[Management of patients with altered mental status in the Emergency Department]. / Medvetanderubbning på akuten: strukturerad utredning viktig.

Patients who present to the Emergency Department with altered mental status may suffer from serious, time-sensitive conditions where appropriate initial management improves prognosis. The differential diagnoses of altered mental status is broad and the assessment is complicated by the patient's inability to provide a detailed history. This article presents a systematic approach to patients with altered mental status in the Emergency Department. Data from a structured physical examination and bedside tests are interpreted in light of the medical background and available history. A checklist is recommended to consider key time-sensitive conditions. The article covers the emergency treatment of selected time-sensitive conditions and provides guidance for when head CT-scan is warranted.

Mobilization of electroosmotic flow markers in capillary zone electrophoresis.

UV-absorbing neutral substances are commonly used as markers of mean electroosmotic flow in capillary electrophoresis for their zero electrophoretic mobility in an electric field. However, some of these markers can interact with background electrolyte components and migrate at a different velocity than the electroosmotic flow. Thus, we tested 11 markers primarily varying in their degree of methylation and type of central atom in combination with five background electrolyte cations differing in their ionic radii and surface charge density, measuring the relative electrophoretic mobility using thiourea as a reference marker. Our results from this set of experiments showed some general trends in the mobilization of the markers based on the effects of marker structure and type of background electrolyte cation on the relative electrophoretic mobility. As an example, the effects of an inadequate choice of marker on analyte identification were illustrated in the electrophoretic separation of glucosinolates. Therefore, our findings may help electrophoretists appropriately select electroosmotic flow markers for various electrophoretic systems.

Heparin-induced thrombocytopenia: a case report and literature overview.

Heparin-induced thrombocytopenia (HIT) is an immunologically-mediated complication, which usually follows heparin exposition, less frequently exposition to other drugs or even occurs spontaneously. The type of heparin, its dose and mode of application as well as the exposition time, major trauma or operation, and obesity represent the main risk factors for HIT. The probability of HIT correlates with so-called 4T-score. A confirmatory laboratory diagnostic should be exclusively reserved for patients with a medium to a high probability of HIT development (more than 3 points in 4T-score). The screening method is based on serological detection of antibodies against heparin-platelet factor-4 complexes; confirmation tests aim to identify the activation of platelets. The treatment of HIT requires an immediate interruption of heparin application and rigorous antithrombotic treatment with an alternative agent. Herein authors describe a clinical case of HIT manifested as an extreme urticarial reaction in the location of nadroparin application as well as thrombosis of deep subcutaneous veins in a polymorbid obese patient with an extensive and infected burn. Due to timely diagnosis and fondaparinux treatment, no more severe thrombotic events occurred in this patient.

Protonation of polyaniline-coated silica stationary phase affects the retention behavior of neutral hydrophobic solutes in reversed-phase capillary liquid chromatography.

Because of its high conductivity when acid doped, polyaniline is known as a synthetic metal and is used in a wide range of applications, such as supercapacitors, biosensors, electrochromic devices, or solar and fuel cells. Emeraldine is the partly oxidized, stable form of polyaniline, consisting of alternating diaminobenzenoid and iminoquinoid segments. When acidified, the nitrogen atoms of emeraldine become protonated. Due to electrostatic repulsion between positive charges, the polarity and morphology of emeraldine chains presumably change; however, the protonation effects on emeraldine have not yet been clarified. Thus, we investigated these changes by reversed-phase capillary liquid chromatography using a linear solvation energy relationship approach to assess differences in dominant retention interactions under a significantly varied mobile phase pH. We observed that hydrophobicity dominates the intermolecular interactions under both acidic and alkaline eluent conditions, albeit to different extents. Therefore, by tuning the mobile phase pH, we can even modulate the retention of neutral hydrophobic solutes, such as aromatic hydrocarbons, because the pH-dependent charge and structure of polymer chains of the emeraldine-coated silica stationary phase show a mixed-mode separation mechanism.

