Validated HPTLC Method for Dihydrokaempferol-4′-O-glucopyranoside Quantitative Determination in Alcea Species

Validated HPTLC Method for Dihydrokaempferol-4′-O-glucopyranoside Quantitative Determination in... Abstract Dihydrokaempferol-4′-O-glucopyranoside, a flavanonol glucoside, is the major compound in the flower of Alcea rosea L. which possesses significant antioxidant and anticancer activity against HepG-2 cell line and thus can be considered a marker compound for A. rosea L. We attempted to establish a new simple, validated high-performance thin-layer chromatographic (HPTLC) method for the quantitation of dihydrokaempferol-4′-O-glucopyranoside to help in the standardization of the hydroalcoholic extracts of A. rosea L. flowers and to evaluate the best method for its extraction from the plant material. The separation was carried out on an HPTLC aluminum plate pre-coated with silica gel 60F-254, eluted with ethyl acetate–methanol–water–acetic acid (30:5:4:0.15 v/v). Densitometric scanning was performed using a Camag TLC scanner III, at 295 nm. A linear relationship was obtained between the concentrations (0.9–3.6 mg) and peak areas with the correlation coefficient (r) of 0.9971 ± 0.0002. The percentage relative standard deviations of intra-day and inter-day precisions were 0.22–1.45 and 0.49–1.66, respectively. The percentage w/w of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. after maceration and sonication for 15 min was found to be 0.733 g/100 g and 0.928 g/100 g, respectively. Introduction Genus Alcea belongs to family Malvaceae; it includes ~60 species primarily of East Mediterranean region. Alcea rosea L. is widely cultivated in gardens of Egypt as an ornamental plant and is known as garden Hollyhock (English name) and in Arabic as Khatma wardi (Khatmi wardi) (1). All parts of the plant have been used in traditional medicin. In India, the flower has been used in the treatment of cough, asthma, throat infection, urinary irritation, boils, kidney pain and jaundice. The root is also used in the treatment of urinary tract burning sensation, dandruff, dermatitis, easy delivery, goiter, gynecological disorders and kidney inflammation. Mucilage (prepared by extracting the raw mucilage by hot extraction method of the plant) is used for prophylaxis and therapy of the respiratory, gastrointestinal tract and urinary complaints. The plant is also used in fever, chest and liver complaints (2, 3). In traditional Chinese medicine, this plant is considered as anti-diabetic ingredient (4). Dihydrokaempferol-4′-O-glucopyranoside was described for the first time in Petunia hybrida and also in the flowers of Althaea officinalis L. and Malva sylvestris L. (5). Dihydrokaempferol-4′-O-glucopyranoside shows significant antioxidant and anticancer activity against HepG-2 cell line with the advantage of being flavonoid glucoside that imparts the compound a good water solubility and hence increases its absorption (6). These findings prompted us to develop a validated high-performance thin-layer chromatography (HPTLC) method for the quantification of the compound in plant extracts and to identify the amount of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. as it can be a commercial source for the production of the compound. It can also serve as a marker compound for A. rosea L. A comparison was drawn between two techniques for the extraction of the compound from A. rosea L.; maceration as a traditional extraction technique and ultrasonication as an advanced extraction technique. The comparison was made to evaluate the effectiveness of sonication as an easy, efficient and time-saving method for the extraction of the compound. View largeDownload slide View largeDownload slide Structure of dihydrokaempferol-4′-O-glucopyranoside HPLC method to quantify dihydrokaempferol-4′-O-glucopyranoside among other constituents in different plants of genus Althaea has been reported (7, 8), but no article related to the HPTLC determination of the compound in plant extracts has been reported in literature or in pharmacopeias. Nowadays, HPTLC is becoming a routine analytical technique as it offers many advantages when compared with other techniques, like HPLC, spectrometric, titrations, etc. One of the major advantages of HPTLC is the ability to analyze several samples simultaneously using a small quantity of mobile phase. This reduces time, cost of the analysis and the possibilities of environmental pollution. Moreover, the plates can be stored for a long time after separation and the detection performed at a later stage to obtain the analytical information. This could be done because the chromatographic development and detection of the separated spots on a plate are generally separate processes in time. It also facilitates scanning in situ and repeated detection (scanning) of the chromatogram with the same or different parameters (9, 10, 11). The aim of the present work is to develop accurate, specific, repeatable and robust HPTLC method for the determination of dihydrokaempferol-4′-O-glucopyranoside in Alcea extracts. Experimental Isolation of dihydrokaempferol-4′-O-glucopyranoside Dihydrokaempferol-4′-O-glucopyranoside was isolated in a previous work (6) from the ethyl acetate fraction of the hydroalcoholic extract of A. rosea L. flowers by evaporation then direct crystallization from methanol. The isolated compound showed a single spot in TLC using the solvent system ethyl acetate–methanol–water–acetic acid (30:5:4:0.15 v/v) and the compound was characterized based on various spectral analysis and reported results in previous work (6). Preparation of standard solutions About 4.5 mg of standard dihydrokaempferol-4′-O-glucopyranoside was accurately weighed, quantitatively transferred into a 10-mL volumetric flask, dissolved in methanol and the volume was adjusted with the same solvent. Preparation of sample solutions About 4 g dried whole flowers of A. rosea L. were exhaustively extracted using 250 mL methanol by maceration for 10 days (Sample A) and 4 g dried whole flowers were extracted using 150 mL methanol by ultrasonication for 15 min (Sample B). The solvent was evaporated producing the crude extracts. The crude extracts were re-dissolved in methanol, filtered, transferred quantitatively to a 50-mL volumetric flask, adjusted to volume with methanol and shaken to mix thoroughly to give samples A and B, respectively. Chromatographic parameters and conditions The chromatographic estimation was performed by spotting standard and sample solutions on pre-coated silica gel aluminum plate 60F-254 (20 cm × 10 cm with 250 μm