A simple, specific, accurate, precise and stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method is developed for estimation of Dapagliflozin (DGF) in bulk and Pharmaceutical dosage form. The method employed, Hypersil BDS C18 250 mm x 4.6 mm, 5 mm column in isocratic mode with mobile phase of 0.1% Ortho phosphoric acid buffer and acetonitrile 50:50% v/v. The flow rate was 1.0 mL min-1 and effluent was monitored at 245 nm using PDA detector. The injection volume was 10 µl and the total runtime was set as 5min. The retention time for DGF was found to be 2.226min.The method was validated in terms of Linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc. in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there was a good linear relationship between response and concentration in the range of 25 - 150 µg/ml respectively. The LOD and LOQ values for HPLC method were found to be 0.04 and 0.121 µg/ml respectively. No chromatographic interference from the tablet excipients was found. The proposed method was successfully used for estimation of Dapagliflozin (DGF) in Bulk and Pharmaceutical dosage form.
CALIBRATION OF HPLC
Pressure Test.
Drift and Noise
Column oven and sample cooler
Pump by flow rate accuracy measurement.
Pump by gradient flow measurement.
UV-Vis / PDA detector by reference energy check.
When designing a pharmaceutical manufacturing facility, the ways in which materials are moved from one stage of production to another should be considered from the very beginning. so the drug industry location and design considered crucial and should be according to GMP standards.
In this slide contains principle of IR spectroscopy and sampling techniques.
Presented by: R.Banuteja (Department of pharmaceutical analysis).
RIPER, anantpur.
CALIBRATION OF HPLC
Pressure Test.
Drift and Noise
Column oven and sample cooler
Pump by flow rate accuracy measurement.
Pump by gradient flow measurement.
UV-Vis / PDA detector by reference energy check.
When designing a pharmaceutical manufacturing facility, the ways in which materials are moved from one stage of production to another should be considered from the very beginning. so the drug industry location and design considered crucial and should be according to GMP standards.
In this slide contains principle of IR spectroscopy and sampling techniques.
Presented by: R.Banuteja (Department of pharmaceutical analysis).
RIPER, anantpur.
Role of Hippophae rhamnoides L. in the Management of Depression by Regulating...BRNSS Publication Hub
Depression is one of the major health burden in almost all the societies particularly in urban community.
As per the World Health Organization, depression will be the second largest disease burden due to the
urban way of lifestyle. The modern era is full of stress and strains the people are living in the most
competitive society than the previous year. Although many antidepressant drugs have been develop
to manage anxiety, stress and depression due to adverse side reaction, most of the drug could show
satisfactory results. Considering the above fact, we have selected a drug Hippophae rhamnoides which
contain many phyto molecules showing preventing role of anxiety and depression by regulating some
of the biochemical parameters.
Development and Validation of an RP HPLC Method for Analysis of Sitagliptinijtsrd
Sitagliptin is a drug used against type 2 diabetes mellitus and it is a member of class of anti diabetic drugs known as dipeptidyl peptidase 4 inhibitors or gliptins . A simple, sensitive and accurate RP HPLC method has been developed for the determination of Sitagliptin in bulk formulation. The max of the Sitagliptin was found to be 267nm in Methanol phosphate buffer 10mM pH 4.8 60 40 v v . The method shows high sensitivity with linearity 10 to 50µg ml regression equation y = 45765x 239272 r2 = 0.9996 . The various parameters according to ICH guidelines and USP are followed for validating and testing of this method. The Detection limit and quantitation limit were found to be 0.743 µg ml-1 and 2.25µgml-1 respectively. The results demonstrated that the procedure is accurate, specific and reproducible RSD 2 , and also being simple, cheap and less time consuming and appropriate for the determination of Sitagliptin in bulk and pharmaceutical formulation. Pradnya Lokhande "Development and Validation of an RP-HPLC Method for Analysis of Sitagliptin" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-6 , October 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29214.pdf Paper URL: https://www.ijtsrd.com/chemistry/analytical-chemistry/29214/development-and-validation-of-an-rp-hplc-method-for-analysis-of-sitagliptin/pradnya-lokhande
Method Development and Validation for Estimation of Oral Hypoglycaemic Drug D...ijtsrd
HPLC is a chromatographic technique employed in active compound chemistry and biochemistry to separate a mixture and substances with the goal of identifying, measuring, and purifying the different components of the mixture. Its a much better variety of column and traditional chromatography. The objective of the research work is to develop and validate a simple and accurate reverse phase chromatographic method to estimate amount of drug in dosage form. The developed method successfully can be applied to estimate the amount of Dapagliflozin in tablet dosage form. After oral administration of dapagliflozin, the maximum plasma concentration Concentration max under two hours. High performance liquid chromatographic system was alleviated according to the chromatographic settings. After attaining the steady base line, to verify the system suitability, a single 40 µg ml of standard solution proportional to 100 test concentration of dapagliflozin was injected into the HPLC system. The gradient mobile phase flow rate programming assisted in optimising the lengthy run duration and resolution of sample analysis, making the approach more cost effective and quick. Validation of the developed and optimized HPLC method was carried out according to ICH guidelines with respect to parameters such as linearity, specificity, precision and accuracy. Junaid Ahmed | Himanchal Sharma | Shiva Teotia "Method Development and Validation for Estimation of Oral Hypoglycaemic Drug Dapagliflozinina Tablet Dosage form by the Employment of Rp-HPLC" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-6 , October 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46395.pdf Paper URL : https://www.ijtsrd.com/pharmacy/analytical-chemistry/46395/method-development-and-validation-for-estimation-of-oral-hypoglycaemic-drug-dapagliflozinina-tablet-dosage-form-by-the-employment-of-rphplc/junaid-ahmed
Analytical method development and validation for the estimation of quinapril ...SriramNagarajan19
A simple and selective LC method is described for the determination of Quinapril and Tolcapone tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a Mixed Phosphate buffer (KH2PO4 +K2HPO4): Acetonitrile 40:60, with detection of 239 nm. Linearity was observed in the range 50 - 150 µg /ml for Quinapril (r2 =0.995) and 62.5- 187.5µg /ml for Tolcapone (r2 =0.999) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.
