Determination of 465 psychoactive substances, drugs and their metabolites in urine by LC-MS/MS.

Due to the emergence of novel psychoactive substances on the drug market, there is a growing demand for analytical methods allowing identification and determination of as many psychoactive substances as possible in the shortest possible time, which can be easily expanded to include additional analytes appearing in street trade. Immunochemical methods are not sufficient to meet constantly growing requirements. Therefore, the aim of the study was to develop an analytical method enabling quick analysis of urine samples for psychoactive substances, drugs and their metabolites. Liquid-liquid extraction (LLE) and liquid chromatography coupled with mass spectrometry (LC-MS/MS) were used for this purpose. Using available analytical standards, the operating parameters of the mass spectrometer were selected for each of the 477 analytes, and MRM (multiple reaction monitoring) acquisition was selected for each of them. The use of analytical standards allowed for the identification of analytes separated on the chromatography column. The exceptions are methylmethcathinone isomers (3-MMC and 4-MMC), which we were unable to separate using the gradient elution method used. Extraction using acetonitrile with the addition of ammonium formate and formic acid allowed us to obtain high recoveries without the use of ß-glucuronidase. Recovery values ranged from 18.43 to 119.94%. The matrix effect was eliminated by obtaining a calibration curve in the matrix. The developed analytical method was validated in accordance with SWGTOX guidelines. Only 12 substances did not meet the validation criteria (CV: ±20% and bias: ±20%). Thus the validated method makes it possible to determine 465 psychoactive substances in just 30 minutes. In the validation process, values such as the limit of detection (LOD) and the limit of quantification (LOQ) were also determined. The LODs are in the range of 3-30 ng mL-1, and the LOQs are in the range of 10-100 ng mL-1. The method was successfully applied to toxicological analyses of urine samples, which was an opportunity to develop it further to meet the needs of toxicology.

Accumulation of polystyrene nanoplastics and triclosan by a model tooth-carp fish, Aphaniops hormuzensis (Teleostei: Aphaniidae).

The presence and effects of nanoplastics (NPs; <1 µm) in the aquatic environment are a growing concern. In this study, a model tooth-carp fish, Aphaniops hormuzensis, has been exposed to different concentrations of fluorescent polystyrene nanoplastics (PS-NP) in its diet (up to 5 mg kg-1) over periods of 28 d and the particle accumulation in different tissues determined. Accumulation was observed in both digestive and non-digestive organs, with concentrations greater in the gut, liver and gill (up to 400 µg kg-1 dw) than in the skin and muscle (<180 µg kg-1 dw), but no dependency on exposure time or dose was evident. The presence of the organic contaminant, triclosan (TCS), in the diet and at concentrations up to 0.5 µg kg-1 did not affect PS-NP uptake by A. hormuzensis, while TCS accumulation in the whole body increased with time (up to 10 µg kg-1) and, likewise, appeared to be unaffected by the presence of PS-NPs. These observations suggest that the two contaminants do not interact with each other or that any interactions have no impact on accumulation. The results of this study add to the growing body of evidence that NPs can be translocated by aquatic organisms after ingestion, and reveal that, for the species and conditions employed, nanoplastics are accumulated more readily than a widely used organic chemical.

Identification and quantification of cyclitols and sugars isolated from different morphological parts of Raphanus sativus L.

Through this study, we aimed to develop a new analytical method for identification and quantification of sugars and cyclitols isolated from different morphological parts of Raphanus sativus L (R. sativus). Accelerated solvent extraction with water was involved for targets extraction. Solid phase extraction was used for purification and preconcentration, while high performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was used for chromatographic analyses. A short method of only 30 min for a single analysis was developed finally. The obtained results, allowed for quantification of eight targets, i.e., three cyclitols (D-pinitol, allo-inositol and scyllo-inositol) and five sugars (xylose, D-mannose, D-fructose, D-glucose and sucrose) that were determined simultaneously using a single analysis. The developed method can be applied in industry as a routine method for analysis of sugars and cyclitols from different sources.

One-step extraction and determination of 513 psychoactive substances, drugs, and their metabolites from hair by LC-MS/MS.

