International Journal of ChemTech Research CODEN( USA): IJCRGG ISSN : 0974-4290 Vol.1, No.4, pp 826-831, Oct-Dec 2009 Development and Validation of HPTLC Method for Determination of Glycyrrhizin in Herbal Extract and in Herbal Gel Varsha M. Jadhav*, Uttam S. Kedar, Sachin B. Gholve, Vilasrao J. Kadam Department of Quality Assurance, Bharati Vidyapeeth’s College of Pharmacy, Sector-8, CBD Belapur, Navi Mumbai-400614, India. *Corres.author: drvmjadhav_bvcop@rediffmail.com Tel No. 91-22-27571122, +91 9869046618,Fax No. +91-22-27571182 ABSTRACT: A new, simple, sensitive, selective, precise and robust high-performance thin-layer chromatographic (HPTLC) method for analysis of glycyrrhizin has been developed and validated for the determination of glycyrrhizin in herbal extracts and in herbal gel. The analyte was extracted with ethanol and applied on TLC aluminium plates along with standard using Linomat 5 spray on sample applicator (CAMAG). Analysis of glycyrrhizin was performed on pre-coated TLC aluminium plates with silica gel as the stationary phase and prewashed with methanol. Linear ascending development was carried out in twin trough glass chamber saturated with mobile phase consisting of ethyl acetate-methanol-water-formic acid (15:2:1:1 v/v/v/v). Spectrodensitometric scanning was performed by TLC scanner III (CAMAG) in absorbance mode at the wavelength of 252nm. The system was found to give compact spots for glycyrrhizin (Rf value of 0.34 ± 0.04). The linear regression analysis data for the calibration plots showed good linear relationship with r2=0.9981 in the concentration range 2-15 µl with respect to peak area. According to the International Conference on Harmonization (ICH) guidelines the method was validated for precision, recovery, stability in solution and robustness. The glycyrrhizin content quantified from herbal extracts and from the formulation was found well within limits. Statistical analysis of the data showed that the method is reproducible and selective for the estimation of glycyrrhizin. Keywords: Glycyrrhiza glabra, Glycyrrhizin, HPTLC, Method development and validation, Monoammonium Glycyrrhizinate (MAG), Gel. INTRODUCTION Glycyrrhiza glabra is a vital herb in nature as it has wide therapeutic applications. Glycyrrhizin is main constituent that has anti-inflammatory, antimicrobial, anti ulcer activity. Glycyrrhizin is saponin glycoside and molecular formula is C42H62016. Gel containing glycyrrhizin is effective for mouth ulcer and aphatae. The novel gel formulation contains carbopol, chitosan, PEG, glycerin. 1, 2 Figure 1. Chemical structure of Glycyrrhizin Various analytical methods reported such as HPLC, HPTLC, and Capillary electrophoresis. The present paper describes precise, accurate, sensitive HPTLC method for determination of glycyrrhizin from the extract and formulation3. MATERIALS AND METHODS Materials A CAMAG TLC system comprising of a Linomat-5 applicator and CAMAG TLC III scanner. Stationary phase used was silica gel G60F254, 20x10 cm TLC plate. The Reference standard of Monoammonium Glycyrrhizinate (MAG) was obtained from Amsar Industries Ltd., Indore, India. Methanol, Glacial Acetic acid, Formic acid and Chloroform were used of AR Grade. The plates were developed in a CAMAG twin trough glass chamber (20 x 10 cm) by ascending method. Distance of solvent front 80mm, band length 6mm, slit dimension 5.00 x 0.45 mm and detection wavelength 252 nm were used for the present study. Varsha M. Jadhav et al /Int.J. ChemTech Res.2009,1(4) Method Preparation of Monoammonium Glycyrrhizinate (MAG) Solutions The standard solution was prepared by dissolving 10 mg MAG in 10 ml methanol solution which gives 1000µg/ml. The working standard of 100 µg/ml was prepared from standard solution by diluting with methanol. Different concentrations of 2, 5, 10, 15, 20 µg/ml were prepared from standard solutions. Chromatographic Conditions Analysis was performed on 20 cm × 10 cm HPTLC silica gel G60F254 plates with fluorescent indicator. The plate cleaned