A novel high-performance liquid chromatography with diode array detector method for the simultaneous quantification of the enzyme-reactivating oximes obidoxime, pralidoxime, and HI-6 in human plasma.

Oximes such as pralidoxime (2-PAM), obidoxime (Obi), and HI-6 are the only currently available therapeutic agents to reactivate inhibited acetylcholinesterase (AChE) in case of intoxications with organophosphorus (OP) compounds. However, each oxime has characteristic agent-dependent reactivating efficacy, and therefore the combined administration of complementary oximes might be a promising approach to improve therapy. Accordingly, a new high-performance liquid chromatography method with diode-array detection (HPLC-DAD) was developed and validated allowing for simultaneous or single quantification of 2-PAM, Obi, and HI-6 in human plasma. Plasma was precipitated using 5% w/v aqueous zinc sulfate solution and subsequently acetonitrile yielding high recoveries of 94.2%-101.0%. An Atlantis T3 column (150 × 2.1mm I.D., 3 µm) was used for chromatographic separation with a total run time of 15 min. Quantification was possible without interferences within a linear range from 0.12 to 120 µg/mL for all oximes. Excellent intra-day (accuracy 91.7%-98.6%, precision 0.5%-4.4%) and inter-day characteristics (accuracy 89.4%-97.4%, precision 0.4%-2.2%) as well as good ruggedness were found. Oximes in processed samples were stable for at least 12 h in the autosampler at 15°C as well as in human plasma for at least four freeze-thaw cycles. Finally, the method was applied to plasma samples of a clinical case of pesticide poisoning.

Development and validation of a FIA/UV-vis method for pK(a) determination of oxime based acetylcholinesterase reactivators.

Acetylcholinesterase reactivators (oximes) are compounds used for antidotal treatment in case of organophosphorus poisoning. The dissociation constants (pK(a1)) of ten standard or promising acetylcholinesterase reactivators were determined by ultraviolet absorption spectrometry. Two methods of spectra measurement (UV-vis spectrometry, FIA/UV-vis) were applied and compared. The soft and hard models for calculation of pK(a1) values were performed. The pK(a1) values were recommended in the range 7.00-8.35, where at least 10% of oximate anion is available for organophosphate reactivation. All tested oximes were found to have pK(a1) in this range. The FIA/UV-vis method provided rapid sample throughput, low sample consumption, high sensitivity and precision compared to standard UV-vis method. The hard calculation model was proposed as more accurate for pK(a1) calculation.

Deer bone extract prevents against scopolamine-induced memory impairment in mice.

Deer bone has been used as a health-enhancing food as well as an antiaging agent in traditional Oriental medicine. Recently, the water extract of deer bone (DBE) showed a neuroprotective action against glutamate or Aß1-42-induced cell death of mouse hippocampal cells by exerting antioxidant activity through the suppression of MAP kinases. The present study is to examine whether DBE improves memory impairment induced by scopolamine. DBE (50, 100 or 200 mg/kg) was administered orally to mice for 14 days, and then scopolamine (2 mg/kg, i.p.) was administered together with DBE for another 7 days. Memory performance was evaluated in the Morris water maze (MWM) test and passive avoidance test. Also, brain acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activity, biomarkers of oxidative stress and the loss of neuronal cells in the hippocampus, was evaluated by histological examinations. Administration of DBE significantly restored memory impairments induced by scopolamine in the MWM test (escape latency and number of crossing platform area), and in the passive avoidance test. Treatment with DBE inhibited the AChE activity and increased the ChAT activity in the brain of memory-impaired mice induced by scopolamine. Additionally, the administration of DBE significantly prevented the increase of lipid peroxidation and the decrease of glutathione level in the brain of mice treated with scopolamine. Also, the DBE treatment restored the activities of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and glutathione reductase to control the level. Furthermore, scopolamine-induced oxidative damage of neurons in hippocampal CA1 and CA3 regions were prevented by DBE treatment. It is suggested that DBE may be useful for memory improvement through the regulation of cholinergic marker enzyme activities and the suppression of oxidative damage of neurons in the brain of mice treated with scopolamine.

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.

Development of an enzyme linked immunosorbent assay and an immunochromatographic assay for detection of organophosphorus pesticides in different agricultural products.

OBJECTIVE: Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. METHODS: A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. RESULTS: Under the optimized conditions, the IC(50) values of the ELISA ranged from 3.7 to 162.2 ng mL(-1) for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC(50) values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL(-1). The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. CONCLUSIONS: The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.