Stability indicating method development and validation for the estimation of ...SriramNagarajan18
Stability indicating method development and validation for the estimation of Doxorubicin by using RP-HPLC method in a bulk and pharmaceutical dosage form
Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-...SriramNagarajan15
A new, simple sensitive, rapid, accurate and precise RP-HPLC method was developed for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Clopidogrel bisulphate was chromatographed on a reverse phase C18column (150 mm x 4.5 mm, i.d 5μm) in a mobile phase consisting of acetonitrile and phosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow rate of 1 ml/min with detection at 224 nm. The detector response was linear in the concentration of 50-150 μg /ml. The limit of detection and limit of quantitation was found to be 1.3 and 4.2 µg/ml, respectively. The intra and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was 99.79%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Key words: Clopidogrel bisulphate, RP-HPLC, Validation, Accuracy, Precision.
Method Development and Validation of Clopidogrel Bisulphate by Reverse Phase-...SriramNagarajan15
A new, simple sensitive, rapid, accurate and precise RP-HPLC method was developed for the estimation of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Clopidogrel bisulphate was chromatographed on a reverse phase C18column (150 mm x 4.5 mm, i.d 5μm) in a mobile phase consisting of acetonitrile and phosphate buffer (pH: 3.0) in the ratio of 60:40 % v/v. The mobile phase was pumped at a flow rate of 1 ml/min with detection at 224 nm. The detector response was linear in the concentration of 50-150 μg /ml. The limit of detection and limit of quantitation was found to be 1.3 and 4.2 µg/ml, respectively. The intra and inter day variation was found to be less than 2%. The mean recovery of the drug from the solution was 99.79%. The proposed method is simple, fast, accurate, precise and reproducible hence, it can be applied for routine quality control analysis of Clopidogrel bisulphate in bulk drug and pharmaceutical formulation. Key words: Clopidogrel bisulphate, RP-HPLC, Validation, Accuracy, Precision.
Similar to Stability indicating RP-HPLC method for estimation of dapagliflozin in bulk and tablet dosage form (20)
A new RP -HPLC method development and validation for simultaneous estimation ...SriramNagarajan19
A simple, accurate, precise method was developed for the simultaneous estimation of the Aspirin and Omeprazole in Tablet dosage form. Chromatogram was run through Discovery 250 x 4.6 mm, 5m. Mobile phase containing Buffer and Acetonitrile in the ratio of 70:30 v/v was pumped through column at a flow rate of 1 ml/min. Temperature was maintained at 30°C. Optimized wavelength for Aspirin and Omeprazole was 241 nm. Retention time of Aspirin and Omeprazole were found to be 2.454 min and 3.168 min %RSD of the Aspirin and Omeprazole were and found to be 1.1 and 0.8 respectively. Percentage recovery was obtained as 99.50% and 99.57%for Aspirin and Omeprazole. LOD, LOQ values were obtained from regression equations of Aspirin and Omeprazole were 0.26ppm, 0.80ppm and 0.06ppm, 0.17ppm respectively. Regression equation of Aspirin is y = 3524x + 3853, and of Omeprazole is y = 10438x+542.2.
Nutrease powder; a natural plant based nutritional shake with co-factors & co...SriramNagarajan19
Nutrease powder is an effective natural vitamin and minerals Nutritional supplementation to improve metabolism. Nutrease powder just ½ serving (1 scoop) Provides 150 calories,18 grams of protein, 12 grams of fiber, and 1 gram of sugar per day. Nutrease powder Supports effective weight management, Reduces hunger and cravings, Promotes energy and positive mood, Promotes loss of fat and preservation of lean body mass, Improves metabolism and insulin sensitivity. This article reviews the current available scientific literature regarding the effect of nutrease powder as an effective supplementation for daily energy needs.
Method development and validation of simultaneous estimation of paracetamol &...SriramNagarajan19
A drug may be defined as a substance meant for diagnosis, cure, mitigation, prevention or treatment of diseases in human beings or animals or for alternating any structure or function of the body of human being or animals. Pharmaceutical chemistry is a science that makes use of general laws of chemistry to study drugs i.e. their preparation, chemical natures, composition, structure, influence on an organism and studies the physical and chemical properties of drugs, the methods of quality control and the conditions of their storage etc. the family of drugs may be broadly classified as.
1. Pharmacodynamic agents.
2. Chemotherapeutic agents.
It is necessary to find the content of each drug either in pure or single, combined dosage forms for purity testing. It is also essential to know the concentration of the drug and it’s metabolites in biological fluids after taking the dosage form for treatment.
The scope of developing and validating analytical methods is to ensure a suitable method for a particular analyte more specific, accurate and precise. The main objective for that is to improve the conditions and parameters, which should be followed in the development and validation.
WELLIA-R tablets; helps to protect brain tissue in cerebral edemaSriramNagarajan19
Boswellia serrate extract in Wellia- R gained significant importance in treatment of cerebral edema in patients with brain tumors, colon cancer, lung cancer, blood cancer, skin cancer, breast cancer, renal cancer, fibro sarcoma, prostate cancer and pancreatic cancer. The medicinal properties of Boswellia serrate extract in Wellia- R have been known and utilized since antiquity. Its current potential as an anti inflammatory and anticancer agent are being investigated and hold great promise. This article reviews the current available scientific literature regarding the effect of wellia-R tablets, from Boswellia serrate extract that Provides long lasting cerebral protection in brain tumor patients
Formulation and invivo evaluation of mucoadhesive microspheres embedded clero...SriramNagarajan19
In this study an attempt was made to prepare mucoadhesive microcapsules of Clerodendrum phlomidis extract using alginate polymers for prolonged release. Encapsulation of extract into sodium alginate polymer was done by ionic-gelation technique. In vivo testing of the mucoadhesive microcapsules in diabetic albino rats demonstrated significant antidiabetic effect of extract. The hypoglycemic effect obtained by mucoadhesive microcapsules was for more than 16 h whereas plain CP extract produced an antidiabetic effect for only 4 h suggesting that mucoadhesive microcapsules are a valuable system for the long term delivery of CP extract. In-vivo data obtained over a 120-h period indicate that CP extract loaded alginate microspheres from batch F7 showed the better glycemic control than control and a commercial brand of the drug.