by predevelopment to the top with methanol, and dried in an oven 1050C for 5 min. Sample and standard zones were applied to the layer as bands by means of a CAMAG. Linomat 5 automated spray-on applicator equipped with a 100 µl syringe and operated with the settings band length 6 mm, application rate 4 µl/sec, distance between bands 4 mm, distance from the plate side edge 6.5 mm, and distance from the bottom of the plate 2 cm. Calibration Curve 2, 5, 10 and 15µl standard solution of Monoammonium Glycyrrhizinate was applied onto TLC plate to generate Calibration curve. The chromatograms were developed using said chromatographic conditions. The plate was dried in air and kept in hot air oven at 105 °C for 5 min. The standard zones were quantified by linear scanning at 254 nm (Fluorescent) by use of a TLC Scanner III CAMAG with a mercury source4. Analysis of glycyrrhizin in herbal extracts The Glycyrrhiza powder 500mg was taken in conical flask and extracted twice with 10 ml of 70% ethanol, and sonicated for 10 min in ultrasonic bath. The solution was filtered through Whatman filter paper No.44 and the filtrate was used as further analysis.5, 6 Analysis of glycyrrhizin in herbal formulations The weighted amount of gel equivalent to 10mg of glycyrrhizin was mixed with 70% ethanol with vigorous shaking and sonicated for 10 min in ultrasonic bath. The solution was filtered through Whatman filter paper No.44 and the filtrate was used as further analysis. 827 METHOD VALIDATION7, 8: Precision Repeatability: Repeatability of sample application and measurement of peak area was carried out using the three replicates of same spot 1000 ng/spot. Repeatability is also termed intra-assay precision. Intermediate precision: The intra-day and inter-day variations for determination of glycyrrhizin were carried out at three different concentration levels 2, 5, 10 µl/spot. Specificity The specificity of method was ascertained by standard MAG and samples (extracted from powder and extracted from formulation). The spots of diluent ethanol, Placebo of formulation, standard MAG, extracted samples (extracted from powder and extracted from formulation) were spotted on TLC plate in duplicate and run. The spots for glycyrrhizin that eluted were confirmed with R f value of MAG.9,10 Recovery Studies Recovery Study was performed by spiking 20, 40 and 60 % of standard drug externally to the pre analyzed samples. The experiment was conducted in triplicate and applied onto the plate in duplicate. This was conducted to check the recovery of drugs at different levels of formulations.11 Robustness The robustness of method can be performed by allowing variation in sample application time to scanning. RESULT AND DISCUSSION Mobile Phase Development The mixtures of several mobile phases were tried to separate spot of glycyrrhizin from other spots and get stable peak. The solvent system used was ethyl acetate: Glacial acetic acid: Formic acid: water (15:2:1:1 v/v/v/v) was selected for estimation of glycyrrhizin, which gave good resolution. Good chromatogram in Figure 2 was attained with Rf value 0.34 ± 0.04. The wavelength of 252 nm was used for quantification of sample. Table no.1. Comparative data of standard, extract and extract from formulation Track Rf Height of Peak Area % Area 1 0.33 31.4 402 6.41 2 0.33 139.4 5532.7 62.97 3 0.33 215.5 8226.3 12 4 0.33 293.8 1179.5 10.39 5 0.33 208.6 7333.7 11.51 6 0.33 230.1 8708.1 29.64 Varsha M. Jadhav et al /Int.J. ChemTech Res.2009,1(4) 828 Figure 2. Chromatogram of representative sample Calibration Curve, Linearity and Range Linear regression data showed a good linear relationship over concentration range. The correlation coefficient r2 was 0.998 shown in Table no.2. y = 137.51x - 15.85 R2 = 0.998 Calibration Curve of MAG 3000 Peak Area AU 2500 Area 2000 Linear (Area) 1500 1000 500 0 0 5 10 15 Conc. mcg/ml Figure 3. Calibration Curve of MAG 20 25 Varsha M. Jadhav et al /Int.J. ChemTech Res.2009,1(4) 829 Table no.2. Linearity regression data for calibration curve Parameters Value Linearity Range 200-1000ng/spot Correlation of Coefficient (According to area) 0.999 Slope 141.97 Precision: The repeatability and intermediate precision were studied sepearatey and shown below in table 3 and 4 respectively. Repeatability It showed very low % RSD of peak area of drug. Table no.3. Repeatability study Sr. No. Sample applied ng Area AU %RSD 1. 