Page5112 Indo American Journal of Pharmaceutical Research, 2016 ISSN NO: 2231-6876 ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF PAIN RELIEF HERBAL FORMULATIONS Rucha J. Shah 1, Ajay I. Patel 1, Kartik V. Vikani 2, Nisha L. Patel 1 1 B. K. Mody Govt. Pharmacy College affiliated to Gujarat Technological University, Ahmadabad, Gujarat, India. 2 Aum Research Laboratories, Rakanpur, Gandhinagar, Gujarat, India. ARTICLE INFO Article history Received 03/04/2016 Available online 30/04/2016 Keywords Menthol, Methyl Salicylate, Linseed Oil, Gas Chromatography. ABSTRACT A rapid, sensitive, precise, and Robust Gas chromatography(gc) method was developed and validated for the simultaneous estimation of Menthol, Methyl salicylate and Linseed oil in Laboratory prepared pain relief herbal formulation and marketed pain relief formulation. The determination was carried out on capillary gas chromatography using flame ionization detector. Good separation of menthol (t R = 10.368), methyl salicylate (t R = 10.795) and linseed oil (t R = 21.912 and 25.180) and terpene hydrate (t R = 13.914) was obtained. The recovery of Menthol, Methyl Salicylate and Linseed oil was found to be 98.34%, 99.1% and 102.74% respectively. The correlation coefficients of linear regression analysis (r 2 ) were found to be 0.999, 0.999 and 0.996 respectively. Thus Proposed GC method provides a good resolution of Menthol. Methyl salicylate and Linseed oil. Corresponding author Rucha J. Shah M.Pharm. Student, Department of Quality Assurance, B. K. Mody Govt. Pharmacy College, Rajkot (Mo): 08734916515 rucha.j.shah@gmail.com Please cite this article in press as Rucha J. Shah et al. Analytical Method Development and Validation of Pain Relief Herbal Formulations. Indo American Journal of Pharmaceutical Research.2016:6(04). 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.
Page5113 INTRODUCTION Plants have been used medicinally for thousands of years by cultures all over the world and are the main source of medicine throughout human history. Herbs and products containing herb(s) have been in trade and commerce and are currently used for a variety of purposes. As a country, India has a rich history of use of herbs, processed herbs and formulations containing herbs both from traditional wisdom as well as cultural usage. [1] All modern medicines are derived originally from traditional herbal sources. These have evolved to produce the conventional medicine, which uses both synthetic drugs and isolated natural compounds. Herbal medicines are popular among the public and improvements in their formulation resulted in a new generation of phytomedicines that are more potent than before. Since time immemorial, various herbs and their derived compounds have been used in treatment of inflammation. [2] Inflammation is a normal, protective response to tissue injury caused by physical trauma, noxious chemicals or microbiologic agents, which is a part of the host defense. Sometimes, inflammation seems to produce events that are quite serious and become chronic like occurrence of rheumatoid arthritis and hay fever which may be life threatening. Rubor (redness), calor (heat), tumor (swelling) and dolor (pain) are the main signs of inflammation. [3] Menthol, Methyl salicylate and Linseed oil are presents in many pain relief, anti-inflammatory ointments. There is no method available for simultaneous estimation of Menthol, Methyl salicylate and Linseed oil. Menthol and Methyl salicylate are volatile in nature while Linseed oil is fatty acid. So, it is required to develop a method in which thre is no heat. In this research work we have developed a simple, optimized and validated GC method for the simultaneous estimation of Menthol, Methyl salicylate and Linseed oil. The method was validated on the basis of its linearity, precision, accuracy, Limit of detection and limit of quantification. EXPERIMENTAL Chromatographic conditions: Gas Chromatography was performed on Shimadzu GC2010 apparatus, equipped with split-splitless injector attached to DB- 01 column (30 m 0.53 mm, film thickness 3 µm) and attached to flame-ionization detector (FID).Operating conditions were set as follow: carrier gas was N 2. Temperature were set as f C/min. Data equisition and integration was performed using GCsolution software. MATERIALS Standard of menthol, methyl salicylate, linseed oil and terpene hydrate were given by Aum research laboratories and formulation was prepared in