The emergence of new psychoactive substances on the market is a significant problem on a global scale. This type of substance in society is associated with many negative consequences, such as traffic accidents, accidents at work, rape, homicide, poisoning, or overdose deaths. The analysis of these substances in biological samples is very important for further legal action and saving lives. Therefore, laboratories face a tremendous challenge in tackling the evolving drug market. The paper describes the optimization of the analytical LC-MS/MS method to identify and determine 513 psychoactive substances in hair samples. A method of chromatographic separation was developed, and the working parameters of the mass spectrometer were selected for each analyte. The method has been validated, and the results are as follows: the limit of quantification of the developed method ranges from 0.025 to 1.25 ng/mg hair. The mean recovery of the tested analytes ranges from 80 to 120%. The achieved coefficient of variation in within-run precision ranged from 1.05 to 19.99%. The results achieved for BIAS are in the range of ± 20%.

Overview of analytical methods for determining novel psychoactive substances, drugs and their metabolites in biological samples.

Recent years have witnessed a growing in interest in psychoactive substances, particularly those available in e-commerce. These have led to an increase in the number of drug-related poisonings, deaths, and road accidents. Illegal drugs are available on an unprecedented scale and cause previously unknown adverse effects, which creates a challenge for analysts to find rapid methods for identifying these substances and taking appropriate action in the shortest possible time. New psychoactive substances (NPSs) can be lethal at very low concentrations, which give particularly serious cause for concern. These drugs are easily accessible and often regarded (or claimed) to be safe, which encourages many people, in particular young people, to try them. The widespread use of these substances is compounded by the awareness that they are difficult to detect with the existing rapid screening tests. Simple, fast, sensitive, and specific methods for determining the largest possible number of black-market psychoactive substances and their metabolites are therefore essential. Such methods will facilitate treatment and increase the effectiveness of measures aiming to reduce drug addiction. The objective of this review article was to critically compare the most commonly used analytical methods for determining NPS and their metabolites in biological material, with special emphasis on the sample preparation process, and to highlight the possibilities offered by the existing analytical methods.

Cytokines-Biogenesis and Their Role in Human Breast Milk and Determination.

Cytokines play a huge role in many biological processes. Their production, release and interactions are subject to a very complex mechanism. Cytokines are produced by all types of cells, they function very differently and they are characterized by synergism in action, antagonism, and aggregation activity, opposing action of one cytokine, overlapping activity, induction of another cytokine, inhibition of cytokine synthesis at the mRNA level as well as autoregulation-stimulation or inhibition of own production. The predominance of pro-inflammatory cytokines leads to a systemic inflammatory response, and anti-inflammatory-to an anti-inflammatory response. They regulate the organism's immune response and protect it against sudden disturbances in homeostasis. The synthesis and activity of cytokines are influenced by the central nervous system through the endocrine system (pituitary gland, adrenal glands).

Determination of Neonicotinoids in Honey Samples Originated from Poland and Other World Countries.

A method development for determination of neonicotinoid residues in honey samples was developed. The proposed methodology consisted in QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe). That was used for sample preparation and UHPLC/UV (ultra-performance liquid chromatography with ultraviolet detection) utilized for chromatographic analysis. The developed method proved to be sensitive, with LOD (Limit of detection) value in the range of 60.80 to 80.98 ng/g hence LOQ (Limit of quantification) value was in the range of 184.26 to 245.40 ng/g. The method has tested on Polish honey and applied to honey from various countries (Bulgaria, Czech Republic, France, Greece, Italy, Portugal, Romania, Australia, Brazil, Cameroon, Russia, USA and Turkey). Several honey types were tested, while physicochemical properties of all honeys and were investigated. The methodology for general characterization of pollen grains originated from selected plants, to confirm the type of honey was also presented. There was a total lack of the mentioned neonicotinoids in sunflower honey. Except of this, only two samples of rapeseed and two samples of acacia honey (from Poland and Romania) were neonicotinoids free. In 19 samples the targeted pesticides were detected above LOQ. In all other investigated samples, the neonicotinoids were found at least at the LOD or LOQ level.