Online screening of α-amylase inhibitors by capillary electrophoresis.

Pancreatic α-amylase plays an important role in dietary starch hydrolysis in the small intestine and participates in enhanced glucose concentration after meals. It seems to be a problem for diabetic patients, who suffer from longer postprandial hyperglycemia after meal consumption than healthy people. There are commercially available drugs that inhibit α-amylase and thus reduce the postprandial hyperglycemia effect. However, these drugs may cause severe side effects. Conversely, some naturally occurring flavonoids were suggested to have an α-amylase-inhibiting effect without any side effects. There had been no rapid, undemanding method in terms of sample and reagent preparation that would enable screening of many potential inhibitors. Therefore, we developed an online capillary electrophoresis method to monitor α-amylase activity in the presence of an inhibitor. Each reaction constituent was introduced separately, directly into a capillary where the reagents were mixed by diffusion, which resulted in a 5-min analysis including conditioning of the capillary. We applied the method to test the inhibitory effect of flavonoid standards and their mixture and we investigated the inhibitory effect of ethanolic extract from Betula pendula bark. The developed method presents a faster and less expensive alternative to previously described offline methods. Graphical abstract Online CE screening of α-amylase inhibitors.

Binding of Divalent Cations to Insulin: Capillary Electrophoresis and Molecular Simulations.

In the present study, we characterize the binding of divalent cations to insulin in aqueous salt solutions by means of capillary electrophoresis and molecular dynamics simulations. The results show a strong pH dependence. At low pH, at which all the carboxylate groups are protonated and the protein has an overall positive charge, all the cations exhibit only weak and rather unspecific interactions with insulin. In contrast, at close to neutral pH, when all the carboxylate groups are deprotonated and negatively charged, the charge-neutralizing effect of magnesium, calcium, and zinc, in particular, on the electrophoretic mobility of insulin is significant. This is also reflected in the results of molecular dynamics simulations showing accumulation of cations at the protein surface, which becomes smaller in magnitude upon effective inclusion of electronic polarization via charge rescaling.

Quantification of paracetamol and 5-oxoproline in serum by capillary electrophoresis: Implication for clinical toxicology.

High anion gap metabolic acidosis frequently complicates acute paracetamol overdose and is generally attributed to lactic acidosis or compromised hepatic function. However, metabolic acidosis can also be caused by organic acid 5-oxoproline (pyroglutamic acid). Paracetamol's toxic intermediate, N-acetyl-p-benzoquinoneimine irreversibly binds to glutathione and its depletion leads to subsequent disruption of the gamma glutamyl cycle and an excessive 5-oxoproline generation. This is undoubtedly an underdiagnosed condition because measurement of serum 5-oxoproline level is not readily available. A simple, cost effective, and fast capillary electrophoresis method with diode array detection (DAD) for simultaneous measurement of both paracetamol (acetaminophen) and 5-oxoproline in serum was developed and validated. This method is highly suitable for clinical toxicology laboratory diagnostic, allowing rapid quantification of acidosis inducing organic acid 5-oxoproline present in cases of paracetamol overdose. The calibration dependence of the method was proved to be linear in the range of 1.3-250µgmL-1, with adequate accuracy (96.4-107.8%) and precision (12.3%). LOQ equaled 1.3µgmL-1 for paracetamol and 4.9µgmL-1 for 5-oxoproline.

Design of experiments for amino acid extraction from tobacco leaves and their subsequent determination by capillary zone electrophoresis.

