Identification of Prednisolone Acetate

Identification of Prednisolone Acetate appointment AND CHARACTERIZATION OF DRUGThe procured prednisolone acetate was identified and characterized found on the following parameters.ORGANOLEPTIC PROPERTIESFor the selected attempts ( dose + excipients) the following organoleptic properties were studied victimisation descriptive ter bitology. Those were nature, odour and saturationPARTITIONING BEHAVIOR OF PA IN DISTINCT LIPIDSThe percent splitter coefficient of Prednisolone acetate in selected lipides was canvass as elucidated elsewhere. On the get off the ground of the study/briefly, a florilegium composed of limpid lipid and 10mL of de-ionized weewee to this 10mg of PA was added. The resultant categorization was agitated reciprocally for one hr hold opened at 50C to a higher place the lipid change state point. The amount of lipid utilized was mentioned in table 1. The resultant statistical distribution was allowed to simmer down. Upon making cool both phases were separated by ultracentrifugation ( ) at 15,000 rev and tryed. The drug issue in supernatant was analyzed spectrophotometrically (240nm). The percent division coefficient was predicted as follows% partition = w1-w2/w1 x 100W1 = Amount of drug addedW2 = Amount of drug in aqueous phaseDETERMINATION OF MELTING POINTPA feeding point was instructd by capillary method. Theoritical appreciate was compared with practical rate. sack ON DRYINGThis method measures the weight of volatile compounds or moisture of any(prenominal) kind which can be driven off nether the undertake conditions. Stoppered, shallow glass bottle was weighed and dried under the specified conditions. 2 grams of sample was accurately weighed and transferred to dried bottle. accordingly the skew-whiff bottle was in electric oven, removed stopper and left near by it in drying chamber at 1050c for 2 hours at atmospheric military press. After devil hours the glass container was interpreted unwrap from oven and w eighed it again.The following formula was apply for the tally of LOD% LOD = Mass of leaven specimen Mass of loaded bottle aft(prenominal) drying/ Mass of sample Mass of empty glass bottleIR SPECTROSCOPY OF DRUGThin pellet was prepared development atomic number 19 bromide and drug in a ratio of 1001 respectively. The molecular state of prednisolone acetate was studied exploitation FTIR spectrometer.DSCPA thermogram was obtained in DSC 200 F3 Maia using empty aluminum scrap as reference. The accurately weighed 2mg of sample was exposed to temperature range 0oc five hundredoc under due north atmosphere (flow rate 60ml/min) at a heating rate of 10oc/min.UV-VISIBLE SPECTROPHOTOMETER (WAVE continuance SELECTION)10mg of drug was dissolved in 100mL of simulated nasal electrolyte solution (SNES) which results in 100g/ml concentration solution. From this 25 g/ml was prepared. A few ml of this solution is transferred into cuvette (path length of 10mm cell) and scanned in the waf ture length range of 200-400nm against SNES as silent person in double glint UV-Vis spectrophotometer.COMPATIBILITY STUDIES OF LIPID AND SURFACTANT MIXTUREThe selected mixtures of lipid and wetter, lipid and co wetting agent were blended at diverse ratios like 11, 21, 31 and 41. The blend was mixed for 10min and mixtures were observed visually for clarity (or) absence of turbidity. solubility STUDIES OF PREDNISOLONE ACETATEThe solubility of drug was estimated in diverse buffers, lipids, surfactant mixture ratios and solvents which are essential during development of cooking.IN DIFFERENT LIPIDS prove tube method was use for analysis of PA solubility in several solid lipids. To determine it 10mg of drug was placed to a test tube and temperature was maintained 50c higher/above the lipid melting point. The lipid was added an increase in quantity of 10mg till PA was solubilized completely and quantity of solid lipid needed for decay of drug was determined.IN divers(a) RATIOS OF S URFACTANT MIXTUREThe solubility of prednisolone acetate was investigated in numerous surfactant and co-surfactant ratios range from 11 to 41(Sur Co-sur- Sur Co-sur). solvability studies were analyzed by adding an excess amount of PA in a 20 mL screw capped containers consisting disparate ratios of 10mL of each surfactant mixture. The mixtures were vortexed on orbital shaker at 50 rpm for 2 days at 37oc to enhance solubilization. After attaining equilibrium the samples were ultracentrifuged at 12,000 rpm for 30 min to separate the undissolved drug (PA) and supernatant was filtered through whatman filter paper. HPLC grade methanol was used to dilute the supernatant sample and these were filtered with 0.45 m membrane filter. consequently, the diluted samples were used to quantify the PA by UV-Vis spectrophotometer at 240nm.IN DIFFERENT SOLVENTS AND BUFFERSThe excess quantity of drug was added to 10ml of diverse solvents includes pH 6, 6.4, 7, 7.4, 8 phosphate buffer, ethanol, doub le distilled water and methanol in a 25ml strengthtric flask. Then flasks were properly capped and agitated at 370.5oc in orbital shaker for 48 hours. The samples were filtered through whatman filter paper. The filtrate was diluted using suitable diluent and again filtered using 0.45m membrane