DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR DETERMINATION OF ANDOGRAPHOLIDE

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1 Page5816 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR DETERMINATION OF ANDOGRAPHOLIDE Mrinalini. Damle *, Namrata Gujar AISSMS College of Pharmacy, Kennedy Road, Near R.T.O., Pune , Maharashtra, India. ARTICLE INFO Article history Received 17/06/2016 Available online 30/06/2016 Keywords Andrographis Paniculata, Andrographolide, High Performance Thin Layer Chromatography, Stability Indicating Method. Corresponding author Mrinalini Damle Department of Quality Assurance, AISSMS College of Pharmacy, Kennedy Road, Near R.T.O., Pune , Maharashtra, India ABSTRACT Andrographis Paniculata (Kalmegh) is used since ancient time for treatment of various diseases viz cancer, malaria, diabetes etc. Andrographolide is one of the major phytoconstituent responsible for its activity, which is a diterpene lactone. An approach for the stress degradation was successfully applied for the development of stability indicating HPTLC method for the determination of Andrographolide was achieved on the plates precoated with silica gel 60F 254. The mobile phase used was Chloroform: Methanol (9:1v/v) and quantification was carried out at wavelength 226 nm. The system showed a peak for Andrographolide at Rf value of 0.34 ± 0.03.The marker was subjected to acid, base, neutral hydrolysis, oxidation, thermal degradation and photolysis. Stress testing of Andrographolide was carried out according to the International Conference of Harmonization (ICH) guideline Q1A (R2). The method was successfully validated according to ICH guidelines Q2 (R1). The data of linear regression analysis indicated a good linear relationship over the range of ng/band concentrations with correlation coefficient value of The accuracy of the method was established based on the recovery studies. The LOD and LOQ of Andrographolide was found to be 13.2 ng/band and ng/band, respectively. Among various stressed conditions, Andrographolide showed considerable degradation under alkali and acid catalyzed hydrolysis, oxidative and photolytic condition. Please cite this article in press as Mrinalini Damle et al. Development and Validation of Stability Indicating HPTLC Method for Determination of Andographolide. Indo American Journal of Pharmaceutical Research.2016:6(06). Copy right 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page5817 INTRODUCTION Andrographis paniculata (Burm. f.) Nees is one of the wildly distributed medicinal plants in India and used since ancient times in traditional ayurvedic systems of medicines [1].It is known to contain diterpenes, lactones and flavonoids. The plant is traditionally used as antibacterial, antioxidant, antidiabetic, antipyretic, hepatoprotective and in treatment of several infectious diseases ranging from malaria to dysentery [2]. The primary bioactive component of the medicinal plant Andrographis Paniculata is Andrographolide, which is colourless crystalline bicylic diterpene lactone [3]. Andrographolide, which is grouped as an unsaturated trihydroxy lactone has the molecular formula of C 20 H 30 O 5. The molecular structure of andrographolide is shown in Fig. 1 [4] Fig 1: Structure of Andrographolide. Literature survey for Andrographis Paniculata reveals HPTLC [5-8], HPLC [9-10], UV [11] methods reported for estimation of Andrographolide, But there is no stability indicating HPTLC method reported. Hence, a Densitometric HPTLC method has been developed in the present work for quantitation of Andrographolide. MATERIALS AND METHODS Chemicals and Reagents Working Standard Andrographolide (Marker), along with the Raw Material (Stems of Andrographis Paniculata) was purchased from Yucca Enterprises, Mumbai. The marker was used as such, without any further purification. Andrographis Paniculata stems were authenticated from Agharker Research Institute, Pune. Aluminum sheets precoated with silica gel (60F 254, 20 cm 20 cm with 250 µm layer thickness) were purchased from E-Merck, Darmstadt, Merck (Germany). Methanol (AR grade), Chloroform (AR grade), Hydrochloric acid (HCl), hydrogen peroxide (H 2 O 2, 30% v/v), NaoH Pellets, were purchased from S. D. fine chemical Laboratories, Mumbai. Chromatographic conditions and instrumentation Chromatographic separation of drug was performed on Aluminum plates precoated with silica gel 60 F 254, (10 cm 10 cm with 250 µm layer thickness). Samples were applied on the plate as a band with 6mm width using Camag 100 μl sample syringe (Hamilton, Switzerland) with Linomat 5 applicator (Camag, Switzerland). The optimized mobile phase comprises of Chloroform:Methanol::9:1 v/v. CAMAG twin trough glass chamber (10 cm 10 cm) was used for linear ascending development of TLC plate with 15 min saturation conditions and 10 ml of mobile phase was used per run, migration distance was 80 mm. Densitometric scanning was performed using Camag TLC scanner 3, operated by win CATS software (Version 1.4.3, Camag). Preparation of standard solutions Standard stock solution was prepared by dissolving 10 mg Andrographolide in 10 ml of methanol to get concentration of 1000 µg/ml. From the standard stock solution, working standard solution was prepared by diluting 1ml of standard stock to 10 ml with methanol to obtain working standard solution containing 100 µg/ml of concentration of Andrographolide. UV spectra of the working standard solution was obtaioned as shown in figure no.2. From the spectrum of Andrographolide, 226 was selected as absorbance maxima. Standard densitogram at λ max 226 is as shown figure No.3.

