Analysis of acetylcholinesterase inhibitors by extraction in choline saccharinate aqueous biphasic systems.

Ionic liquid-based aqueous biphasic systems (IL-ABS) formed by ILs composed of ions of low toxicity, choline ([Chol]+) coupled with saccharinate ([Sac]-) and acesulfamate ([Ace]-), and inorganic salts with distinct water-structuring properties were employed for simultaneous extraction and concentration of acetylcholinesterase (AChE) inhibitors - galantamine (gal), N-desmethyl galantamine (des) and ungiminorine (ung). Comprehensive experiments aimed to assess the influence of salt and IL type and concentration, as well as the pH and temperature on the phase-forming ability and distribution of the target alkaloids between the two phases formed reveled that the IL anion and pH are the most important factors. At the optimal conditions found a quantitative recovery into the IL-rich phase of gal, des and ung was achieved in a single extractive step. These results were further used as a platform for the development of a simple and safer sample pretreatment method for analysis of the three analytes, followed by RP-HPLC/UV detection. The method showed satisfactory analytical performance, the latter allowing quantitative determination of these AChE inhibitors in pharmaceutical dosage form and in human urine.

Highly sensitive HPLC method for the determination of galantamine in human plasma and urine through derivatization with dansyl chloride using fluorescence detector.

A new method based on fluorescence derivatization with 5-(dimethylamino) naphthalene-1-sulfonyl chloride (dansyl chloride) was developed for the quantitative determination of galantamine in human plasma and urine using high-performance liquid chromatography. The reaction between galantamine and dansyl chloride was optimally realized in 30 min at room temperature and pH 10.5, with a reagent to galantamine molar ratio of 2.13. The derivative was extracted with dichloromethane, and the extract was dried under a nitrogen stream and dissolved in the mobile phase. Chromatographic analysis was performed with an Inertsil C18 column and a mobile phase comprising 40% acetonitrile and 60% 10 mM o-phosphoric acid, 1.2 ml/min. The injection volume was 20 µl. The derivatives were detected with a fluorescence detector (excitation 375 nm/emission 537 nm). The retention time for the dansyl derivative of galantamine was 16.8 min. Linearity was observed between 125 and 2000 ng/ml in water, urine and plasma. The limit of detection and limit of quantification for the developed method were 6.27-70.99 and 18.81-212.97 ng/ml, respectively. Per cent recovery was calculated as 95.15 for urine and 95.78 for plasma. Interday repeatability values for urine and plasma samples (n = 6) at three different concentrations were calculated as a per cent relative standard deviation of 0.24-0.59 and 0.35-0.56. The corresponding per cent relative standard deviation values for intraday repeatability were 0.13-0.51 and 0.04-0.15, respectively.

Determination of therapeutics with growth-hormone secretagogue activity in human urine for doping control purposes.

The administration of growth-promoting agents such as human growth hormone as well as compounds with respective secretagogue activity is prohibited in sports according to the regulations of the World Anti-Doping Agency. Acetylcholine esterase inhibitors have been demonstrated to stimulate growth-hormone secretion in elderly humans, and new orally active drugs have been developed to provide alternatives to therapeutic injections of growth-hormone preparations. Preventive anti-doping strategies include method development for emerging drugs and potentially misused compounds. Hence, the mass spectrometric dissociation behavior of three acetylcholine esterase inhibitors (donepezil, galantamine and rivastigmine) and a structural analogue to the growth-hormone secretagogue SM-130686 were studied using high-resolution/high-accuracy orbitrap mass spectrometry. These data provided substantial information for screening procedures, complementing common methods of sports drug testing. Using liquid-liquid extraction and subsequent liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, the four target analytes were determined at urinary concentrations of 15-20 ng/mL, recoveries ranged from 55-97%, and assay precisions were calculated at 5.2-15.8% (intraday) and 10.2-21.6% (interday) for all compounds. The applicability of the developed assay to authentic urine specimens was tested using two administration study urine samples after application of Reminyl (galantamine) and Aricept (donepezil). In both cases, the administered drug and the respective desmethylated metabolites were detected.

The metabolism and excretion of galantamine in rats, dogs, and humans.