In this study, we optimized a method for the determination of free amino acids in Nicotiana tabacum leaves. Capillary electrophoresis with contactless conductivity detector was used for the separation of 20 proteinogenic amino acids in acidic background electrolyte. Subsequently, the conditions of extraction with HCl were optimized for the highest extraction yield of the amino acids because sample treatment of plant materials brings some specific challenges. Central composite face-centered design with fractional factorial design was used in order to evaluate the significance of selected factors (HCl volume, HCl concentration, sonication, shaking) on the extraction process. In addition, the composite design helped us to find the optimal values for each factor using the response surface method. The limits of detection and limits of quantification for the 20 proteinogenic amino acids were found to be in the order of 10-5 and 10-4 mol l-1, respectively. Addition of acetonitrile to the sample was tested as a method commonly used to decrease limits of detection. Ambiguous results of this experiment pointed out some features of plant extract samples, which often required specific approaches. Suitability of the method for metabolomic studies was tested by analysis of a real sample, in which all amino acids, except for L-methionine and L-cysteine, were successfully detected. The optimized extraction process together with the capillary electrophoresis method can be used for the determination of proteinogenic amino acids in plant materials. The resulting inexpensive, simple, and robust method is well suited for various metabolomic studies in plants. As such, the method represents a valuable tool for research and practical application in the fields of biology, biochemistry, and agriculture.

Characterization of polyaniline-coated stationary phases by using the linear solvation energy relationship in the hydrophilic interaction liquid chromatography mode using capillary liquid chromatography.

A polyaniline coating was used to modify the surface of bare silica gel and octadecyl silica stationary phases to characterize the properties of altered materials. It was assumed that the mixed-mode retention was established on the basis of the polyaniline chemical structure and its combination with the original sorbents. Polyaniline was deposited onto the original surfaces during the chemical polymerization of aniline hydrochloride. The prepared materials were slurry packed into capillary columns and systematic chromatographic characterization was performed using the linear solvation energy relationship, also employing descriptors that allow inclusion of ionic interactions in the proposed retention mechanism. The retention times of 80 solutes with various chemical structures were measured in the hydrophilic interaction liquid chromatography mode. The obtained results demonstrated the significant contribution of the polyaniline coating to the retention mechanism under the given conditions; the assumed mixed-mode retention was confirmed. The dominant retention interaction for both modified stationary phases was based on the protonation of nitrogen atoms in the polyaniline structure, leading to suitable retention and selectivity for the hydrophilic analytes, especially anionic and zwitterionic species. Thus, especially, the polyaniline-coated bare silica gel sorbent seems to be promising for potential applications related to the separation of polar compounds.

Determination of dasatinib in the tablet dosage form by ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis.

Dasatinib is a novel oral prescription drug proposed for treating adult patients with chronic myeloid leukemia. Three analytical methods, namely ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis, were developed, validated, and compared for determination of the drug in the tablet dosage form. The total analysis time of optimized ultra high performance liquid chromatography and capillary zone electrophoresis methods was 2.0 and 2.2 min, respectively. Direct ultraviolet detection with detection wavelength of 322 nm was employed in both cases. The optimized sequential injection analysis method was based on spectrophotometric detection of dasatinib after a simple colorimetric reaction with folin ciocalteau reagent forming a blue-colored complex with an absorbance maximum at 745 nm. The total analysis time was 2.5 min. The ultra high performance liquid chromatography method provided the lowest detection and quantitation limits and the most precise and accurate results. All three newly developed methods were demonstrated to be specific, linear, sensitive, precise, and accurate, providing results satisfactorily meeting the requirements of the pharmaceutical industry, and can be employed for the routine determination of the active pharmaceutical ingredient in the tablet dosage form.

Gabapentin, Pregabalin and Vigabatrin Quantification in Human Serum by GC-MS After Hexyl Chloroformate Derivatization.

A simple, sensitive and robust method for simultaneous determination of antiepileptic drugs (gabapentin, pregabalin and vigabatrin) in human serum using GC-MS was developed and validated for clinical toxicology purposes. This method employs an emerging class of derivatization agents - alkyl chloroformates allowing the efficient and rapid derivatization of both the amino and carboxylic groups of the tested antiepileptic drugs within seconds. The derivatization protocol was optimized using the Design of Experiment statistical methodology, and the entire sample preparation requires less than 5 min. Linear calibration curves were obtained in the concentration range from 0.5 to 50.0 mg/L, with adequate accuracy (97.9-109.3%) and precision (<12.1%). The method was successfully applied to quantification of selected γ-aminobutyric acid analogs in the serum of patients in both therapeutic and toxic concentration ranges.

