To the best of our knowledge, this is the frst work presenting a GC–MS/MS method for the determination of NPS in blood samples. The presented procedure was applied to authentic samples from forensic cases, demonstrating its utility in the quantifcation of a wide number of psychoactive substances in routine toxicological analyses. The developed procedure can also be easily expanded to additional compounds.