3 Page Abs Wavelength [nm] Fig.2: UV Spectrum of Andrographolide. Fig.3: Densitogram of Andrographolide. STRESS DEGRADATION STUDY OF ANDROGRAPHOLIDE Stress degradation studies were carried under condition of acid/ base/ neutral hydrolysis, oxidation, dry heat and photolysis as per ICH Q1A (R2) guideline [12-13]. For each study, samples were prepared as follows: 1. The blank was subjected to stress in the same manner as the marker solution. 2. Andrographolide working standard solution subjected to stress condition. Stress condition were optimized in terms of strength of reagent and time of exposure to attain about 10-30% degradation. Alkaline hydrolysis To 1 ml of 1000 µg/ml solution of Andrographolide, 1 ml of 0.1 N NaoH was added. The volume was made up to 10 ml with methanol. The above solution was kept 30mins at room temperature. Acidic hydrolysis To 5 ml standard stock solution of Andrographolide (1000 µg/ml) with 5 ml of 1N HCl was added. The volume made up to 50 ml with methanol. Solution was refluxed for 1 hr. Neutral Hydrolysis To 5 ml standard stock solution of Andrographolide (1000 µg/ml) with 5 ml water and volume made up to 50 ml with methanol. The solution was refluxed for 1.

4 Page5819 Oxidation To 5 ml standard stock solution of Andrographolide (1000 µg/ml), 5 ml of 30% v/v solution of H 2 O 2 was added and volume made up to 10 ml with methanol. Solution was refluxed for 1 hr. Degradation under dry heat Dry heat studies were performed by keeping drug sample in oven (60 0 C) for 8 hrs. Photo-degradation studies Photolytic studies were carried out by exposure of drug to UV light up to 200 watt hours/square meter and subsequently to cool fluorescent light to achieve an illumination of 1.2 million Lux hrs. Sample was weighed, dissolved in methanol and further dilutions were made to get final concentration of 100 µg/ml. RESULT AND DISCUSSION Under optimized chromatographic conditions retention factor of Andrographolide was found to be 0.34 ± Degradation was observed for Andrographolide during stress conditions like Acid hydrolysis, Base hydrolysis, Neutral Hydrolysis, oxidation, dry heat and photolysis but the peak of degradation product of Andrographolide was observed only under alkali hydrolysis. Andrographolide showed two degradation products in alkaline hydrolysis at Rf 0.50 and 0.59, Summary of stress degradation results is given in Table No.1. Peak purity results greater than indicate that Andrographolide peaks are homogeneous in all stress conditions tested indicating noninterference of product of degradation. The unaffected assay of Andrographolide confirms the stability indicating power of the method. Fig.4:-Densitogram of Alkali Treated Standard Andrographolide with degradation products.

5 Page5820 Fig.5:- Overlain UV spectrum of standard Andrographolide and its degradation products. Table No.1: Summary of stress degradation of Andrographolide. Stress degradation condition Peak area % recovery Rf of Degradation Product Peak Purity r ( s,m) r (m,e) Alkaline hydrolysis and (0.1 N NaoH, 30mins) Acidic hydrolysis (1 N HCl, Reflux for 1Hr) Neutral hydrolysis (Reflux for 1Hr) H 2 O 2 30%v/v (Reflux for 1 Hr) Heat dry (60 0 C,6 hrs) Photo stability UV-200Watt hrs/m 2 Fluroscence-1.2 million lux hrs Method Validation: For the developed method, validation parameters checked were specificity, Linearity, Accuracy, Precision, Sensitivity i.e., LOD and LOQ, Robustness as per ICH Q2A (R1) guideline [14]. Specificity The specificity of the method was ascertained by peak purity profiling studies. The peak purity values were found to be more than 0.997, indicating the noninterference of any other peak Linearity Linearity studies were performed in the concentration range of ng/band an accurate correlation was found between peak area and amount spotted. Result are tabulated in Table No.2 Table No.2 Linearity of Andrographolide. Sr. no Concentration (ng/band) Peak area

