Targeting the Aryl Hydrocarbon Receptor with Microbial Metabolite Mimics Alleviates Experimental Colitis in Mice.

Targeting the aryl hydrocarbon receptor (AhR) is an emerging therapeutic strategy for multiple diseases (e.g., inflammatory bowel disease). Thermosporothrix hazakensis microbial metabolite 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a putative AhR endogenous ligand. To improve the chemical stability, we synthesized a series of ITE chemical mimics. Using a series of in vitro assays, we identified 2-(1H-indole-3-carbonyl)-N-methyl thiazole-4-carboxamide (ITE-CONHCH3) as a highly potent (EC50 = 1.6 nM) AhR agonist with high affinity (Ki = 88 nM). ITE-CONHCH3 triggered AhR nuclear translocation and dimerization of AhR-ARNT, enhanced AhR binding in the CYP1A1 promoter, and induced AhR-regulated genes in an AhR-dependent manner. The metabolic stability of ITE-CONHCH3 in a cell culture was 10 times higher than that of ITE. Finally, we observed protective effects of ITE-CONHCH3 in mice with DSS-induced colitis. Overall, we demonstrate and validate a concept of microbial metabolite mimicry in the therapeutic targeting of AhR.

Electroanalysis of Fentanyl and Its New Analogs: A Review.

The review describes fentanyl and its analogs as new synthetic opioids and the possibilities of their identification and determination using electrochemical methods (e.g., voltammetry, potentiometry, electrochemiluminescence) and electrochemical methods combined with various separation methods. The review also covers the analysis of new synthetic opioids, their parent compounds, and corresponding metabolites in body fluids, such as urine, blood, serum, and plasma, necessary for a fast and accurate diagnosis of intoxication. Identifying and quantifying these addictive and illicit substances and their metabolites is necessary for clinical, toxicological, and forensic purposes. As a reaction to the growing number of new synthetic opioid intoxications and increasing fatalities observed over the past ten years, we provide thorough background for developing new biosensors, screen-printed electrodes, or other point-of-care devices.

Acclimations to Cold and Warm Conditions Differently Affect the Energy Metabolism of Diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.).

The European corn borer Ostrinia nubilalis is a pest species, whose fifth instar larvae gradually develop cold hardiness during diapause. The physiological changes underlying diapause progression and cold hardiness development are still insufficiently understood in insects. Here, we follow a complex of changes related to energy metabolism during cold acclimation (5°C) of diapausing larvae and compare this to warm-acclimated (22°C) and non-diapause controls. Capillary electrophoresis of nucleotides and coenzymes has shown that in gradually cold-acclimated groups concentrations of ATP/ADP and, consequently, energy charge slowly decrease during diapause, while the concentration of AMP increases, especially in the first months of diapause. Also, the activity of cytochrome c oxidase (COX), as well as the concentrations of NAD+ and GMP, decline in cold-acclimated groups, until the latter part of diapause, when they recover. Relative expression of NADH dehydrogenase (nd1), coenzyme Q-cytochrome c reductase (uqcr), COX, ATP synthase (atp), ADP/ATP translocase (ant), and prohibitin 2 (phb2) is supressed in cold-acclimated larvae during the first months of diapause and gradually increases toward the termination of diapause. Contrary to this, NADP+ and UMP levels significantly increased in the first few months of diapause, after gradual cold acclimation, which is in connection with the biosynthesis of cryoprotective molecules, as well as regeneration of small antioxidants. Our findings evidence the existence of a cold-induced energy-saving program that facilitates long-term maintenance of larval diapause, as well as gradual development of cold hardiness. In contrast, warm acclimation induced faster depletion of ATP, ADP, UMP, NAD+, and NADP+, as well as higher activity of COX and generally higher expression of all energy-related genes in comparison to cold-acclimated larvae. Moreover, such unusually high metabolic activity, driven by high temperatures, lead to premature mortality in the warm-acclimated group after 2 months of diapause. Thus, our findings strongly support the importance of low temperature exposure in early diapause for gradual cold hardiness acquisition, successful maintenance of the resting state and return to active development. Moreover, they demonstrate potentially adverse effects of global climate changes and subsequent increase in winter temperatures on cold-adapted terrestrial organisms in temperate and subpolar regions.

A synergy of liquid chromatography with high-resolution mass spectrometry and coagulation test for determination of direct oral anticoagulants for clinical and toxicological purposes.

