Ubiquitous Selfish Toxin-Antidote Elements in Caenorhabditis Species.

Toxin-antidote elements (TAs) are selfish genetic dyads that spread in populations by selectively killing non-carriers. TAs are common in prokaryotes, but very few examples are known in animals. Here, we report the discovery of maternal-effect TAs in both C. tropicalis and C. briggsae, two distant relatives of C. elegans. In C. tropicalis, multiple TAs combine to cause a striking degree of intraspecific incompatibility: five elements reduce the fitness of >70% of the F2 hybrid progeny of two Caribbean isolates. We identified the genes underlying one of the novel TAs, slow-1/grow-1, and found that its toxin, slow-1, is homologous to nuclear hormone receptors. Remarkably, although previously known TAs act during embryonic development, maternal loading of slow-1 in oocytes specifically slows down larval development, delaying the onset of reproduction by several days. Finally, we found that balancing selection acting on linked, conflicting TAs hampers their ability to spread in populations, leading to more stable genetic incompatibilities. Our findings indicate that TAs are widespread in Caenorhabditis species and target a wide range of developmental processes and that antagonism between them may cause lasting incompatibilities in natural populations. We expect that similar phenomena exist in other animal species.

Antioxidant studies of citric acid and citrus fruits towards paraquat by cyclic voltammetry: An antidote of paraquat poisoning.

Voltammetric parameters are studied to confirm the antioxidant activity of citric acid towards reduced form of methyl viologen dication (1, 1'-dimethyl-4, 4'-bipyridinium, MV 2+, also known as paraquat). In this study the kinetics of the reaction of citric acid with reduced form of methyl viologen is also reported. Out of two oxidative peaks (i.e. MVo to MV+˖ and MV+˖ to MV+2) the first peak is almost removed after interaction with citric acid. Shifting in second cathodic peak potential is also obvious and possibility of citric acid to scavenge or making adduct with paraquat is identified. Some real samples (fruit juices) as a rich source of citric acid are also studied. This study presents a simple, relevant and fast voltammetric method by which quick quantitation and monitoring of antioxidant/ scavenging activity of food, herbs and other spices towards paraquat poisoning is possible. It may constitute a new wave of treatment or therapy for the disease caused by paraquat.

Analysis of potential cyanide antidote, dimethyl trisulfide, in whole blood by dynamic headspace gas chromatography-mass spectroscopy.

Cyanide is a rapidly acting and highly toxic chemical. It inhibits cytochrome c oxidase in the mitochondrial electron transport chain, resulting in cellular hypoxia, cytotoxic anoxia and potentially death. In order to overcome challenges associated with current cyanide antidotes, dimethyl trisulfide (DMTS), which converts cyanide to less toxic thiocyanate in vivo, has gained much attention recently as a promising next-generation cyanide antidote. While there are three analysis methods available for DMTS, they each have significant disadvantages. Hence, in this study, a dynamic headspace (DHS) gas chromatography-mass spectroscopy method was developed for the analysis of DMTS from rabbit whole blood. The method is extremely simple, involving only acidification of a blood sample, addition of an internal standard (DMTS-d6) and DHS-GC-MS analysis. The method produced a limit of detection of 0.04 µM for DMTS with dynamic range from 0.2 to 50 µM. Inter- and intraassay accuracy (100 ± 15% and 100 ± 9%, respectively), and precision (<10% and <9% relative standard deviation, respectively) were good. The validated method performed well during pharmacokinetic analysis of DMTS from the blood of rats treated with DMTS, producing excellent pharmacokinetic parameters for the treatment of cyanide exposure. The method produced significant advantages over current methods for analysis of DMTS and should be considered as a "gold standard" method for further development of DMTS as a potential next-generation cyanide countermeasure.

Fluorescent MUA-stabilized Au nanoclusters for sensitive and selective detection of penicillamine.

In this study, we have developed a facile method for preparation of highly fluorescent Au nanoclusters (AuNCs) using 11-mercaptoundecanoic acid (MUA) as both the reducing and stabilizing agent. The as-prepared MUA functionalized AuNCs (MUA-AuNCs) have good water solubility, excellent photostability, and strong fluorescence emission at 610 nm with a quantum yield of 7.28% in water. The fluorescence of MUA-AuNCs was first quenched by copper ions through electron transfer, subsequently caused obvious restoration by competitive effect after adding penicillamine, making it a potential fluorescent sensor for penicillamine with a detection limit of 0.08 µM. Furthermore, the newly designed fluorescence "on-off-on" assay was explored for the measurement of penicillamine in complex real water and urine samples with satisfactory results.