thickness, E. Merck, Darmstadt, Germany) using a Camag Linomat IV sample applicator (Camag, Muttenz, Switzerland) and a 100 μL Hamilton syringe. The samples, in the form of bands of length 6 mm, were spotted 15 mm from the bottom, 10 mm from left margin of the plate and 4 mm apart, at a constant application rate of 15 s/μL using nitrogen aspirator. Plates were developed using a mobile phase consisting of ethyl acetate–methanol–water–acetic acid (30:5:4:0.15, v/v/v/v). The volumes applied for routine analysis were triplicate 2.0 , 4.0 and 6.0 μL of the TLC dihydrokaempferol-4′-O-glucopyranoside standard (0.9, 1.8, 2.7 μg) and triplicate 4 μL aliquots of sample solution. Linear ascending development was carried out in 20 cm × 20 cm twin-trough glass chamber (Camag Muttenz, Switzerland) equilibrated with mobile phase. About 25 mL of mobile phase (system III) was used for development and the optimized chamber saturation time for mobile phase was 30 min at room temperature. The length of chromatogram run was 7 cm. The development time was 11 min. After development, the plates were air-dried for 10 min. Densitometric scanning was performed on Camag TLC scanner III in the reflectance-absorbance mode at λ 295 nm and operated by WINCATS software (V. 3.1). The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm. The slit dimension was kept at 6 mm × 0.1 mm. Concentrations of the samples and standards chromatographed were determined from the intensity of diffusely reflected light. Evaluation was via peak areas with linear regression. Calibration curve construction As recommended by the International Committee on Harmonization guidelines (12, 13), a calibration curve was established using five analyte concentrations (2.0, 3.0, 4.0, 6.0 and 8 μl/band) of the TLC standard applied in triplicate, representing 0.9–3.6 μg of dihydrokaempferol-4′-O-glucopyranoside. For routine analytical procedures, a three-point calibration curve within this range was used, produced by applying duplicate 2.0, 4.0 and 6.0 μL (0.9, 1.8 and 2.7 μg) of the HPTLC standard on each plate. Instrumentation and reagent Instrumentation Sample solutions for HPTLC analyses were applied by means of Camag Linomat IV sample applicator (Camag, Muttenz, Switzerland) and a 100 μL Hamilton syringe. Linear ascending development was carried out in 20 cm × 20 cm twin-trough glass chamber (Camag Muttenz, Switzerland). Zones were quantified by scanning at 295 nm with a Camag TLC Scanner 3 with a deuterium source in the reflection mode; slit dimension settings of length 6 and width 0.1, monochromator bandwidth of 20 nm, and a scanning rate of 10 mm/s operated by WINCATS software (V. 3.1). Solvents and reagents HPTLC analyses were performed on plates pre-coated with silica gel 60F-254 (20 × 10 cm; 0.25 mm layer thickness) plates that were obtained from Merck (Darmstadt, Germany). All the reagents used in the experiment were of analytical grade and were supplied by Merck (Darmstadt, Germany) and El-Nasr Chemicals Ltd (Egypt). Plant material A. rosea L. flowers were collected from El-Shalalat Park, Alexandria, Egypt, in March 2016 at the flowering stage. A voucher sample is deposited in the department of Pharmacognosy, Faculty of Pharmacy, Alexandria, Egypt. Results Validation of HPTLC method Selectivity It is the ability of an analytical method to unequivocally assess the analyte in the presence of other components (impurities, degradents and excipients) (14). The scan-densitogram obtained from a representative sample (Figure 1) showed selective baseline separation between dihydrokaempferol-4′-O-glucopyranoside and other components in the sample. Figure 1. View largeDownload slide In situ absorption spectra obtained from the band of standard dihydrokaempferol-4′-O-glucopyranoside and the corresponding band obtained from the flower extract of Alcea rosea L. Figure 1. View largeDownload slide In situ absorption spectra obtained from the band of standard dihydrokaempferol-4′-O-glucopyranoside and the corresponding band obtained from the flower extract of Alcea rosea L. Linearity The relationship between the peak areas and the amount of substance applied showed linearity over the range 0.9–3.6 μg/spot. The regression equation data, correlation coefficient (r-value) and other statistical parameters are listed in Table I. The three-point calibration was repeated many times and was also found to have a linear regression correlation coefficient of 0.9971. Table I. Linear Regression Data for the Calibration Curve of Dihydrokaempferol-4′-O-Glucopyranoside (n = 5) Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Table I. Linear Regression Data for the Calibration Curve of Dihydrokaempferol-4′-O-Glucopyranoside (n = 5) Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Range The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical method has suitable levels of precision, accuracy and linearity. The proposed method was applied for the determination of Dihydrokaempferol-4′-O-glucopyranoside in the concentration range of 50–150% of the working concentrations. The results are shown in Table I. Limit of detection and limit of quantitation Detection limit (DL) is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. Quantitation limit (QL) is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. According to the ICH (international conference on harmonization of drugs) guidelines, the DL may be expressed as: DL = 3.3 σ/S, while the QL may be expressed as: QL = 10 σ/S where σ = the standard deviation of the response and S = the slope of the calibration curve. The slope S is estimated from the calibration curve. The estimate of σ was carried out by studying the calibration curves where the standard deviations of y-intercepts of regression lines were used as the standard deviation. Limit of detection (LOD) and limit of quantitation (LOQ) were experimentally verified by diluting known concentrations of standard dihydrokaempferol-4′-O-glucopyranoside solutions. The results are shown in Table I. Precision The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree (15). In accordance with the ICH guidelines, precision was determined by independent repeated analysis of five different dihydrokaempferol-4′-O-glucopyranoside standard solutions and repeated analysis of a homogenous sample (3 μg/spot) by the use of the same equipment and the same analytical procedure in the same laboratory and on the same plate. Intermediate precision was performed by the analysis of six different concentrations of the standard solution, each repeated three times in the same day for intra-day precision and on different