Comparison of DAD and FLD Detection for Identification of Selected Bisphenols in Human Breast Milk Samples and Their Quantitative Analysis by LC-MS/MS.

BACKGROUND: Determination of bisphenols released from packaging material is undoubtedly a difficult and tricky task, requiring the chemical analyst to develop an individual approach to obtain reliable analytical information. OBJECTIVE: QuECHERS (Quick, Easy, Cheap, Effective, Rugged, and Safe)/dispersive solid-phase extraction (d-SPE) technique and high performance liquid chromatography (HPLC) coupled with modern detection techniques such as diode-array detector (DAD), fluorescence detector (FLD) or tandem mass spectrometry (MS/MS) for the determination of bisphenols such as bisphenol A (BPA), bisphenol S (BPS), bisphenol F (BPF), bisphenol B (BPB), 2-[[4-[2-[4-(Oxiran-2-ylmethoxy)phenyl]propan-2yl]phenoxy] methyl]oxirane (BADGE), 3-[4-[2-[4-(Oxiran-2-ylmethoxy)phenyl]propan-2-yl]phenoxy]propane-1,2-diol (BADGE*H2O), 3-[4-[2-[4-(2,3-Dihydroxypropoxy)phenyl]propan-2-yl]phenoxy]propane-1,2-diol (BADGE*2H2O), 1-Chloro-3-[4-[2-[4-(3-chloro-2-hydroxypropoxy)phenyl] propan-2-yl]phenoxy]propan-2-ol (BADGE*2HCl) in human breast milk samples have been performed. METHODS: For the analysis of total analytes, prior to the extraction with acetonitrile, a deconjugation step was implemented in a tube by adding 1 mL of the enzymatic solution with the ß-Glucuronidase to 5 mL of sample. The mix was homogenized and incubated for 17 h at 37°C. Ten milliliters of acetonitrile, and a QuEChERS salt packet with 4 g anhydrous MgSO4 and 1 g NaCl were added. During the d-SPE step the extract was transferred into tube with 30 mg Z-Sep and 50 mg PSA (and also 150 mg MgSO4 for LC-MS/MS analysis). MeOH-water (20:80, v/v) were added to the dry residue and the extract was reconstituted in 150 µL (25-fold analytes pre-concentration is achieved). Next bisphenols were identified by HPLC-DAD-FLD and quantified by LC-MS/MS equipment. CONCLUSIONS: During the bisphenols HPLC-DAD-FLD analysis, from 6 min a reinforcement of 15 was used, which allowed analytes to be identified at 750 pg/mL. Application of LC-MS/MS allowed quantification of bisphenols in the range from 2.12 to 116.22 ng/mL in a total 27 human breast milk samples. HIGHLIGHTS: First QuEChERS/d-SPE coupled with HPLC-DAD-FLD or LC-MS/MS method for the quantification of bisphenols and its analogues in breast milk Faster and cheaper alternative to traditional extraction methods The method was applied for the first biomonitoring of bisphenols and its analogues in breast milk.

Synthesis of Magnetic Molecularly Imprinted Polymer Sorbents for Isolation of Parabens from Breast Milk.

Magnetic molecularly imprinted polymers (MMIPs) are an invaluable asset in the development of many methods in analytical chemistry, particularly sample preparation. Novel adsorbents based on MMIPs are characterized by high selectivity towards a specific analyte due to the presence of a specific cavity on their polymer surface, enabling the lock-key model interactions to occur. In addition, the magnetic core provides superparamagnetic properties that allow rapid separation of the sorbent from the sample solution. Such a combination of imprinted polymers with a magnetic core has an innovative influence on the development of separation techniques. Hence, the present study describes the synthesis of MMIPs with 17ß-estradiol used as a template molecule in the production of imprinted polymers. The as-prepared sorbent was used for a sorption/desorption study of five parabens from breast milk samples. The obtained results were characterized by sorption efficiency exceeding 92%, which shows the high affinity of the analytes to the functional groups on the sorbent. The final determination of the selected analytes was done with high-performance liquid chromatography using a fluorometric detector. The determined linearity ranges for selected parabens were characterized by high determination coefficients (r2 from 0.9992 to 0.9999), and the calculated limit of detection (LOD) and limit of quantification (LOQ) for the identified compounds were low (LOD from 1.1-2.7 ng mL-1; LOQ from 3.6-8.1 ng mL-1), which makes their quantitative analysis in real samples feasible.

