Development and validation of a method for the simultaneous analysis of fatty acid ethyl esters, ethyl sulfate and ethyl glucuronide in neonatal meconium: application in two cases of alcohol consumption during pregnancy.

Alcohol consumption during pregnancy constitutes one of the leading preventable causes of birth defects and neurodevelopmental disorders in the exposed children. Fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG) and ethyl sulfate (EtS) have been studied as potential biomarkers of alcohol consumption. However, most analytical approaches proposed for their analysis in meconium samples consist of separated extraction procedures requiring the use of two meconium aliquots, which is costly in terms of both time and materials. Therefore, the aim of this study was to develop and validate a method for the simultaneous extraction of 9 FAEEs, EtG and EtS from one meconium aliquot. The sample was homogenized using methanol, and then FAEEs were extracted with hexane while EtG and EtS were isolated using acetonitrile. Then, extracts were applied to solid-phase extraction columns and analysed by gas chromatography mass spectrometry (FAEEs) and liquid chromatography tandem mass spectrometry (EtG and EtS). Calibration curves were linear with r values greater than 0.99. The LODs ranged from 0.8 to 7.5 ng/g for FAEEs and were 0.2 ng/g and 0.8 ng/g for EtS and EtG, respectively. LOQs ranged from 5 to 25 ng/g for FAEEs and were 1 ng/g and 2.5 ng/g for EtS and EtG, respectively. Accuracies and precisions were between 93.8 and 107% and between 3.5 and 9.7%, respectively. The recovery values ranged from 89.1 to 109%. The method proved to be sensitive, specific, simple and fast and allowed for the reduction of the amount of organic solvent used for extraction compared to other published data while higher recoveries were obtained. The method was used for analysis of meconium samples in two cases of mothers who were consuming alcohol during pregnancy.

New potent STS inhibitors based on fluorinated 4-(1-phenyl-1H-[1,2,3]triazol-4-yl)-phenyl sulfamates.

A series of fluorinated analogs based on the frameworks of 4-(1-phenyl-1H-[1,2,3]triazol-4-yl)-phenyl sulfamates have been synthesized as steroid sulfatase (STS) inhibitors. The design of chemical structures of new potential STS inhibitors was supported by molecular docking techniques to identify potential interactions between inhibitors and amino acid residues located in the STS active site. The STS inhibitory potency was evaluated on STS isolated from human placenta. We found that compounds substituted with fluorine atom at the meta position demonstrated the highest inhibitory effects in enzymatic STS assay. The most active analog 12e - inhibited STS enzyme with the IC50 value of 36 nM.

Differences between selected volatile aromatic compound concentrations in sludge samples in various steps of wastewater treatment plant operations.

Sewage sludge, one of the main wastes generated during wastewater treatment, constitutes an important source of emissions of volatile chemical compounds such as volatile aromatic compounds These substances may undergo various changes as a result of operations and unit processes, which affects their concentrations in sewage sludge. An important factor determining the potential hazardousness of volatile organic compounds is the quality of wastewater delivered to wastewater treatment plants and the technical and equipment solutions applied to wastewater. In this study, a rapid and sensitive headspace gas chromatography method, coupled with tandem mass spectrometry using the standard addition method, was developed for the determination of selected volatile aromatic compounds in sewage sludge samples collected at different stages from three wastewater treatment plants located in Poland. This study attempted to assess the relationship between differences in the emissions of representative VACs and the given stage of the technological process within three analysed wastewater treatment plants. Toluene was detected with the highest frequency in analysed samples, at concentrations varying from 0.234 ± 0.035 ng/g of sludge to 28.3*102±3.2*102 ng/g of sludge. The highest concentration levels were determined for p-cresol, with concentrations ranging from 44.0*101±5.6*101 ng/g of sludge (sludge from aerobic chamber, wastewater treatment plant no.2) to 47.7*102±6.9*102 ng/g of sludge (sludge from aerobic chamber, wastewater treatment plant no.1), while the lowest concentration levels were observed for chlorobenzene, with concentrations ranging from 0.1300 ±â€¯0.0030 ng/g of sludge (sludge from anaerobic chamber, wastewater treatment plant no.2), to 0.2606 ±â€¯0.0046 ng/g of sludge (primary sludge, wastewater treatment plant no.1). The repeatability of the method was better than 10%, with accuracy levels in the ranges 89%-108%.Wastewater treatment technologies and residual sludge management in the selected wastewater treatment plantsinfluenced volatile aromatic compounds emission. Furthermore, the diversity of the wastewater quality, depending on the catchment area, is also an important factor determining the differentiation in volatile aromatic compounds emission. The microbial composition of raw wastewater highly influenced not only the treatment effectiveness of wastewater treatment plants but also the production of intermediate products, such as volatile aromatic compounds, which may contribute to odour emissions.