Brian stroke memory impairment and treatment strategiesSriramNagarajan19
A brain stroke occurs when one of the brain parts are deprived form oxygen-rich blood due to various mechanism. Usually a brain stroke occurs when one of the arteries is blocked either because of narrowing of small arteries with in the brain or the hardening of the arteries that lead to atherosclerosis strokes can be either ischemic (85%) or Hemorrhagic (15%). Forget fullness is a common complaint among older people. Age –related memory changes are not the same thing as dementia. Preventing memory loss is by exercise regularly staying social, manage stress, get plenty of sleep and don’t smoke. Eat plenty of fruits and vegetable and take food contain antioxidant in abundance; will reduce your risk of stroke. Walking regularly is an easy to fight memory loss and also brain exercises to prevent loss and boost brainpower. Research is going no to enhance memory power in brain in patient with brain stroke.
A new analytical method development and validation for the estimation of lenv...SriramNagarajan19
A simple and selective LC method is described for the determination of Lenvatinib dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of Phosphate buffer (KH2PO4): Acetonitrile (80:20) with detection of 240nm. Linearity was observed in the range 60-140 µg /ml for Lenvatinib (r2 =0.996) for the amount of drug estimated by the proposed methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.
A new analytical method development and validation for the simultaneus estima...SriramNagarajan19
A simple and selective LC method is described for the determination of LEDIPASVIR and SOFOSBUVIR in tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of Mixed Phosphate Buffer:ACN (55:45) with detection of 213 nm. Linearity was observed in the range 60-140 µg/ml for LEDIPASVIR oxalate (r2 =0.999) and 6-14 µg /ml for SOFOSBUVIR (r2 =0.996) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.
A new analytical method development and validation for the simultaneus estima...SriramNagarajan19
A simple and selective LC method is described for the determination of Ibuprofen and Tramadol in tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of 60 volumes of Triethylamine buffer, 40 volumes of acetonitrile with detection of 227 nm. Linearity was observed in the range 50-150 µg/ml for Ibuprofen (r2 =0.983) and 50-150 µg /ml for Tramadol (r2 =0.985) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.
Method development and validation of escitalopram and estizolam in tablet dos...SriramNagarajan19
A simple and selective LC method is described for the determination of Escitalopram oxalate and Etizolam in tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of 30 volumes of ammonium acetate buffer, 40 volumes of acetonitrile and 30 volumes of Methanol with detection of 238 nm. Linearity was observed in the range 60-140 µg/ml for Escitalopram oxalate (r2 =0.999) and 6-14 µg /ml for Etizolam (r2 =0.996) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.
Analytical method development and validation for the estimation of aspirin an...SriramNagarajan19
A simple and selective LC method is described for the determination of Aspirin and Omeprazole in tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of 30 volumes of ammonium acetate buffer, 40 volumes of acetonitrile and 30 volumes of Methanol with detection of 233 nm. Linearity was observed in the range 18-42 µg/ml for Aspirin (r2 =0.983) and 6-14 µg /ml for Omeprazole (r2 =0.970) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim. The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.
A new analytical method development and validation for the simultaneus estima...SriramNagarajan19
A simple and selective LC method is described for the determination of Albuterol and Ipratropium Bromide in tablet dosage forms. Chromatographic separation was achieved on a c18 column using mobile phase consisting of a mixture of 80 volumes of methanol and 20 volumes of water with detection of 239 nm. Linearity was observed in the range 36-84 µg /ml for Albuterol (r2 =0.996) and 6-14 µg /ml for Ipratropium Bromide (r2 =0.997) for the amount of drugs estimated by the proposed methods was in good agreement with the label claim.
The proposed methods were validated. The accuracy of the methods was assessed by recovery studies at three different levels. Recovery experiments indicated the absence of interference from commonly encountered pharmaceutical additives. The method was found to be precise as indicated by the repeatability analysis, showing %RSD less than 2. All statistical data proves validity of the methods and can be used for routine analysis of pharmaceutical dosage form.
Formulation and evaluation of rosiglitazone nanosuspensionSriramNagarajan19
The main aim of this study is to formulate and evaluate Rosiglitazone Nano suspension. Nano suspensions are colloidal dispersion of Nano sized drug particles stabilized by surfactants. They can also be defined as a biphasic system consisting of pure drug particles dispersed in an aqueous vehicle in which the diameter of the suspended particle is less than 1micro meter in size. Rosiglitazone is an oral rapid and short –acting anti-diabetic drug from the sulfonylurea class. It is classified as a second generation sulfonylurea, which means that it undergoes enter hepatic circulation. Rosiglitazone Nano suspension was prepared by precipitation technique. After preparation of Nano suspension various characterization studies were done such as drug content, %yield, FTIR, DSC, TEM, and Invitro drug release.PVPK30,polaxomer are used as stabilizers. From the dissolution study F4 formulation which containts PVPK30 as stabilizer was considered as optimized formulation. It showed maximum drug release at 30min.FTIR and DSC studies revealed that good stability in dispersion.