Direct oral anticoagulants are an alternative to anticoagulants based on vitamin K antagonists. Monitoring of direct oral anticoagulant concentration levels is necessary in specific cases (e.g. in emergency conditions, for determination of the cause of bleeding, adverse effects, risk of drug-direct oral anticoagulants interaction); therefore, a sensitive and specific method is needed. A methanol protein precipitation method followed by liquid chromatography with high-resolution mass spectrometry was developed for simultaneous separation and determination of apixaban, betrixaban, edoxaban, dabigatran, rivaroxaban and ximelagatran. The proposed method was fully validated in terms of linearity, the limits of detection and quantification, intra- and inter-day trueness and precision, recovery, matrix effect, process efficiency and stability. The method shows a strong correlation (Pearson's correlation coefficients > 0.92) with coagulation assays of apixaban, dabigatran and rivaroxaban (dilute thrombin time for gatrans and anti Xa factor (anti-Xa) activity for xabans). In addition, the developed method was applied for the identification and determination of apixaban and dabigatran in post-mortem serum samples. The developed method is a good alternative to coagulation tests which may show various interferences.

Fatal intoxication by intravenous injection of castor bean (Ricinus communis L.) extract-a case study.

A case report of a 25-year-old man who committed suicide by intravenous injection himself of an aqueous home-made castor bean extract is presented. The patient was hospitalized and treated symptomatically and was released at its own request fourth day after intoxication. The next day, the patient's condition deteriorated, and he died 6 days after intoxication even though he was given medical care. Case history, autopsy, and toxicological investigation of ante- and post-mortem collected materials are described. Blood and urine collected from the patient ante-mortem and other several biological materials (namely blood from the upper and lower limb, blood from the right and left ventricle, pericardial fluid, vitreous humour, liver, kidney, and spleen) were collected post-mortem during autopsy. Liquid-liquid extraction procedure followed by high-performance liquid chromatography tandem mass spectrometry analysis for identification and determination of ricinine as a biomarker of ricin/castor seed intoxication was developed and validated. The method was applied on analysis of collected ante- and post-mortem biological materials. The post-mortem contents of ricinine in organs (namely the liver, kidney, and spleen) are firstly reported. The obtained results indicated approximately uniform distribution of ricinine (concentration level about 1 ng mL-1) in the body after death. In addition, the GC-MS method was also applied for the analysis of extract of castor seed and the patient's urine, to demonstrate alternative possibility for identification of ricinine for clinical and forensic purposes.

A direct LA-ICP-MS screening of elemental impurities in pharmaceutical products in compliance with USP and ICH-Q3D.

The novel laser ablation inductively coupled plasma mass spectrometry methodology for the rapid screening of elemental impurities in solid pharmaceutical samples with the daily dose less than 2.0 g has been developed in accordance with requirements of established USP <232/233> chapters and ICH-Q3D guideline. The LA-ICP-MS methodology covering the determination of Cd, Pb, As, Hg, Co, V, Ni, Tl, Au, Pd, Ir, Os, Rh, Ru, Se, Ag, Pt was successfully validated in terms of linearity, limit of quantification, accuracy, precision, intermediate precision, specificity and range. Moreover, the presented 'in-house' matrix-matched standards preparation methodology helps to overcome crucial analytical problem connected with unavailability of commercial certified matrix-matched reference material suitable for the direct elemental impurities analysis in various kinds of solid pharmaceutical products. A two step homogeneity study of prepared matrix-matched calibration standards is also reported to investigate the homogeneity of distribution of elemental impurities and internal standards across the pressed pellet. The validated LA-ICP-MS method was applied on analysis of several types of solid pharmaceutical samples (active pharmaceutical ingredients, excipients, placebo and final drug products). The proposed method allowed the accurate, precise and fast screening of elemental impurities without necessity of time and labour consuming solutions preparation and thus it can be used in routine practice as an alternative to conventional ICP-MS or ICP-OES for the rapid quality control of different stages of pharmaceutical production.

Laser ablation inductively coupled plasma mass spectrometry imaging: A personal identification based on a gunshot residue analysis on latent fingerprints.

