Investigating the degradation of the sympathomimetic drug phenylephrine by electrospray ionisation-mass spectrometry.

The frequently used sympathomimetic drug phenylephrine has been studied by electrospray ionisation-mass spectrometry. The stability of the adrenoceptor agonist was examined by investigations of the pharmaceutically used salts phenylephrine hydrochloride and phenylephrine bitartrate. Photostability has been studied by use of an irradiation equipment emitting a solar radiation spectrum. The experiments were carried out by analysis of aqueous drug solutions before and after irradiation treatment. The phenylephrine derivative with unsaturated side chain originating from the drug by loss of one water molecule has been detected as the major degradation product of both phenylephrine salts the hydrochloride and the bitartrate. Further degradation and oxidation products were detectable already in the full scan mode demonstrating a low stability of the drug. Tandem mass spectrometry and multiple stage mass spectrometry experiments enabled the establishment of fragmentation schemes of both salts for the first time. Irradiation treatment indicated that phenylephrine bitartrate is more prone to degradation than the hydrochloride because of an additional decomposition sensitivity of the tartaric acid counter ion. An interaction between phenylephrine and its counter ion degradation products via a nucleophilic addition mechanism is suggested to be the explanation for the detected ion signals after irradiation treatment of phenylephrine bitartrate.

Further investigations on the role of ascorbic acid in stratum corneum lipid models after UV exposure.

This study is the continuation of our research into vitamin C and its possible effects on human skin after topical administration. The effects of ascorbic acid, iron ions and UV irradiation on stratum corneum lipid models were investigated. The lipid models used were: a simple system (linolenic acid dispersion), a complex system (liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol and linolenic acid) and complex systems with additionally incorporated ceramides (types III and IV). The lipid peroxidation was quantified by the thiobarbituric acid assay. A human adult low-calcium high-temperature (HaCaT) keratinocytes cell culture was used as a second in-vitro model. The amount of intracellular peroxides was determined by measuring the fluorescence intensity using the dihydrorhodamine 123 assay. Electron paramagnetic resonance spectroscopy was used to study the influence of ascorbic acid and iron ions on the signal intensity of 5-doxylstearic acid during UV exposure. Ascorbic acid showed prooxidative properties in the thiobarbituric acid assay whereas cell protection was measured in the HaCaT keratinocytes experiments. Electron paramagnetic resonance investigations revealed different extents of free radical production generated by iron ions, ascorbic acid and UV irradiation. In evaluating the results from this study new aspects of the mechanism of lipid damage caused by these three factors were suggested, transcending the simple redox behaviour of ascorbic acid.

The examination of polysaccharides as potential antioxidative compounds for topical administration using a lipid model system.

Aim of this study was the detection of polysaccharides with antioxidative properties as potential lipid protectors for topical administration. The effects of eight different polysaccharides on UV irradiation induced lipid peroxidation were investigated in a concentration dependent manner. An aqueous linolenic acid dispersion was used as an in vitro test system to examine the influences of acacia gum, agar agar, alginic acid, guar gum, novelose 330 and xanthan gum on the lipid peroxidation level after UV exposure. Four different samples of pectin and locust bean gum resulting from a swing mill grinding series were tested as well. Iron ions were added as transition metal catalysts. A UV irradiation device was used to create high level radiation. The amount of lipid peroxidation secondary products was quantified by the thiobarbituric acid assay detecting malondialdehyde. All of the tested polysaccharides showed antioxidative effects at least at one concentration. For acacia and xanthan gum, a concentration dependency of the protective effects was measured. The samples of agar agar, guar gum and novelose 330 acted antioxidatively without showing any concentration dependency. For alginic acid, prooxidative effects were determined. A correlation between grinding time and the effects of pectin and locust bean gum on the model lipid was not observed. The administration of lipid protective polysaccharides in cosmetic formulations or sunscreens could be helpful for the protection of the human skin against UV induced damage. In vivo experiments with the lipid protective polysaccharides found in this study should follow.

Screening for new antioxidative compounds for topical administration using skin lipid model systems.