Galantamine is a competitive acetylcholine esterase inhibitor with a beneficial therapeutic effect in patients with Alzheimer's disease. The metabolism and excretion of orally administered (3)H-labeled galantamine was investigated in rats and dogs at a dose of 2.5 mg base-Eq/kg body weight and in humans at a dose of 4 mg base-Eq. Both poor and extensive metabolizers of CYP2D6 were included in the human study. Urine, feces, and plasma samples were collected for up to 96 h (rats) or 168 h (dogs and humans) after dosing. The radioactivity of the samples and the concentrations of galantamine and its major metabolites were analyzed. In all species, galantamine and its metabolites were predominantly excreted in the urine (from 60% in male rats to 93% in humans). Excretion of radioactivity was rapid and nearly complete at 96 h after dosing in all species. Major metabolic pathways were glucuronidation, O-demethylation, N-demethylation, N-oxidation, and epimerization. All metabolic pathways observed in humans occurred in at least one animal species. In extensive metabolizers for CYP2D6, urinary metabolites resulting from O-demethylation represented 33.2% of the dose compared with 5.2% in poor metabolizers, which showed correspondingly higher urinary excretion of unchanged galantamine and its N-oxide. The glucuronide of O-desmethyl-galantamine represented up to 19% of the plasma radioactivity in extensive metabolizers but could not be detected in poor metabolizers. Nonvolatile radioactivity and unchanged galantamine plasma kinetics were similar for poor and extensive metabolizers. Genetic polymorphism in the expression of CYP2D6 is not expected to affect the pharmacodynamics of galantamine.

Capillary zone electrophoresis determination of galanthamine in biological fluids and pharmaceutical preparatives: experimental design and artificial neural network optimization.

Galanthamine is a third-generation cholinesterase inhibitor used against Alzheimer's disease. New analytical methods for the determination of galanthamine in pharmaceutical preparatives and biological fluids, such as urine and serum, were developed. An experimental design and artificial neural network approach were used for method optimization. Thirty-five ppb of galanthamine were determined in serum samples (with addition of 10 mM magnesium chloride and using solid-phase preconcentration).

The O-demethylation of the antidementia drug galanthamine is catalysed by cytochrome P450 2D6.

Galanthamine proved effective in symptomatic treatment of senile dementia of Alzheimer's type. The aim of this study was to elucidate the metabolism of galanthamine. Two novel metabolites of galanthamine have been isolated from the urine of eight young men after single doses of 10-15 mg. Some 19.8% of the doses were excreted as O-demethylgalanthamine glucuronide, 5% as N-demethylgalanthamine, 25.1% as galanthamine, and 0.8% as epigalanthamine. After coadministration of quinidine hydrogen sulfate, which inhibits cytochrome P450 2D6 (CYP2D6) selectively, O-demethylgalanthamine glucuronide was highly diminished in urine. In vitro, human liver microsomes metabolized galanthamine to O-demethylgalanthamine with Vmax 5.2 nmol/mg protein/h and Km 187 microM. Ki of quinidine to inhibit O-demethylation was 28 nM. To inhibit cholinesterases, O-demethylgalanthamine was 10-fold more selective for acetylcholinesterase (AChE) versus butyrylcholinesterase (BuChE) than galanthamine. After glucuronidation, O-demethylgalanthamine failed to inhibit AChE and BuChE. N-Demethylgalanthamine inhibited cholinesterases less potently than galanthamine.

Pharmacokinetics of galanthamine (a long-acting anticholinesterase drug) in anaesthetized patients.

The pharmacokinetics of the long-acting anticholinesterase drug, galanthamine, were investigated in eight patients. After i.v. injection of 0.3 mg kg-1, the decrease in the serum concentration of galanthamine followed a biexponential curve. The serum concentration decreased rapidly from 543 +/- 47 ng ml-1 to 128 +/- 14 ng ml-1 between 2 and 30 min with a T1/2 alpha of 6.42 +/- 2.15 min, and then declined more slowly with a T1/2 beta of 264 +/- 28 min. Total serum clearance of galanthamine amounted to 5.37 +/- 0.87 ml min-1 kg-1, and the renal clearance was 1.36 +/- 0.10 ml min-1 kg-1. The cumulative urinary excretion of galanthamine between 0 and 48 h after injection amounted to 28.0 +/- 5.4% of the administered dose. The biliary excretion of galanthamine during 24 h amounted to 0.2 +/- 0.1% of the dose. There was no evidence of glucuronide or sulphate conjugation of galanthamine.

High-performance liquid chromatographic determination of galanthamine, a long-acting anticholinesterase drug, in serum, urine and bile.

The anticholinesterase drug galanthamine is obtained from alkalinized serum by repeated liquid--liquid extraction. The resulting extract is approximately 100 times concentrated with respect to the original sample. Quantitative determination of galanthamine is performed with normal-phase liquid chromatography using a mixture of dichloromethane--n-hexane and ethanolamine as an eluent. Phenacetin is used as internal standard. The absorption of the column effluent is monitored at 235 nm. No endogenous sources of interference have been observed. A galanthamine serum level of 5 ng/ml is found as the minimum detectable concentration; the coefficient of variation at this level is 37.8% (n = 4). For the assay of galanthamine in the concentration range 10-100 ng/ml, standard deviations vary between 18.9 and 2.5% (n = 32).