Chromatography
Analytical Method Development and Validation for the Identifi cation of Spiraeoside Using RP-HPLC in Pharmaceutical Gel Formulation Vildan Tombayoglu1
, Ozlem Kenar1 , Arzu Palantoken1 , Ali Turkyilmaz1 1Sanovel Pharmaceutical, Research and Development Department, 34580 Silivri, Istanbul, Turkey
The aim of this study was to validate a Reversed-Phase High Performance Liquid Chromatographic (RP-HPLC) method for the qualitative determination of spiraeoside contained in a gel preparation. The chromatography column used was a stainless steel column (25 cm x 4.6 mm, dp=5 µm) packed with silica surface covered with cross-linked diol groups for polar selectivity (Luna HILIC). Mobile phase consisting buffer containing 1 mL of 10% Tetra methyl ammonium hydroxide aqueous solution (pH 2.5) and acetonitrile and isocratic system was used. The fl ow rate was 0.4 ml/min and the detective wavelength was 365 nm. Injection value for each time was 100 µl and separations were carried out isothermally at 35°C in a heated chamber and sample temperature was 25°C. System suitability parameters were studied by injecting the standard solution six times and results were well under the acceptance criteria. The proposed validated method could separate spiraeoside peak was confi rmed using photo-diode array detector.
Introduction
Meadowsweet Filipendula ulmaria (L.) Maxim. (Rosacea fam.) is generally used as an analgesic, corroborant, anti-infl ammatory, wound-healing, antiulcerogenic, hypoglycaemic, sedative, antihemorrhoid, astringent and diuretic drug in some European countries and in Russia. This plant is composed of fl avonoids, tannins, salicylates, volatile oils, coumarin, mucilage, carbohydrates and ascorbic acid (vitamin C) and catechins [1–3]. From 3-4% in the fl owering herb up to 6% in the fresh fl owers contain fl avonoids. Some fl avonoids are spiraeoside (quercetin-4’- glucoside), hyperoside, other quercetin and kaempferol derivatives and kaempferol-4’-glucoside [4]. Many of these components have benefi cial properties to human health.
The major and typical fl avonoid of Filipendula ulmaria fl owers is spiraeoside (quercetin-4’-glucoside) [5]. The chemical structures of the reported fl avonoid glycosides from Filipendula ulmaria are shown in Figure 1. The effi cacy of the fl uid extract from onion is not yet fully elucidated. Therefore, the herbal preparation is regarded as the active substance in its entirity. The herbal preparation is composed of a complex mixture of constituents; it cannot be given a structural formula.
Background
Active ingredients of plant extracts are very diffi cult to identify in pharmaceutical dosage forms. Quantifi cation of these compounds
Figure 1. Chemical structures of the reported fl avonoid glycosides from Filipendula ulmaria
is usually performed by HPLC with UV detection [6]. The widely accepted technique for separating complex mixtures of plant extracts in pharmaceutical dosage forms is RP-HPLC. Derya Algul et al., reported this method for the quantitative determination of hyperoside in a cream preparation [7]. Geetha M. et al., reported to fi nd out the quercetin and cyanidin present in red onion peel and big onion peel by using HPLC [8]. Xiuling Yang et al., established an RP-HPLC method for the simultaneous determination of four major constituents from Folium
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