Chemical stability of reactive skin decontamination lotion (RSDL®).

Reactive Skin Decontamination Lotion (RSDL®) is used for the decontamination of Chemical Warfare Agents and Toxic Industrial Compounds after dermal exposure. It has to be stockpiled over a long period and is handled in all climatic zones. Therefore stability is an essential matter of concern. In this work we describe a study to the chemical stability of RSDL® as basis for an estimation of shelf life. We analysed RSDL® for the active ingredient 2,3-butandione monoxime (diacetylmonooxime, DAM), the putative degradation product dimethylglyoxime (DMG) and unknown degradation products by means of a reversed phase high pressure liquid chromatography (HPLC). Calculations were done according to the Arrhenius equation. Based on the temperature dependent rate constants, the time span was calculated, until defined threshold values for DAM and DMG subject to specification and valid regulations were exceeded. The calculated data were compared to the ones gathered from stockpiled samples and samples exposed during foreign mission. The decline of DAM followed first order kinetics, while formation of DMG could be described by zero order kinetics. The rate constants were distinctively temperature dependent. Calculated data were in good accordance to the measured ones from stockpile and mission. Based on a specified acceptable DAM-content of 90% and a valid threshold value of 0.1% (w/w) for the degradation product DMG, RSDL® proved to be stable for at least four years if stored at the recommended conditions of 15°C-30°C. If continuously stored at higher temperatures shelf life will decrease markedly. Therefore RSDL® is an object for risk orientated quality monitoring during storage.

Recognition of D-Penicillamine Using Schiff Base Centered Fluorescent Organic Nanoparticles and Application to Medicine Analysis.

Schiff base centered fluorescent organic compound 1,1'-[(1E,2E)-hydrazine-1,2-diylidenedi(E)methylylidene]- dinaphthalen-2-ol (HN) was synthesized followed by spectral characterization viz., NMR, IR and Mass spectroscopy. The fluorescent nanoparticles of HN prepared using reprecipitation method shows red shifted aggregation induced enhanced emission (AIEE) with respect to HN solution in acetone. The average particle size of nanoparticles (HNNPs) is of 67.2 nm shows sphere shape morphology. The surfactant cetyltrimethyl ammonium bromide (CTAB) used to stabilize HNNPs induces positive charge surface with zeta potential of 11.6 mV. The positive charge of HNNPs responsible to adsorb oppositely charged analyte on its surface with binding interactions. The fluorescence experiments performed with and without addition of different analytes to the aqueous suspension of HNNPs shows selective fluorescence quenching of HNNPs by D-Penicillamine (D-PA). The effect of other coexisting analytes does not affect the selective sensing behavior of D-PA. The mechanism of binding between HNNPs and D-PA was discussed on the basis of electrostatic interaction and adsorption phenomenon. The results interpreted by using DLS-Zeta sizer, Fluorescence lifetime measurements, conductometric titration supports the electrostatic adsorption between HNNPs and D-PA. The method has extremely low limit of detection (LOD) value 0.021 ppm is of significant as compared to reported methods. The proposed fluorescence quenching method was effectively used for quantitative estimation of D-PA from pharmaceutical medicine. Graphical Abstract The fluorescence quenching based selective recognition of D-Penicillamine (D-PA) by using Schiff base centered fluorescent organic nanoparticles was developed and successfully applied to quantitative determination of D-PA from pharmaceutical samples viz. capsule and tablet.

Inhibición de los efectos coagulante, fosfolipasa A2 y proteolítico del veneno de Bothrops asper por plantas usadas tradicionalmente en Centroamérica / Inhibition of the coagulant, phospholipase A2 and proteolytic effects of Bothrops asper venom by plants traditionally used in Central America