lab. Preparation of standard solution: Weigh accurately 200 mg of menthol, 300 mg of methyl salicylate, 200 mg of linseed oil and 200 mg of terpene hydrate as internal standard. Transfer it in 100 ml volumetric flask. Add 50 ml of 0.05 M methanolic KOH (700 mg KOH in 250 ml methanol). Keep this solution on magnetic stirrer for 1 hr. After 1 hr add 280 mg of tartaric acid for precipitation of KOH. Shake it well. Centrifuge it for 3 min. and then filter it with whatmann filter paper. Take this filter for GC injection. Preparation of sample solution Weigh accurately 10 gm of laboratory prepare ointment and 4.5 gm of marketed ointment in 100 ml volumetric flask. Add 50 ml of 0.05 M methanolic KOH (700 mg KOH in 250 ml methanol). Keep this solution on magnetic stirrer for 1 hr. After 1 hr add 280 mg of tartaric acid for precipitation of KOH. Shake it well. Centrifuge it for 3 min. and then filter it with whatmann filter paper. Take this filter for GC injection. Figure 1: chromatogram of Standard mixture.
Page5114 Validation parameters The method was validated as per ICH guideline for system Suitability, Linearity, Accuracy, Precision, Robustness, Limit of Detection and Limit of Quantification. System suitability was performed using standard mixture (n=6) and test (n=1). Linearity of the method was performed by analyzing standard solution of analytes by the method in concentration range 1500 µg/ml to 9000 µg/ml. The accuracy of the proposed method was determined by a recovery study, carried out by adding known quantity of standards in placebo with three different concentrations and analyzed under previously optimized conditions. Precision was determined by repeatability (n=6 test) and intermediate precision (chemist change) (n=6 std and test both). Robustness of the method was evaluated by changing in column flow rate. RESULTS AND DISCUSSIONS Selection and optimization of chromatographic conditions Several trials have been taken in different solvents and different column oven temperature programming. A satisfactory result and good resolution obtained in Methanolic KOH and follow C/min.GC chromatogram for laboratory formulation and marketed formulation was developed. Figure: 2 Chromatogram of laboratory formulation. Figure: 3 Chromatogram of Marketed formulation. Validation of the proposed method The proposed method has been validated for the simultaneous determination of Menthol, Methyl salicylate and Linseed oil incommercial herbal formulation using following parameters. System suitability test Table 1: System suitability parameters for Menthol, Methyl salicylate and Linseed oil. Parameters Data obtained(n=6) Menthol ± RSD Methyl salicylate ± RSD Linseed oil(1) ± RSD Linseed oil(2) ± RSD Retention time 10.258 ± 0.03 10.654 ± 0.03 18.950 ± 0.02 22.122 ± 0.02 Area 4412068.5 ± 1.76 4691688.7 ± 1.86 327973.7 ± 1.69 5823797.3 ± 1.70 Theoritical plates 57663.8 ± 1.07 55474.7 ± 1.58 192628.0 ± 1.77 96751.5 ± 1.77 Tailing factor 1.632 ± 1.07 1.918 ± 1.45 1.135 ± 1.83 0.950 ± 1.15
Page5115 Linearity Linear correlation was obtained between peak area Vs concentrations of Menthol and Linseed oil in the concentration ranges of 2000-6000 µg/ml while for Methyl salicylate 1500-9000 µg/ml. Regression parameters are mentioned in table and the calibration curves of these three markers are shown in Fig 4, Fig. 5 and Fig. 6. Figure: 4 Calibration curve of menthol. Figure: 5 Calibration curve of methyl salicylate. Figure: 6 calibration curve of linseed oil. Table 2: Regression parameter, Linearity, Limit of Detection and Limit of Quantification of the proposed GC method. Marker Concentration Range(µg/ml) Regression equation R 2 LOD (µg/ml) LOQ (µg/ml) Menthol 2000-6000 y = 1177.x - 49650 0.999 194.58 589.64 Methyl salicylate 1500-9000 y = 856.5x - 77387 0.999 223.20 676.38 Linseed oil 2000-6000 y = 1261.x + 92553 0.996 380.32 1152.49 Range Range is the interval between upper and lower concentration of analyte in sample for which it has been demonstrated that the analytical method has suitable precision, accuracy and linearity. The linearity response was observed over a range of 2000-6000 µg/ml for menthol and linseed oil and 1500-9000 µg/ml for methyl salicylate. Accuracy The recovery experiment was performed by the standard addition method. The summary of the result and average mean of recovery data for each level within accepted range as shown in table 3.The low value of standard deviation indicates that proposed method is accurate.
