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Chandrasekar R, J. Global Trends Pharm Sci, 2020; 11 (4): 8657 - 8664 Chandrasekar R, J. Global Trends Pharm Sci, 2020; 11 (4): 8657 - 8664 An Elsevier Indexed Journal ISSN-2230-7346 Journal of Global Trends in Pharmaceutical Sciences DEVELOPMENT AND VALIDATION OF HESPERIDIN FROM ORANGE PEEL CITRUS AURANTIUM BY RP-HPLC METHOD Chandrasekar R*, Sivagami B2, Rushitha K, Harshitha B, Sowber Sadhik A, Vamsi P, Sivakumar N, Vinay Kumar M Seven Hills College of Pharmacy, Tirupati, Chitoor, Andhra Pradesh, India *Corresponding author E-mail: [email protected] ARTICLE INFO ABSTRACT Key Words The present study was investigated to develop and validate a reversed phase high performance liquid chromatography method for the determination of Hesperidin Hesperidin, Citrus from orange peel of Citrus aurantium belonging to rutaceae family. Hesperidin was aurantium, Method isolated from orange peel. A new method was developed and validated for development, Validation, Hesperidin using methanol and water as mobile phase. The separation was achieved RP-HPLC on a reversed-phase C18 column (Chromatopak (250mm×4.6×5micron) using a mobile phase composed of water (0.1% orthophosphoric acid): methanol (65:35) at a Access this article online flow rate of 1.0 ml min-1. The peak response time for hesperidin was observed at Website: 5.669 minutes the detection was carried out on a UV detector at 280 nm. The https://www.jgtps.com/ developed method was validated according to the requirements for International Quick Response Code: Conference on Harmonisation (ICH) guidelines, which includes specificity, linearity, precision, accuracy, limit of detection and limit of quantitation. The developed method validates good linearity with excellent correlation coefficient (R2 > 0.999). In repeatability and intermediate precision, the percentage relative standard deviation (% RSD) of peak area was less than 1% shows high precision of the method. The developed HPLC method is a simple, rapid, precise, accurate and widely accepted and it is recommended for efficient assays in routine work. INTRODUCTION Hesperidin is a flavanone glycoside From mast cells. The possible anti-cancer (flavonoid) (C28H34O15) found abundantly in activity of hesperidin could be explained by the citrus fruits. Its aglycone form is called inhibition of polyamine synthesis. Sources of Hesperidin. Hesperidin, an abundant and hesperidin include citrus fruits, berries, onions, inexpensive bioflavonoid in (Citrus aurantium) parsley and green tea. Hesperidin is a flavanone peel, has been reported to possess a wide range glycoside found abundantly in citrus fruits, of pharmacological properties like antioxidant, vegetables and medicinal plants possess antiinflammatory, hypolipidemic, vasoprotective antioxidant activity [1, 2]. It has breast cancer and anticarcinogenic and cholesterol lowering activity [3]. Anti-allergic activity. [4] Hesperidin actions. Hesperidin is also an enzyme inhibitor has anti-inflammatory and analgesic effects [5– and inhibits phospholipase A2, lipoxygenase, 7]. In addition, the results revealed that HMG-CoA reductase and cyclo-oxygenase8. hesperidin exhibited pronounced anticancer Hesperidin improves the health of capillaries by activity against the selected cell lines [8]. In reducing the capillary permeability. Hesperidin literature, several analytical methods for the is used to reduce hay fever and other allergic determination of hesperidin have been reported. conditions by inhibiting the release of histamine Several high performance liquid 8657 © Journal of Global Trends in Pharmaceutical Sciences Chandrasekar R, J. Global Trends Pharm Sci, 2020; 11 (4): 8657 - 8664 chromatography (HPLC) methods were and filtered through 0.2 μm pore size filter. The developed for the estimation of hesperidin either column was maintained at 40°C throughout alone or in mixture with other flavonoids in analysis, and the UV detector was set at 280 nm. plant juices and pharmaceutical formulations [9– The total LC run time was 30 min. 15]. Literature survey revealed that only few analytical methods were reported for hesperidin Extraction of Crude Hesperidin: by RP-HPLC method. [16-22] Some RP-HPLC methods were not economical in terms of mobile 800 mL petroleum ether (40 – 60°C) was phase composition, column dimensions and run filled in a 250 mL round bottom flask with times. Therefore the aim of present investigation magnetic stir bar. 250g dried and powdered was to develop a simple, precise and accurate dried orange peel was placed in the extraction high performance liquid chromatography sleeve of a Soxhlet extractor and covered with a (HPLC) method for the identification and little glass wool. A reflux condenser is put on determination quantity of hesperidin in citrus the Soxhlet extraction unit, and then the