days for inter-day precision (13, 16). The percent relative standard deviation (RSD%) was calculated, and the results are shown in Tables II and III. The results shown in the tables meet the acceptance criterion for RSD% specified by the ICH which is a precision of <2%. Table II. Precision of the HPTLC Method for the Determination of Dihydrokaempferol-4′-O-Glucopyranoside (Repeatability) Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Table II. Precision of the HPTLC Method for the Determination of Dihydrokaempferol-4′-O-Glucopyranoside (Repeatability) Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Table III. Results of Replicate Analysis of a Homogenous Sample by HPTLC Method Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Table III. Results of Replicate Analysis of a Homogenous Sample by HPTLC Method Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Accuracy and recovery The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. Recovery is expressed as the amount/weight of the compound of interest analyzed as a percentage to the theoretical amount present in the medium. The accuracy of the method was validated by a standard addition analysis. The sample solutions were spiked with three different, known, concentrations of dihydrokaempferol-4′-O-glucopyranoside corresponding to 34, 102 and 170% of calculated concentration had been added. The three fortified sample solutions (mix.1, 2 and 3) were analyzed on the same plate by application of triplicate 4.0 μL volumes, respectively, in addition to the three standards described earlier for routine analyses which were applied in triplicate. The difference between the expected concentrations and the found ones was calculated to determine the accuracy of the method. The results of the experiments are presented in Table IV. Table IV. Results of the Standard Addition Experiments Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 aEach concentration is the average of three determinations. Table IV. Results of the Standard Addition Experiments Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 aEach concentration is the average of three determinations. Robustness Robustness tests examine the effect of the operational parameters on the analysis results. ICH defines robustness as a measure of the method′s capability to remain unaffected by small, but deliberate variations in method parameters (13). By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different composition like (ethyl acetate:methanol:water:acetic acid 3:5:4:(three drops), 30:5:5:(six drops) and 25:5:4:(three drops)) were tried. Time from spotting to chromatography and from chromatography to scanning was varied from 1, 2 and 24 h. Robustness of the method was carried out at three concentration levels 1, 2 and 3 μg/spot and the results are represented in Table V. Table V. Robustness Testing Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 aAverage of three concentrations 1, 2 and 3 μg/spot. Table V. Robustness Testing Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 aAverage of three concentrations 1, 2 and 3 μg/spot. Quantification of dihydrokaempferol-4′-O-glucopyranoside in Alcea rosea L. Quantification of dihydrokaempferol-4′-O-glucopyranoside was performed according to the procedure described in the ‘Chromatographic parameter and conditions’ section. The HPTLC profile of the two samples (A and B) at 295 nm showed acceptable separation of dihydrokaempferol-4′-O-glucopyranoside from the other components in the samples under the specified conditions (Figure 2). The amount of dihydrokaempferol-4′-O-glucopyranoside in the extracts were determined from the calibration graphs and the percentage w/w of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. after maceration and sonication for 15 min was found to be 0.733 g/100 g and 0.928 g/100 g, respectively, which is comparable to the amount present in different plants of genus Althaea reported in literature (0.839–1.934 g/100 g) (8). Figure 2. View largeDownload slide HPTLC scan-densitogram showing the separation of dihydrokaempferol-4′-O-glucopyranoside (6) from other components in the sample at 295 nm. Figure 2. View largeDownload slide HPTLC scan-densitogram showing the separation of dihydrokaempferol-4′-O-glucopyranoside (6) from other components in the sample at 295 nm. Discussion Method optimization Experimental conditions, such as mobile phase composition, scan mode, scan speed and wavelength of detection were optimized to provide accurate and precise results. Development with the mobile phase described above on the HPTLC silica gel layers produced compact, flat, dark bands of dihydrokaempferol-4′-O-glucopyranoside (Rf 0.58) when viewed under a 295 nm UV light. The dihydrokaempferol-4′-O-glucopyranoside band showed base peak separation from other spots in the sample as shown in Figure 2. Sample extraction Extraction of dihydrokaempferol-4′-O-glucopyranoside from the flowers of A. rosea L. was performed using two different extraction techniques; maceration as a traditional extraction technique and ultrasonication as an advanced one. The extracts prepared by maceration and ultrasonication were compared with identify the best method for the extraction of the compound from A. rosea L. It is obvious from the data that ultrasonication is a more efficient extraction technique for the compound from Alcea rosea L. The mechanical effect of ultrasound provides a greater penetration of solvent into the cellular materials and result in the disruption of biological cell walls to facilitate the release of contents. The advantages of ultrasonic extraction are increase in both the extraction yield and extraction rate, resulting in reduced extraction time and a higher throughput (17). Method validation It has been demonstrated above that validation data for the new quantitative HPTLC method meet the acceptance criteria for accuracy, precision, linearity, detection and quantification limits set by the International Conference on Harmonization (12, 13). The method specificity was assessed by performing a peak purity test of dihydrokaempferol-4′-O-glucopyranoside by comparing the UV absorption spectrum of the standard compound with the corresponding band in the sample (Figure 1). No interference was observed regarding the densitograms of the sample, confirming the selectivity of the method (Figure 2) (18). Conclusion It has been demonstrated above that validation data for the new quantitative HPTLC method meet the acceptance criteria for accuracy, precision, linearity, detection and quantification limits set by the International Conference on