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Introduction: Depression is a mental disease causing large personal and socio-economic problems, and new improved drugs are therefore needed. Selective monoamine oxidase A (MAO-A) inhibitors are potential anti-depressants, but discovering new MAO-A inhibitors from natural sources by bioassay-guided approaches are a lengthy and time-consuming process. New analytical technologies that allow simultaneously chemical and biological screening of extracts are therefore urgently needed., Method: In the present study we describe coupling of a photometric microplate-based high-resolution MAO-A inhibitor assay with a hyphenated system consisting of high-performance liquid chromatography, solid-phase extraction and tube transfer nuclear magnetic resonance (HPLC-SPE-ttNMR). The standard compound clorgyline, and an extract of black pepper (Piper nigrum L.), representing a complex plant matrix, were used for proof-of-concept., Results: The work with clorgyline showed that the microplate-based high-resolution assay produced MAO-A inhibition profiles that easily allowed detection of submicrogram amounts of this selective MAO-A inhibitor. Furthermore, the HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform allowed identification of piperine and two piperine analogues as the main MAO-A inhibitors in the black pepper petroleum ether extract., Conclusion: The HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform is a powerful tool for fast and efficient identification of new MAO-A inhibitors from complex extracts, and promise future advancement in the search for new anti-depressants from natural sources., (Copyright © 2012 John Wiley & Sons, Ltd.)

(E)-beta-Fluoromethylene-m-tyrosine (FMMT, MDL 72394) represents a prodrug approach to site-selective, irreversible inhibition of monoamine oxidase. A sensitive and specific method for the quantification of FMMT and its active metabolite (MDL 72392) in human plasma and urine has been developed for future pharmacokinetic studies. The procedure consists of a liquid-liquid extraction of plasma and urine samples, esterification with HCl/butanol and, subsequently, N-acylation with pentafluoropropionic anhydride. This is followed by gas chromatography/mass spectrometry employing negative ion chemical ionization. The precision and the accuracy of the method have been optimized by using two internal standards: MDL 72661 (a methyl homologue of FMMT) for the quantification of FMMT, and MDL 72761 (a methyl homologue of MDL 72392) for the quantification of MDL 72392. The method has been validated for FMMT and MDL 72392 over the concentration range 2.5-400 pmol ml-1 with a limit of quantification for both compounds of 2.5 pmol ml-1. Sample clean-up and selected ion monitoring by mass spectrometry ensured the specificity and sensitivity required for the pharmacokinetic evaluation of FMMT and MDL 72392.

1. Accumulation of noradrenaline in extraneuronal tissue of the guinea-pig trachea has been studied by the use of the fluorescence histochemical technique of Falck and Hillarp.2. After incubation in solutions of noradrenaline, fluorescence developed in cellular structures (tracheal smooth muscle, vascular smooth muscle and endothelium, fibroblasts and chondroblasts), in the intercellular matrix of the cartilage and throughout the loose connective tissue of the adventitia and submucosa.3. The effect of various experimental procedures on the development of this fluorescence has been examined i.e. incubation with noradrenaline at reduced temperature (0 degrees C), removal of fluorescence by washing with Krebs solution at 37 degrees C or 0 degrees C, incubation with phenoxybenzamine (10(-4)M) or metanephrine (10(-4)M).4. From these observations it has been concluded that noradrenaline accumulates in the trachea:(a) in cellular structures where it is firmly bound, i.e. not easily removed by washing at 0 degrees C, and where the accumulation is prevented by phenoxybenzamine, metanephrine or cold.(b) in the intercellular matrix of the cartilage where it is also firmly bound, but where the accumulation is not prevented by phenoxybenzamine, metanephrine or cold. This probably represents binding to sulphated mucopolysaccharides.(c) in the adventitia and submucosa where it is loosely bound and easily removed by washing.5. Some implications of these findings in pharmacological experiments with guinea-pig trachea are discussed.