Supramolecular structures based on regioisomers of cinnamyl-α-cyclodextrins - new media for capillary separation techniques.

This work focuses on the preparation and application of supramolecular structures based on mono-cinnamyl-α-cyclodextrins (Cin-α-CD). Pure regioisomers of Cin-α-CD having the cinnamyl moiety at the 2-O- or at the 3-O-position, respectively, were prepared, characterized and applied in capillary electrophoresis as additives to the background electrolyte. These new monomer units with a potential to self-organize into supramolecular structures were synthesized via a straightforward one-step synthetic procedure and purified using preparative reversed-phase chromatography allowing a large scale separation of the regioisomers. The ability of the monomers to self-assemble was proved by various methods including NMR spectroscopy and dynamic light scattering (DLS). The light scattering experiments showed that the monomer units have distinguishable ability to form supramolecular structures in different solvents and the size distribution of the aggregates in water can be easily modulated using different external stimuli, such as temperature or competitive guest molecules. The obtained results indicated that the two regioisomers of Cin-α-CD formed different supramolecular assemblies highlighting the fact that the position of the cinnamyl group plays an important role in the intermolecular complex formation.

Kinetic analysis of decreased sperm fertilizing ability by fluorides and fluoroaluminates: a tool for analyzing the effect of environmental substances on biological events.

Fluorides and fluoroaluminates decrease mouse sperm fertilizing potential by modifying the process of sperm preparation for fertilization, so-called capacitation, followed by acrosome reaction (AR). Capacitation was monitored by protein tyrosine phosphorylation (pTyr), and AR was induced consequently. The aim of this study was to apply kinetic analysis to the previously obtained dependences of pTyr and AR at capacitation times, and propose a mathematical theory for a mechanism when sperm maturation ability is amended by external stimuli. The experimental input data, previously obtained, are consistent with the proposed theory and the results of kinetic analysis show that sperm capacitation runs as two subsequent first-order steps. Firstly, an unstable intermediate is formed and then gradually decomposes. The time corresponding to the maximal production of the unstable intermediate is probably most suitable for sperm obtaining the ability to fertilize the egg. The presented calculations indicate that the application of kinetic analysis can serve as a tool to predict or confirm a course of biological events that are modified by external factors, and therefore the proposed theory shall be of interest to a broad scientific audience.

Sample pretreatment for the capillary electrophoretic determination of organic acids in chromium(III) plating baths.

This work deals with the development and optimization of the sample pretreatment and consequent electrophoretic analysis of two modern plating baths containing chromium(III) and either citric acid or oxalic acid. Some model mixtures containing known amounts of components of industrial baths have been prepared to simulate simplified bath matrices. Prior to analysis, a sample pretreatment consisting of the addition of some agents that could release acid from the stable chromium complex was tested. Determination of organic anions was accomplished by indirect UV detection. The best results were achieved by precipitation of chromium(III) hydroxide. The content of oxalate and citrate in real samples was calculated as 96.5% (SD 2.3%) and 97.3% (SD 0.8%), respectively, of the declared amount. Very good robustness of the method and satisfactory repeatability of migration time and peak area were obtained. This simple inexpensive method is suitable for routine determination of citric and oxalic acid in chromium(III)-based plating baths.

Identification and quantification of acidosis inducing metabolites in cases of alcohols intoxication by GC-MS for emergency toxicology.