Page5116 Table 3: Recovery data of Menthol, Methyl salicylate and Linseed oil. Drug spiked(%) Amount found Recovery Menthol Methyl salicylate Linseed oil Menthol Methyl salicylate Linseed oil 50 96.87 137.69 95.94 98.21±0.21 99.07±0.92 98.20±0.30 100 197.38 282.04 198.67 98.69±0.42 98.14±0.07 99.04±0.55 150 296.28 441.21 298.28 98.79±0.72 98.47±0.39 99.38±0.28 Precision The low %RSD values of repeatability and intermediate precision parameter reveals that proposed parameter is precise. Table 4: Precision data for Menthol, Methyl salicylate and Linseed oil. Marker Repeatability(%RSD) Chemist change(%rsd) Menthol 0.49 0.85 Methyl salicylate 0.52 0.53 Linseed oil 0.73 0.72 LOD and LOQ LOD values for Menthol, Methyl salicylate and Linseed oil were found to be 194.58 µg/ml, 223.20 µg/ml and 380.32 µg/ml respectively. LOQ values for Menthol, Methyl salicylate and Linseed oil were found to be 589.64 µg/ml, 676.39 µg/ml and 1152.49 µg/ml respectively. These data shows that the proposed method is sensitive for the determination of Menthol, Methyl salicylate and Linseed oil. Robustness To evaluate robustness of the method was checked by changing in column flow rate. The average value of % RSD for determination of Menthol, Methyl salicylate and Linseed oil less than 2% revealed the robustness of the method. The average value of %RSD for determination of menthol, methyl salicylate and linseed oil less than 2% revealed the robustness of the method. Table 5: Robustness of the method for Menthol, Methyl salicylate and Linseed oil. Flow rate(ml/min) Menthol ± RSD Methyl salicylate ± RSD Linseed oil± RSD 4.5 4166485.5 ± 0.02 4565338.3 ± 0.04 6217026 ± 0.05 5.5 4413450.5 ± 0.05 4709377.3 ± 0.08 6106581.7 ± 0.03 Quantification of markers present in marketed formulation The three markers were found in marketed formulation and they were quantified with respect to Menthol (5%), Methyl salicylate (10%) and Linseed oil (3%) Marker % Assay Menthol 103.15% Methyl salicylate 99.55% Linseed oil 98.5% CONCLUSION Method development for simultaneous estimation in herbal formulation is difficult due to presence of so many phytoconstituents. In the present case an attempt was made to develop method for simultaneous estimation of menthol, methyl salicylate and linseed oil using GC. The developed GC method was found to be simple, precise and reliable. The method was applied to commercial polyherbal formulation and was found to be applicable for quantification of biological markers. The method can also be used for multicomponent formulation. The proposed method can be further improved by changes in column. ACKNOWLEDGEMENT Authors would like to thank Aum Research Laboratories for providing technical facilities to complete our research work.
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