A pendant droplet-based sensor for the detection of acetylcholinesterase and its inhibitors.

In this work, a pendant droplet-based sensor is developed for the rapid and label-free detection of acetylcholinesterase (AChE) and its inhibitors. The detection limit of AChE reaches 0.17 mU mL-1. The pIC50 values of AChE inhibitors such as neostigmine, rivastigmine and galantamine are determined to be 0.45 µM, 0.64 µM and 4.93 µM, respectively.

Modified carbon paste sensor for the potentiometric determination of neostigmine bromide in pharmaceutical formulations, human plasma and urine.

A novel, simple, rapid, selective and sensitive method for the determination of neostigmine (Ns) ion in its bulk powder, different pharmaceutical dosage forms, and biological fluids (plasma and urine) using four modified carbon paste electrodes was developed. Sensor 1 is based on ion-association Ns-TPB, sensor 2 used Ns-PT, sensor 3 comprises a mixture of (Ns-PT+Ns-TPB) and sensor 4 was constructed using (Ns-PT+ß-CD). Solvent mediator 2-NPPE exhibited a proper behavior including Nernstian slope ranging from 61.5±0.5 to 64.5±0.5 mV per decade over the pH range of 3.8-10 for the four sensors. Linear responses of Ns within the concentration range 1.0×10(-7)-1.0×10(-2) mol/L were obtained. The response time is very short (≤10s) with a detection limit 6.3×10(-8) M. In flow injection analysis (FIA), sensor 3 shows a Nernstian slope value 75.5±0.5 mV per decade within the concentration range of 1×10(-6)-1×10(-2) mol/L and with a detection limit 7.5×10(-7) mol/L. The utility of mixed or additives of ß-CD had a significant influence on increasing the sensitivity of sensors 3 and 4 compared to sensors 1 and 2. The sensors were applied for the determination of neostigmine (Ns) ion in its bulk powder, different pharmaceutical dosage forms, and biological fluids (plasma and urine). The results obtained were satisfactory with excellent percentage recovery comparable with official method for the assay based on non-aqueous titration using perchloric acid as a titrant.

Acetylcholinesterase biosensor for carbamate drugs based on tetrathiafulvalene-tetracyanoquinodimethane/ionic liquid conductive gels.

A highly sensitive acetylcholinesterase biosensor was developed for detection of carbamate drugs based on TTF-TCNQ-ionic liquid gel thiocholine sensor. The TTF-TCNQ-ionic/ionic liquid gel was characterized by FT-IR and scanning electron microscopy. The electrocatalytic behavior of TTF-TCNQ-ionic liquid gels toward oxidation of thiocholine was thoroughly investigated. 1-Ethyl-3-methylimidazolium tetracyanoborate gel based sensor allowed amperometric detection of thiocholine at +400 mV vs. Ag/AgCl with a high sensitivity of 55.9±1.2 µA mM(-1)cm(-2) and a low detection limit equal to 7.6 µM. The catalytic rate constant and diffusion constant of thiocholine were estimated from chronoamperometric data. The proposed biosensor based on AChE immobilized in sol-gel matrix was used for the detection of two carbamate therapeutic drugs. Very low detection limits of 26 pM eserine and 0.3 nM neostigmine were achieved. The analysis of spiked tap water proved the biosensor capability to be used as a screening method for detection of carbamate drugs in wastewaters.

Comparative study of 2-hydroxy propyl beta cyclodextrin and calixarene as ionophores in potentiometric ion-selective electrodes for neostigmine bromide.

Three novel neostigmine bromide (NEO) selective electrodes were investigated with 2-nitrophenyl octyl ether as a plasticiser in a polymeric matrix of polyvinyl chloride (PVC). Sensor 1 was fabricated using tetrakis(4-chlorophenyl)borate (TpClPB) as an anionic exchanger without incorporation of an ionophore. Sensor 2 used 2-hydroxy propyl ß-cyclodextrin as an ionophore while sensor 3 was constructed using 4-sulfocalix-8-arene as an ionophore. Linear responses of NEO within the concentration ranges of 10(-5) to 10(-2), 10(-6) to 10(-2) and 10(-7) to 10(-2) mol L(-1) were obtained using sensors 1, 2 and 3, respectively. Nernstian slopes of 51.6 ± 0.8, 52.9 ± 0.6 and 58.6 ± 0.4 mV/decade over the pH range of 4-9 were observed. The selectivity coefficients of the developed sensors indicated excellent selectivity for NEO. The utility of 2-hydroxy propyl ß-cyclodextrin and 4-sulfocalix[8]arene as ionophores had a significant influence on increasing the membrane sensitivity and selectivity of sensors 2 and 3 compared to sensor 1. The proposed sensors displayed useful analytical characteristics for the determination of NEO in bulk powder, different pharmaceutical formulations, and biological fluids (plasma and cerebrospinal fluid (CSF)) and in the presence of its degradation product (3-hydroxyphenyltrimethyl ammonium bromide) and thus could be used for stability-indicating methods.