Effect of hydrophilic polymers on solubility of some antihypertentives drugs ...SriramNagarajan19
The main aim of the present study is to carried out to enhance solubility of Felodipine. Felodipine.was selected as model drug and different carriers like Vitamin-E, Polyethylene Glycol 8000, Polyvinyl pyrrolidone K-30 were used in drug to carrier ratio 1:1, 1:2, 1:4 by weight respectively. Solid dispersions were prepared by physical mixing method and solvent evaporation method. Solid dispersions were evaluated by drug content, in-vitro release, FT-IR, DSC and XRD. The obtained data of solid dispersion prepared by solvent evaporation method were compared with physical mixing method. The result showed decrease in melting point change from crystalline to amorphous form and improved dissolution rate as compared to physical mixing as well as pure Felodipine. The finding of present study proposes that solid dispersion approach is beneficial in enhancing solubility of drug and bioavailability as well.
A review on plants act on both antidiabetic and antihyperlipidemic plantsSriramNagarajan19
Since ancient times, plants have been an exemplary source of medicine. Ayurveda and other Indian literature mentioned the use of plants in treatment of various human ailments. Medical plants play an important role in the management of diabetes mellitus especially in developing countries where resources are meager. Oral hypoglycemic agents like sulphonylureas and biguanides are still the major players in the management of the disease but there is growing interest in herbal remedies due to the side effects associated with the oral hypoglycemic agents. Herbal medicines have been the highly esteemed source of medicine throughout human history. Hyperlipidemia has been ranked as one of the greatest risk factors contributing to prevalence and severity of coronary heart diseases. Hyperlipidemia is a condition when abnormally high levels of lipids i.e. the fatty substances are found in the blood. Hypolipidemic drugs are extensively used as prophylactic agents to prevent such atherosclerosis induced disorders. But these hypolipidemic drugs are not free from adverse effects. Many plant derivatives and domestic remedies have been screened for their hypolipidemic action. More than 70 medicinal plants have been documented to have significant hypolipidemic action. During the last decade, an increase in the use of medicinal plants has been observed in metropolitan areas of developed countries. Medicinal plants play a major role in diabetes and hypolipidemic activity. The advantages of herbal medicines reported are effectiveness, safety, affordability and acceptability, this review focus on diabeties and hyperlipidemia and the role of plants used for the treatment of diabeties and hyperlipidemia.
FORMULATION AND CHARACTERISATION OF TRANSDERMAL PATCHES OF PERINDOPRILSriramNagarajan19
Transdermal drug delivery system (TDDS) has been an increased interest in the drug administration via the skin for both local therapeutic effects on diseased skin (topical delivery) as well as for systemic delivery of drugs. The skin as a site of drug delivery, has a number of significant advantages over many other routes of drug administration, including the ability to avoid problems of gastric irritation, pH and emptying rate effects, avoid hepatic first-pass metabolism thereby increasing the bioavailability of drug, reduce the risk of systemic side effects by minimizing plasma concentrations compared to oral therapy, provide a sustained release of drug at the site of application; rapid termination of therapy by removal of the device or formulation, the reduction of fluctuations in plasma levels of drugs, and avoid pain associated with injections. The transdermal delivery can also eliminate pulsed entry into the systemic circulation, which might often cause undesirable side effects. Main objective of formulating the transdermal system was to prolong the drug release time, reduce the frequency of administration and to improve patient compliance. In the present study, five formulations were prepared using single polymer in different ratios, along with plasticizers and penetration enhancer. Finally it was concluded that Some formulations show formation of brittle patch due to insufficient amount of polymer and in some patches texture of patch is not elegant due to plasticizer concentration for patch preparation. So by increasing concentration of polymer and plasticizer, finally formulation-5 was considered as optimized formula for preparing transdermal patch of Perindopril, where it shown best drug release profile.
Intercontinental journal of pharmaceutical Investigations and ResearchSriramNagarajan19
Anti-inflammatory activity of the ethanolic extract of Portulaca quadrifida Linn. was studied in wister rats using the carrageenan induced left hind paw edema, carrageenan induced pleurisy and cotton pellet induced granuloma model. The ethanolic extract (200 mg/kg, p.o.,) produced the inhibition of carrageenan induced rat paw edema. It also showed an inhibitory effect on leukocyte migration and a reduction on the pleural exudates as well as reduction on the granuloma weight in the cotton pellet granuloma method. The results indicated that the ethanolic extract produced significant (P<0.001) anti-inflammatory activity when compared with the standard and untreated control.
ANTI-BACTERIAL ACTIVITY OF EXTRACTS OF TACHYSPERMUM AMMI FRUITSSriramNagarajan19
This study was carried out with an objective to investigate the antibacterial activity of Tachyspermum ammi fruits extracts. In the present study, the anti-bacterial activity of aqueous and ethanolic extracts of Tachyspermum ammi fruits was evaluated for potential antimicrobial activity against medically important bacterial and fungal strains. The antimicrobial activity was determined using agar disc diffusion method. The antibacterial and antifungal activities of extracts were tested against Gram-positive—Staphylococcus aureus and Gram-negative—Escherichia coli human pathogenic bacteria. Zone of inhibition of extracts were compared with that of different standard drugs. The results showed that the remarkable inhibition of the bacterial growth was shown against the tested organisms. The phytochemical analyses of the plants were carried out. The antibacterial activity of the Tachyspermum ammi fruits was due to the presence of various secondary metabolites. Hence, these plants can be used to discover bioactive natural products that may serve as leads in the development of new pharmaceuticals research activities.
Live Longer, Stay healthy, Feel better with AstashinecapsulesSriramNagarajan19
ASTASHINE capsule contains natural astaxanthin from Haematococcus pluvialis Astaxanthin has exceptional antioxidant activity to combat singlet oxygen when compared to other antioxidants. In particular, Astaxanthin can be used to defend against singlet oxygen damage, which are especially susceptible to aging effects.