Conformational and configurational analysis of an N,N carbonyl dipyrrinone-derived oximate and nitrone by NMR and quantum chemical calculations.

The geometries and relative energies of new N,N carbonyl dipyrrinone-derived oxime molecules (E/Z-s-cis 4a and E/Z-s-cis 4b) have been investigated. The calculated energies, molecular geometries, and (1) H/(13) C NMR chemical shifts agree with experimental data, and the results are presented herein. The E-s-cis conformations of 4a and 4b and the Z-s-cis conformation of 5b were found to be the thermodynamically most stable isomers with the oxime hydrogen atom or the methyl functional group adopting an anti-orientation with respect to the dipyrrinone group. This conformation was unambiguously supported by a number of 2D NMR experiments.

Hydrophobicity/hydrophilicity descriptors obtained from extrapolated chromatographic retention data as modeling tools for biological distribution: application to some oxime-type acetylcholinesterase reactivators.

Chromatographic retention data obtained from interactions between some oxime-type compounds and different stationary phases (involving hydrophobic interaction, ion pairing formation availability, pi-pi, H-bonding, dipole-dipole, ion-dipole, electrostatic interaction and glycoprotein binding ability) have been studied. The logarithms of the capacity factors extrapolated at 0% or 100% organic solvent, resulting from the functional dependencies between retention and mobile phase composition, were used for estimation of different kind of hydrophobicity or hydrophilicity descriptors (HHDs) of these compounds. The conditions of the chromatographic separation were chosen as close as possible to in-vivo conditions (the aqueous component of the mobile phase has a pH in the physiologic interval 6.8-7.2, 0.9% sodium chloride was added to reproduce ionic strength and isotonic character, and the temperature was set at 37 degrees C). These descriptors characterizing the partition between stationary/mobile phases through specific interactions may be directly used for correlation to biological distribution processes, such as penetration of the blood/brain barrier. Oxime-type compounds used as acetylcholinesterase (AChE, E.C.3.1.1.7) reactivators have been considered for the retention study. The choice is supported by their use in the therapy of acute intoxication with organophosphorus AChE inhibitors (OPIs, especially nerve agents and pesticides), a rather complicated chemistry in solution and a relative lack of data about computational molecular descriptors used for modeling biological partition/distribution. Some correlations between the determined descriptors and computational values have also been discussed.

Liquid chromatography-tandem mass spectrometry method for determination of the pyridinium aldoxime 4-PAO in brain, liver, lung, and kidney.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was validated and applied to the in vitro determination of 4-[(hydroxyimino)methyl]-1-octylpyridinium cation (4-PAO), which can penetrate the blood-brain barrier and reactivate acetylcholinesterase (AChE) inhibited by alkylphosphonate in the brain, liver, lung, and kidney. The limit of detection (LOD) was 0.235 microg cation/g wet weight, and the quantification range and linearity of the calibration curve extended over a range of 0.470-941 microg cation/g wet weight. For the proof of applicability, when 4-PAO was administrated intravenously via the rat tail vein at 10% LD(50), we were able to quantify the 4-PAO concentration in the tissues: brain 7.60+/-1.32 microg cation/g wet weight (mean+/-SD, n=5), liver 26.8+/-2.82 microg cation/g, lung 76.4+/-24.9mugcation/g, and kidney 638+/-266 microg cation/g. In addition, the methods for 4-[(hydroxyimino)methyl]-1-decylpyridinium bromide (4-PAD) and 4-[(hydroxyimino)methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) were partly validated referring to the findings of the 4-PAO full validation. Thus, the LC-MS/MS method described in this study can be useful for quantification of pyridinium aldoxime methiodide (PAM)-type oximes in biological samples.

A quantitative NMR protocol for the simultaneous analysis of atropine and obidoxime in parenteral injection devices.

A rapid selective and accurate quantitative (1)H NMR method was developed for the simultaneous analysis of obidoxime chloride and atropine sulfate, the active components in parenteral injection devices (PID) used for the emergency treatment of poisoning by toxic organophosphates. The spectra were acquired in 90% H(2)O-10% D(2)O using sodium 3-(trimethylsilyl)-1-propane sulfonate hydrate as the internal standard. Both synthetic mixtures and dosage forms were assayed. The results were compared with those obtained from a reported HPLC method.

RP-HPLC determination of the lipophilicity of bispyridinium reactivators of acetylcholinesterase bearing a but-2-ene connecting linker.