A simple, cost effective, and fast gas chromatography method with mass spectrometry detection (GC-MS) for simultaneous measurement of formic acid, glycolic acid, methoxyacetic acid, ethoxyacetic acid and 2-hydroxyethoxyacetic acid in serum and urine was developed and validated. This multi-analyte method is highly suitable for clinical and emergency toxicology laboratory diagnostic, allowing identification and quantification of five most common acidosis inducing organic acids present in cases of alcohol intoxication. Furthermore, when patients are admitted to emergency unit at late stage of toxic alcohol intoxication, the concentration of parent compound may be already low or not detectable. This new method employs a relatively less used class of derivatization agents - alkyl chloroformates, allowing the efficient and rapid derivatization of carboxylic acids within seconds. The entire sample preparation procedure is completed within 5 min. The optimal conditions of derivatization procedure have been found using chemometric approach (design of experiment). The calibration dependence of the method was proved to be quadratic in the range of 25-3000 mg L(-1), with adequate accuracy (97.3-108.0%) and precision (<12.8%). The method was successfully applied for identification and quantification of the selected compounds in serum of patients from emergency units.

A new, rapid, stability-indicating UPLC method for separation and determination of impurities in amlodipine besylate, valsartan and hydrochlorothiazide in their combined tablet dosage form.

A new rapid stability-indicating UPLC method for separation and determination of impurities in amlodipine besylate, valsartan and hydrochlorothiazide in their combined tablet dosage form was developed. The separation of Ph. Eur. related substances of amlodipine besylate (A, B, D, E, F, G), hydrochlorothiazide (A, B, C), valsartan (B, C), two other valsartan impurities (S)-2-(N-{[2'-cyanobiphenyl-4-yl]methyl}pentanamido)-3-methylbutanoic acid and (S)-3-methyl-2-{[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methylamino}butanoic acid and several unknown impurities was achieved by reversed phase liquid chromatography with UV detection. The detection wavelengths were set as follows: 225nm for valsartan, its impurities and for the impurity D of amlodipine, 271nm for hydrochlorothiazide and its impurities and 360nm for amlodipine and its impurities except for impurity D. Zorbax Eclipse C8 RRHD (100mm×3.0mm, 1.8µm) was used as a separation column and the analytes were eluted within 11min by a programmed gradient mixture of 0.01M phosphate buffer pH 2.5 and acetonitrile. The method was successfully validated in accordance to the International Conference of Harmonization (ICH) guidelines for amlodipine besylate and its impurity D, valsartan and its impurity C and hydrochlorothiazide and its impurities A, B and C. The triple-combined tablets were exposed to thermal, higher humidity, acid, alkaline, oxidative and photolytic stress conditions. Stressed samples were analyzed by the proposed method. All the significant degradation products and impurities were satisfactory separated from each other and from the principal peaks of drug substances. The peak purity test complied for peaks of amlodipine, valsartan and hydrochlorothiazide in all the stressed samples and indicated no co-elution of degradation products. The method was found to be precise, linear, accurate, sensitive, specific, robust and stability-indicating and could be used as a routine purity test method for amlodipine besylate, valsartan, hydrochlorothiazide and their pharmaceutical combinations.

Analysis of 1,2-diol diesters in vernix caseosa by high-performance liquid chromatography - atmospheric pressure chemical ionization mass spectrometry.

Fatty acid diesters of long-chain 1,2-diols (1,2-DDE), or type II wax diesters, were analyzed in the vernix caseosa of a newborn girl. 1,2-DDE were isolated from the total lipid extract by the semipreparative TLC using plates coated with silica gel. Chromatographic separation of the 1,2-DDE molecular species was achieved on the non-aqueous reversed-phase HPLC with two Nova-Pak C18 columns connected in series (a total length of 45cm) and using an acetonitrile-ethyl acetate gradient. 1,2-DDE eluted from the column in the order of their equivalent chain number. The analytes were detected as ammonium adducts by an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionization source. Their structures were elucidated using tandem mass spectrometry with MS, MS(2) and MS(3) steps in a data-dependent mode. More than two thousand molecular species of 1,2-DDE were identified in 141 chromatographic peaks. The most abundant 1,2-DDE were monounsaturated lipids consisting of a C22 diol and a C18:1 fatty acid together with C16:0, C14:0 or C15:0 fatty acids. The positions of double bonds were characterized by the fragmentation of [M+C3H5N](+) formed in the ion source.

Separation and determination of impurities in paracetamol, codeine and pitophenone in the presence of fenpiverinium in combined suppository dosage form.

