HPLC/DAD determination of rosmarinic acid in Salvia officinalis: sample preparation optimization by factorial design

Abstract

A sálvia (Salvia officinalis) contém altos teores de ácido rosmarínico (RA), uma substância bioativa, bem como outros polifenóis. RA é facilmente oxidável e pode sofrer degradação durante a preparação da amostra para análise. O objetivo deste trabalho foi desenvolver e validar um método analítico para a determinação do RA em sálvia, usando planejamento fatorial para a otimização da preparação de amostras. Inicialmente foram determinadas as variáveis estatisticamente significativas para melhorar o rendimento da extração do RA, as quais foram depois otimizadas usando planejamento composto central (CCD). O método analítico foi validado e aplicado na análise de amostras comerciais de sálvia. O procedimento otimizado consistiu na extração com metanol aquoso (40%) contendo uma mistura antioxidante (ácido ascórbico e ácido etileno diamino tetracético (EDTA)), com sonicação a 45 o C por 20 min. As amostras foram injetadas em um sistema contendo coluna C 18 , usando metanol (A) e ácido fosfórico aquoso 0.1% (B) em gradiente (45A:55B, 0-5 min; 80A:20B, 5-10 min) com fluxo de 1.0 mL min −1 e deteção em 330 nm. Utilizando estas condições, as concentrações do RA foram 50% maiores quando comparadas com extração na ausência de antioxidante (recuperação de 98,94 ± 1,07%). Auto-oxidação do RA durante a preparação da amostra foi evitada pelo uso de antioxidante levando a resultados analíticos mais confiáveis. O método foi então aplicado na análise de amostras comerciais de sálvia.Sage (Salvia officinalis) contains high amounts of the biologically active rosmarinic acid (RA) and other polyphenolic compounds. RA is easily oxidized, and may undergo degradation during sample preparation for analysis. The objective of this work was to develop and validate an analytical procedure for determination of RA in sage, using factorial design of experiments for optimizing sample preparation. The statistically significant variables for improving RA extraction yield were determined initially and then used in the optimization step, using central composite design (CCD). The analytical method was then fully validated, and used for the analysis of commercial samples of sage. The optimized procedure involved extraction with aqueous methanol (40%) containing an antioxidant mixture (ascorbic acid and ethylenediaminetetraacetic acid (EDTA)), with sonication at 45 o C for 20 min. The samples were then injected in a system containing a C 18 column, using methanol (A) and 0.1% phosphoric acid in water (B) in step gradient mode (45A:55B, 0-5 min; 80A:20B, 5-10 min) with flow rate of 1.0 mL min −1 and detection at 330 nm. Using this conditions, RA concentrations were 50% higher when compared to extractions without antioxidants (98.94 ± 1.07% recovery). Auto-oxidation of RA during sample extraction was prevented by the use of antioxidants resulting in more reliable analytical results. The method was then used for the analysis of commercial samples of sage.

Recent Advances in the Analysis of Rosmarinic Acid From Herbs in the Lamiaceae Family

“…The acid concentration can vary from 0.01% to 6%, 93 and the buffer salts concentration from 5 to 50 mM. 86 Most commonly, gradient techniques are used to identify RA, 16,83,94,95 but an isocratic method has also been reported. 77,[96][97][98][99]…”

Rosmarinic Acid – Pharmaceutical and Clinical Aspects

“…Extraction, Isolation, and Analytical Methods ! Several extraction protocols [7][8][9][10][11], separation [12][13][14], and chromatographic methods have been developed to optimize the extraction and identification of RA [7,12,15]. The type of solvents, temperature, extraction time, particle size of the material to be extracted, and ratio of the solvents are some of the parameters studied [16][17][18][19].…”

“…To provide one-step isolation and purification of RA from S. miltiorrhiza, an online adsorption chromatography-countercurrent chromatography was carried out after UAE, adsorbing the plant extract on the resin and immediately submitting it to high-speed countercurrent chromatography (HSCCC), which led to the isolation of RA with a purity of about 88% [12]. A template-assisted process using the supramolecular technique has also been used to extract RA from P. frutescens leaves.…”

Voltammetric Determination of Rosmarinic Acid on Chitosan/Carbon Nanotube Composite-Modified Carbon Paste Electrode Covered with DNA

“…Comparison with other reported methods.-To evaluate the efficiency of the biosensor presented here, it has been compared with some other methods 23,[40][41][42][43][44] reported for the determination of RA. The results of this comparison given in Table II, show that the concentration range and detection limit of the presented approach are more acceptable than those of the others with the exception of the pretreated glassy carbon electrode.…”