New acetylcholinesterase reactivators with either a (E) or (Z)-but-2-ene connecting linker were recently prepared. The purity of the compounds was checked by HPLC and was found to be sufficient for in-vitro screening. All the discussed bispyridinium reactivators were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) to measure lipophilicity. The procedure was performed under isocratic conditions with methanol as organic modifier in the mobile phase using an end-capped non-polar C(18) stationary phase RP column. Relationships between the lipophilicity (logarithm of the RP-HPLC capacity factor, log k) and chemical structures of the studied compounds are discussed. Lipophilicity was different for the (E) and (Z) compounds and varied among the compounds in each of these groups. The lipophilicity differences also indicated an apparent influence of intramolecular interactions. Lipophilicity calculations (log P/Clog P) by means of commonly used software were not successful due to the presence of quaternary nitrogen atoms in the molecules of the reactivators.

Measurement of K-27, an oxime-type cholinesterase reactivator by high-performance liquid chromatography with electrochemical detection from different biological samples.

K-27 is a bisquaternary asymmetric pyridinium aldoxime-type cholinesterase reactivator of use in the treatment of poisoning with organophosphorous esterase inhibitors. A sensitive, simple and reliable reverse-phase high-performance liquid chromatographic method with electrochemical detection was developed for the measurement of K-27 concentrations in rat brain, cerebrospinal fluid, serum and urine samples. Male Wistar rats were treated intramuscularly with K-27 and the samples were collected 60 min later. Separation was carried out on an octadecyl silica stationary phase and a disodium phosphate solution (pH 3.7) containing citric acid, octane sulphonic acid and acetonitrile served as mobile phase. Measurements were carried out at 30 degrees C at E(ox) 0.65 V. The calibration curve was linear through the range of 10-250 ng/mL. Accuracy, precision and the limit of detection calculated were satisfactory according to internationally accepted criteria. Limit of quantitation was 10 ng/mL. The method developed is reliable and sensitive enough for monitoring K-27 levels from different biological samples including as little as 10 microL of cerebrospinal fluid. The method--with slight modification in the composition of the mobile phase--can be used to measure a wide range of other related pyridinium aldoxime-type cholinesterase reactivators.

Efecto del tiempo y las condiciones de almacenamiento sobre la actividad de las colinesterasas / Effect of time and the conditions of storage in the activity of the cholinesterases

Se evaluó la estabilidad de la colinesterasa plasmática y la colinesterasa eritrocítica a temperatura ambiente, 4§C y -20 §C, durante un periodo de tres meses. Se determinó que la colinesterasa plasmática es estable en refrigeración y congelación. A temperatura ambiente la actividad de esta enzima disminuye significativamente en una semana. Por su parte la colinesterasa eritrocítica es estable únicamente en refrigeración. El proceso de congelamiento y descongelamiento provoca disminución importante en la actividad de la enzima. En las muestras conservadas por un día a -20 §C, la actividad de la enzima eritrocitaria fue enre 10 y 20 por ciento más baja que la actividad basal. A temperatura ambiente la actividad de esta enzima disminuye en forma importante en una semana. Palabras clave: colinesterasa plasmática, colinesterasa eritrocítica, plaguicidas, organofosforados, carbamatos, toxicología

[The pharmacokinetic interaction of cholinolytics and cholinesterase reactivators as a reflection of the modulation of their binding in blood plasma and in brain tissue]. / Farmakokineticheskoe vzaimodeistvie kholinolitikov i reaktivatorov kholinésterazy kak otrazhenie moduliatsii ikh sviazyvaniia v plazme krovi i v tkani mozga.

Radioligand assay showed that the cholinesterase (ChE) reactivators dipiroxime and benzyxime, but not carboxime, modulate selective absorption of some cholinolytics (tributam, pediphen, aprophen) in rat brain. Significant suppression of the specific binding of muscarine antagonists was recorded after chlorophos (2.LD50) intoxication. Under such conditions, the ChE reactivators induce increase in the number of binding sites and in the parameters of the constant of cholinolytic absorption on the brain membranes. It was also established by equilibrium dialysis that the binding of cholinolytics in blood plasma under the effect of ChE reactivators is reduced, leading to redistribution of their free and bound fractures, which is most favorable for tissue sorption.

Development and optimization of reactivation techniques for carbamate-inhibited brain and plasma cholinesterases in birds and mammals.