A new HPLC method for separation and determination of impurities in paracetamol, codeine phosphate hemihydrate and pitophenone hydrochloride in the presence of fenpiverinium bromide in combined suppository dosage form was developed and validated. The separation of paracetamol and its impurities 4-aminophenol, 4-nitrophenol, 4-chloracetanilid; codeine and its impurities methylcodeine, morphine, codeine dimer and 10-hydroxycodeine; pitophenone and its impurities 2-[4-[2-(1-piperidinyl)ethoxy]benzoyl] benzoic acid, 2-[4-[2-(1-piperidinyl)ethoxy]benzoyl]benzoic acid 2-(1-piperidinyl)-ethyl ester, methyl ester of 2-(4-hydroxybenzoyl) benzoic acid and fenpiverinium was achieved by using ion-pair reversed phase liquid chromatography with UV detection. Validation parameters such as the precision, accuracy, linearity, limit of detection (LOD), limit of quantification (LOQ) and robustness were verified for all the mentioned impurities of codeine phosphate hemihydrate and 4-aminophenol and 2-[4-[2-(1-piperidinyl)ethoxy]benzoyl] benzoic acid as the main degradation products of paracetamol and pitophenone hydrochloride, respectively. The described method was found to be useful for analysis of the stability samples and therefore suitable for routine purity testing of the drug product.

Purification and enzymatic characterization of tobacco leaf ß-N-acetylhexosaminidase.

The kinetic properties of ß-N-acetylhexosaminidase purified from tobacco (Nicotiana tabacum L.) leaves have been investigated. In addition to chromogenic pNP derivates, N,N'-diacetylchitobiose and N,N',N″-triacetylchitotriose were also used as substrates of ß-N-acetylhexosaminidase. The highest reaction rate and the affinity for the substrate were observed for pNP-GlcNAc; however, an excess of this substrate inhibits the reaction. The reaction rate with pNP-GalNAc as the substrate was found to be about 85% of that obtained with pNP-GlcNAc. The hydrolysis of acetylated chitooligomers by ß-N-acetylhexosaminidase followed by separation and quantification using capillary electrophoresis was slower compared to pNP-GlcNAc. The pH optimum of ß-N-acetylhexosaminidase for individual substrates was found at 4.3-5.0 and the temperature optimum was 50-55 °C. Gel permeation chromatography and red native electrophoresis determined the relative molecular weight as 280 000 and the isoelectric point as 5.3. The inhibition of ß-N-acetylhexosaminidase by monosaccharides GlcN, GalN, GlcNAc, GalNAc in combination with substrates pNP-GlcNAc and pNP-GalNAc was studied and the type of inhibition and the inhibition constants were determined.

Newborn boys and girls differ in the lipid composition of vernix caseosa.

Vernix caseosa protects the skin of a human fetus during the last trimester of pregnancy and of a newborn after the delivery. Besides its cellular and proteinaceous components, an important constituent and functional agent is a complex lipid fraction, implicated in a multitude of salubrious effects of vernix caseosa. Little is known about how the chemical composition of vernix caseosa lipids is affected by various biological characteristics of the baby, such as the gestational age, birth weight, and, last but not least, the gender of the newborn. This study reports on the chemical variability of lipids contained in the vernix caseosa of twenty newborn girls and boys and shows that the quantitative patterns of the lipids are sex-specific. The specificity of lipids was investigated at the level of fatty acids in the total lipid extracts and intact lipids of several neutral lipid classes. Hydrocarbons, wax esters, cholesteryl esters, diol diesters and triacylglycerols were isolated using optimized semipreparative thin-layer chromatography, and the molecular species within each class were characterized using matrix-assisted laser desorption/ionization mass spectrometry. Statistical evaluation revealed significant quantitative sex-related differences in the lipid composition of vernix caseosa among the newborns, pronounced in the two lipid classes associated with the activity of sebaceous glands. Higher proportions of wax esters and triacylglycerols with longer hydrocarbon chains were observed in newborn girls.