In this study, Astaxanthin extracted from Haematococcus microalgae powerfully quenched singlet oxygen. Results show that the quenching effect of Astaxanthin is 800 times greater than coenzyme Q10. Astaxanthin was also about 75 times greater than alpha lipoic acid, about 550 times greater than green tea catechins and about 6000 times greater than Vitamin C.the present Article reviews the role of ASTASHINE capsules as World’s most powerful Antioxidant and Anti-aging Nutrient.
Astashine capsules: an excellent choice for eye fatique relieveSriramNagarajan19
Scientists long ago discovered that a class of naturally-occurring pigments called carotenoids held powerful antioxidant properties that are crucial for eye health. This carotenoid is called astaxanthin. Astaxanthin is produced by the microalgae Haematococcus pluvialis when its water supply dries up, forcing it to protect itself from ultraviolet radiation. Astaxanthin is leaps and bounds more powerful than beta-carotene, alpha-tocopherol, lycopene, and lutein--other members of its chemical family. Astaxanthin exhibits very strong free radical scavenging activity, and protects eyes from oxidative damage. Astaxanthin is by far the most powerful carotenoid antioxidant when it comes to free radical scavenging: it is 65 times more powerful than vitamin C, 54 times more powerful than beta-carotene, and 14 times more powerful than vitamin E. Astaxanthin is far more effective than other carotenoids at "singlet oxygen quenching," which is a particular type of oxidation. The damaging effects of sunlight and various organic materials are caused by this less-stable form of oxygen. Astaxanthin is 550 times more powerful than vitamin E and 11 times more powerful than beta-carotene at neutralizing this singlet oxygen. Astaxanthin crosses the blood-brain barrier and the blood-retinal barrier which has huge implications for the health of eyes.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
HOT NEW PRODUCT! BIG SALES FAST SHIPPING NOW FROM CHINA!! EU KU DB BK substit...GL Anaacs
Contact us if you are interested:
Email / Skype : kefaya1771@gmail.com
Threema: PXHY5PDH
New BATCH Ku !!! MUCH IN DEMAND FAST SALE EVERY BATCH HAPPY GOOD EFFECT BIG BATCH !
Contact me on Threema or skype to start big business!!
Hot-sale products:
NEW HOT EUTYLONE WHITE CRYSTAL!!
5cl-adba precursor (semi finished )
5cl-adba raw materials
ADBB precursor (semi finished )
ADBB raw materials
APVP powder
5fadb/4f-adb
Jwh018 / Jwh210
Eutylone crystal
Protonitazene (hydrochloride) CAS: 119276-01-6
Flubrotizolam CAS: 57801-95-3
Metonitazene CAS: 14680-51-4
Payment terms: Western Union,MoneyGram,Bitcoin or USDT.
Deliver Time: Usually 7-15days
Shipping method: FedEx, TNT, DHL,UPS etc.Our deliveries are 100% safe, fast, reliable and discreet.
Samples will be sent for your evaluation!If you are interested in, please contact me, let's talk details.
We specializes in exporting high quality Research chemical, medical intermediate, Pharmaceutical chemicals and so on. Products are exported to USA, Canada, France, Korea, Japan,Russia, Southeast Asia and other countries.
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
micro teaching on communication m.sc nursing.pdfAnurag Sharma
Microteaching is a unique model of practice teaching. It is a viable instrument for the. desired change in the teaching behavior or the behavior potential which, in specified types of real. classroom situations, tends to facilitate the achievement of specified types of objectives.
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Stability indicating RP-HPLC method for estimation of dapagliflozin in bulk and tablet dosage form
1. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~69~
Available online at www.icjpir.com ISSN: 2349-5448
Intercontinental journal of pharmaceutical
Investigations and Research
ICJPIR |Volume 2 | Issue 4 | Oct – Dec- 2015 Research Article
Stability indicating RP-HPLC method for estimation of dapagliflozin in
bulk and tablet dosage form
Jeyabaskaran M,1*
Prof. Rambabu C,2
and Lakshmi Maneka S
1
Research scholar, Department of Chemistry, Acharya Nagarjuna University, Nagarjuna
Nagar,Guntur (Dist), Andhra Pradesh, India.
2
Professor and Head, Department of Chemistry, Acharya Nagarjuna University, Nagarjuna
Nagar,Guntur (Dist), Andhra Pradesh, India.
Corresponding Author: Jeyabaskaran M
ABSTRACT
A simple, specific, accurate, precise and stability-indicating reverse phase high performance liquid
chromatography (RP-HPLC) method is developed for estimation of Dapagliflozin (DGF) in bulk and
Pharmaceutical dosage form. The method employed, Hypersil BDS C18 250 mm x 4.6 mm, 5 m column in
isocratic mode with mobile phase of 0.1% Ortho phosphoric acid buffer and acetonitrile 50:50% v/v. The flow
rate was 1.0 mL min-1
and effluent was monitored at 245 nm using PDA detector. The injection volume was 10
µl and the total runtime was set as 5min. The retention time for DGF was found to be 2.226min.The method was
validated in terms of Linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc.
in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there
was a good linear relationship between response and concentration in the range of 25 - 150 µg/ml respectively.
The LOD and LOQ values for HPLC method were found to be 0.04 and 0.121 µg/ml respectively. No
chromatographic interference from the tablet excipients was found. The proposed method was successfully used
for estimation of Dapagliflozin (DGF) in Bulk and Pharmaceutical dosage form.
Keywords: Dapagliflozin, RP-HPLC, Stability indicates, Stress conditions
INTRODUCTION
Dapagliflozin (DGF) is a potent and selective
SGLT-2(SLC5A2) inhibitor with EC50 of 1.1nM.