Two biochemical assays were developed which promote and measure the induced reactivation of carbamate-inhibited cholinesterases in avian and mammalian brain and plasma samples. The effects of inhibitor concentration, temperature, and the extent of dilution on the achievement of a steady state equilibrium and the subsequent level and rate of recovery of brain cholinesterase activity were investigated. A similar procedure for reactivation of carbamate-inhibited plasma cholinesterase activity involved the removal of excess carbamate from a small sample volume (< 400 microliters). Both methods begin by measuring cholinesterase activity immediately following dilution and involve an incubation period during which conditions for spontaneous reactivation of the inhibited enzymes are maximized. Both assays are suitable for large-scale, rapid use and appear able to restore inhibited cholinesterase activity to levels closely approximating that of control values for each species tested. These methods will not only maximize the usefulness of cholinesterases in monitoring carbamate pesticide exposure but should prove to be extremely useful tools in the forensic assessment of carbamate exposure in human and wildlife pesticide incidents.

The diacetylmonoxime assay of urea, its application to the assay of diacetylmonoxime and a comparison with other methods for the analysis of diacetylmonoxime.

The experiments reported herein were designed to identify the best assay of diacetylmonoxime (DAM) in biological fluids, with particular emphasis on detection limits. Initially, four variations of the assay of urea with excess DAM were compared. The best method, published in 1977, was one that includes thiosemicarbazide, 4-aminoantipyrine and ceric ammonium sulphate in the acid reagent; it is fast, gives a reasonably stable chromophore and displays good linearity. However, the reaction was two or more times less sensitive when applied to the assay of DAM, with urea in excess, by interchanging the amounts of urea and DAM. Further, the calibration graphs did not pass through the origin, and one of the methods gave a mixture of two chromophores. None approached the sensitivity of a DAM-urea reaction specifically designed to assay biacetyl (formed from DAM by acid hydrolysis) and published in 1968. This method, using antipyrine and arsenicosulphuric acid, is also fast, with good linearity and a stable chromophore, but is sensitive to interference by plasma and urine. An alternative photometric assay that does not involve urea was equally sensitive. It had the advantage of less interference by plasma and urine but was more time-consuming. Both of these photometric methods had a limit of detection of ca. 0.2 microgram DAM, similar to that of a high-performance liquid chromatography (HPLC) assay. Sample clean-up is necessary before application of the HPLC assay.

The acetylcholinesterase reactivator HI-6 (1-[[[4-(aminocarbonyl)pyridinio]methoxyl]methyl]- 2-[(hydroxyimino)methyl]-pyridinium dichloride): a comparative study of HI-6 samples from various sources.

A comparison of the chemical purity, toxicology and potency of HI-6 (1-[[[4-(aminocarbonyl)pyridinio]methoxy]methyl]-2- [(hydroxyimino)methyl]-pyridinium dichloride) obtained from various sources (Canada, Israel, Yugoslavia, The Netherlands, United Kingdom) was performed. There were no significant differences between HI-6 obtained from Israel, Yugoslavia, The Netherlands and Canada regarding their potency, when combined with atropine, as an antidote of organophosphate poisoning. HI-6 obtained from the United Kingdom was significantly more toxic and less potent than any of the other HI-6 samples. In addition, the results of this study showed that there was no significant difference between HI-6 prepared as a laboratory batch and HI-6 prepared commercially with regards to chemical purity, toxicology or potency.

Stability studies of bis(pyridiniumaldoxime) reactivators of organophosphate-inhibited acetylcholinesterase.

Relative stability studies of three organophosphate-inhibited acetylcholinesterase reactivators, 1-(2-hydroximinomethyl-1-pyridinium)-3-(4-carbamoyl-1-pyridinium)- 2-oxapropane dichloride (HI-6), 1,1'-methylenebis(4-hydroximinomethylpyridinium) dichloride (MMB-4), and 1,1'-trimethylenebis(4-hydroximinomethylpyridinium) dibromide (TMB-4) were carried out by semiquantitative TLC and NMR methods. TMB-4 appears to be the most, and HI-6 the least stable of the three compounds. The extent of hydrolysis of HI-6, MMB-4, and TMB-4 in 0.05 M, pH 7 phosphate buffer was approximately 50, 25, and less than 1%, respectively, after 20 d at room temperature. The hydrolysis products of HI-6 were identified by NMR and MS (electron impact) as 2-pyridinealdoxime, picolinamide, and isonicotinamide, whereas that of MMB-4 was identified as 4-pyridinealdoxime. The stability of these reactivators decreases with increasing pH. TMB-4 was stable under both neutral and basic conditions at room temperature. Deuterium exchange of the methylene protons of MMB-4 in D2O and of the protons at the 2- and 6-positions of the pyridinium ring of TMB-4 in NaOD/D2O were observed.