The direct electron transfer between covalently immobilized glucose oxidase (EC 1.1.3.4) and a cyanuric chloride modified graphite electrode Is observed by use of differential pulse voltammetry. A well-defined peak at -0.51 V vs. Ag/AgCI, due to the reduction of flavin adenine dinucleotide (FAD), Is obtained which Is 100 mV more positive than the free enzyme. Comparisons to adsorbed FAD and glucose oxidase Indicate that the peak In covalently attached enzyme Is due to the reduction of the prosthetic group as part of the enzyme molecule, rather than FAD adsorbed on the electrode surface. Suggestions for the analytical utility of direct electron transfer at modified electrodes are discussed.

Chemically modified graphite electrodes containing covalently Immobilized xanthine oxidase (E.C. 1.2.3.2) have been employed for the potentlometric and amperometrlc detection of xanthine. Potassium hexacyanoferrate( III) Is used as the

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ABSTRACT:The metabolism and pharmacokinetics of moxonidine, a potent central-acting antihypertensive agent, were studied in four healthy subjects after a single oral administration of approximately 1 mg (ϳ60 Ci) of [ 14 C 3 ]moxonidine. Moxonidine was rapidly absorbed, with peak plasma concentration achieved between 0.5 to 2 h postdose. The maximal plasma concentration and the area under the curve of unchanged moxonidine are lower than those determined for radioactivity, indicating presence of circulating metabolite(s). The total recovery of radiocarbon over 120 h ranged from 99.6 to 105.2%, with 92.3 to 103.3% of the radioactivity excreted in the urine and only 1.9 to 7.3% of the dose excreted in the feces. Thus, renal elimination represented the principal route of excretion of radioactivity. Metabolites of moxonidine were identified in urine and plasma samples by high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Oxidation of moxonidine on the methyl group or on the imidazoline ring resulted in the formation of hydroxymethyl moxonidine, hydroxy moxonidine, dihydroxy moxonidine, and dehydrogenated moxonidine. Metabolite profiling results indicated that parent moxonidine was the most abundant component in the urine. The dehydrogenated moxonidine was the major urinary metabolite as well as the major circulating metabolite. Moxonidine also underwent phase II metabolism, generating a cysteine conjugate. In summary, moxonidine is well absorbed after oral administration. The major clearance pathway for moxonidine in humans is via renal elimination. Furthermore, seven metabolites were identified with three metabolites unique to humans.Moxonidine is a new antihypertensive agent that acts on central nervous system imidazoline receptors to decrease sympathetic nervous system tone (Ernsberger et al., 1992;Prichard and Graham, 1996). It has been marketed throughout Europe for the treatment of hypertension. The safety and efficacy of moxonidine in hypertension patients has been established (Ollivier et al., 1992;Sides et al., 1998). In comparison to clonidine and rilmenidine, moxonidine has fewer adverse effects such as dry mouth and sedation because it is considerably more selective for the I 1 receptor than the ␣ 2 receptor that is associated with these indicated side effects (Yu and Frishman, 1996). In elderly patients, the clearance of moxonidine is reduced, and the area under the curve of unchanged drug is greater in comparison with younger patients suggesting the existence of an age-related decrease in metabolism (Theodor et al., 1996). Moxonidine has a relatively short half-life (about 2-3 h). However, it has long duration of effect and is given only once daily clinically. There is no clear correlation between the pharmacokinetics and pharmacodynamic effect of moxonidine.To determine whether the long duration of moxonidine is attributed to the production of active metabolite(s), efforts were undertaken to identify and synthesize major metabolites of moxonidine and test their ph...

Xanomeline, (3-[4-(hexyloxy)-1, 2, 5-thiadiazol-3-yl]-1, 2, 5, 6-tetrahydro-1-methylpyridine) is a selective M 1 muscarinic agonist currently under investigation for the potential symptomatic treatment of Alzheimer's disease. The characterization of the metabolites of xanomeline has been accomplished utilizing electrospray tandem mass spectrometry (ES-MS/MS). The use of ES-MS/MS has been employed in the identification of numerous oxidative metabolites of xanomeline resulting from extensive first pass metabolism. In addition, on-line liquid chromatography (LC) coupled with ES-MS/MS has been utilized to further characterize the metabolites of xanomeline after subcutaneous dosing in rats and transdermal dosing in humans. The advantages and limitations of on-line LC/ES-MS/MS for biotransformation studies are demonstrated in this application.Electrospray (ionspray) ionization coupled with tandem mass spectrometry (ES-MS/MS) has proven to be an invaluable tool in the characterization of trace level mammalian metabolites of pharmaceutical candidates (i, 2). Furthermore, the combination of on-line LC with ES-MS/MS has become the method of choice for analysis of polar, thermolabile drug candidates and their respective metabolites in complex biological matrices (3, 4). The utilization of this methodology from the initial discovery phase of a potential drug candidate through human clinical trials and beyond can enhance a drug's rapid development (5, 6). Electrospray typically generates abundant [M+H] + species and avoids the use of high temperatures which may cause thermal degradation of metabolites (particularly polar conjugates). In addition, tandem mass spectrometry provides both the selectivity and sensitivity required for detection of trace level (ng/mL) metabolites in the presence of large concentrations of endogenous components.Identification of the metabolites from a new drug substance typically follows three steps in our laboratories. Initially animal urine, plasma or microsomal incubates are examined to tentatively identify major metabolites. Second, in vivo metabolism and balance studies are performed in different animal species utilizing radiolabeled drug substance and third, human studies are performed with