1000 255.6 0.056 2. 1000 251.6 0.064 3. 1000 258.2 0.056 Intermediate precision Table no. 4. Intraday and Interday Precision Sr. No. Sample applied ng Intraday % RSD Interday RSD 1. 200 2.028 2.17 2. 400 1.98 1.9 3. 1000 1.54 1.6 Specificity The mobile phase was optimized and it showed good result. Glycyrrhizin was found to be well separated from other constituents present in extracted sample. There was no interference of diluent, placebo and other constituent’s peaks from extracted sample found at the Rf value of MAG peak, indicates specificity of the method. Robustness of method The time from sample application to scanning varied from 0, 20, 40, 60 mins. The standard deviation of peak areas was calculated for each parameter and % RSD was found to be less than 3 %. Recovery studies The proposed method when used for extraction and subsequent estimation of glycyrrhizin from gel after spiking with 20, 80, 120% standard MAG. The afforded recovery is listed below in Table no.5. Estimation of glycyrrhizin in herbal extracts and in herbal gel The optimized solvent system was used for the estimation of the glycyrrhizin from herbal extract and from herbal gel. There was no interference in analysis from other components present in extract. The resolution was good and components were observed at different Rf value. The total glycyrrhizin present in herbal extract was found to be 18.52% w/w, 17.92% w/w and 18.26% w/w. There was no interference from in analysis from other active components from extract and inactive components from formulation. The total glycyrrhizin present in formulations are 17.96%, w/w 17.85% w/w, and 18.1% w/w. CONCLUSION The developed HPTLC method is fast, simple, precise, specific and accurate. Statistical analysis proved that method is repeatable and selective for determination of Glycyrrhizin. Varsha M. Jadhav et al /Int.J. ChemTech Res.2009,1(4) 830 Figure 4. Comparision of Extracts peaks with Standard. Table no. 5: Recovery studies Sample Amount added % % Recovery %RSD 20 40 60 Amount recovered (mg)±SD 19.12 39.2 58.9 Sample extracted from glycyrrhiza powder 95.60 98.00 98.16 0.169 0.298 0.275 Sample extracted from Gel 20 40 60 18.14 38.38 57.65 90.7 95.95 96.08 0.198 0.254 0.312 ACKNOWLEDGEMENT Authors would like to thanks Amsar Industries, Indore (India) for providing gift sample of Monoammonium Glycyrrhizinate. REFERENCES 1) Bernard Fried, Joseph Sherma, Practical Thinlayer Chromatography: A Multidisciplinary Approach, CRC Press, Published 1996, pp.233234. 2) C. K. Kokate, A.P. Purohit, S. B. Gokhale, Pharmacognosy, ninetieth edi., Nirali Prakashan, Pune, 2002, pp 3) Vampa, G : Benvenuti, S : Rossi, T, Farmaco. 1992 May; 47(5 Suppl): 825-32. Varsha M. Jadhav et al /Int.J. ChemTech Res.2009,1(4) 4) Validation of analytical Procedures, Methodology, ICH harmonized triapartite guidelines, 1996. 5) Isao Kitagawa, Pure Appl. Chem., Vol. 74, No. 7, pp. 1189–1198, 2002. 6) M. Saeedi, K. Morteza-Semnani, M.R. Ghoreishi, Journal of Dermatological Treatment, 2003 (14) 153-157 7) Chauhan SK, Singh BP, Kimothi GP, Agarwal S, Indian Journal of Pharmaceutical sciences, 1998, Volume 60, Issue: 4; 251-252 ***** 831 8) Hayashi M, Kadowaki E, Takamatsu T, Matsuoka M., Yakugaku Zasshi. 1992 Jul;112(7):496-502. 9) Valéria M. Di Mambro, Journal of Pharmaceutical and Biomedical Analysis · Volume 37, Issue 2, 23 February 2005 10) Cristina Fiorea,, Journal of Ethnopharmacology · Volume 99, Issue 3, 14 July 2005, Pages 317324 11) Ali Nokhodchi, etal al, IL Farmaco 59, 155-161 12) Teruko Imaia, International Journal of Pharmaceutics · Volume 294, Issues 1-2, 27 April 2005, Pages 11-21