Vol-3, Issue-4, Suppl-1, Nov 2012 ISSN: Ghodasara et al PHARMA SCIENCE MONITOR
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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES HPTLC METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF DIOSGENIN AND GALLIC ACID IN MARKETED FORMULATION Ghodasara T*, Gaudani R, Nayak B and Shingala D Shree Swaminarayan Sanskar Pharmacy College, At & post Zundal, Ta. Dist. Gandhinagar ABSTRACT The stationary phase pre-coated silicagel 60 F254 plate was selected for separation and the sample was developed using a mixture of Toluene: Ethyl acetate: Formic acidin the ratio 6:5:1 v/v as mobile phase. Quantification was carried out densitometricallyat 425 nm for Diosgenin and 254 nm for Gallic acid. The Rf value of Diosgenin and Gallic acid was found to be 0.8 ± 0.2 and 0.45 ±0.1 respectively. Linearity was found to be in the concentration range of 400 to 2400 ng/spot of Diosgenin and 100 to 600 for Gallic acid the correlation coefficient value is and The results of analysis were validated in terms of accuracy and precision. The LOD was found to be 49.45ng and 34.83ng of Diosgenin and Gallic acid respectively. LOQ was found to be ng and ng/spot of Diosgenin and Gallic acidrespectively. The proposed HPTLC method provides a faster and cost effective quantitative control for routine analysis of Diosgenin and Gallic acid. Keywords: Simultaneous estimation, Diosgenin, Gallic acid, validation. INTRODUCTION For Past few decades compounds from natural sources have been gaining importance because of the vast chemical diversity that they offer. This has led to phenomenal increase in the demand for herbal medicines in the last two decades and a need has been felt for ensuring the quality, safety and efficacy of herbal drugs. Phytochemical evaluation is one of the tools for the quality assessment, which includes preliminary phytochemical screening; chemo profiling and marker compound analysis using modern analytical techniques. In the last two decades (HPTLC) has emerged as an important tool for the qualitative semi-quantitative and quantitative phytochemical analysis of herbal drugs and formulations. This includes developing TLC fingerprint profiles and estimation of chemical markers and biomarkers. The major advantage of HPTLC is that several samples can be analyzed simultaneously using a small quantity of mobile phase [1,2]. IC Value
2 Diosgenin present in Fruits of Tribulus terrestris Linn. Family: Zygophyllaceae [3] and Gallic acid presents in dried rhizomes of Bergenia ligulata wall. Family: Saxifragaceae [4]. Both the herb is important plant in the treatment of renal disorders including kidney stone, calculations, cystitis, and nephritis [5,6]. Effective formulations are present in market for both plant extracts individually and in combination. Literature survey showed no HPTLC method present for the simultaneous estimation of Diosgenin and Gallic acid in marketed formulation. However, individual analytical methods have so far been reported for its determination [1,7]. Most of these methods are not precisely validated. Hence this paper reports a simple, precise, rapid and cost effective HPTLC method for the estimation of Diosgenin and Gallic acid. MATERIALS AND METHODS: Apparatus: A CAMAG TLC system comprising of a Linomat V automatic sample spotter and CAMAG TLC scanner and single pan balance of Shimadzu model was used, for the present study. Chemicals: Analytical grade Toluene, Ethyl acetate, Formic acid, was obtained from Rankem Pvt. Ltd. Stationary phase used was HPTLC plates: cm, 0.2 mm thickness pre-coated with silica gel 60 F254; Merck KgaA (Darmstadt, Germany). Diosgenin and Gallic acid are collected from Eucca Enterprise. Sample Preparation: Stosun tablet-a polyherbal formulation was taken for quantitative estimation of biomarkers like Diosgenin and Gallic acid. 20 tablets were weighed and the total weigh was determined. The capsule granules are subjected to fine powder with the help of mortar and pestle. A 5 gmpowder was refluxed with 50 ml