New approach for e-cigarette aerosol collection by an original automatic aerosol generator utilizing melt-blown non-woven fabric.

Currently, there is lack of standardized conditions for the collection and analysis of e-cigarette (EC) aerosol. Considering the urgent need for the development of these guidelines, a procedure for EC aerosol analysis was developed. A novel automatic e-cigarette aerosol generator was designed. For the first time, melt-blown non-woven fabric was applied for the effective uptake of compounds released from vaporized e-liquid. The extraction procedure was optimized in terms of type of extraction solvent, amount of sorbent and solvent volume. For optimization, a model e-liquid containing flavour additives belonging to various chemicals group with various chemical properties was investigated. The aerosol trapping efficiency was satisfactory and was equal to 92 ±â€¯7%. Final determination was performed by GC-MS/MS. Quantitation was based on the mass change tracking approach (MCT), which assumes the monitoring of e-liquid mass changes before and after vaping. The combination of non-woven fabric and sampling approach (MCT) was proven to be effective in acquisition of reliable data. Thus, the concentrations in aerosol and emission factors were calculated for aerosols collected during the vaping of both model e-liquids and real samples. Validation was performed by evaluating key analytical parameters, such as linearity, accuracy, precision, limit of detection (LOD) and quantitation (LOQ). For all investigated compounds, recoveries from 70% to 118% together with precision and reproducibility below 12% were achieved. The applicability of the described approach was examined by analysing EC refill solutions commercially available on the Polish market.

Influence of Diosmin Treatment on the Level of Oxidative Stress Markers in Patients with Chronic Venous Insufficiency.

Oxidative stress plays an important role in the pathophysiology of many human disorders, while antioxidants prevent the development of various adverse symptoms. Diosmin is a natural flavonoid applied in vascular system disorders, especially in chronic venous insufficiency (CVI), and it plays a significant part in the alleviation of CVI symptoms. Due to antioxidant activity, it also has the ability to scavenge the oxygen free radicals and hence decreases the level of oxidative stress biomarkers, such as prostaglandins and their precursors-isoprostanes. In the study, the influence of diosmin treatment on the level of isoprostanes in plasma samples of patients suffering from CVI was examined. The qualitative analysis was performed using high-performance liquid chromatography with spectrometry detection (LC-MS). The statistically significant decrease of isoprostane content after 3 months of treatment was observed within the studied group; however, the most significant changes were observed in patients who smoke.

Evaluation of flavour profiles in e-cigarette refill solutions using gas chromatography-tandem mass spectrometry.

Many flavour compounds that are present in e-liquids for e-cigarettes are responsible for specific tastes and smoking sensations for users. Data concerning content and specific types of flavours is often limited and unknown to users. The aim of the research was to define and compare flavour profiles of e-liquids with the same group taste from different manufacturers. Gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was used to separate and identify 90 popular compounds (98, including isomers) of interest. The developed method was validated in terms of accuracy (88-113%) for three spiking levels and the intra-day (0.2-13%) and inter-day precision (1-10%). Limits of quantitation were in the range of 10-816 ng/mL, while the matrix effects for 80% of the compounds were at negligible levels. The proposed method is rapid, simple and reliable and uses a green and modern GC-MS/MS technique. Twenty-five samples of five different flavours (tobacco, strawberry, cherry, menthol and apple) from five different producers were analysed, and the determined compounds were categorized and differentiated. The approach proposed in this study allowed for the evaluation of which compounds/group of compounds are responsible for taste and to distinguish common flavour compounds among the investigated brands for each flavour. Furthermore, the presented research can be considered in future toxicological studies.

