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.

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.

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.

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.

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.

Enantioseparation of tartaric acid by ligand-exchange capillary electrophoresis using contactless conductivity detection.

A method for the determination of tartaric acid enantiomers using CE with contactless conductivity detection has been developed. Cu(II) as a central metal ion together with L-hydroxyproline were used as a chiral selector, the BGE was composed of 7 mM CuCl2, 14 mM trans-4-hydroxy-L-proline, and 100 mM ε-aminocaproic acid; the pH was adjusted to 5 by hydrochloric acid. Separation with a resolution of 1.9 was achieved in 9 min in a polyacrylamide-coated capillary to suppress the EOF. Various counterions of the BGE were studied, and migration order reversal was achieved when switching from ε-aminocaproic acid to L-histidine. With detection limits of about 20 µM, the method was applied to the analysis of wine and grape samples; only L-tartaric acid was found.

Enantiomeric separation of R,S-tolterodine and R,S-methoxytolterodine with negatively charged cyclodextrins by capillary electrophoresis.

The methods for separation of R,S-tolterodine and R,S-methoxytolterodine enantiomers using sulfated α-, ß-CD and phosphated-γ-CD by CE in acidic BGE based on Tris/phosphate pH 2.5 buffer were developed. Sulfated α- and ß-CD allow anodic detection while phosphated-γ-CD allows only cathodic detection of the separated enantiomers. The influence of chiral selector (CS)'s concentration as well as the influence of composition and concentration of BGE on resolutions were studied. Reversal migration order of tolterodine and methoxytolterodine enantiomers was observed, when sulfated-α- and sulfated-ß-CD were used. The developed methods with all three studied CSs, were validated and compared. All proposed methods enable determination of 0.2% of S-tolterodine as an optical impurity in pills, however the method with phosphated-γ-CD provided lower detection limit, better repeatability of peak areas and migration times, and also lower consumption of CS. Developed method employing phosphated-γ-CD that was applied for the determination of optical purity of R-tolterodine in commercial pills.

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis.

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on-line preconcentration. 5 mmol.L⁻¹ naphthalene-1-sulfonic acid and 10 mmol.L⁻¹ triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large-volume sample stacking and the field-amplified sample injection were applied and compared. Large-volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L⁻¹, respectively. Field-amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L⁻¹, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid-phase extraction, which further improved LOD toward 5.6 × 10⁻¹° mol.L⁻¹ for PFOS and 6.4 × 10⁻¹° mol.L⁻¹ for PFOA and allows the method to be used for river water contamination screening or decomposition studies.

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.

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.

Determination of rosiglitazone and metformin in human serum by CE-ESI-MS.

A new method for the determination of anti-diabetic drugs metformin and rosiglitazone based on the use of capillary electrophoresis with electrospray mass spectrometry was developed. The proposed method allowed their separation within 11 min by using 50 mM formic acid at +20 kV. Positive electrospray ionization and selected ion monitoring [M+H](+) of metformin (m/z=130) and rosiglitazone (m/z=358) were performed. Several important experimental parameters influencing electrospray ionization of metformin and rosiglitazone were studied. The final composition of sheath liquid was water/methanol/formic acid (50:49.5:0.5, v/v/v), at a flow rate of 2 µL/min. The developed method was applied for the determination of metformin and rosiglitazone simultaneously in human serum after protein precipitation with acetonitrile. The limits of detection of developed method were 4.42 and 2.14 ng/mL for rosiglitazone and for metformin, respectively, which is sufficient for therapeutic serum concentration levels monitoring for both studied drugs.

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.

Study of electromigration effects on a pH boundary during the on-line electrokinetic preconcentration by capillary electrophoresis.

A contribution to the description of electrokinetic effects on the pH boundary formed by sodium borate pH 9.5 and sodium phosphate pH 2.5 electrolytes for on-line preconcentration of weak acids is presented in this article. Simulations of electrokinetic injections together with experimental studies using contactless conductivity detection verified that the preconcentration is induced mainly by dissociation changes of analytes on the pH boundary and transient ITP state. Moreover, a study of the addition of organic solvent to the injection electrolyte was performed with impressive results. Subnanomolar LODs of hydroxybenzoic acids were achieved with 80% of methanol in the injection electrolyte which represents more than 70 000-fold preconcentration in comparison with classical CZE method.