Existen pocos estudios científicos que demuestren el valor terapéutico de las plantas usadas en la medicina tradicional centroamericana para tratar el envenenamiento ofídico. En este estudio se evaluó la capacidad de los extractos etanólicos de nueve plantas de uso etnomédico en Centroamérica (Acacia hindsii, Aristolochia maxima, Bursera simaruba, Cissampelos pareira, Eryngium foetidum, Hamelia patens, Pimenta dioica, Piper peltatum y Sansevieria hyacinthoides) para inhibir el efecto coagulante del veneno de Bothrops asper. Tres de ellas (B. simaruba, E. foetidum y P. dioica) también fueron evaluadas en cuanto a su capacidad inhibitoria de los efectos fosfolipasa A2 (PLA2) y proteolítico del veneno. Las plantas fueron colectadas en Guatemala, secadas, extraídas con etanol y los efectos inhibitorios evaluados in vitro después de preincubar concentraciones variables de extracto con concentraciones fijas de veneno. Los resultados demostraron que ninguno de los extractos logró inhibir los efectos coagulante y PLA2, pero los extractos clorofilados de P. dioica y E. foetidum inhibieron efectivamente la actividad proteolítica del veneno. El tamizaje fitoquímico, mediante ensayos macro y semimicrométricos de cromatografía en capa fina, demostró la presencia de metabolitos secundarios reportados con actividad antiproteolítica (flavonoides, antocianinas, catequinas y taninos) en la composición química de los extractos de E. foetidum y P. dioica. Su efecto sobre el veneno se evaluó mediante electroforesis SDS-PAGE, demostrándose que no está mediado por degradación proteolítica de los componentes del veneno. El aislamiento y caracterización específica de sus metabolitos secundarios en futuros estudios, permitirá determinar el mecanismo de acción inhibitoria ejercido por estos extractos.

Medicinal plants have been traditionally used in Central America to treat snakebite envenomations, however, very few scientific studies aimed to demonstrate their efficacy and safety have been performed. In this study, ethanolic extracts of nine plants used in the region by traditional healers in snakebite cases (Acacia hindsii, Aristolochia maxima, Bursera simaruba, Cissampelos pareira, Eryngium foetidum, Hamelia patens, Pimenta dioica, Piper peltatum and Sansevieria hyacinthoides) were evaluated for their ability to inhibit the coagulant effect induced by the venom of the snake Bothrops asper. Three of these extracts (B. simaruba, E. foetidum and P. dioica) were also evaluated for their inhibitory effect on the phospholipase A2 (PLA2) and proteolytic activities of the venom. Plants were collected in Guatemala, dried, extracted with ethanol, and their inhibitory effects were evaluated in vitro after pre-incubation of several amounts of each extract with a challenge concentration of venom. Results showed that none of the extracts inhibited the coagulant and PLA2 effects; however, chlorophyllated extracts of E. foetidum and P. dioica effectively inhibited the proteolytic activity of the venom. Phytochemical analysis of these extracts, conducted by macrometric assays and semimicroanalysis by thin layer chromatography, identified secondary metabolites (flavones, anthocyanins, catequines and tannins) whose anti-proteolytic activities have been widely reported. SDS-PAGE analysis demonstrated that the mechanism of inhibition is not related to proteolytic degradation of the venom proteins by the plant extracts. Further studies are needed to isolate and identify the active venom inhibitory compounds of these plants, aimed to understand their mechanism of action.

Inactivated antithrombins as fondaparinux antidotes: a promising alternative to haemostatic agents as assessed in vitro in a thrombin-generation assay.

In the absence of specific antidote to fondaparinux, two modified forms of antithrombin (AT), one recombinant inactive (ri-AT) and the other chemically inactivated (chi-AT), were designed to antagonise AT-mediated anticoagulants, e. g. heparins or fondaparinux. These inactive ATs were previously proven to effectively neutralise anticoagulant activity associated with heparin derivatives in vitro and in vivo, as assessed by direct measurement of anti-FXa activity. This study was undertaken to evaluate in vitro the effectivity of inactive ATs to reverse anticoagulation by heparin derivatives and to compare them with non-specific fondaparinux reversal agents, like recombinant-activated factor VII (rFVIIa) or activated prothrombin-complex concentrate (aPCC), in a thrombin-generation assay (TGA). Addition of fondaparinux (3 µg/ml) to normal plasma inhibited thrombin generation by prolonging lag time (LT) as much as 244 % and lowering endogenous thrombin potential (ETP) to 17 % of their control (normal plasma) values. Fondaparinux-anticoagulant activity was reversed by ri-AT and chi-AT, as reflected by the corrections of LT up to 117 % and 114 % of its control value, and ETP recovery to 78 % and 63 %, respectively. Unlike ri-AT that had no effect on thrombin generation in normal plasma, chi-AT retained anticoagulant activity that minimises its reversal capacity. However, both ATs were more effective than rFVIIa or aPCC at neutralising fondaparinux and, unlike non-specific antidotes, inactive ATs specifically reversed AT-mediated anticoagulant activities, as suggested by their absence of procoagulant activity in anticoagulant-free plasma.