Harmonization. The percentage w/w of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. after maceration and sonication for 15 min was found to be 0.733 g/100 g and 0.928 g/100 g, respectively, which is comparable to the amount present in different plants of genus Althaea reported in literature (0.839–1.934 g/100 g). The described method is suitable for routine use for determination of the compound in the plant samples. Compared with HPLC, the proposed method is simpler and faster as up to six samples (applied in duplicate singly with a minimum of three standard concentrations) can be analyzed simultaneously on each plate rather than performing sequential injection of the samples and standards in HPLC. Only 15 mL of mobile phase is required in the chamber trough containing the plate for development, and an additional 10 mL for vapor saturation in the other trough, thus minimizing the cost for solvent purchase and disposal. The processing of samples and standards together at the same time (in-system calibration) leads to improved reproducibility and accuracy. Acknowledgments We wish to thank the Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University for the financial support. References 1 Shaheen , N. , Khan , M.A. , Yasmin , G. , Hayat , M.Q. , Munsif , S. , Ahmad , K. ; Foliar epidermal anatomy and pollen morphology of the genera Alcea and Althaea (Malvaceae) from Pakistan ; International Journal of Agriculture and Biology , ( 2010 ); 12 ( 3 ): 329 – 334 . 2 Jain , S.K. ; Ethnobotany and research on medicinal plants in India ; Ciba Foundation symposium [Internet] , ( 1994 ); 185 : 153 – 64-8 . Available from http://www.ncbi.nlm.nih.gov/pubmed/7736852. 3 Gairola , S. , Sharma , J. , Bedi , Y.S. ; A cross-cultural analysis of Jammu, Kashmir and Ladakh (India) medicinal plant use ; Journal of Ethnopharmacology , ( 2014 ); 155 ( 2 ): 925 – 986 . Google Scholar CrossRef Search ADS PubMed 4 Zhang , Y. , Jin , L. , Chen , Q. , Wu , Z. , Dong , Y. , Han , L. , et al. . ; Hypoglycemic activity evaluation and chemical study on hollyhock flowers ; Fitoterapia , ( 2015 ); 102 : 7 – 14 . Google Scholar CrossRef Search ADS PubMed 5 Matławska , I. , Sikorska , M. , Bylka , W. ; Flavonoid compounds in Lavatera thuringiaca L. (Malvaceae) flowers ; Acta Poloniae Pharmaceutica—Drug Research , ( 1999 ); 56 ( 6 ): 453 – 458 . 6 Abdel-salam , N.A. , Ghazy , N.M. , Sallam , S.M. , et al. . ; Flavonoids of Alcea rosea L. and their immune stimulant, antioxidant and cytotoxic activities on hepatocellular carcinoma HepG-2 cell line ; Natural Product Research , ( 2017 ); 1 – 5 . 7 Dzido , T.H. , Soczewiński , E. , Gudej , J. ; Computer-aided optimization of high-performance liquid chromatographic analysis of flavonoids from some species of the genus Althaea ; Journal of Chromatography A , ( 1991 ); 550 ( C ): 71 – 76 . Google Scholar CrossRef Search ADS 8 Gudej , J. , Bieganowska , M.L. ; Chromatographic investigations of flavonoid compounds in the leaves and flowers of some species of the genus Althaea ; Chromatographia , ( 1990 ); 30 ( 5–6 ): 333 – 336 . Google Scholar CrossRef Search ADS 9 Abou-Donia , A.H. , Toaima , S.M. , Hammoda , H.M. , Shawky , E. ; New rapid validated HPTLC method for the determination of galanthamine in Amaryllidaceae plant extracts ; Phytochemical Analysis , ( 2008 ); 19 ( 4 ): 353 – 358 . Google Scholar CrossRef Search ADS PubMed 10 Shewiyo , D.H. , Kaale , E. , Risha , P.G. , Dejaegher , B. , Smeyers-Verbeke , J. , Vander Heyden , Y. ; HPTLC methods to assay active ingredients in pharmaceutical formulations: a review of the method development and validation steps ; Journal of Pharmaceutical and Biomedical Analysis , ( 2012 ); 66 : 11 – 23 . Google Scholar CrossRef Search ADS PubMed 11 Tayade , N.G. , Nagarsenker , M.S. ; Validated HPTLC method of analysis for artemether and its formulations ; Journal of Pharmaceutical and Biomedical Analysis , ( 2007 ); 43 ( 3 ): 839 – 844 . Google Scholar CrossRef Search ADS PubMed 12 Ich . ICH Topic Q2 (R1) Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization, ( 2005 ) 1994 (November 1996), 17. 13 ICH . Guidance for industry: Q2B validation of analytical procedures: methodology. International conference on harmonisation of technical requirements for registration tripartite guideline, ( 1996 ) (November), 13. 14 Rathore , A.S. , Sathiyanarayanan , L. , Mahadik , K.R. ; Development of Validated HPLC and HPTLC Methods for Simultaneous Determination of Levocetirizine Dihydrochloride and Montelukast Sodium in Bulk Drug and Pharmaceutical Dosage Form ; Pharmaceutica Analytica Acta , ( 2010 ); 1 : 106 . Google Scholar CrossRef Search ADS 15 Kamal , A. , Singh , M. , Ahmad , F.J. , Saleem , K. , Ahmad , S. ; A validated HPTLC method for the quantification of podophyllotoxin in Podophyllum hexandrum and etoposide in marketed formulation ; Arabian Journal of Chemistry , ( 2017 ); 10 : S2539 – S2546 . Google Scholar CrossRef Search ADS 16 Aboul-Ela , M.A. , El-Lakany , A.M. , Eldin , S.M.S. , Hammoda , H.M. ; A new validated HPTLC method for quantitative determination of 1, 5-dicaffeoylquinic acid in Inula crithmoides roots ; Pakistan Journal of Pharmaceutical Sciences , ( 2012 ); 25 ( 4 ): 721 – 725 . Google Scholar PubMed 17 Eh , A.L.S. , Teoh , S.G. ; Novel modified ultrasonication technique for the extraction of lycopene from tomatoes ; Ultrasonics Sonochemistry , ( 2012 ); 19 ( 1 ): 151 – 159 . Google Scholar CrossRef Search ADS PubMed 18 Shawky , E. ; Determination of synephrine and octopamine in bitter orange peel by HPTLC with densitometry ; Journal of Chromatographic Science , ( 2014 ); 52 ( 8 ): 899 – 904 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Validated HPTLC Method for Dihydrokaempferol-4′-O-glucopyranoside Quantitative Determination in Alcea Species

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Abstract