6 Page5821 Fig.4: Calibration curve of Andrographolide. Precision Interday precision Precision of the system was evaluated by analyzing three independent standard preparations on three different days and % RSD value obtained was calculated to determine system precision. Result are tabulated in Table No.3 Table No.3: Interday precision of andrographolide. Concentration (ng/band) Area SD RSD Intraday precision Precision of the system was evaluated by analyzing six independent standard preparations in a day and % RSD value obtained was calculated to determine system precision. Result are tabulated in Table No.4 Table No.4: Intraday precision of Andrographolide. Replicate Intraday Precision Mean Area Standard Deviation 1.50 % RSD 1.56 Assay:- Assay was performed by dissolving 10mg of Methanolic extract in 10 ml methanol and further spotting was done of 10 µl to obtain assay percent of 10.5%. Accuracy Accuracy studies were performed by adding 80%, 100%, and 120% solutions with respect to target assay concentration (400ng/band).The amount of Andrographolide was calculated and % recovery is tabulated in Table No.5

7 Page5822 Table No.5:%Recovery. Level% Sample Standard Mean area %Recovery Limit of detection (LOD) and limit of quantification (LOQ) The LOD of Andrographolide was found to be 13.2ng/ band and 40.11ng/band respectively. Robustness Robustness was determined by carrying out the analysis under conditions during which mobile phase ratio, time form application to development and time form development to scanning, chamber saturation time were altered. Impact on peak area was assessed and it was found that method is robust. Result are tabulated in Table No.6 Table No.6: Results of Robustness. Sr no Parameter %RSD 1 Mobile phase composition- Chloroform:Methanol (9:1) ± 0.2mlChamber Saturation time(15) Time from spotting to development (immediate& after 2hrs) Time from development to scanning (immediate& after 2hrs) 0.80 RESULT AND DISCUSSION This method is stability indicating, whereas other methods in literature are simple chromatographic methods. Also the method developed is more sensitive. Current study confirms that andrographolide does not degrade at mild conditions and degrades after refluxing with HCl, H 2 O 2, and neutral condition. Whereas it is thermolabile and degrades at higher temperature. Andrographolide is sensitive to UV radiation. CONCLUSION The developed method was found to be simple, time saving, economic, accurate and precise. This method can be used for stability study of Andrographolide. ACKNOWLEDGEMENT Authors are thankful to the Principal, AISSMS College of Pharmacy, Pune for providing the necessary facilities for research work. REFERENCES 1. Sharma M, Sharma R.G. Identification, Purification and Quantification of Andrographolide from Andrographis Paniculata (Burm. F.) Nees By HPTLC At Different Stages Of Life Cycle Of Crop.J.Curr.Chem.Sci.2013;3(1): Das P, Srivastav A. Phytochemical Extraction and Characterization of the Leaves of Andrographis Paniculata for Its Anti- Bacterial, Anti-Oxidant, Anti-Pyretic and Anti- Diabetic Activity. International Journal of Innovative Research in science, Engineering and Technology.2014; 3(8): Jarukamjorn K., Nemoto Pharmacological Aspects of Andrographis Paniculata on Health and Its Major Diterpenoid Constituent Andrographolide. Journal of Health Science.2008; 54(4): Jadhao D., Thorat B. Purification (Crystallization) Of Bioactive Ingredient Andgrographolide from Andrographis Paniculata. World Journal of Pharmacy and Pharmaceutical sciences.2014; 3(10): Manvitha. Determination of Andrographolide in Andrographis Paniculata Extract with and Without Human Serum by High Performance Thin Layer Chromatography.Inetrnational Research Journal of Pharmacy.2013; 4(12): Jadhao M, Estimation of Andrographolide in Herbal Powder and Polyherbal Asava by HPTLC.International Journal of Pharma and BioScience.2010; 1(4): Mamatha A. Quantitative HPTLC Analysis of Andrographolide in Andrographis Paniculata Obtained from Different Geographical Sources (India).International. Journal of Pharmacy and Pharmaceutical Science.2011; 3(2): Zade S, Bhanu P Bagmar U.Development and Validation of HPTLC Method for Estimation of Hepatoprotective Diterpenoid Andrographolide in Polyherbal Formulations. International. Journal of Pharmacy and Pharmaceutical Science.2013;5(3): Patarapanich C,Laungcholatan S, Chaiyo Chaichantipayuth.HPLC Deteremination Of Active Diterpene Lactones From Andrographis Paniculata Nees Planted In Various Seasons And Regions In Thailand.Thailand Journal of Pharmaceutical Science.2007;31: Sharma M, Sharma A, Tyagi S.Quantitative HPLC Analysis Of Andrographolide In Andrographis Paniculata At Two Different Stages Of Life Cycle Of Plant. Acta Chimica.Pharmaceutica Idica.2012;2(1):1-7.

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