Antídotos de los nuevos anticoagulantes orales: realidad y expectativas / Antidotes for the new oral anticoagulants: reality and expectations

No disponible

Determination of flumazenil in serum by liquid chromatography-mass spectrometry: Application to kinetics study in acute diazepam overdose.

BACKGOUND/AIM: Flumazenil is benzodiazepine receptor antagonist. It has been studied for a various indications, including reversal of sedation after surgery or diagnostic procedures, awakening of comatose patients in benzodiazepine overdose, or for symptomatic treatment of hepatic encephalopathy. Some drugs, like theophylline, may prolong its elimination half-life. Considering the long half-life of diazepam and its metabolites, concomitant use of theophylline may reduce the need for repeated dosing of flumazenil in patients with acute diazepam poisoning. The aim of this study was to introduce a reliable and accurate method for determining the concentration of flumazenil after therapeutic application in patients with acute poisoning, and using that method to assess whether the kinetics of flumazenil change in the presence of aminophylline (combination of theophylline and ethylenediamine in a 2:1 ratio) applied as concomitant therapy. METHODS: Blood samples from patients with acute diazepam poisoning that received flumazenil at the dose of 0.5 mg, or the same dose with 3 mg/kg of body weight of aminophylline, were collected 1, 3, 10, 30, 60, 120 and 240 min after its intravenous administration. Samples were prepared by solid-phase extraction on Oasis HLB cartridges with ethylacetate as extracting agens. Flumazenil was determined by liquid chromatography with mass spectrometry (LC-MS) in single ionmonitoring mode at m/z 304. Separation of flumazenil from matrix compound was performed on Lichrospher RP-8 column usingthe mixture of acidic acetonitrile and 20 mM of ammonium acetatein water (55 : 45) as a mobile phase. RESULTS: The applied analitycal method showed excellent recovery (94.65%). The obtained extracts were much cleaner than the extracts obtained by the sameextractant in the process of liquid-liquid extraction. The limit ofdetection of the LC-MS method described in this paper was 0.5 ng/mL and the limit of quantitation was 1 ng/mL. In the patientstreated with both flumazenil and aminophylline, the eliminationconstant for flumazenil was significantly lower and the elimination half-life was longer (p < 0.05) in comparison with the same parameters in.the patients who received flumazenil alone. CONCLUSION: The applied LC-MS method for the determination of flumazenil in serum samples of patients with acute diazepam poisoning is rapid, sensitive, precise and specific. Concomitant use with theophylline significantly prolonged elimination of flumazenil during the treatment of acute poisonings with diazepam.

Reversed-phase ion-pair chromatography-diode array detection of the bispyridinium compound MB327: plasma analysis of a potential novel antidote for the treatment of organophosphorus poisoning.

In the case of poisoning by organophosphorus nerve agents or pesticides, there is still a lack of pharmacological treatment of the cholinergic crisis selectively targeting the nicotinic acetylcholine receptor. Recently, the compound MB327 was identified as a potential novel lead structure to close this gap, thus demanding a quantitative assay for initial pharmacokinetic (PK) studies. MB327 is a salt consisting of the dicationic bispyridinium compound (BPC) 1,1´-(propane-1,3-diyl)bis(4-tert-butylpyridinium) and two iodide counter ions. Due to the permanent positive charge of the BPC, an isocratic reversed-phase ion-pair chromatographic separation (RPIPC) was developed using heptanesulfonic acid as ion-pairing reagent and 45% v/v methanol as organic modifier (1 mL/min). Selective UV-detection (230 nm) was done by a diode array detector (DAD) for reliable, rugged, precise (RSD < 7%) and accurate (96-104%) quantitative analysis of 50 µL swine plasma (linear range 1-1000 µg BPC/mL plasma, lower limit of quantification 2 µg/mL). During method validation, diverse parameters essential for the chromatographic process were investigated to generate van´t Hoff, van Deemter and width plots allowing calculation of thermodynamic data like the distribution constant K (5.7 ± 0.3), change in enthalpy, ΔH(0) : -23.66 kJ/mol, and entropy, ΔS(0) : -65 J/(mol*K). In addition, RPIPC-DAD analysis enabled calculation of molar absorptivities of the BPC, ε230 : 17 400 ± 1100 L/(mol*cm), and iodide, ε230 : 9900 ± 400 L/(mol*cm), which determination was hampered by interference with each other in conventional cuvette UV-spectrophotometric measurements. Finally, the RPIPC-DAD procedure was applied to samples from an in vivo study of swine.