Abstract Dihydrokaempferol-4′-O-glucopyranoside, a flavanonol glucoside, is the major compound in the flower of Alcea rosea L. which possesses significant antioxidant and anticancer activity against HepG-2 cell line and thus can be considered a marker compound for A. rosea L. We attempted to establish a new simple, validated high-performance thin-layer chromatographic (HPTLC) method for the quantitation of dihydrokaempferol-4′-O-glucopyranoside to help in the standardization of the hydroalcoholic extracts of A. rosea L. flowers and to evaluate the best method for its extraction from the plant material. The separation was carried out on an HPTLC aluminum plate pre-coated with silica gel 60F-254, eluted with ethyl acetate–methanol–water–acetic acid (30:5:4:0.15 v/v). Densitometric scanning was performed using a Camag TLC scanner III, at 295 nm. A linear relationship was obtained between the concentrations (0.9–3.6 mg) and peak areas with the correlation coefficient (r) of 0.9971 ± 0.0002. The percentage relative standard deviations of intra-day and inter-day precisions were 0.22–1.45 and 0.49–1.66, respectively. The percentage w/w of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. after maceration and sonication for 15 min was found to be 0.733 g/100 g and 0.928 g/100 g, respectively. Introduction Genus Alcea belongs to family Malvaceae; it includes ~60 species primarily of East Mediterranean region. Alcea rosea L. is widely cultivated in gardens of Egypt as an ornamental plant and is known as garden Hollyhock (English name) and in Arabic as Khatma wardi (Khatmi wardi) (1). All parts of the plant have been used in traditional medicin. In India, the flower has been used in the treatment of cough, asthma, throat infection, urinary irritation, boils, kidney pain and jaundice. The root is also used in the treatment of urinary tract burning sensation, dandruff, dermatitis, easy delivery, goiter, gynecological disorders and kidney inflammation. Mucilage (prepared by extracting the raw mucilage by hot extraction method of the plant) is used for prophylaxis and therapy of the respiratory, gastrointestinal tract and urinary complaints. The plant is also used in fever, chest and liver complaints (2, 3). In traditional Chinese medicine, this plant is considered as anti-diabetic ingredient (4). Dihydrokaempferol-4′-O-glucopyranoside was described for the first time in Petunia hybrida and also in the flowers of Althaea officinalis L. and Malva sylvestris L. (5). Dihydrokaempferol-4′-O-glucopyranoside shows significant antioxidant and anticancer activity against HepG-2 cell line with the advantage of being flavonoid glucoside that imparts the compound a good water solubility and hence increases its absorption (6). These findings prompted us to develop a validated high-performance thin-layer chromatography (HPTLC) method for the quantification of the compound in plant extracts and to identify the amount of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. as it can be a commercial source for the production of the compound. It can also serve as a marker compound for A. rosea L. A comparison was drawn between two techniques for the extraction of the compound from A. rosea L.; maceration as a traditional extraction technique and ultrasonication as an advanced extraction technique. The comparison was made to evaluate the effectiveness of sonication as an easy, efficient and time-saving method for the extraction of the compound. View largeDownload slide View largeDownload slide Structure of dihydrokaempferol-4′-O-glucopyranoside HPLC method to quantify dihydrokaempferol-4′-O-glucopyranoside among other constituents in different plants of genus Althaea has been reported (7, 8), but no article related to the HPTLC determination of the compound in plant extracts has been reported in literature or in pharmacopeias. Nowadays, HPTLC is becoming a routine analytical technique as it offers many advantages when compared with other techniques, like HPLC, spectrometric, titrations, etc. One of the major advantages of HPTLC is the ability to analyze several samples simultaneously using a small quantity of mobile phase. This reduces time, cost of the analysis and the possibilities of environmental pollution. Moreover, the plates can be stored for a long time after separation and the detection performed at a later stage to obtain the analytical information. This could be done because the chromatographic development and detection of the separated spots on a plate are generally separate processes in time. It also facilitates scanning in situ and repeated detection (scanning) of the chromatogram with the same or different parameters (9, 10, 11). The aim of the present work is to develop accurate, specific, repeatable and robust HPTLC method for the determination of dihydrokaempferol-4′-O-glucopyranoside in Alcea extracts. Experimental Isolation of dihydrokaempferol-4′-O-glucopyranoside Dihydrokaempferol-4′-O-glucopyranoside was isolated in a previous work (6) from the ethyl acetate fraction of the hydroalcoholic extract of A. rosea L. flowers by evaporation then direct crystallization from methanol. The isolated compound showed a single spot in TLC using the solvent system ethyl acetate–methanol–water–acetic acid (30:5:4:0.15 v/v) and the compound was characterized based on various spectral analysis and reported results in previous work (6). Preparation of standard solutions About 4.5 mg of standard dihydrokaempferol-4′-O-glucopyranoside was accurately weighed, quantitatively transferred into a 10-mL volumetric flask, dissolved in methanol and the volume was adjusted with the same solvent. Preparation of sample solutions About 4 g dried whole flowers of A. rosea L. were exhaustively extracted using 250 mL methanol by maceration for 10 days (Sample A) and 4 g dried whole flowers were extracted using 150 mL methanol by ultrasonication for 15 min (Sample B). The solvent was evaporated producing the crude extracts. The crude extracts were re-dissolved in methanol, filtered, transferred quantitatively to a 50-mL volumetric flask, adjusted to volume with methanol and shaken to mix thoroughly to give samples A and B, respectively. Chromatographic parameters and conditions The chromatographic estimation was performed by spotting standard and sample solutions on pre-coated silica gel aluminum plate 60F-254 (20 cm × 10 cm with 250 μm thickness, E. Merck, Darmstadt, Germany) using a Camag Linomat IV sample applicator (Camag, Muttenz, Switzerland) and a 100 μL Hamilton syringe. The samples, in the form of bands of length 6 mm, were spotted 15 mm from the bottom, 10 mm from left margin of the plate and 4 mm apart, at a constant application rate of 15 s/μL using nitrogen aspirator. Plates were developed using a mobile phase consisting of ethyl acetate–methanol–water–acetic acid (30:5:4:0.15, v/v/v/v). The volumes applied for routine analysis were triplicate 2.0 , 4.0 and 6.0 μL of the TLC dihydrokaempferol-4′-O-glucopyranoside standard (0.9, 1.8, 2.7 μg) and triplicate 4 μL aliquots of sample solution. Linear ascending development was carried out in 20 cm × 20 cm twin-trough glass chamber (Camag Muttenz, Switzerland) equilibrated with mobile phase. About 25 mL of mobile phase (system III) was used for development and the optimized chamber saturation time for mobile phase was 30 min