Application of gas chromatography-tandem mass spectrometry for the determination of amphetamine-type stimulants in blood and urine.

Amphetamine, methamphetamine, phentermine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) are the most popular amphetamine-type stimulants. The use of these substances is a serious societal problem worldwide. In this study, a method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) with simple and rapid liquid-liquid extraction (LLE) and derivatization was developed and validated for the simultaneous determination of the six aforementioned amphetamine derivatives in blood and urine. The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. The most important advantage of the method is the minimal sample volume (as low as 200µL) required for the extraction procedure. The validation parameters, i.e., the recovery (90.5-104%), inter-day accuracy (94.2-109.1%) and precision (0.5-5.8%), showed the repeatability and sensitivity of the method for both matrices and indicated that the proposed procedure fulfils internationally established acceptance criteria for bioanalytical methods The procedure was successfully applied to the analysis of real blood and urine samples examined in 22 forensic toxicological cases. To the best of our knowledge, this is the first work presenting the use of GC-MS/MS for the determination of amphetamine-type stimulants in blood and urine. In view of the low limits of detection (0.09-0.81ng/mL), limits of quantification (0.26-2.4ng/mL), and high selectivity, the procedure can be applied for drug monitoring in both fatal and non-fatal intoxication cases in routine toxicology analysis.

Comprehensive determination of flavouring additives and nicotine in e-cigarette refill solutions. Part I: Liquid chromatography-tandem mass spectrometry analysis.

Liquid chromatography-tandem mass spectrometry with electrospray ionization (HPLC-ESI-MS/MS) methods were developed for the simultaneous determination of 42 flavouring compounds and nicotine in liquids for e-cigarettes. The chromatographic separation was performed using an Ace® Ultracore™ SuperC18™ (100×2.1mm, 2.5µm) column in both acidic and alkaline pH conditions to separate all the compounds. A simple "dilute & shoot" approach was used for the sample preparation. The method validation was performed by evaluating key analytical parameters such as linearity, accuracy, selectivity, precision, limit of detection (LOD) and limit of quantification (LOQ). The calibration curves showed good linearity within the specific ranges for the investigated compounds with correlation coefficients greater than 0.990 in each case. The recovery for all the investigated compounds varied from 89% to 110%. The intra- and inter-day precision were within the acceptable limits (±15%) at all tested concentrations. The applicability of the methods was examined by analysing 25 liquid samples from e-cigarettes commercially available on the Polish market.

Comprehensive determination of flavouring additives and nicotine in e-cigarette refill solutions. Part II: Gas-chromatography-mass spectrometry analysis.

Flavouring compounds are an essential part of e-liquid products for cigarettes. In general, they are regarded as safe for ingestion, but they may have unrecognized risks when they are inhaled. In some cases, manufactures do not currently abide by the Tobacco Products Directive (2014/40/EU) and do not declare the detailed contents of e-liquids on their labels. To help evaluate the health impact of flavouring substances, there is a need for comprehensive approaches to determine their concentrations in e-liquids. For this purpose, a GC-EI-MS method was developed and validated for the simultaneous determination of 46 commonly used flavour additives in e-liquids. The proposed method performed well in terms of the key validation parameters: accuracy (84-113%), inter-/intra-day precision: 0.1-10% and 1-11%, respectively, and sensitivity (limit of detection: 3-87ng/mL). The sample preparation step was based on a simple "dilute & shoot" approach. This study is a complementary method to the LC-MS/MS procedure described in Part I. Both approaches are suitable for the comprehensive determination of 88 flavouring compounds and nicotine and can be used as tools for the rapid evaluation of the quality and safety of e-cigarette products.

Novel liquid chromatography method based on linear weighted regression for the fast determination of isoprostane isomers in plasma samples using sensitive tandem mass spectrometry detection.