methanol and filtered while hot. Filtrate was evaporated to get 1 mg of extract, further diluted to 10 ml to get concentration of 100mg/ml. A stock solution of Gallic acid was prepared by dissolving 5 mg of accurately weighed Gallic acid in methanol and the volume was made up to 50 ml with methanol to get the final concentration of 0.1mg/ml and a stock solution of Diosgenin was prepared by dissolving 4 mg of accurately weighed diosgenin in methanol and the volume was made up to10 ml with methanol to get the final concentration of 0.4 mg/ml. HPTLC method for estimation of Diosgenin and Gallic acid: IC Value
3 Preparation of calibration curve for Diosgenin and Gallic acid: 1-6 µl of each of the standard solution of Diosgeninand Gallic acid were applied in triplicate on TLC plates. The plates was developed in a solvent system of toluene: ethyl acetate: formic acid (6: 5: 1) at 25 ±20ºC temperature and 40% relative humidity and allowed to travel up to a distance of 8 cm. After development the plates were dried in air and scanned densitometrically at 425 nm for Diosgenin and 254 nm for Gallic acid. The peak areas were recorded. A calibration curve of were prepared by plotting peak areas vs concentration. (Fig. 1, 2) Figure 1 Calibration Graph for Diosgenin Calibration curve for standard Diosgenin (conc. vs peak area) *Correlation coefficient: 0.999, Slope: Intercept: TABLE 1: CALIBRATION CURVE DATA FOR DIOSGENIN AND GALLIC ACID (CONCENTRATION VS PEAK AREA) Conc. of Diosgenin (ng/spot) R f Peak area ± SD (n=3) % CV ± ± ± ± ± ± IC Value
4 Conc. of Gallic acid (ng/spot) R f Peak area ± SD % CV (n=3) ± ± ± ± ± ± *Values are expressed as mean ± SD (n=3) Figure 2 Calibration Graph for Gallic acid Calibration curve for standard Gallic acid (conc. vs peak area) *Correlation coefficient: 0.998, Slope: Intercept: 7526 Method Specification: Different concentration (10 µl) of Stosun powder and tablet was applied on HPTLC plate for estimation of Diosgenin and Gallic acid in Stosun tablet. The HPTLC plates was developed in a solvent system Toluene: Ethyl acetate: Formic acid (6: 5: 1) and dried in air and scanned densitometrically at 425 nm for Diosgenin and 254 nm for Gallic acid. The peak areas were recorded. Stosun tablet shows the peak area ± 58.9for Diosgenin and ± 82.8for Gallic acid. The concentration of Diosgeninis 0.42 ± IC Value
5 0.0041% and Gallic acid is 0.24 ± % in methanolic extract of Stosun tablet. Relative standard deviations (% CV) for Diosgeninand Gallic acid are 0.96 and 0.97 respectively for Stosun tablet. (Table 2) The identity of the bands in the sample extracts were confirmed by comparing the R f and the absorption spectra by overlaying their UV absorption spectra with those of their respective standards. The purity of the bands due to Gallic acid and Diosgenin in the sample extracts were confirmed by overlaying the absorption spectra recorded at start, middle and end position of the band in the sample tracks. TABLE 2: ESTIMATION OF DIOSGENIN AND GALLIC ACID IN STOSUN TABLET BY HPTLC ANALYSIS Formulation Concentration of Formulation (µg/ml) Tablet 100 Formulation Concentration of formulation (µg/ml) Mean peak area±sd (n=3) ± 58.9 Mean peak area±sd (n=3) Average amount of Diosgenin (ng/spot) in Formulation Average % w/w ± SD Diosgenin in Formulation %CV 422 ± ± Average amount of Gallic acid (ng/spot) in formulation ± 2.43 Average % w/w ± SD Gallic acid in formulation %CV Tablet ± ± 82.8 *Values are expressed as mean ± SD (n=3) Validation of Method: The developed method was validated in terms of Linearity, Accuracy, Precision, Limit of detection, Limit of quantification [8,9]. RESULT AND DISCUSSION A solvent system that would give dense and compact spots with significant Rf values was desired for quantification of Diosgenin and Gallic acid in Stosun tablet. The mobile phase consists of Toluene: ethyl acetate: Formic acid (6:5:1: v/v) gave Rf values of 0.8 ± 0.2 and 0.45±0.1 for Diosgenin and Gallic acid respectively (Table 1). The linear regression IC Value