Chemically, DGZ is (2S,3R,4R,5S,6R)-2-{4-
chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-6-
(hydroxymethyl)oxane-3,4,5-triol with empirical
formula of C21H25ClO6 and Molecular weight is
408.87 g/mol.2
It has good permeability across
Caco-2 cell membranes and is a substrate for P-
glycoprotein (P-gp). Dapagliflozin is not a
significant P-gp inhibitor.1
Dapagliflozin is
indicated for the management of diabetes mellitus
type 2, and functions to improve glycemic control
in adults when combined with diet and exercise.
2. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~70~
Dapagliflozin is a sodium-glucose cotransporter 2
inhibitor, which prevents glucose reabsorption in
the kidney. Using dapagliflozin leads to heavy
glycosuria (glucose excretion in the urine), which
can lead to weight loss and tiredness.3
The
chemical structure of DGZ was shown in Fig.01.
The drug product in a stability test sample needs to
be determined using a stability indicating method,
as recommended by the International Conference
on Harmonization (ICH) guidelines4
and U.S.
Pharmacopoeia (USP) 265
. Although stability
indicating methods have been reported for assay of
various drugs in drug products, most of them
describe assay procedures for drug products
containing only one active drug substance. Only
few stability indicating methods are reported for
assay of drug products containing two or more
active drug substances. The objective of this work
was to develop a simple, precise, and rapid column
liquid chromatography (LC) procedure that would
serve as stability indicating assay method for drug
product of DGF.
An extreme literature survey revealed that very
few analytical methods have been reported such as
HPLC6-8
for DGF in individual and combination
with other drugs. In order to minimize the batch –
to- batch variation, it is very important to develop
suitable analytical methods for day –to- day
analysis of drugs. It was found that one attempt has
been made to develop stability indicating studies
and estimation of DGF by RP-HPLC at the starting
of my work. Therefore, it was thought of interest in
development and validating an advanced new
sensitive, specific, precise, accurate stability
indicating RP-HPLC method for estimation of SOF
in bulk drug and in pharmaceutical dosage form.
We here in report a simple, rapid and reliable
HPLC for the estimation of DGF in bulk and
pharmaceutical dosage forms as per ICH
guidelines.9-13
Fig 01: Structure of Dapagliflozin.
MATERIALS AND METHODS
Chemicals and reagents
Pure standard of DGF was obtained as gift
sample from Dr,Reddy’s Laboratory in Hyderabad.
HPLC grade acetonitrile, HPLC grade water, Ortho
phosphoric acid HPLC (Merck. Mumbai, India),
Potassium dihydrogen phosphate and Triethylamine
(RANKEM, Mumbai, India.) and All solvents used
in this work are HPLC grade. Forxiga tablets (Astra
Zeneca Ab) Containing Dapagliflozin marketed
formulation was purchased from local market, high
precision weighing balance (wensar instruments,
hyderabad), micro pipette (in labs 10-100 µl) were
employed in the study. All the glassware employed
in the work cleaned with hot water followed acetic
anhydride then acetone and dried in hot air oven
whenever required. Working environment was
maintained in between 25o
C.
HPLC and chromatographic conditions
The analysis was performed on Waters 2695
RP-HPLC separation module (Waters Corporation,
Milford, USA) equipped with PDA detector having
back pressure 5000psi, automatic injector and
Hypersil BDS C18 (250 mm × 4.6 mm, 5 µm) were
used as stationary phase. Single pan Balance
(Shimadzu, model AY-120), Control Dynamics pH
meter (Mettler Toledo), Sonicator (Labindia
Instruments). The chromatographic separation was
achieved by using 0.1 % Ortho phosphoric acid
buffer and acetonitrile 50:50 %v/v as mobile phase
at a flow rate of 1ml/min. The mobile phase was
filtered through 0.45µm nylon membrane filter and
3. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~71~
degassed before use. The injection volume was 10
µl and the total runtime was set as 5min. The
determination of analytes was carried out at 245 nm
using PDA detector.
HPLC METHOD
Preparation of stock solution
Accurately Weighed and transferred 10mg
Dapagliflozin working Standard into a 10 ml clean
dry volumetric flask, add 5 ml of diluents (Water
and acetonitrile 50:50), sonicated for 30 minutes
and make up to the final volume with diluents.
Preparation of standard solution
From the above stock solution, 1 ml was pipette
out into a 10ml volumetric flask and then make up
to the final volume with diluent. HPLC spectrum of
DGF in optimized conditions is shown in Fig.2.
Fig.02; A typical chromatogram of DGF
Method validation
The accuracy of the proposed method was
determined by standard addition method. It is the
closeness of the analytical results obtained by the
analysis to the true value. A known amount of
standard drug was added to the fixed amount of
injection solution. Accuracy was expressed as
percentage recovery. Recovery test was performed
with three different concentrations i.e. 50 µg/ml,
100µg/ml and 150 µg/ml for Dapagliflozin. The %
recovery results were calculated and given in
Table.1.
Table.1: % Recovery results of Dapagliflozin
Conc. Dapagliflozin
Amount added (µg/ml) Amount recovered
(µg/ml)
% Recovery
50% 50 50.07 100.14
50 50.30 100.59
50 49.94 99.87
100% 100 99.77 99.77
100 100.23 100.23
100 101.72 101.72
150% 150 148.43 98.95
150 148.57 99.04
150 151.12 100.75
4. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~72~
Precision
Repeatability or Precision of the method was
determined by injecting six replicates of standard
solution at 100µg/ml of dapagliflozin HPLC
system. From the results obtained it was found that
the proposed method was precise shown in Table.2.
Table.2: Precision data
Injection Dapagliflozin concentration Area
1
100µg/ml
1539364
2 1541556
3 1545028
4 1548226
5 1523848
6 1545504
Mean 1540588
STDV 8773.6
%RSD 0.6
Linearity
A series of six concentrations in the range of 25
to 150µg/ml of Dapagliflozin has been prepared
and peak areas were recorded at 245 nm. A
calibration curve was plotted between peak area
versus concentration of respective Dapagliflozin
and the response of the drug was found to be linear.