Direct detection of acetylcholinesterase inhibitor binding with an enzyme-based surface plasmon resonance sensor.

Acetylcholinesterase (AChE) inhibitors are potentially lethal but also have applications as therapeutic drugs for neurodegenerative diseases such as Alzheimer's. Enzyme inhibitor binding are difficult to be detected directly by surface plasmon resonance (SPR) due to their small molecular weight. In this article, we describe the detection of AChE inhibitor binding by SPR without the use of competitive binding or antibodies. AChE was immobilized on the gold surface of an SPR sensor through covalent attachment to a self-assembled monolayer (SAM) of a COOH-terminated alkanethiol. The activity of the immobilized protein and the surface density were determined by using a standard photometric assay. Binding of two reversible inhibitors, which are used as therapeutic drugs, was detectable by SPR without the need to further modify the surface or the use of other reagents. The binding affinities (K(A)) obtained from the fits were 3.8 × 10(3)M(-1) for neostigmine and 1.7 × 10(3)M(-1) for eserine, showing a higher affinity of the sensor for neostigmine. We believe that the SPR sensor's ability to detect these inhibitors is due to conformational changes of the enzyme structure on inhibitor binding.

Simultaneous determination of active ingredients in an ophthalmic solution by isocratic tandem-mode HPLC connected reverse phase column and strong cation exchange column.

A tandem-mode high performance liquid chromatography (HPLC) system is described here, which employs reversed phase liquid chromatography (RPLC) followed by strong cation exchange liquid chromatography (SCX), was used to determine the mixture of six ingredients in an ophthalmic solution. As a result of investigations, isocratic HPLC methods that using two columns in tandem-mode; Atlantis dC18 (75 mm x 4.6 mm i.d., 3 microm, ODS) and CAPCELL PAK SCX UG80 (75 mm x 4.6 mm i.d., SCX), which have different separation modes, and control of mixture of methanol/ammonium dihydrogenphosphate buffer as used for the eluent, allowed for six target ingredients to be determined simultaneously. And all ingredients separated perfectly and were determined efficiently and rapidly. Validation of the method was accomplished with respect to linearity (r>0.999), recovery (99.4-100.4%), precision (R.S.D. 0.1-0.9%) and specificity. These results suggest that the fusion of different separation modes can be used for the simultaneous determination of ingredients in ophthalmic solutions, and this can be accomplished rapidly and with high precision.

High-performance liquid chromatography/tandem mass spectrometry assay for the rapid high sensitivity measurement of basal acetylcholine from microdialysates.

A high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the analysis of acetylcholine (ACh) in brain dialysates. This separation of ACh is based on cation exchange chromatography with elution buffer consisting of a mixture of ammonium acetate, ammonium formate and acetonitrile. Using isocratic separation conditions, ACh was resolved within a minute and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for ACh was found to be 1 fmol on column with a S/N ratio of 3:1. The assay has been used routinely for the measurement of ACh in brain dialysates from awake freely moving rats. Furthermore, separation conditions were modified to allow simultaneous measurement of ACh and the acetylcholine esterase inhibitor, neostigmine.

Chip-based capillary electrophoresis with an electrodeless nanospray interface.

A sheathless and electrodeless nanospray interface has been used to interface a polycarbonate capillary electrophoresis (CE) chip to a mass spectrometer (MS). The chip was made of two flat polycarbonate plates which were bolted together. Channels were imprinted in one of the plates with metal wires, using a hydraulic press. A short tapered capillary connected to the chip was used as the nanospray emitter. The advantage of this electrodeless interface is that it was not necessary to apply a electrospray voltage to the chip or the nanospray emitter. Instead, the CE voltage already applied to the buffer compartment on the chip, to drive the electrophoresis, was used to generate the spray also. A low conductivity buffer of 1.25 mmol/L ammonium acetate in 80% methanol was used to obtain a large electric field across the buffer channel. The performance of the device was evaluated by analyzing a mixture of three beta-agonists Relative standard deviation (RSD) values obtained were between 4.8 and 5.0%. A sample concentration of 40 nmol/L resulted in a signal-to-noise ratio of 2 to 5 for the different components. Compared to a conventional CE analysis in a fused silica capillary with UV detection, only a minor loss of resolution was observed, which can be attributed to the design of the chip.