Uso de antídotos en un servicio de urgencias pediátricas / Antidote use in a pediatric emergency department

INTRODUCCIÓN: La intoxicación es un motivo de consulta poco frecuente en un servicio de urgencias pediátricas (SUP) pero potencialmente grave. Conviene que el pediatra conozca el uso adecuado de los antídotos disponibles. OBJETIVOS: Analizar el uso de antídotos en un SUP y evaluar la idoneidad de su indicación. MATERIALES Y MÉTODOS: Estudio retrospectivo de los pacientes que consultaron, entre enero del 2008 y junio del 2012, por sospecha de intoxicación por una sustancia para la cual existe antídoto. La evaluación de la idoneidad de la indicación del antídoto se basó en las recomendaciones de la Sociedad Española de Urgencias de Pediatría. RESULTADOS: Se recogieron 1.728 consultas por sospecha de intoxicación (0,4% de las visitas). En 353 (20,4%) el tóxico implicado podía ser tratado con un antídoto. Recibieron antídoto 67 pacientes (3,9% de las consultas por sospecha de intoxicación) y se realizaron en total 69 administraciones de antídoto: oxígeno 100% (46), N-acetilcisteína (10), flumazenilo (4), naloxona (3), desferroxamina (2), vitamina K (2), bicarbonato (1) y carnitina (1). En 3 casos no existía indicación del antídoto: flumazenilo sin depresión respiratoria (2) y vitamina K tras exposición a cumarínico (1). Como efecto secundario se objetivó agitación psicomotriz tras uso de flumazenilo y disminución del tiempo de protrombina durante la infusión de N-acetilcisteína. CONCLUSIONES: La administración de antídotos en este SUP es infrecuente, mayoritariamente acorde a las recomendaciones y sin efectos secundarios importantes. Debe insistirse en la necesidad de limitar el uso de flumazenilo a los casos claramente indicados, y comprobando siempre la ausencia de contraindicaciones

INTRODUCTION: Poisoning is an infrequent cause of consultation in a pediatric emergency department (PED), but it can be potentially serious. Pediatricians should know how to use the available antidotes properly. OBJECTIVES: To analyze the use of antidotes in a PED and to assess the suitability of their indications. MATERIALS AND METHODS: A retrospective review of antidote use in a PED between January 2008 and June 2012. Inclusion criteria were age younger than 18 years and consultation for suspicious poisoning by a substance that could be treated with an antidote. The adequacy of antidote indication was based on the recommendations of the Spanish Society of Pediatric Emergencies (SSPE). RESULTS: A total of 1728 consultations for suspicious poisoning (0.4% of the total visits in the PED) were recorded. In 353 cases (20.4%) the involved poison could be treated with an antidote. Sixty-seven patients received an antidote (3.9% of consultations for suspicious poisoning), and a total of 69 administrations of an antidote were made: 100% oxygen (46), N-acetylcysteine (10), flumazenil (4), naloxone (3), deferoxamine (2), vitamin K (2), bicarbonate (1), and carnitine (1). In 3 cases there was no indication for administration: flumazenil without respiratory depression, and vitamin K following coumarin exposure. As side effects, agitation was noted after the use of flumazenil, and a decrease in the prothrombin time during infusion of N-acetylcysteine. CONCLUSIONS: The administration of antidotes in this PED is uncommon and, mainly, in accordance with the SSPE recommendations, and without serious side effects. The use of flumazenil needs to be limited to the cases with a clear indication and without any contraindication

Antidote removal during haemodialysis for massive acetaminophen overdose.