A simple, fast, sensitive and accurate methodology based on a LLE followed by liquid chromatography-tandem mass spectrometry for simultaneous determination of four regioisomers (8-iso prostaglandin F2α, 8-iso-15(R)-prostaglandin F2α, 11ß-prostaglandin F2α, 15(R)-prostaglandin F2α) in routine analysis of human plasma samples was developed. Isoprostanes are stable products of arachidonic acid peroxidation and are regarded as the most reliable markers of oxidative stress in vivo. Validation of method was performed by evaluation of the key analytical parameters such as: matrix effect, analytical curve, trueness, precision, limits of detection and limits of quantification. As a homoscedasticity was not met for analytical data, weighted linear regression was applied in order to improve the accuracy at the lower end points of calibration curve. The detection limits (LODs) ranged from 1.0 to 2.1pg/mL. For plasma samples spiked with the isoprostanes at the level of 50pg/mL, intra-and interday repeatability ranged from 2.1 to 3.5% and 0.1 to 5.1%, respectively. The applicability of the proposed approach has been verified by monitoring of isoprostane isomers level in plasma samples collected from young patients (n=8) subjected to hyperbaric hyperoxia (100% oxygen at 280kPa(a) for 30min) in a multiplace hyperbaric chamber.

Identification of novel psychoactive substances 25B-NBOMe and 4-CMC in biological material using HPLC-Q-TOF-MS and their quantification in blood using UPLC-MS/MS in case of severe intoxications.

This paper describes cases of poisoning caused by new psychoactive substances such as: 25B-NBOMe (2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine) and 4-CMC (1-(4-chlorophenyl)-2-(methylamino)-1-propanone). The analytical procedure includes rapid and selective method for the extraction and determination of 4-CMC and 25B-NBOMe in blood samples using UPLC-MS/MS technique. To the best of our knowledge, this is the first report, that involves a fully validated method for quantification of new-designer drug - 4-CMC in postmortem blood samples. The biological material was also analyzed with the use of routine analytical methods: immunochemical techniques, gas chromatography with flame ionization detection and gas chromatography with electron impact mass spectrometry. The results of real samples analyses correspond to possible toxicological effects: death resulting from 25B-NBOMe - mediated hallucinations (661ng/mL of 25B-NBOMe and 0.887ng/mL of 4-CMC), fatal overdose of 25B-NBOMe and 4-CMC (66.5ng/mL of 25B-NBOMe and 2.14ng/mL of 4-CMC) and non-fatal intoxication of these drugs (38.4ng/mL of 25B NBOMe and 0.181ng/mL of 4-CMC). Additionally, O-demethylathed O, O-bis-demethylathed and glucuronidated metabolites of 25B-NBOMe in biological specimens were detected.

The use of HPLC-Q-TOF-MS for comprehensive screening of drugs and psychoactive substances in hair samples and several "legal highs" products.

ABSTRACT: Non-targeted screening of drugs present in herbal products, known as "legal high" drugs and in hair as a biological matrix commonly used in toxicological investigations was accomplished with the use of high pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). In total, 25 and 14 therapeutical drugs and psychoactive substances/metabolites were detected in investigated hair samples and herbal products, respectively. We demonstrate that the HPLC-Q-TOF methodology seems to be a powerful tool in the qualitative analysis applied in identification of these designer drugs, thus enabling a laboratory to stay-up-to-date with the drugs that are being sold as legal high products on black market.

Inspired by Nature: The Use of Plant-derived Substrate/Enzyme Combinations to Generate Antimicrobial Activity in situ.

The last decade has witnessed a renewed interest in antimicrobial agents. Plants have received particular attention and frequently rely on the spontaneous enzymatic conversion of an inactive precursor to an active agent. Such two-component substrate/enzyme defence systems can be reconstituted ex vivo. Here, the alliin/alliinase system from garlic seems to be rather effective against Saccharomyces cerevisiae, whilst the glucosinolate/myrosinase system from mustard appears to be more active against certain bacteria. Studies with myrosinase also confirm that enzyme and substrate can be added sequentially. Ultimately, such binary systems hold considerable promise and may be employed in a medical or agricultural context.