Determination of compounds with anticholinesterase activity in commercial drugs by a new enzyme sensor.

A suitable enzyme sensor for the analysis of anticholinesterase compounds of pharmaceutical interest is described. It is based on the competitive inhibiting properties of these compounds on the enzyme butyrylcholinesterase and it is constituted by a hydrogen peroxide amperometric electrode modified by a superimposed Nylon membrane containing two chemically immobilized biological mediators (butyrylcholinesterase and choline oxidase). Some applications to the analysis of several pharmaceutical forms containing different compounds showing anticholinesterase activity are also reported and evaluated.

[The kinetics of neostigmine degradation in aqueous solution]. / Zur Kinetik des Neostigminabbaus in wässriger Lösung.

The concentration course of the degradation products of aqueous temperature-stressed solutions of neostigmine bromide (1) shows, that 1 simultaneously is hydrolyzed to 3-hydroxyphenyltrimethylammonium bromide (2) and demethylated to 3-dimethylaminophenyldimethylcarbamate (4). The ester hydrolysis is the rate-determining predominant reaction and of pseudo first-order. Under temperature-stressed conditions only, the demethylation to 4 followed by consecutive reactions proceeds at a measurable rate. The kinetic parameters of the ester hydrolysis calculated from the results of isothermic short-term tests permit the prediction of a five-year stability of 2% neostigmine solutions in the therapeutically relevant pH-value range from 3 to 7. On the basis of the kinetic results the possible reaction mechanisms and the influence of selected components on the rate of the hydrolysis and demethylation are discussed.

[The structure of degradation products of neostigmine bromide]. / Zur Struktur der Abbauprodukte von Neostigminbromid.

In aqueous temperature-stressed solutions of neostigmine bromide besides the known product of hydrolysis additional degradation products were discovered, detected by TLC and HPLC. By means of spectroscopic studies (1H-NMR, IR, UV, MS) and elemental analysis the isolated decomposition products were identified as 3-hydroxyphenyltrimethylammonium bromide (2), 3-dimethylaminophenol (3), 3-dimethylaminophenyl-dimethylcarbamate (4), 3-methylaminophenyl-dimethylcarbamate (5), 3-aminophenyl-dimethylcarbamate (6) and 4 (resp. 6)-bromo-3-dimethylaminophenyl-dimethylcarbamate (7). The structures of 2-7 were confirmed by comparison with synthesized authentic compounds.

Synthesis and preliminary pharmacology of an internal standard for assay of neostigmine.

The synthesis of the diethyl analog of neostigmine, its preliminary pharmacology, and its use as an internal standard for the GLC assay of neostigmine are described. Both the diethyl analog and neostigmine undergo thermal demethylation in the injection port. The column selected produced satisfactory resolution and short retention times for neostigmine and the diethyl analog. The diethyl analog apparently possesses acetylcholinesterase-inhibiting properties, as evidenced by potentiation of the contractile response to acetylcholine in the ileum. In addition, acetylcholine levels in the brain were elevated slightly. Water solutions of the diethyl analog appeared to lose biological activity with time. the diethyl analog appears to be suitable for use as an internal standard for the GLC assay of neostigmine.

Reversed-phase, ion-pair liquid chromatography of quaternary ammonium compounds: determination of pyridostigmine, neostigmine and edrophionium in biological fluids.

A reversed-phase, ion-pair liquid chromatographic method for the quantitative determination of quaternary acetylcholinesterase inhibitors is described. The method uses an ion-pair extraction to isolate the drugs from biological material prior to liquid chromatographic separation and online UV detection at 214 nm. Quantiation down to 5 ng/ml and within-day precison with coefficient of variation (C.V.) of 1.5% (n = 10, x = 100 ng/ml) for neostigmine, C.V., 1.7% (n = 10, x = 80 ng/ml) for pyridostigmine and C.V., 1.5% (n = 10, x = 100 ng/ml) for edrophonium have been achieved. The assay was designed for pharmacokinetic studies of these drugs in anesthetized patients.