CONTEXT: Haemodialysis is sometimes used for patients with massive acetaminophen overdose when signs of "mitochondrial paralysis" (lactic acidosis, altered mental status, hypothermia and hyperglycaemia) are present. The role of haemodialysis is debated, in part because the evidence base is weak and the endogenous clearance of acetaminophen is high. There is also concern because the antidote acetylcysteine is also dialyzable. We prospectively measured serum acetylcysteine concentrations during haemodialysis in three such cases. CASE DETAILS: Three adults each presented comatose and acidemic 10 to ~18 h after ingesting > 1000mg/kg of acetaminophen. Two were hypothermic and hyperglycaemic. Serum lactate concentrations ranged from 7 mM to 12.5 mM. All three were intubated, and initial acetaminophen concentrations were as high as 5980 µM (900 µg/mL). An intravenous loading dose of 150 mg/kg acetylcysteine was initiated between 10.8 and ~18 h post ingestion, and additional doses were empirically administered during haemodialysis to compensate for possible antidote removal. A single run of 3-4 h of haemodialysis removed 10-20 g of acetaminophen (48-80% of remaining body burden), reduced serum acetaminophen concentrations by 56-84% (total clearance 3.4-7.8 mL/kg/min), accelerated native acetaminophen clearance (mean elimination half-life 580 min pre-dialysis, 120 min during and 340 min post-dialysis) and corrected acidemia. Extraction ratios of acetylcysteine across the dialysis circuit ranged from 73% to 87% (dialysance 3.0 to 5.3 mL/kg/min). All three patients recovered fully, and none developed coagulopathy or other signs of liver failure. DISCUSSION: When massive acetaminophen ingestion is accompanied by coma and lactic acidosis, emergency haemodialysis can result in rapid biochemical improvement. As expected, haemodialysis more than doubles the clearance of both acetaminophen and acetylcysteine. Because acetylcysteine dosing is largely empirical, we recommend doubling the dose during haemodialysis, with an additional half-load when dialysis exceeds 6 h.

An antidote for imazalil-induced genotoxicity in vitro: the lichen, Dermatocarpon intestiniforme (Körber) Hasse.

Imazalil (IMA), a commonly used fungicide in both agricultural and clinical domains, is suspected to produce serious toxic effects in vertebrates. In recent years, a number of studies have suggested that lichens might be easily accessible sources of natural drugs that could be used as a possible food supplement. Extensive research is being performed to explore the importance of lichen species, which are known to contain a variety of pharmacological active compounds. In this context, the antigenotoxic effect of aqueous Dermatocarpon intestiniforme (Körber) Hasse. extract (DIE) was studied against the genotoxic damage induced by IMA on cultured human lymphocytes (n = 6) using chromosomal aberration (CA) and micronucleus (MN) as cytogenetic endpoints. Human peripheral lymphocytes were treated in vitro with varying concentrations of DIE (0, 25, 50 and 100 µg/ml), tested in combination with IMA (336 µg/ml). DIE alone were not genotoxic and when combined with IMA treatment, it reduced the frequency of CAs and the rate of MNs. A clear dose-dependent decrease in the genotoxic damage of IMA was observed, suggesting a genoprotective role of DIE. The results of the present study suggest that this plant extract per se does not have a genotoxic potential, but can alleviate the genotoxicity of IMA on cultured human lymphocytes. In conclusion our findings may have an important application for the protection of cultured human lymphocyte from the genetic damage and side effects induced by medical and agricultural chemicals hazardous for people.

Rapid resolution liquid chromatography for monitoring the quality of stockpiled atropine preparations for injection.

We describe a rapid resolution liquid chromatography (RRLC) method for analyzing atropine sulfate, its degradation products (tropic acid, apoatropine, atropic acid) and other components (e.g. phenol, methylparaben) in injectable medicines that are used by the German armed forces in emergency situations. Chromatography is performed using an acetonitrile/phosphate buffer gradient (pH = 1.0) and an RP 18 column (50 x 4.6 mm, 1.8 µm) with the detection wavelength set at 220 nm. The concentration of the active ingredient (atropine sulfate) in the tested products ranges from about 1 mg•ml(-1) to 10 mg•ml(-1) . The concentrations of the detected degradation products range from 0.2% to 4.7% (tropic acid) in relation to the active pharmaceutical ingredient (API). Using shorter separation columns and smaller particle sizes of the stationary phase improved analysis time from 40 to 10 min and reduced the consumption of solvents by approximately 75%. Owing to the pressure conditions (< 200 bar), UHPLC (ultra high performance liquid chromatography) systems are not needed. Comparison of the atropine and tropic acid results obtained with the previously used HPLC (high performance liquid chromatography) method of the MAH (marketing authorization holder) show that there is no indication of a significant difference between the two methods.