6 data (n=5, Table 1) showed a good linear relationship over a concentration range of ng/spot for and ng/spot for Diosgenin and Gallic acid respectively. Standard Diosgenin and Gallic acid showed single peak in HPTLC chromatogram (Fig. 3, 5). The HPTLC chromatogram of Stosun tablet with Gallic acid and Diosgenin showed marker peak (Fig. 4, 6). The calibration curve of Diosgenin and Gallic acid were obtained by spotting standard Diosgenin and Gallic acid on HPTLC plate. After development the plate was scanned at 425 nm and 254 nm respectively. The calibration curve was prepared by plotting the concentration of Diosgenin versus average area of the peak (Fig.1). Stosun tablet extract were analyzed by the proposed method. The amount of Diosgenin and Gallic acid were computed from calibration curve and calibration curve were shown in Fig. 1 and 2. The observed percentage recoveries were 99.72% and 99.76% for Diosgenin and Gallic acid respectively which shows that the method is free from interference from excipient present in the formulation, as given in table 3. TABLE 3: RECOVERY STUDY OF MARKER COMPOUND IN STOSUN TABLET BY PROPOSED HPTLC METHOD. Biomarker compound Amount present in the sample (ng) Peak area Amount added (ng) Amount found (ng) Recovery (%) Gallic acid ± Average recovery (%) ± ± Diosgenin ± ± ± IC Value
7 Figure 3 HPTLC chromatogram for Gallic acid Figure 4 HPTLC chromatogram for Gallic acid in Stosun tablet Figure 5 HPTLC chromatogram for Diosgenin Figure 6 HPTLC chromatogram for Diosgenin in Stosun tablet CONCLUSION There is significant difference between the RF values of the Diosgenin and Gallic acid therefore this analytical method can be utilizes for the simultaneous estimation of this substances. The extraction procedure utilized for the crude drugs has to be optimized. Because industry the large scale extraction of crude drugs were carried out by using commercial solvents which may be aqueous alcohols; if the constituent is non-polar, it will ultimately reduce its yield in the active extracts. In the validation study it is observed that LOD and LOQ determination by using signal to noise ratio is unreliable as compare to its determination by the method involving calculation of RSD within the responses, IC Value
8 however it can provide the guidance for selecting the starting range for the study (Table 4). TABLE 4: SUMMARY OF VALIDATION PARAMETERS FOR GALLIC ACID AND DIOSGENIN Parameters Gallic acid Diosgenin Linearity (correlation-coefficient) Repeatability of measurement Limit of detection 34.83ng/spot 49.45ng/spot Limit of quantification ng/spot ng/spot Accuracy in tablet Range Specificity Specific Specific REFERENCES: 1. Chaudhury RR.Herbal medicine for human health;world Health Organization, Geneva, CBS Publishers and Distributors Limited, New Delhi,1999, pp Raina MK, Quality control of herbal and herbo-mineral formulations. Ind. J.Nat. Product. 2003, 19, Anonyms.Ayurvedic Pharmacopoeia of India; 1st Edn, Government of IndiaMinistry of Health and Family Welfare,2007, Part-I, 1,pp Anonyms. Ayurvedic Pharmacopoeia of India; 1stEdn,Government of IndiaMinistry of Health and Family Welfare,2007, Part-I, 1, pp Anand R, Patnaik GK, Kulshreshtha DK and Dhawan BN, Activity of certain fractions of Tribulusterrestris fruits against experimentally induced urolithiasis in rats. Ind. J.Exp. Bio.1994, 32, Harsoliya MS, Pathan JK, KhanN, Bhatt D and Patel VM, Effect of ethanolic extracts of Bergenialigulata, Nigella sativa and combination on calcium oxalate urolithiasis in rats. Int. J. Drg. Formulation and Res.2011, 2, Anonyms. World Health Organization Guidelines; Validation of analytical procedures used in examination of pharmaceutical materials, 32nd report,aitbs Publisher and Distributors, Delhi, IC Value
9 8. Anonyms. ICH Harmonized Tripartite Guidelines; Validation of analytical procedures, Text and Methodology, 2005, pp Mukherjee PK. Quality control of Herbal drugs. Published by Business Horizons 1st edition, For Correspondence: Ghodasara Trupti IC Value
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