The linear regression equation (y=mx+c) was found
to be y = 15079x + 4232.3 (Fig.3) for
Dapagliflozin. The linearity results were given in
Table.3.
Fig.3: Calibration curve of Dapagliflozin
Table.3: Linearity results of Dapagliflozin
Concentration
(µg/ml)
Area Average area % RSD
25
385637
381984
0.82
380158
380158
50
757872
761708
0.44
764259
762992
75
1120567
1124143
0.55
1120527
1131335
y = 15079x + 4232.3
R² = 0.9998
0
500000
1000000
1500000
2000000
2500000
0 50 100 150 200
5. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~73~
100
1522931
1529862
0.79
1543878
1522777
125
1870059
1869125
0.15
1871352
1865965
150
2272551
2275299
0.20
2272709
2280636
Robustness
Influence of small changes in chromatographic
conditions such as change in flow rate (
0.1ml/min), Temperature ( 50
C), mobile phase (
5%) studied to determine the robustness of the
method are also in favour of (Table.4, % RSD <
2%) the developed RP-HPLC method for the
analysis of dapagliflozin ( API).
Table. 4: Robustness of method
Parameter Variation Average Area %RSD
Standard - 1540588 0.60
Flow rate 0.9 ml 1571462 0.81
1.1 ml 1457151 0.59
Mobile phase 55:45 1612324 0.38
65:35 1572705 0.66
Temperature -5o
C 1556328 1.27
+5o
C 1372587 1.90
Limit of Detection (LOD)
Limit of detection is the known concentration of
Dapagliflozin and establishing minimum
concentration at which the Dapagliflozin can be
reliably detected. It was calculated based on the
standard deviation of the response and the slope of
the standard calibration curve. The LOD was found
to be 0.040 µg/ml of Dapagliflozin.
Limit of Quantification (LOQ)
Limit of quantification is the known
concentration of Dapagliflozin and establishing
minimum level at which the Dapagliflozin can be
quantified with acceptable accuracy and precision.
The LOQ was found to be 0.121 µg/ml of
Dapagliflozin. The LOD and LOQ results were
given Table.5.
Table.5: LOD and LOQ results of Dapagliflozin
Sample LOD LOQ
Dapagliflozin 0.040 µg/ml 0.121 µg/ml
Specificity and stability in analytical solution
The results of specificity indicated that the peak
was pure in presence of degraded sample. It is
important to mention here that the dapagliflozin
was stable in solution form up to 24 hrs at 250
C.
The results of linearity, precision, inter & intraday
assays, method robustness, LOD, LOQ, specificity
and stability in analytical solution established the
validation of the developed RP-HPLC method for
analysis of dapagliflozin.
Assay of DGF in dosage form
10 tablets were weighed and average weight
was calculated. Then from the transferred the
equivalent to one tablet to 100ml volumetric flask,
70ml of diluent was added and sonicated for 15
min, further the volume was made up with diluent.
From the filtered solution, 1ml was pipette out into
6. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~74~
10ml volumetric flask and made up to 10ml with
diluent. From the solution, 10µl was injected into
HPLC system and peak area was recorded with
detector at 245nm. The % assay was calculated
with obtained peak area of detector response. This
indicates that developed method can be used for
routine analysis. (Fig.4)
Fig.4: Chromatogram showing the assay of DGF marketed dosage form.
Optimization of chromatographic conditions
The chromatographic conditions were optimized
by different means. (Using different column,
different mobile phase, different flow rate, different
detection wavelength & different diluents for
sample preparation etc.(Table.6)
Table.6: Optimized chromatographic conditions
Parameter Condition
RP-HPLC Water 2695 separation module with PDA detector
Mobile phase 0.1% OPA(Ortho phosphoric acid) :ACN 50:50 v/v
Column Hypersil BDS 250 mm x 4.6 mm, 5 µm.
Column
Temperature
250
C
Wavelength 245nm
Diluent First dissolved in methanol and make up quantity with Acetonitrile: Water in the
ratio of (50:50)
Injector volume 10µl
Flowrate 1ml min-1
Runtime 5min
Retention time 2.226min
Stability indicating studies
The API (Dapagliflozin) was subjected to stress
conditions in various ways to observe the rate and
extent of degradation that is likely to occur in the
course of storage and/or after administration to
body. This is one type of accelerated stability
studies that helps us determining the fate of the
drug that is likely to happen after along time
storage, within a very short time as compare to the
real time or long term stability testing. The various
degradation pathways studied are acid hydrolysis,
alkali hydrolysis, oxidative degradation, Photolytic
degradation and dry heat degradation.
7. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~75~
Preparation of Acid induced degradation
Product
10ml (10mg) of Dapagliflozin stock solution
was taken into 100ml volumetric flask and refluxed
with 10ml 2N Hydrochloric acid at 800
C for 30
min. The resultant solution was collected, diluted
with diluent to get the concentration of 100µg/ml
and 10µl solution was injected into HPLC system
and chromatograms were recorded. (Fig.5)
Preparation of Alkali induced Degradation
Product
10ml of Dapagliflozin stock solution was taken
into 100ml volumetric flask and refluxed with
0.1N sodium hydroxide at 80 0
C. After 30 min the
resultant solutions was diluted with diluent to get
the concentration of 100µg/ml and 10µl solution
was injected into HPLC system and chromatograms
were recorded.(Fig.6)
Preparation of hydrogen peroxide induced
degradation product
10ml of Dapagliflozin stock solution was taken
into 10ml volumetric flask. 10 ml of freshly
prepared 10% H2O2 solution was added into
volumetric flask and solution was kept at 80 0
C for
30min. Then the resultant solution was injected into
HPLC system to get the chromatograms were
recorded. (Fig.7)
Preparation of water induced degradation
product
10ml of Dapagliflozin stock solution was taken
into 10ml volumetric flask. 10ml of water was
added into volumetric flask and solution was kept
at 80ºC for 8 hrs. the resultant solution was diluted
and 10µl solution was injected into the HPLC
system to get the chromatogram were recorded.