Qualitative analysis of enzymatic and chemical depolymerized low molecular weight heparins by UHPLC coupled with electrospray ionization quadrupole time-of-flight-mass spectrometry.

Complete heparin digestion with heparin lyase I and II results in a mixture of hexasaccharides and tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Because these tetrasaccharides are derived from antithrombin III-binding sites of heparin, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. Therefore, this paper presents a new low molecular weight heparin sample preparation method-chemical depolymerization. Qualitative analysis of the studied compounds and a comparison of their composition are an important contribution to the structural analysis of low molecular weight heparins, which has not been fully conducted so far. Qualitative on-line liquid chromatography-mass spectrometric analysis of these resistant oligosaccharides is also described in this paper.

Carbon dots as rapid assays for detection of mercury(II) ions based on turn-off mode and breast milk.

In this research, nitrogen co-doped carbon dots were synthesized by solid thermal method with citric acid used as the precursor of carbon, and melamine as nitrogen source. Such carbon dots show high quantum yield of 44%. Furthermore, the native fluorescence of CDs can be reduced by mercury(II), while other metals had no significant influence on fluorescence intensity. During the study, the optimal parameters were selected, such as pH or time for incubation with analyte. Under the optimal conditions, quenching effect caused by mercury ions was evaluated. It was observed that with increasing mercury concentration, the fluorescence of the carbon dots decreased proportionally. The response was characterized by linearity within the range from 2 to 14 µM. Moreover, the limit of detection was 0.44 µM. It was the first time that human milk was used as a real sample to test the applicability of carbon dots. The study results demonstrated good recovery in the 74-111% range (RSD < 6%) As a novel carbon material, CDs show promise for broader applications in analyzing complicated biological samples.

QuEChERS extraction coupled to GC-MS for a fast determination of polychlorinated biphenyls in breast milk from Polish women.

Polychlorinated biphenyls (PCBs) in breast milk has been determined. Therefore, it was necessary to develop and adapt an analytical method to analyze PCB compounds. The whole procedure was applied to 31 breast milk samples, which were collected from Polish mothers. The QuEChERS method was optimized as a fast and cheap sample preparation method. The procedure allowed us to obtain recovery values between 96.46% and 119.98% with acceptable relative standard deviations (3.36-12.71%). Gas chromatography with mass spectrometry (GC-MS) was used for final determination. The method was validated using parameters such as linearity, limit of detection and quantification, intra-day precision, and reproducibility. The mean concentration of ∑iPCBs in this study was 30.94 ng/g of lipid. Assigned daily intake of PCBs was lower than the tolerable daily intake, which shows that the analyzed milk is safe to the infants. However, the monitoring of PCBs in milk is still important, and the QuEChERS method with GC-MS can be an effective tool for tracking organic impurities in breast milk.

Molecularly imprinted polymer film grafted from porous silica for efficient enrichment of steroid hormones in water samples.

Steroid hormones as endocrine disrupting compounds can interfere with the functioning of hormonal systems of organisms and thus affect the health and reproduction of humans and wildlife. Unfortunately, these types of harmful endocrine disrupting compounds have been found in a variety of environmental samples at very low concentrations. Therefore, a simple, fast, and efficient method for enrichment of water samples is needed. A molecularly imprinted solid-phase extraction combined with high performance liquid chromatography coupled with diode array detection was developed for the determination of six steroid hormones, such as estrone, 17-ß-estradiol, estriol, 17-α-ethinylestradiol, progesterone, and testosterone in water samples. The recoveries obtained in the proposed method were in the range of 78.7-101.3%. Matrix effect below 20% suggests that the quantitative and qualitative results of the analysis were not significantly affected by the matrix. The results show that molecularly imprinted polymers based on spherical silica gel had the potential to be a highly innovative and selective sorbent. The proposed method was proved to be applicable for molecularly imprinted solid-phase extraction in selective and reliable extraction and enrichment of steroid hormones in environmental water samples.