at room temperature. The length of chromatogram run was 7 cm. The development time was 11 min. After development, the plates were air-dried for 10 min. Densitometric scanning was performed on Camag TLC scanner III in the reflectance-absorbance mode at λ 295 nm and operated by WINCATS software (V. 3.1). The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm. The slit dimension was kept at 6 mm × 0.1 mm. Concentrations of the samples and standards chromatographed were determined from the intensity of diffusely reflected light. Evaluation was via peak areas with linear regression. Calibration curve construction As recommended by the International Committee on Harmonization guidelines (12, 13), a calibration curve was established using five analyte concentrations (2.0, 3.0, 4.0, 6.0 and 8 μl/band) of the TLC standard applied in triplicate, representing 0.9–3.6 μg of dihydrokaempferol-4′-O-glucopyranoside. For routine analytical procedures, a three-point calibration curve within this range was used, produced by applying duplicate 2.0, 4.0 and 6.0 μL (0.9, 1.8 and 2.7 μg) of the HPTLC standard on each plate. Instrumentation and reagent Instrumentation Sample solutions for HPTLC analyses were applied by means of Camag Linomat IV sample applicator (Camag, Muttenz, Switzerland) and a 100 μL Hamilton syringe. Linear ascending development was carried out in 20 cm × 20 cm twin-trough glass chamber (Camag Muttenz, Switzerland). Zones were quantified by scanning at 295 nm with a Camag TLC Scanner 3 with a deuterium source in the reflection mode; slit dimension settings of length 6 and width 0.1, monochromator bandwidth of 20 nm, and a scanning rate of 10 mm/s operated by WINCATS software (V. 3.1). Solvents and reagents HPTLC analyses were performed on plates pre-coated with silica gel 60F-254 (20 × 10 cm; 0.25 mm layer thickness) plates that were obtained from Merck (Darmstadt, Germany). All the reagents used in the experiment were of analytical grade and were supplied by Merck (Darmstadt, Germany) and El-Nasr Chemicals Ltd (Egypt). Plant material A. rosea L. flowers were collected from El-Shalalat Park, Alexandria, Egypt, in March 2016 at the flowering stage. A voucher sample is deposited in the department of Pharmacognosy, Faculty of Pharmacy, Alexandria, Egypt. Results Validation of HPTLC method Selectivity It is the ability of an analytical method to unequivocally assess the analyte in the presence of other components (impurities, degradents and excipients) (14). The scan-densitogram obtained from a representative sample (Figure 1) showed selective baseline separation between dihydrokaempferol-4′-O-glucopyranoside and other components in the sample. Figure 1. View largeDownload slide In situ absorption spectra obtained from the band of standard dihydrokaempferol-4′-O-glucopyranoside and the corresponding band obtained from the flower extract of Alcea rosea L. Figure 1. View largeDownload slide In situ absorption spectra obtained from the band of standard dihydrokaempferol-4′-O-glucopyranoside and the corresponding band obtained from the flower extract of Alcea rosea L. Linearity The relationship between the peak areas and the amount of substance applied showed linearity over the range 0.9–3.6 μg/spot. The regression equation data, correlation coefficient (r-value) and other statistical parameters are listed in Table I. The three-point calibration was repeated many times and was also found to have a linear regression correlation coefficient of 0.9971. Table I. Linear Regression Data for the Calibration Curve of Dihydrokaempferol-4′-O-Glucopyranoside (n = 5) Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Table I. Linear Regression Data for the Calibration Curve of Dihydrokaempferol-4′-O-Glucopyranoside (n = 5) Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Dihydrokaempferol-4′-O-glucopyranoside Linearity range (μg/spot) 0.9–3.6 Intercept (a ± SD) 11258 ± 14.95 Slope (b ± SD) 2165.7 ± 217.6 Correlation coefficient (r ± SD) 0.9971 ± 0.000197 Sa standard deviation of x 1.083743512 Sb standard deviation of y 2353.802 Standard error 205.0697 LOD (μg/spot) 0.312 LOQ (μg/spot) 0.94689 Range The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analyte in the sample for which it has been demonstrated that the analytical method has suitable levels of precision, accuracy and linearity. The proposed method was applied for the determination of Dihydrokaempferol-4′-O-glucopyranoside in the concentration range of 50–150% of the working concentrations. The results are shown in Table I. Limit of detection and limit of quantitation Detection limit (DL) is the lowest concentration of analyte in a sample that can be detected, but not necessarily quantitated, under the stated experimental conditions. Quantitation limit (QL) is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions. According to the ICH (international conference on harmonization of drugs) guidelines, the DL may be expressed as: DL = 3.3 σ/S, while the QL may be expressed as: QL = 10 σ/S where σ = the standard deviation of the response and S = the slope of the calibration curve. The slope S is estimated from the calibration curve. The estimate of σ was carried out by studying the calibration curves where the standard deviations of y-intercepts of regression lines were used as the standard deviation. Limit of detection (LOD) and limit of quantitation (LOQ) were experimentally verified by diluting known concentrations of standard dihydrokaempferol-4′-O-glucopyranoside solutions. The results are shown in Table I. Precision The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree (15). In accordance with the ICH guidelines, precision was determined by independent repeated analysis of five different dihydrokaempferol-4′-O-glucopyranoside standard solutions and repeated analysis of a homogenous sample (3 μg/spot) by the use of the same equipment and the same analytical procedure in the same laboratory and on the same plate. Intermediate precision was performed by the analysis of six different concentrations of the standard solution, each repeated three times in the same day for intra-day precision and on different days for inter-day precision (13, 16). The percent relative standard deviation (RSD%) was calculated, and the results are shown in Tables II and III. The results shown in the tables meet the acceptance criterion for RSD% specified by the ICH which is a precision of <2%. Table II. Precision of the HPTLC Method for the Determination of Dihydrokaempferol-4′-O-Glucopyranoside (Repeatability) Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Table II. Precision