(Fig.8)
Photochemical Stability induced Product
The photochemical stability study of the drug
was studied by exposing the sample concentration
of 100µg/ml to UV light in UV chamber for 7 days
or 200 Watt hours/m2.
Then the resultant solution
was diluted and 10µl solution was injected into the
HPLC system to get the chromatograms were
recorded. (Fig.9)
Dry heat induced degradation product
To study the dry heat degradation studies, the
standard drug solution of Dapagliflozin was placed
in oven for 6hrs at 1050
C. The resultant solutions
were diluted to get the concentration of 100µg/ml
and 10µl solution was injected into HPLC system
and chromatograms were recorded to assess the
stability studies. (Fig.10)
Results of degradation studies
The specificity was successfully performed
under various stress conditions like acid, base,
oxidative, dry heat and photolytic and all the
degraded products were separated from sample
peaks. It was found that no interference of the
degraded products was seen with the drug products.
All Dapagliflozin peaks were tested for purity test
by comparing purity of angle and purity of
threshold. Purity of angle and purity of threshold
was estimated by chromatographic software, where
in all the conditions purity of angle was always less
than purity of threshold. This indicates that the
proposed method was specific. The results of
forced degradation shown in Table.7.
Table.7: Forced degradation studies of Dapagliflozin
Stress condition Dapagliflozin
Purity of
angle
Purity of
Threshold
Acid degradation 1.656 1.925
Base degradation 0.766 1.442
Peroxide degradation
Water degradation
0.476
0.434
0.530
0.608
Dry heat degradation 0.367 0.548
Photolytic degradation 0.419 0.583
8. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~76~
RESULTS AND DISCUSSION
To develop a precise, linear, specific & suitable
stability indicating RP-HPLC method for analysis
of dapagliflozin, different chromatographic
conditions were applied & the results observed are
presented. Isocratic elution is simple, requires only
one pump & flat baseline separation for easy and
reproducible results. So, it was preferred for the
current study over gradient elution.
In case of RP-HPLC various columns are
available, but here Hypersil BDS C18 (250mm × 4.6
mm, 5µm) column was preferred because using this
column peak shape, resolution and absorbance were
good. Mobile phase & diluent for preparation of
various samples were finalized after studying the
solubility of API in different solvents of our
disposal (methanol, acetonitrile, dichloromethane,
water, 0.1M NaOH, 0.1M HCl). The drug was
found to be highly soluble in acetonitrile & ethanol.
Drug was insoluble in water. Using these solvents
with appropriate composition newer methods can
be developed and validated.
The result shows the developed method is yet
another suitable method for assay and stability
studies which can help in the analysis of
dapagliflozin in different formulations.
Fig.5: Chromatogram of Acid degradation
Fig.6: Chromatogram of base degradation
9. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~77~
Fig.7: Chromatogram of oxidative degradation
Fig.8: Chromatogram of water degradation
Fig.9: Chromatogram of dry heat degradation
10. Jeyabaskaran M et al, ICJPIR 2015, 2(4), 69-78
www.icjpir.com
~78~
Fig.10: Chromatogram of photolytic degradation
CONCLUSION
A sensitive & selective RP-HPLC method has
been developed & validated for the analysis of
Dapagliflozin (API). Further the proposed RP-
HPLC method has excellent sensitivity, precision
and reproducibility. The result shows the developed
method is yet another suitable method for assay,
impurity studies which can help in the analysis of
Dapagliflozin in different formulations.
Acknowledgement
I am thankful to Dr.Reddy’s laboratory for
providing the drug sample for this project.
REFERENCES
[1]. http://en.wikipedia.org/wiki/Dapagliflozin.
[2]. http://www.scbt.com/datasheet-461432-26-8.html.
[3]. http://www.drugbank.ca/drugs/DB06292.
[4]. ICH, Q2B. (1993) Validation of analytical procedures methodology, In proceedings of The International
Conference on Harmonization, Geneva.
[5]. The United states Pharmacopoeia Convention, Inc, Rockville,MD,2007, 2287-2288, 3102.
[6]. Manasa. Sanagapati, K. Dhanalakshmi, G. Nagarjunareddy and S. Sreenivasa, development and
validation of a rp-hplc method for the estimation of dapagliflozin in api.ijpsr, 2014; vol. 5(12): 5394-
5397.
[7]. Shyamala, Nidhi B, Kavitha M, Pooja and JVC Sharma, validated rp-hplc method for simultaneous
estimation of metformin hdrochloride and dapagliflozin in tablet dosage formajbpr. 2015; 2(2):109-113.
[8]. Mohammad yunoos, Gowri sankar D, a validated stability indicating high-performance liquid
chromatographic method for simultaneous determination of metformin hcl and Dapagliflozin bulk drug
and tablet dosage form asian j pharm clin res, vol 8, issue 3, 2015, 320-326.
[9]. ICH, Stability testing of new Drug substances and products, International Conference on Harmonisation,
IFPMA, Geneva, 1993.
[10]. ICH, Impurities in new drug products, International Conference on Harmonisation, IFPMA, Geneva,
1996.
[11]. ICH, Specifications: Test procedures and acceptance criteria for new drug substances andnew drug
products: Chemical substances. International Conference on Harmonisation, IFPMA, Geneva, 1999.
[12]. ICH, Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products,
International Conference on Harmonisation, IFPMA, Geneva, 1995.
[13]. FDA, Guideline for Submitting Documentation for the Stability of Human Drugs and Biologics. Food
and Drug Administration, Rockville, MD.