Towards A New Approach for the Description of Cyclo⁻2,4-Dihydroxybenzoate, A Substance Which Effectively Mimics Zearalenone in Imprinted Polymers Designed for Analyzing Selected Mycotoxins in Urine.

A method of purifying cyclododecyl 2,4-dihydroxybenzoate as a potential replacement template molecule for preparation of molecularly-imprinted polymers for isolation of zearalenone in urine was developed. Full physicochemical characteristics of cyclododecyl 2,4-dihydroxybenzoate for the first time included crystallographic analysis and molecular modelling, which made possible the determination of the similarity between the cyclododecyl 2,4-dihydroxybenzoate and zearalenone molecules. The obtained molecularly-imprinted polymers show very high in vitro selectivity towards zearalenone due to specific interactions (e.g., hydrogen bonding, molecular recognition interaction). The achieved extraction recovery exceeds 94% at the tested concentration levels (20⁻500 ng·mL-1) with a relative standard deviation below 2%. Immunosorbents were found to have lower recoveries (below 92.5%) and RSD value between 2 and 4% for higher concentrations of the studied substance (400 ng·mL-1).

Toxic effects of single animal hormones and their mixtures on the growth of Chlorella vulgaris and Scenedesmus armatus.

In their environments, aquatic organisms are simultaneously exposed to mixtures of several endocrine disrupting compounds, including hormones. However, most of the toxicity studies so far focused on effects of single contaminants. The available information on the potential toxicity of combined hormones on microalgae is extremely limited. For these reasons the aim of this study was to evaluate the individual and mixture effect of estrone (E1), ß-estradiol (E2), estriol (E3), 17-α-ethinylestradiol (EE2), progesterone (PRO), 5-pregnen-3ß-ol-20-one (PRE), levonorgestrel (LG) and testosterone (TST) on Chlorella vulgaris and Scenedesmus armatus. Green algae cells were exposed to different concentrations (0.1-100 mg L-1) of hormones for 14 days. Biomass in the form of dry weight and chlorophyll a was examined. The decreasing order of toxicity (based on EC50, 14d) to Chlorella vulgaris and Scenedesmus armatus was: EE2>PRO > E2>PRE > TST > E3>LG > E1 and EE2>PRO > TST > E2>PRE > LG > E1>E3, respectively. Chlorella vulgaris was more sensitive to the effects of hormones than Scenedesmus armatus. Although mixed hormones were more toxic to green algae than single hormones, in the ecosystem mixtures can pose higher ecological risk than single pollutants. Therefore, data on the toxicology of both single and mixed hormones is very valuable for assessment of the possibility of adverse ecological effects caused by these pollutants. Furthermore, these results suggest that environmental exposure to hormone mixtures may cause toxicity levels different to the sum of those of the single hormones and provides a basic understanding of their toxic effect on algae.

The impact of environmental pollution on the quality of mother's milk.

Breastfeeding is a gold standard of neonate nutrition because human milk contains a lot of essential compounds crucial for proper development of a child. However, milk is also a biofluid which can contain environmental pollution, which can have effects on immune system and consequently on the various body organs. Polychlorinated biphenyls are organic pollutants which have been detected in human milk. They have lipophilic properties, so they can penetrate to fatty milk and ultimately to neonate digestive track. Another problem of interest is the presence in milk of heavy metals-arsenic, lead, cadmium, and mercury-as these compounds can lead to disorders in production of cytokines, which are important immunomodulators. The toxicants cause stimulation or suppression of this compounds. This can lead to health problems in children as allergy, disorders in the endocrine system, end even neurodevelopment delay and disorder. Consequently, correlations between pollutants and bioactive components in milk should be investigated. This article provides an overview of environmental pollutants found in human milk as well as of the consequences of cytokine disorder correlated with presence of heavy metals. Graphical abstract.

Recent Developments in the Separation of Low Molecular Weight Heparin Anticoagulants.