of the HPTLC Method for the Determination of Dihydrokaempferol-4′-O-Glucopyranoside (Repeatability) Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Experiment number Intra-day precision Inter-day precision Concentration (μg/spot) Mean ± SD RSD % Concentration (μg/spot) Mean ± SD RSD% 1 0.893 0.891 0.889 0.891 ± 0.002 0.22 0.86 0.87 0.88 0.87 ± 0.012 1.35 2 1.295 1.285 1.299 1.29 ± 0.007 0.56 1.359 1.393 1.4 1.384 ± 0.022 1.603 3 1.765 1.763 1.797 1.77 ± 0.019 1.07 1.89 1.86 1.836 1.866 ± 0.031 1.66 4 2.68 2.699 2.72 2.7 ± 0.02 0.75 2.81 2.8 2.73 2.778 ± 0.044 1.599 5 3.598 3.703 3.637 3.65 ± 0.05 1.45 3.65 3.64 3.67 3.653 ± 0.018 0.492 Table III. Results of Replicate Analysis of a Homogenous Sample by HPTLC Method Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Table III. Results of Replicate Analysis of a Homogenous Sample by HPTLC Method Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Measurement Concentration (μg/spot) 1 3.05 2 3.019 3 3.02 4 3.02 5 2.95 6 2.95 Mean 3.055 SD 0.043 RSD% 1.432 Accuracy and recovery The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. Recovery is expressed as the amount/weight of the compound of interest analyzed as a percentage to the theoretical amount present in the medium. The accuracy of the method was validated by a standard addition analysis. The sample solutions were spiked with three different, known, concentrations of dihydrokaempferol-4′-O-glucopyranoside corresponding to 34, 102 and 170% of calculated concentration had been added. The three fortified sample solutions (mix.1, 2 and 3) were analyzed on the same plate by application of triplicate 4.0 μL volumes, respectively, in addition to the three standards described earlier for routine analyses which were applied in triplicate. The difference between the expected concentrations and the found ones was calculated to determine the accuracy of the method. The results of the experiments are presented in Table IV. Table IV. Results of the Standard Addition Experiments Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 aEach concentration is the average of three determinations. Table IV. Results of the Standard Addition Experiments Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 Concentration of dihydrokaempferol-4′-O-glucopyranoside in sample Concentration of dihydrokaempferol-4′-O-glucopyranoside added Concentration of dihydrokaempferol-4′-O-glucopyranoside found in mixturea Recovery (%) ± SD RSD% 1 0.733 0.25 0.948 96.45 ± 0.017 1.8 2 0.733 0.75 1.4197 95.73 ± 0.014 1.46 3 0.733 1.25 1.981 99.89 ± 0.003 0.33 aEach concentration is the average of three determinations. Robustness Robustness tests examine the effect of the operational parameters on the analysis results. ICH defines robustness as a measure of the method′s capability to remain unaffected by small, but deliberate variations in method parameters (13). By introducing small changes in the mobile phase composition, the effects on the results were examined. Mobile phases having different composition like (ethyl acetate:methanol:water:acetic acid 3:5:4:(three drops), 30:5:5:(six drops) and 25:5:4:(three drops)) were tried. Time from spotting to chromatography and from chromatography to scanning was varied from 1, 2 and 24 h. Robustness of the method was carried out at three concentration levels 1, 2 and 3 μg/spot and the results are represented in Table V. Table V. Robustness Testing Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 aAverage of three concentrations 1, 2 and 3 μg/spot. Table V. Robustness Testing Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 Parameter SDa RSD %a Mobile phase composition 181.65 1.93 Time from spotting to chromatography 89.07 0.676 Time from chromatography to scanning 178.19 1.514 aAverage of three concentrations 1, 2 and 3 μg/spot. Quantification of dihydrokaempferol-4′-O-glucopyranoside in Alcea rosea L. Quantification of dihydrokaempferol-4′-O-glucopyranoside was performed according to the procedure described in the ‘Chromatographic parameter and conditions’ section. The HPTLC profile of the two samples (A and B) at 295 nm showed acceptable separation of dihydrokaempferol-4′-O-glucopyranoside from the other components in the samples under the specified conditions (Figure 2). The amount of dihydrokaempferol-4′-O-glucopyranoside in the extracts were determined from the calibration graphs and the percentage w/w of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. after maceration and sonication for 15 min was found to be 0.733 g/100 g and 0.928 g/100 g, respectively, which is comparable to the amount present in different plants of genus Althaea reported in literature (0.839–1.934 g/100 g) (8). Figure 2. View largeDownload slide HPTLC scan-densitogram showing the separation of dihydrokaempferol-4′-O-glucopyranoside (6) from other components in the sample at 295 nm. Figure 2. View largeDownload slide HPTLC scan-densitogram showing the separation of dihydrokaempferol-4′-O-glucopyranoside (6) from other components in the sample at 295 nm. Discussion Method optimization Experimental conditions, such as mobile phase composition, scan mode, scan speed and wavelength of detection were optimized to provide accurate and precise results. Development with the mobile phase described above on the HPTLC silica gel layers produced compact, flat, dark bands of dihydrokaempferol-4′-O-glucopyranoside (Rf 0.58) when viewed under a 295 nm UV light. The dihydrokaempferol-4′-O-glucopyranoside band showed base peak separation from other spots in the sample as shown in Figure 2. Sample extraction Extraction of dihydrokaempferol-4′-O-glucopyranoside from the flowers of A. rosea L. was performed using two different extraction techniques; maceration as a traditional extraction technique and ultrasonication as an advanced one. The extracts prepared by maceration and ultrasonication were compared with identify the best method for the extraction of the compound from A. rosea L. It is obvious from the data that ultrasonication is a more efficient extraction technique for the compound from Alcea rosea L. The mechanical effect of ultrasound provides a greater penetration of solvent into the cellular materials and result in the disruption of biological cell walls to facilitate the release of contents. The advantages of ultrasonic extraction are increase in both the extraction yield and extraction rate, resulting in reduced extraction time and a higher throughput (17). Method validation It has been demonstrated above that validation data for the new quantitative HPTLC method meet the acceptance criteria for accuracy, precision, linearity, detection