Rapid analysis of melatonin in plant tissues by micellar electrokinetic capillary chromatography (MEKC)

MESSINA, G.; STEGE, P.; SOMBRA, L.; MARTINEZ, L. D.; SILVA, M. F.

Santiago de Chile

Simposio; XIII Simposio Latinoamericano en Aplicaciones de la Electroforesis Capilar y Tecnolog¨ªa del Microchip en Biotecnolog¨ªa, Biomedicina , Biofarmacia e Industria; 2007

R.W. Johnson- Universidad de Chile

Melatonin (MT) is an ubiquitous, highly conserved molecule which may function as a hormone, protective antioxidant, and a mediator of light/dark signalling. The occurrence of MT in higher plants was reported by Dubbels et al. (1995) and Hattori et al. (1995) in two separate studies aimed at determining the natural sources of MT in human nutritional diets. In 1997, MT was found at higher than expected levels in medicinal plants traditionally used in the treatment of human neurological diseases, and therefore, this hormone may be a key factor in their therapeutic actions. Not only medicinal plants but some examples of everyday edible have been reported to show significant levels of MT. Further studies using radioisotope tracer techniques indicated that tryptophan is the common precursor for melatonin, serotonin and indole-3-acetic acid (IAA) in higher plants,. Endogenous MT has also been shown to play an important role in plant physiology that may be related to auxin metabolism. Micellar electrokinetic capillary chromatography (MEKC) has been established as a powerful technique for the separation of a large variety of neutral and charged analytes, with higher peak efficiency and resolution than high performance liquid chromatography and comparable to those of gas chromatography. Additionally, MEKC offers fast analysis times and is less costly. The purpose of this study was to develop a simple, accurate, fast and reliable MEKC methodology for the determination of MT in Salvia officinalis. The effects of pH, buffer concentration, and sampling and separation modes were investigated.Salvia officinalis. The effects of pH, buffer concentration, and sampling and separation modes were investigated. Sample preparation: plant tissues were dried under nitrogen gas, grounded, accurately weighed about 0.5 g and transferred to a 50 ml beaker. An aliquot (10 ml) of methanol was added to each sample and extraction was completed by sonication in an ultrasonic bath for 45 min in darkness. The supernatant was decanted and centrifuged at 4500¡Ág for 10 min. The resulting extract was filtered through a 0.45 ¦Ìm filter and combined with the filtered supernatant of two subsequent methanol washes of the tissue residue. The entire extraction was dried to complete dryness with nitrogen gas in total darkness and resuspended in 1ml methanol for injection. For quantification, repeated injections of 50 ¦Ìl were made under each of the optimized detection conditions.plant tissues were dried under nitrogen gas, grounded, accurately weighed about 0.5 g and transferred to a 50 ml beaker. An aliquot (10 ml) of methanol was added to each sample and extraction was completed by sonication in an ultrasonic bath for 45 min in darkness. The supernatant was decanted and centrifuged at 4500¡Ág for 10 min. The resulting extract was filtered through a 0.45 ¦Ìm filter and combined with the filtered supernatant of two subsequent methanol washes of the tissue residue. The entire extraction was dried to complete dryness with nitrogen gas in total darkness and resuspended in 1ml methanol for injection. For quantification, repeated injections of 50 ¦Ìl were made under each of the optimized detection conditions. Separation Performance: separation was carried out in less than 10 minutes with a 20 mM sodium tetraborate buffer, 20 mM SDS and 10 % acetonitrile pH 9.50. The carrier electrolyte gave baseline separation with good resolution, great reproducibility and accuracy.separation was carried out in less than 10 minutes with a 20 mM sodium tetraborate buffer, 20 mM SDS and 10 % acetonitrile pH 9.50. The carrier electrolyte gave baseline separation with good resolution, great reproducibility and accuracy. Results: excellent linearity was observed between peak area and concentration of analytes in the range from 7.9 ¡¤10-7 - 2.25 ¡¤10-3 mol L-1 for MT. The detection limits was 3.7¡¤10-7excellent linearity was observed between peak area and concentration of analytes in the range from 7.9 ¡¤10-7 - 2.25 ¡¤10-3 mol L-1 for MT. The detection limits was 3.7¡¤10-7-7 - 2.25 ¡¤10-3 mol L-1 for MT. The detection limits was 3.7¡¤10-7 mol L-1. Detection was performed by UV absorbance at wavelengths of 215 nm. Quantification of the MT was calculated against the responses of prepared freshly external standard solutions. The method was validated and met all analysis requirements of quality assurance and quality control.-1. Detection was performed by UV absorbance at wavelengths of 215 nm. Quantification of the MT was calculated against the responses of prepared freshly external standard solutions. The method was validated and met all analysis requirements of quality assurance and quality control.