Quantification of pralidoxime (2-PAM) in urine by ion pair chromatography-diode array detection: application to in vivo samples from minipig.

Pralidoxime (2-PAM) is a monopyridinium oxime used as an antidote for the treatment of poisoning with organophosphorus (OP) compounds, for example, pesticides and nerve agents, reactivating OP-inhibited acetylcholinesterase. However, appropriate dosing and efficacy remains a matter of discussion requiring experimental data. Therefore, we developed and validated an ion pair chromatography-diode array detection (IPC-DAD) method suitable for quantitative analysis of 2-PAM in human and porcine urine. Before injection of 20 µl, urine was acidified with trichloroacetic acid, mixed with internal standard (pyridine-4-aldoxime, 4-PAO), and diluted with IPC solvent yielding a total dilution of 1:49.5 and a 100% recovery. Isocratic separation was carried out at 25 °C on a LiChrospher 60 RP-select B column (125 x 4.0 mm I.D.) using phosphate buffer (7.5 mM Na(2) HPO(4) , 7.5 mM KH(2) PO(4) , pH 2.6) mixed with octanesulfonate (2.5 mM) as ion pair reagent and acetonitrile (6% v/v) as organic modifier (1 ml/min). 2-PAM was detected at 293 nm and 4-PAO at 275 nm. The method is rugged, selective, and characterized by good intra-day and inter-day precision (RSD, 1.3-6.0%) and accuracy (88-100%) with a limit of detection at 4.9 µg/ml, a limit of quantification at 9.8 µg/ml, and a broad calibration range from 4.9-2500 µg/ml. The procedure was applied to urine samples obtained from dimethoate poisoned minipigs receiving 2-PAM therapy (intravenous bolus injection and infusion). Results indicate that 60-80% of infused 2-PAM is rapidly (within 1-2 h) excreted in the urine.

Review of UV spectroscopic, chromatographic, and electrophoretic methods for the cholinesterase reactivating antidote pralidoxime (2-PAM).

Pralidoxime (2-PAM) belongs to the class of monopyridinium oximes with reactivating potency on cholinesterases inhibited by phosphylating organophosphorus compounds (OPC), for example, pesticides and nerve agents. 2-PAM represents an established antidote for the therapy of anticholinesterase poisoning since the late 1950s. Quite high therapeutic concentrations in human plasma (about 13 µg/ml) lead to concentrations in urine being about 100 times higher allowing the use of less sensitive analytical techniques that were used especially in the early years after 2-PAM was introduced. In this time (mid-1950s until the end of the 1970s) 2-PAM was most often analyzed by either paper chromatography or simple UV spectroscopic techniques omitting any sample separation step. These methods were displaced completely after the establishment of column liquid chromatography in the early 1980s. Since then, diverse techniques including cation exchange, size-exclusion, reversed-phase, and ligand-exchange chromatography have been introduced. Today, the most popular method for 2-PAM quantification is ion pair chromatography often combined with UV detection representing more than 50% of all column chromatographic procedures published. Furthermore, electrophoretic approaches by paper and capillary zone electrophoresis have been successfully used but are seldom applied. This review provides a commentary and exhaustive summary of analytical techniques applied to detect 2-PAM in pharmaceutical formulations and biological samples to characterize stability and pharmacokinetics as well as decomposition and biotransformation products. Separation techniques as well as diverse detectors are discussed in appropriate detail allowing comparison of individual preferences and limitations. In addition, novel data on mass spectrometric fragmentation of 2-PAM are provided.

FT-IR and XRD analysis of natural Na-bentonite and Cu(II)-loaded Na-bentonite.

Na-bentonite has been studied extensively because of its strong adsorption capacity and complexation ability. In this work, surface area, total pore volume, mean pore diameter, TG, DTA, FT-IR and XRD were carried out in order to reveal the characteristics of natural Na-bentonite. XRD and FT-IR of natural Na-bentonite (China) and Cu-loaded Na-bentonite as a function of Na-bentonite dosage and temperature using batch technique were characterized in detail, respectively.