PURPOSE: The effects of forty seven different substances (drugs, plant extracts, plant ingredients and polysaccharides) on UV irradiation induced lipid peroxidation were investigated. METHODS: Two lipid systems of different complexity were used as in vitro screening models. Iron ions were added as transition metal catalysts. A UV irradiation device was used to create high level radiation. The amount of lipid peroxidation secondary products was quantified by the thiobarbituric acid assay detecting malondialdehyde. RESULTS: The screening for antioxidative compounds for topical administration resulted in new, interesting findings. In the drug testings amantadine, bufexamac, tryptophan, melatonin, propranolol and hyaluronic acid were found to act antioxidatively whereas for ascorbic acid pro-oxidative effects were determined. Buckwheat extract significantly reduced the level of irradiation induced lipid peroxidation as well as the extracts of St. John's Wort, melissa and sage. The resistant starch novelose 330 and the samples of locust bean gum from a swing mill grinding series showed lipid protection after UV irradiation in the polysaccharide test rows. CONCLUSIONS: Human skin is constantly exposed to UV light and oxygen. Therefore, the administration of protectors in cosmetic formulations or sunscreens, as found in this study, may be helpful for the protection of the human skin against UV induced damage. In vivo experiments with substances found as protectors should follow to allow in vitro-in vivo correlation and clinical interpretation of the data.

Examinations of the antioxidative properties of the topically administered drug bufexamac reveal new insights into its mechanism of action.

The effect of bufexamac on UV-irradiation-induced lipid peroxidation was investigated. Linolenic acid was used as a model lipid. Bufexamac was shown to be capable of reducing the amount of lipid peroxidation. The quantification was carried out by the thiobarbituric acid assay. Irradiation experiments were also performed using HaCaT keratinocytes as a model system. The oxidative changes were quantified by DNA synthesis measurements and cell viability determinations. Bufexamac was found to act antioxidatively again. To investigate free radical involvement, electron paramagnetic resonance studies were carried out. The influence of bufexamac on the concentration of hydroxyl radicals generated by the Fenton system was examined using the spin trapping technique. Moreover, the hydroxamic acid's ability to react with stable radicals was checked. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate showed no concentration changes of the stable radical caused by bufexamac. In the Fenton assay antioxidative effects were measured after the addition of the drug. The qualitative changes after irradiating bufexamac were studied at a molecular level by electrospray mass spectrometry. Multiple-stage mass spectrometry experiments enabled the establishment of fragmentation schemes. Phenolic degradation products were detected. The results suggest a new interpretation of the controversially debated mechanism of action of bufexamac and indicate possible reasons for its eczema provoking potential.

The effects of hyaluronan and its fragments on lipid models exposed to UV irradiation.

The effects of hyaluronan and its degradation products on irradiation-induced lipid peroxidation were investigated. Liposomal skin lipid models with increasing complexity were used. Hyaluronan and its fragments were able to reduce the amount of lipid peroxidation secondary products quantified by the thiobarbituric acid (TBA) assay. The qualitative changes were studied by mass spectrometry. To elucidate the nature of free radical involvement electron paramagnetic resonance (EPR) studies were carried out. The influence of hyaluronan and its fragments on the concentration of hydroxyl radicals generated by the Fenton system was examined using the spin trapping technique. Moreover, the mucopolysaccharide's ability to react with stable radicals was checked. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) showed no concentration changes of the stable radical caused by hyaluronan. Hyaluronan was found to exhibit prooxidative effects in the Fenton assay in a concentration dependent manner. A transition metal chelation was proposed as a mechanism of this behavior. Considering human skin and its constant exposure to UV light and oxygen and an increased pool of iron in irradiated skin the administration of hyaluronan or its fragments in cosmetic formulations or sunscreens could be helpful for the protection of the human skin.

Role of ascorbic acid in stratum corneum lipid models exposed to UV irradiation.

PURPOSE: The effects of ascorbic acid on Stratum corneum lipid models following ultraviolet irradiation were studied adding iron ions as transition metal catalysts. METHODS: Lipid peroxidation was quantified by the thiobarbituric acid assay. The qualitative changes were studied on a molecular level by mass spectrometry. To elucidate the nature of free radical involvement we carried out electron paramagnetic resonance studies. The influence of ascorbic acid on the concentration of hydroxyl radicals was examined using the spin trapping technique. Moreover, we checked the vitamin's ability to react with stable radicals. RESULTS: Ascorbic acid was found to have prooxidative effects in all lipid systems in a concentration dependent manner. The degradation products of ascorbic acid after its prooxidative action were detected. The concentration of the hydroxyl radicals in the Fenton assay was decreased by ascorbic acid. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate showed reduced concentration levels of the stable radical caused by ascorbic acid. CONCLUSIONS: Considering human skin and its constant exposure to UV light and oxygen, an increased pool of iron ions in irradiated skin and the depletion of co-antioxidants, the administration of ascorbic acid in cosmetic formulations or in sunscreens could unfold adverse effects among the Stratum corneum lipids.