The general function of anticoagulants is to prevent blood clotting and growing of the existing clots in blood vessels. In recent years, there has been a significant improvement in developing methods of prevention as well as pharmacologic and surgical treatment of thrombosis. For over the last two decades, low molecular weight heparins (LMWHs) have found their application in the antithrombotic diseases treatment. These types of drugs are widely used in clinical therapy. Despite the biological and medical importance of LMWHs, they have not been completely characterized in terms of their chemical structure. Due to both, the structural complexity of these anticoagulants and the presence of impurities, their structural characterization requires the employment of advanced analytical techniques. Since separation techniques play the key role in these endeavors, this review will focus on the presentation of recent developments in the separation of LMWH anticoagulants.

Cadmium-induced changes in the production of siderophores by a plant growth promoting strain of Pseudomonas fulva.

The impact of increasing Cd2+ exposure on the quality and quantity of siderophores produced by a plant growth promoting Pseudomonas fulva strain was tested to gain insight into the degree of change. P. fulva was cultured in the increasing concentrations of Cd2+ (0, 0.5, 1.0, 2.0 mM). The secreted siderophores were separated by HPLC and characterized by UHPLC-QTOF/MS. In the presence of 2 mM Cd2+ synthesis of siderophores (hydroxamates, catecholates, phenolates) was mitigated compared to the treatments with lower concentrations of Cd2+ (0.5 and 1 mM). Increased synthesis of catecholates in 0.5 and 1 mM Cd2+ and of phenolates in 0.5-2 mM Cd2+ was revealed compared to the variant without Cd2+ . Out of seven different hydroxamates, the secretion of ferrioxamine E was significantly decreased in the highest Cd2+ concentration. Two additional ferrioxamines, X2 and D2, were secreted independent of the presence or absence of Cd2+ . Exposure to Cd2+ change the composition of siderophores secreted by P. fulva with selective promotion of catecholates and phenolates at the expense of hydroxamates. Successful adaptation in a Cd-contaminated soil in the frame of practical applications to promote phytoremediation can be assumed.

The determination of zearalenone and its major metabolites in endometrial cancer tissues.

Endometrial cancer is one of the most commonly diagnosed cancers in women. The search for factors that contribute to the development of cancer cells in reproductive organs should involve the detection of xenoestrogens, in particular zearalenone (ZEA) and its metabolites. Xenoestrogens are endocrine disruptors-ZEA and its metabolites are structurally similar to estrogens (macrocyclic lactone ring) and show high affinity for estrogen receptors. This study proposes a new method for the preparation of samples of human tissues with endometrial cancer by the use of the QuEChERS technique. Analytical parameters such as centrifugation temperature, extraction solvent, and adsorbents were modified to obtain satisfactory recovery for ZEA (R = 82.6%, RSD = 2.9%) and one of its metabolites, α-zearalenol (R = 50.1%, RSD = 3.2%). High-performance liquid chromatography (HPLC) with fluorescence detection and tandem mass spectrometry (LC-QTOF-MS) were used for the identification and quantitative determination of the analyzed compounds. The developed procedure was applied for analyses of human tissues with endometrial cancer. The presence of α-zearalenol was detected in 47 out of the 61 examined tissue samples. Graphical Abstract Methodology for isolation and identification of zearalenone and its major metabolites.

Separation of aflatoxin B1 from synthetic physiological fluids using talc and diatomite: Kinetic and isotherm aspects.

The objective of the study was to examine adsorption of the aflatoxin B1 from synthetic gastric fluid and synthetic intestinal fluid by talc, raw and calcined diatomite. The kinetic and equilibrium adsorption processes were studied in the batch adsorption experiments applying high performance liquid chromatography for the aflatoxin B1 determination. The kinetic study showed a very fast adsorption of the aflatoxin B1 onto the selected adsorbents from the both physiological fluids with reaching equilibrium within 1-15min. The aflatoxin B1 was almost completely adsorbed in initial linear step of the kinetic process that can be described well by the zero-order kinetics model. The experimental data of the equilibrium adsorption were characterized using the Langmuir and Freundlich isotherm models. The high adsorption effectiveness was found in a range of 90%-100% and 60%-100% for the diatomite samples and the talc respectively at the initial concentrations of the aflatoxin B1 as 31-300ng/mL. The possible mechanisms of the aflatoxin adsorption onto the used mineral adsorbents are also discussed in the work.