Analysis of buserelin in urine by online combination of capillary zone electrophoresis with electrospray mass spectrometry.

A fast and precise analysis of the synthetic peptide buserelin in urine using CZE-ESI-MS method has been demonstrated. Formic acid at 50 mmol/L concentration served as background electrolyte in CZE stage and it is compatible with MS detection in positive ionization mode. Two injection modes were tested, i.e. pressure (50 mbar for 5 s) and electrokinetic injection (5 kV for 5 s), of which electrokinetic injection provided better calibration parameters. Buserelin LODs were 0.47 microg/mL in water and 0.63 microg/mL in ten times diluted urine samples using pressure injection, while they were 0.32 microg/mL in water and 0.34 microg/mL in ten times diluted urine samples using electrokinetic injection. Repeatability of buserelin migration times was below 6% (pressure injection mode) and 1% (electrokinetic injection mode). Repeatability of buserelin peak area in SIM mode (m/z=620.5+/-0.5) was less than 12% (pressure injection mode) and 5.8% (electrokinetic injection mode). In this work, no interferences were observed during the analyses of spiked urine samples.

On-line combination of CE and microscopy: an insight into the migration of microorganisms.

Identification of microbial contamination by CE has interested many researchers mainly because of the high speed of CE analysis. However, the CE separation of such big structures brings a lot of questions mainly about the behavior of microorganisms and about the mechanism of separation. In this work, we constructed a simple apparatus where a microscope was used as one detector and a UV detector was used as the second one and we made the comparison of three typical setups for CE of microorganisms. Saccharomyces cerevisiae was chosen as a model microorganism and was analyzed in bare fused-silica capillaries, covalently modified capillaries and dynamically modified capillaries by poly(ethylene oxide) or CTAB. Results showed that the use of CE instrument directly connected with a microscope could be advantageous for the study of separation mechanisms and/or migration behavior.

Assessment of CE for the identification of microorganisms.

The assessment of capillary electrophoresis for separation and identification of microorganisms is described in this article. The work brings a comparison of the application of uncoated capillaries modified with poly(ethylene oxide) and coated capillaries for the separation of model microorganisms Saccharomyces cerevisiae and Escherichia coli with Tris-borate-EDTA and Tris-citrate-fructose as electrolytes. The best separation was achieved in the coated capillary using 1 mM Tris-citrate-fructose buffer, pH 6.9. A simple identification based on the migration time and UV spectrum was found unsatisfactory and thus a method based on a post-separation cultivation and sequential analysis was developed. Off-line combination with mass spectrometric analysis with the use of desorption electrospray was shown to be an interesting alternative in a study of microorganisms. Mass spectra allowed distinguishing among analyzed cells.

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.

Determination of some phenolic acids in Majorana hortensis by capillary electrophoresis with online electrokinetic preconcentration.

An online accumulation/mobilization preconcentration technique based on a dynamic pH junction technique and electrokinetic injection was employed for analysis of phenolic acids (sinapic, ferulic, coumarinic, caffeic, syringic, vanillic, and 4-hydroxybenzoic acid) in extracts from Majorana hortensis leaves. Samples were extracted by pressurized solvent extraction with acetone at 150 degrees C and 15 MPa. The capillary electrophoretic method employed 50 mmol.L (-1) sodium borate, pH 9.5, as the sample electrolyte, 50 mmol.L (-1) sodium phosphate, pH 2.5, as the background electrolyte, and 50 mmol.L (-1) sodium phosphate, pH 2.5, with 60 mmol.L (-1) sodium dodecyl sulfate as the mobilization electrolyte. The method allowed 720-fold to 5560-fold preconcentration of the phenolic acids during 30 min of electrokinetic accumulation with detection limits from 0.38 to 4.22 ng.mL (-1).