reaction fruits. mixture is stirred and heated for 4 hours under strong reflux. The petroleum ether extract was MATERIALS AND METHODS discarded. In order to remove the adherent petroleum ether, the content of the extraction Chemicals and Reagents: Hesperidin 80% pure sleeve was laid out in an extensive was procured from Sigma-Aldrich, ortho- crystallisation dish. Afterwards the substance phosphoric acid from Merck GR Grade. was placed again in an extraction sleeve and, Deionised water was obtained by means of a like before, but with 800 mL methanol, extracted Milli-Q Water Purification system supplied by unless the solvent leaving the extraction sleeve Millipore HPLC Grade. All the other chemicals was colour less (1 to 2 hours). After complete were procured from SDFCL s d fine-chem Soxhlet extraction and maceration the filtrate limited. was then acidifying (pH 3-4) with 6% acetic acid, Keep the concentrated residual liquid in Instrumentation and Chromatographic refrigerator (4-6 ◦C) over night when a solid Conditions: HPLC was performed on a system crystalline substance appears. It was again equipped with a quaternary low-pressure filtered and the crude hesperidin was separated gradient solvent delivery P3000A HPLC pump, out on buchner funnel as amorphous powder. a high-sensitivity UV3000 ultraviolet (UV) The hesperidin was further characterized and detector. The system controlled and data identified according to various Physical and analyzed by HPLC workstation software. analytical test. Sonicator Model: 1.5L 50 degasser units, Preparation of standard solution (25ppm): Double Beam UV Spectrophotometer-Analytical 50 mg of Hesperidin was weighed accurately into a 100 ml volumetric flask technologies Limited, Model 212R RI, LVDVE, dissolved and diluted to the volume with mobile Superfit Rotavap, Model-PBU-6. Servewell phase. Further 5 ml of the above solution was Instruments Pvt. Ltd. diluted to 100 ml with mobile phase. Collection of Material: The fruits of Citrus Preparation of sample solution (25ppm): aurantium (Orange) were collected from local 50 mg of Hesperidin (sample) was area of Tirupati, Andhrapradesh and they were weighed accurately into a 100 ml volumetric peeled off and peels were dried under shade. flask dissolved and diluted to the volume with mobile phase. Further 5 ml of the above solution Optimized Chromatographic Conditions: A was diluted to 100 ml with mobile phase. separation was carried out in C18 Chromatopak Preparation of spiked sample solution: (250mm×4.6×5micron). The mobile phase The accuracy for the method was consists of isocratic elution with a low-pressure determined by spiking the blank samples at gradient using double-distilled HPLC grade standard concentrations (10, 50 and 100 ppm) Water: HPLC grade methanol (A: B): (65:35) and analyzing their recoveries. with a flow rate of 1.0 ml/min and the injection volume of 20 μl. All solutions were degassed 8658 © Journal of Global Trends in Pharmaceutical Sciences Chandrasekar R, J. Global Trends Pharm Sci, 2020; 11 (4): 8657 - 8664 Validation of the method: The validation of the Flow rate: flow rate of 1.0 ml/min and the developed analytical method was done injection volume of 20 μl. according to the International Conference on Harmonisation (ICH) guidelines. The method is Degassing: All solutions were degassed and validated for specificity, linearity, recovery, filtered through 0.2 μm pore size filter. precision, and the limits of detection (LOD) and Column Temperature: The column was limits of quantification (LOQ). maintained at 40°C throughout analysis, Linearity: Linearity was determined by different known concentrations of hesperidin Detector nm: UV detector was set at 280 nm. standard solution in triplicate by diluting the standard stock solution. For the determination of Run time: The total LC run time was 30 min. linearity, five aliquots were pipetted out from Accuracy: The accuracy for the method was standard stock solution (i.e., 60 ppm). 2-10 ml of determined by spiking the blank samples at standard stock solution was pipetted out in to a standard concentrations (10, 50 and 100 ppm) series of 10 ml volumetric flasks and volume and analyzing their recoveries. Shown in Table 3 was made up with the solvent to obtain concentration ranging from 10 ppm to 60 ppm of Robustness: The Robustness of the method was hesperidin. The calibration curve was determined by making slight changes in the constructed by plotting the peak areas against experimental conditions such as change in the concentration, and the linear regression flow rate, mobile phase and wave length. equations were calculated. The correlation Ruggedness: Ruggedness is the degree or coefficient was also computed. Shown in Table measure of reproducibility under different 1 and Figure 6 situations such as in
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