9
Rhododendri Micranthi [9]. An HPLC method for identifi cation and determination of myricetin, quercetin, kaempferol and total fl avonoids in ten herbal drugs of Macedonian origin is presented by Kulevanova et al. [10].
Concerning literature data, there were many studies about the pharmacological effects of Filipendula ulmaria, yet only a few methods for fl avonoid content in onions (Allium cepa L.). Possible alternatives to high-performance liquid chromatography (HPLC) analysis were investigated; these are spectrophotometery [11], thin layer chromatography [2], [12] and HPLC combined with mass spectrometric (LC/MS) detection [6]. However in 1992, Poukens-Renwart et al. quantifi ed the spiraeoside-containing extract by HP-TLC densitometry after derivatisation with diphenylboric acid-2-aminoethylester [5].
According to Lombard et al., the spectrophotometer does not replace the precision achieved with HPLC for the separation and quantifi cation of individual quercetin conjugates, the spectrophotometer represents a cheap and effi cient method for analysis of high volumes of onions for total quercetin concentration [11].
It is claimed that used thin layer chromatography (TLC), which is more easily available chromatographic method for different laboratories and is more effective compared to HPLC by L. Pobłocka-Olech et al. [2] Brazilian Homeopathic Pharmacopoeia published Allium Cepa monograph which has a TLC method and visual control of colour after chemical reaction [12]
Pemp et al. utilised LC/MS in order to save time and detect fl avonoids in the extracts without expensive sample purifi cation [6]. In literature, there is no such an identifi cation method for spiraeoside in gel formulation. The scope of the present study was to develop and validate a new identifi cation method of Spiraeoside in liquid extract from onion which is contain in a gel formulation using a RP-HPLC method.
Experimental Materials
Spiraeoside was purchased from Finzelberg (Germany). Tetra methyl ammonium hydroxide, phosphoric acid, sodium chloride, ethyl acetate were purchased from Merck Ltd. HPLC grade Acetonitrile was purchased from JT. Baker. High purity deionised water was obtained from Millipore, Milli-Q (Bedford, MA, USA) purifi cation system. Gel formulation (Sanovel, Turkey) was used.
Spiraeoside working standard was purchased from Phytolab. Instrumentation
RP-HPLC system (Waters, USA) equipped with inbuilt autosampler and quaternary gradient pump with an on-line degasser was used. The column compartment having temperature control and photodiode array (PDA) detector was employed throughout the analysis. Chromatographic data was acquired using empower software.
Chromatographic conditions
Luna 5µ HILIC 200A 250x4.6 (Phenomenex, USA) column was used as stationary phase maintained at 35°C. The mobile phase involved a variable composition of (10 mL of 10% Tetra methyl ammonium hydroxide aqueous solution are removed in 1000 ml of water, adjusted to pH 2.5 with orthophosphoric acid) and
Mobile phase composition
Different proportions of mobile phase were tried to obtain enough selectivity and retention time of spiraeoside at the gel sample. Tetramethylammonium hydroxide aqueous solution, the inductive
120 mL of this solution is taken up and stirred with 880 mL of acetonitrile. The mobile phase was pumped through the column with at a fl ow rate of 0.4 ml / min. Pure water, sodium chloride - ethyl acetate mixture solution and mobile phase solution; are used as dilution solution.
Injection volume was 100 µL and the optimum wavelength selected was 365 nm which represents the wavelength of maximum response for spiraeoside in gel. The samples were analysed using a PDA detector covering the range of 200–600 nm.
Preparation of Solutions Dilution Solution (Sodium chloride - Ethyl acetate solution)
5.0 g of sodium chloride is weighed into a 150 mL volumetric fl ask. Add 30 mL of distilled water and stir until dissolved. 100 mL of ethyl acetate is added and the mixture is stirred for 10 min. mixed.
This solutions and mobile phase solution are used as a dilution solutions all of the preparation of samples.
Standard Solution
Take 0.5 mL of the Extractum Cepae working standard and transfer it to a 100 mL volumetric fl ask. Add 70 mL of distilled water and vortex for 15 minutes (min.) to mixed. Diluting it to volume with Sodium chloride - Ethyl acetate solution and stirred for about 30 min. After phase separation, take 1.0 mL of the upper phase and mix to volume with 10 mL volumetric fl ask of mobile phase.
Test solution
5.0 gr of gel was weighed into 100 ml volumetric fl ask. Add 70 mL of distilled water and vortex for 15 min. The ethyl acetate mixture was brought to volume with the solvent and stirred for 30 min. The completing volume with sodium chloride - ethyl acetate solution and stirred for 30 min. After phase separation, take 1.0 mL of the upper phase and mix to volume with 10 mL volumetric fl ask of mobile phase.
Results and Discussion Optimisation of the chromatographic conditions
Different chromatographic conditions have been tried to optimise HPLC parameters.
To optimise the reverse phase HPLC parameters and a good separation, several chromatographic conditions were tried.
Column selection Different columns for injections were used to achieve best partition of spiraeoside with other blank and placebo peaks. The appropriate peak shape, retention time, tailing factor, and column effi ciency were good with Luna HILIC 200A (25 cm x 4.6 mm, 5 µm).
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68 |
Page 69 |
Page 70 |
Page 71 |
Page 72 |
Page 73 |
Page 74 |
Page 75 |
Page 76 |
Page 77 |
Page 78 |
Page 79 |
Page 80 |
Page 81 |
Page 82 |
Page 83 |
Page 84 |
Page 85 |
Page 86 |
Page 87 |
Page 88 |
Page 89 |
Page 90 |
Page 91 |
Page 92 |
Page 93 |
Page 94 |
Page 95 |
Page 96 |
Page 97 |
Page 98 |
Page 99 |
Page 100 |
Page 101 |
Page 102 |
Page 103 |
Page 104 |
Page 105 |
Page 106 |
Page 107 |
Page 108 |
Page 109 |
Page 110 |
Page 111 |
Page 112 |
Page 113 |
Page 114 |
Page 115 |
Page 116 |
Page 117 |
Page 118 |
Page 119 |
Page 120 |
Page 121 |
Page 122 |
Page 123 |
Page 124 |
Page 125 |
Page 126 |
Page 127 |
Page 128