A new laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP-MSI) method has been developed to visualize latent fingerprint of suspect persons possibly related to a criminal offence committed with a gun. Metallic impurities and metallic gunshot residues (GSRs) adhered on a latent fingerprint can link biometric information and shooting. The identification and spatial distribution of characteristic gunshot-related metals (Cu, Zn, Sb, Ba, Hg, Pb) was studied in detail. Observed limits of detection for the corresponding metals were below 0.3 ng cm-2. The distributions of the selected metals were simply transferred to images of fingerprints that can reveal a person somehow manipulating with a gun while the presence of characteristic and consistent GSR particles provided the evidence of the shooting. LA-ICP-MSI has been proved to be a unique tool in dactyloscopic identification of a suspect person from latent and commonly visualized fingerprints.

Simultaneous determination of designer benzodiazepines in human serum using non-aqueous capillary electrophoresis - Tandem mass spectrometry with successive multiple ionic - Polymer layer coated capillary.

A novel non-aqueous capillary electrophoresis - tandem mass spectrometry method for the simultaneous separation, identification and quantification of nine designer benzodiazepines (bentazepam, etizolam, deschloroetizolam, diclazepam, flubromazepam, flubromazolam, nimetazepam, phenazepam, and pyrazolam) was developed. A non-aqueous running electrolyte consisting of 25mM ammonium acetate with 100mM trifluoroacetic acid in acetonitrile was used. The separation was carried out using a semipermanent coated capillary (successive multiple ionic-polymer coating) with a strong anodic electroosmotic flow at a negative separation voltage within twelve minutes. Electrospray ionization with a triple quadrupole mass spectrometry was utilized for the identification and quantification of selected designer benzodiazepines in a positive ionization mode. The developed method was validated and applied on the analysis of spiked serum sample following a simple liquid-liquid extraction. The LODs of the designer benzodiazepines were between 1.5 and 15.0ngmL-1.

Pre-caecal digestible phosphorus in maize and wheat for broiler chickens.

1. The objective of this study was to determine the coefficient of pre-caecal digestion of P in maize (3.9 g/kg of total P, 0.83 g/kg of phytate P, 138 FTU [phytase units]/kg) and wheat (3.17 g/kg of total P, 1.94 g/kg of phytate P, 666 FTU/kg) in broilers according to the WPSA protocol. 2. For the diets, monosodium phosphate was used as an additional P supplement. Two sets of diets containing 200, 460 and 740 g/kg of wheat or 200, 500 and 740 g/kg of maize were formulated. A total of 288 21-d-old male broilers (Ross 308) were assigned to 24 cages (8 birds per cage) and the 6 test diets were assigned to cages. The coefficient of pre-caecal digestion of P was determined by the indicator method and linear regression. 3. In both ingredients, pre-caecal digestible P increased linearly with increasing inclusion levels of maize or wheat (P < 0.05). The coefficients of digestion of pre-caecal P were estimated to be 0.18 for wheat and 0.33 for maize.

Analysis of selected designer benzodiazepines by ultra high performance liquid chromatography with high-resolution time-of-flight mass spectrometry and the estimation of their partition coefficients by micellar electrokinetic chromatography.

A new ultra high performance liquid chromatography with electrospray ionization time of flight mass spectrometry method for the selective and sensitive separation, identification, and determination of selected designer benzodiazepines (namely, pyrazolam, phenazepam, etizolam, flubromazepam, diclazepam, deschloroetizolam, bentazepam, nimetazepam, and flubromazolam) in human serum was developed. The separation of the studied designer benzodiazepines was achieved on C18 chromatographic column using gradient elution within 6 min without any significant matrix interferences. Liquid-liquid extraction with butyl acetate was applied for serum samples cleanup and preconcentration of studied designer benzodiazepines. The method was validated in terms of linearity, limit of detection, limit of quantification, matrix effects, specificity, precision, accuracy, recovery, and sample stability. The limit of detection values were 0.10-0.15 ng/mL. The method was applied to a spiked serum sample to demonstrate its applicability for systematic toxicology analysis. Furthermore, a capillary chromatographic method with micellar electrokinetic chromatography was used for the estimation of partition coefficients of studied designer benzodiazepines as important parameters to evaluate their pharmacological and toxicological properties.

Development of separation methods for the chiral resolution of hexahelicenes.

In this short communication we report optimized procedures for the chiral separation of non-charged [6]helicene (1) and cationic derivative 1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide (2) using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) methods. The possibility of using capillary electrophoresis (CE) was also tested. The satisfactory results were obtained with SFC, where the highly selective resolution of four enantiopure 1 and 2 helicenes was achieved in a single run within 5min. The semi-preparative procedure for the isolation of P and M enantiomers of compound 2, including circular dichroism data, is reported here for the first time. The results could be used in further separations and analytical applications targeting carbohelicenes vs. positively charged helicene derivatives.