and quantification limits set by the International Conference on Harmonization (12, 13). The method specificity was assessed by performing a peak purity test of dihydrokaempferol-4′-O-glucopyranoside by comparing the UV absorption spectrum of the standard compound with the corresponding band in the sample (Figure 1). No interference was observed regarding the densitograms of the sample, confirming the selectivity of the method (Figure 2) (18). Conclusion It has been demonstrated above that validation data for the new quantitative HPTLC method meet the acceptance criteria for accuracy, precision, linearity, detection and quantification limits set by the International Conference on Harmonization. The percentage w/w of dihydrokaempferol-4′-O-glucopyranoside in the flowers of A. rosea L. after maceration and sonication for 15 min was found to be 0.733 g/100 g and 0.928 g/100 g, respectively, which is comparable to the amount present in different plants of genus Althaea reported in literature (0.839–1.934 g/100 g). The described method is suitable for routine use for determination of the compound in the plant samples. Compared with HPLC, the proposed method is simpler and faster as up to six samples (applied in duplicate singly with a minimum of three standard concentrations) can be analyzed simultaneously on each plate rather than performing sequential injection of the samples and standards in HPLC. Only 15 mL of mobile phase is required in the chamber trough containing the plate for development, and an additional 10 mL for vapor saturation in the other trough, thus minimizing the cost for solvent purchase and disposal. The processing of samples and standards together at the same time (in-system calibration) leads to improved reproducibility and accuracy. Acknowledgments We wish to thank the Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University for the financial support. References 1 Shaheen , N. , Khan , M.A. , Yasmin , G. , Hayat , M.Q. , Munsif , S. , Ahmad , K. ; Foliar epidermal anatomy and pollen morphology of the genera Alcea and Althaea (Malvaceae) from Pakistan ; International Journal of Agriculture and Biology , ( 2010 ); 12 ( 3 ): 329 – 334 . 2 Jain , S.K. ; Ethnobotany and research on medicinal plants in India ; Ciba Foundation symposium [Internet] , ( 1994 ); 185 : 153 – 64-8 . Available from http://www.ncbi.nlm.nih.gov/pubmed/7736852. 3 Gairola , S. , Sharma , J. , Bedi , Y.S. ; A cross-cultural analysis of Jammu, Kashmir and Ladakh (India) medicinal plant use ; Journal of Ethnopharmacology , ( 2014 ); 155 ( 2 ): 925 – 986 . Google Scholar CrossRef Search ADS PubMed 4 Zhang , Y. , Jin , L. , Chen , Q. , Wu , Z. , Dong , Y. , Han , L. , et al. . ; Hypoglycemic activity evaluation and chemical study on hollyhock flowers ; Fitoterapia , ( 2015 ); 102 : 7 – 14 . Google Scholar CrossRef Search ADS PubMed 5 Matławska , I. , Sikorska , M. , Bylka , W. ; Flavonoid compounds in Lavatera thuringiaca L. (Malvaceae) flowers ; Acta Poloniae Pharmaceutica—Drug Research , ( 1999 ); 56 ( 6 ): 453 – 458 . 6 Abdel-salam , N.A. , Ghazy , N.M. , Sallam , S.M. , et al. . ; Flavonoids of Alcea rosea L. and their immune stimulant, antioxidant and cytotoxic activities on hepatocellular carcinoma HepG-2 cell line ; Natural Product Research , ( 2017 ); 1 – 5 . 7 Dzido , T.H. , Soczewiński , E. , Gudej , J. ; Computer-aided optimization of high-performance liquid chromatographic analysis of flavonoids from some species of the genus Althaea ; Journal of Chromatography A , ( 1991 ); 550 ( C ): 71 – 76 . Google Scholar CrossRef Search ADS 8 Gudej , J. , Bieganowska , M.L. ; Chromatographic investigations of flavonoid compounds in the leaves and flowers of some species of the genus Althaea ; Chromatographia , ( 1990 ); 30 ( 5–6 ): 333 – 336 . Google Scholar CrossRef Search ADS 9 Abou-Donia , A.H. , Toaima , S.M. , Hammoda , H.M. , Shawky , E. ; New rapid validated HPTLC method for the determination of galanthamine in Amaryllidaceae plant extracts ; Phytochemical Analysis , ( 2008 ); 19 ( 4 ): 353 – 358 . Google Scholar CrossRef Search ADS PubMed 10 Shewiyo , D.H. , Kaale , E. , Risha , P.G. , Dejaegher , B. , Smeyers-Verbeke , J. , Vander Heyden , Y. ; HPTLC methods to assay active ingredients in pharmaceutical formulations: a review of the method development and validation steps ; Journal of Pharmaceutical and Biomedical Analysis , ( 2012 ); 66 : 11 – 23 . Google Scholar CrossRef Search ADS PubMed 11 Tayade , N.G. , Nagarsenker , M.S. ; Validated HPTLC method of analysis for artemether and its formulations ; Journal of Pharmaceutical and Biomedical Analysis , ( 2007 ); 43 ( 3 ): 839 – 844 . Google Scholar CrossRef Search ADS PubMed 12 Ich . ICH Topic Q2 (R1) Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization, ( 2005 ) 1994 (November 1996), 17. 13 ICH . Guidance for industry: Q2B validation of analytical procedures: methodology. International conference on harmonisation of technical requirements for registration tripartite guideline, ( 1996 ) (November), 13. 14 Rathore , A.S. , Sathiyanarayanan , L. , Mahadik , K.R. ; Development of Validated HPLC and HPTLC Methods for Simultaneous Determination of Levocetirizine Dihydrochloride and Montelukast Sodium in Bulk Drug and Pharmaceutical Dosage Form ; Pharmaceutica Analytica Acta , ( 2010 ); 1 : 106 . Google Scholar CrossRef Search ADS 15 Kamal , A. , Singh , M. , Ahmad , F.J. , Saleem , K. , Ahmad , S. ; A validated HPTLC method for the quantification of podophyllotoxin in Podophyllum hexandrum and etoposide in marketed formulation ; Arabian Journal of Chemistry , ( 2017 ); 10 : S2539 – S2546 . Google Scholar CrossRef Search ADS 16 Aboul-Ela , M.A. , El-Lakany , A.M. , Eldin , S.M.S. , Hammoda , H.M. ; A new validated HPTLC method for quantitative determination of 1, 5-dicaffeoylquinic acid in Inula crithmoides roots ; Pakistan Journal of Pharmaceutical Sciences , ( 2012 ); 25 ( 4 ): 721 – 725 . Google Scholar PubMed 17 Eh , A.L.S. , Teoh , S.G. ; Novel modified ultrasonication technique for the extraction of lycopene from tomatoes ; Ultrasonics Sonochemistry , ( 2012 ); 19 ( 1 ): 151 – 159 . Google Scholar CrossRef Search ADS PubMed 18 Shawky , E. ; Determination of synephrine and octopamine in bitter orange peel by HPTLC with densitometry ; Journal of Chromatographic Science , ( 2014 ); 52 ( 8 ): 899 – 904 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. 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Journal of Chromatographic ScienceOxford University Press

Published: Apr 4, 2018

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