Enantioseparation of d,l-2-hydroxyglutaric acid by capillary electrophoresis with tandem mass spectrometry-Fast and efficient tool for d- and l-2-hydroxyglutaracidurias diagnosis.

A novel capillary electrophoresis-tandem mass spectrometry method for the enantioseparation and identification of 2-hydroxyglutaric acid enantiomers without derivatization for clinical purposes was described. Vancomycin chloride was used as an efficient chiral selector for the discrimination of 2-hydroxyglutaric acid enantiomers by capillary electrophoresis employed complete capillary filling method. The obtained resolution was 2.05. Hyphenation of CE with tandem mass spectrometry allows a reliable identification of separated enantiomers as well as their quantification. The method was validated and applied for the separation, identification and determination of 2-hydroxyglutaric enantiomers in urine samples obtained from healthy patients and two urine samples obtained from child patients suffering from high urine excretion of 2-hydroxyglutaric acid. Abnormal excretion of d-hydroxyglutaric acid was found in both child urine samples (104.5±2.1 and 2200.0±12.6mmol/mol of creatinine, respectively). The limits of detection for d- and l-hydroxyglutaric acid were 31 and 38nmol/L, respectively.

Novel cationic coating agent for protein separation by capillary electrophoresis(†).

A novel positively charged surfactant N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride enabled the reversal of the electro-osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.

Determination of selected synthetic cannabinoids and their metabolites by micellar electrokinetic chromatography--mass spectrometry employing perfluoroheptanoic acid-based micellar phase.

Perfluoroheptanoic acid was employed as a volatile micellar phase in background electrolyte for micellar electrokinetic chromatography-tandem mass spectrometry separation and determination of 15 selected naphthoyl- and phenylacetylindole- synthetic cannabinoids and main metabolites derived from JWH-018, JWH-019, JWH-073, JWH-200 and JWH-250. The influence of concentration of perfluoroheptanoic acid in background electrolytes on the separation was studied as well as the influence of perfluoroheptanoic acid on mass spectrometry detection. The background electrolyte consisted of 75 mM perfluoroheptanoic acid, 150 mM ammonium hydroxide pH 9.2 with 10% (v/v) propane-2-ol allowed micellar electrokinetic chromatography separation together with mass spectrometry identification of the studied parent synthetic cannabinoids and their metabolites. The limits of detection of studied synthetic cannabinoids and metabolites were in the range from 0.9 ng/mL for JWH-073 to 3.0 ng/mL for JWH-200 employing liquid-liquid extraction. The developed method was applied on the separation and identification of studied analytes after liquid-liquid extraction of spiked urine and serum samples to demonstrate the potential of the method applicability for forensic and toxicological purposes.

Analysis of Amphetamine-Derived Designer Drugs by Gas Chromatography with Mass Spectrometry.

The 22 amphetamine-derived synthetic drugs (ADSDs), mostly cathinones, were examined by gas chromatography with mass spectrometry using two different derivatization methods with (i) heptafluorobutyric anhydride (HFBA) and (ii) pentafluorobenzoyl chloride (PFBCl). Both developed derivatization approaches were evaluated and compared for urine and serum samples. Extraction procedures proved to give satisfactory results with regard to recoveries and extract purity, even though both derivatization methods reached acceptable sensitivity for the intended use. The derivatization with PFBCl showed better results with respect to retention and response stability, thus the PFBCl method was selected for validation. Calibration curves were linear over the tested concentration range of 20-1,000 ng/mL with the R(2) values ranging from 0.994 to 0.998. Intra- and interday precisions and accuracies were within 20% for all concentrations in the linear range. The limit of detection was determined to be lower than 2 ng/mL for all 22 analytes. The method proved to be a useful analytical tool in the course of systematic toxicological analysis.

The empirical comparison of cyclofructans and cyclodextrins as chiral selectors in capillary electrophoretic separation of atropisomers of R,S-1,1'-binaphthalene-2,2'-diyl hydrogen phosphate.

Native cyclofructans and their isopropyl derivatives were studied as chiral selectors in capillary electrophoresis and compared with α- and ß-cyclodextrin. R,S-1,1'-Binaphthalene-2,2'-diyl hydrogen phosphate was used as a model chiral compound. The empirical observation of the enantioselectivity of native cyclofructans and isopropyl derivatives of cyclofructans was described and compared with the cyclodextrins. The influence of methanol and acetonitrile, as the most commonly used organic solvents, and sodium dodecyl sulfate as a micelle forming additive on the separation of R,S-1,1'-binaphthalene-2,2'-diyl hydrogen phosphate atropisomers was achieved. The different enantiorecognition abilities resulting from unlike interaction mechanism with R,S-1,1'-binaphthalene-2,2'-diyl hydrogen phosphate were observed for the studied cyclodextrins and cyclofructans, especially when methanol or sodium dodecyl sulfate were used as modifiers of the separation conditions.

Enantiomeric separation of 1,3-dimethylamylamine by capillary electrophoresis with indirect UV detection using a dual-selector system.

The CE method employing an indirect UV detection for the enantioseparation of 1,3-dimethylamylamine (DMAA), widely used in various preworkout and dietary supplements labeled as a constituent of geranium extract has been developed. The dual-selector system consisting of negatively charged sulfated α-CD (1.1% w/v) and sulfated ß-CD (0.2% w/v) in 5 mM phosphate/Tris buffer (pH 3.0) containing the addition of 10 mM benzyltriethylammonium chloride (BTEAC) as the chromophoric additive was used for the enantiomeric separation of DMAA stereoisomers with the LODs in the range of 7.82-9.24 µg/mL. The method was partly validated and applied for the determination of the stereoisomeric composition of DMAA in commercial dietary supplements to verify the potential natural origin of DMAA.

Determination and identification of synthetic cannabinoids and their metabolites in different matrices by modern analytical techniques - a review.

Synthetic cannabinoids have gained popularity due to their easy accessibility and psychoactive effects. Furthermore, they cannot be detected in urine by routine drug monitoring. The wide range of active ingredients in analyzed matrices hinders the development of a standard analytical method for their determination. Moreover, their possible side effects are not well known which increases the danger. This review is focused on the sample preparation and the determination of synthetic cannabinoids in different matrices (serum, urine, herbal blends, oral fluid, hair) published since 2004. The review includes separation and identification techniques, such as thin layer chromatography, gas and liquid chromatography and capillary electrophoresis, mostly coupled with mass spectrometry. The review also includes results by spectral methods like infrared spectroscopy, nuclear magnetic resonance or direct-injection mass spectrometry.

Enantiomeric separation of tapentadol by capillary electrophoresis--study of chiral selectivity manipulation by various types of cyclodextrins.

The chiral recognition of the centrally acting analgesic agent tapentadol and its isomers with various cyclodextrins (CDs) was studied by capillary electrophoresis, focusing on the migration order of four stereoisomers. In the case of non-charged hydroxypropylated CDs (2-hydroxypropyl-ß-CD, 2-hydroxypropyl-γ-CD) the beta derivative was able to discriminate the S,R- and R,S-isomers in acidic background electrolyte, whereas the gamma allowed the separation of S,S- and R,R-tapentadol, respectively. Dual CD system containing both hosts was used to separate all of four isomers. Negatively charged sulfated-α-CD at 1.0% (w/v) concentration in 100mM sodium borate buffer (pH 9.5) was capable of separating the isomers with favorable enantiomer migration order and the optimized method was able to determine 0.15% of chiral impurities of tapentadol in the presence of the last migrating clinically important R,R-isomer.

Utilization of micellar electrokinetic chromatography-tandem mass spectrometry employed volatile micellar phase in the analysis of cathinone designer drugs.

A micellar electrokinetic chromatography method with tandem mass spectrometry has been developed for the selective separation, identification and determination of twelve new designer drugs from the group of synthetic cathinones. Ammonium salt of perfluorooctanoic acid at various concentrations as a volatile background electrolyte (BGE) to create micellar phase was studied for separation of selected synthetic cathinones with direct tandem mass spectrometry without significant loss of detection sensitivity. The optimized BGE contained 100 mM perfluorooctanoic acid with 200 mM ammonium hydroxide providing acceptable resolution of studied drugs in the MEKC step. In order to minimize interferences with matrix components and to preconcentrate target analytes, solid phase extraction was introduced as a clean-up step. The method was linear in the concentration range of 10-5000 ng mL(-1) and the limits of detection were in the range of 10-78 ng mL(-1). The method was demonstrated to be specific, sensitive, and reliable for the systematic toxicological analysis of these derivatives in urine samples.