filters. Then samples were analyzed at 240nm by UV spectrophotometer. dsCOMPATIBILITY STUDIES OF DRUG AND EXCIPIENTSThe selected excipients were listed in GRAS (Generally recognized as safe by FDA) and used in various pharmaceutical formulations. Which are procured from reputed national and multinational manufacturers. The study of compatibility between drug and excipients was followed as.DSC system Differential scanning calorimetry was carried out using DSC F3 Maia instrument to analyze the melting transitions and heat mental ability changes of drug alone and tangible mixtures. The physical mixtures were prepared by triturating the PA and excipients in the ratio of 110 in a trench morta r for five minutes. The samples (5mg) were loaded in aluminum pans, sealed hermitically and heated under inert gas i.e. nitrogen at the flow rate of 60mL /min at 10oc/min heating rate ranges/set from 0 oc to 500 oc. The standard reference was an empty alumina crucible. The above said parameters/ conditions were used to analyze the samples and thermograms of DSC recorded/ data recorded using proteus software.Samples analyzed for DSC and FTIR were followed asThe above physical mixtures compatibility was also found by adding desire quantity of mixture in glass vials and subjected to 60 oc for 30 days. The physical appearance of mixture was not changed at the end of the study.FTIRAPI was mixed with different excipients separately and spectral analysis was carried out using FTIR to study the placebo interference. The touch pellet method was used to study the FTIR spectral studies. In this analysis, test-excipient mixture and potassium bromide were taken in the ratio of 1100. The mixtur e when placed in hydraulic press under vacuum pressure of 800 mPa resulted in the formation of compressed thin transparent disc. The molecular states of samples were traced/determined between 4000- thou cm-1 using Bruker FTIR spectrophotometer. The obtained IR spectras were evaluated to determine interactions. The data mentioned in table . Graphical representation shown/given in practice . mode DEVELOPMENT OF PREDNISOLONE ACETATEA survey of literature had showed that different instrumental methods like UV, HPLC, and HPTLC were reported for the quantification of prednisolone acetate. The present research build focused on development of UV spectroscopic method for prodigality samples estimation and HPLC for drug content, dissolution and bioanalytical quantification.Preparation of simulated nasal electrolyte solution(SNES)A solution of SNES was prepared by adding 8.77g of sodium chloride, 2.98g of potassium chloride, 0.45g of calcium chloride in water and finally made the volume to 100 mL resulting in pH 5.5.UV-Vis METHODStock solution prepartion100mg of test sample was accurately weighed, transferred into 100 mL volumetric flask. To this little quantity of ethanol was added to dissolve the sample and made the volume up to 100ml using SNES (pH5.5) to get 1000g/ml concentration stock solution.Prepartion of Linearity plotFrom the above stock solution 0.5, 1, 1.5, 2, 2.5ml were transferred in separate 100 mL volumetric flasks and diluted up to the mark with SNES to produce 5, 10,15, 20, 25 g/ml concentrations respectively. The absorbance of each concentration was recorded in 1cm cell with SNES as unfilled at 240nm using Shimadzu, UV spectrophotometer. This bit was done over for 3 times. With concentration on x-axis and absorbance on y-axis a calibration graph was constructed. lay to rest day and intraday studies were carried out for the determination of accuracy and precision using 1,5,10 g/ml standard concentration solutions. The readings were tabulated in ta ble and figure .HPLC METHODPSEUDOTERNARY PHASE DIAGRAM CNSTRUCTIONTo construct the phase plots (prosim 1.0 software) hot water titration method was used. Through these phase diagrams components concentration range was investigated which can be results in existence of large microemulsion area. The surfactants used were tween 80,60,20, Cremophor RH40 and cosurfactants were ethanol, PEG 400. The surfactant and cosurfactants was blended in fixed weight ratios such as 11, 21, 31, 41. Solid lipids were tristearin, tripalmitin, GMS, stearic acid, Palmitic acid, cetyl palmitate. Lipid was melted at 65oc to this demand quantity of hot surfactant mixture was added followed by adding water thieve by drop to mixture under stirring with magnetic stirrer until turbidity formed.DEVELOPMENT OF DRUG LOADED MICROEMULSIONSFrom each phase diagram ME area was calculated by using CAD software. From each lipid, based on the above area values highest area of phase diagram was selected then the ME formu lations were taken at required component weight ratios. The procedure of desired microemulsion was as follows. Required amount of lipid heated at 65oc in this 10mg of PA was dissolved. Heated SM and water was added to the above melted lipid mixture under stirring. The mixture was categorized as microemulsions if melt was appeared clear. Then mixture was considered as microemulsion.SOLID LIPID NANO subdivisionS PREPAR TION USING study SONICATORPA SLNs were prepared by hot lipid microemulsion technique. Drug loaded ME procedure was aforementioned in page No . 2mL of loaded ME was taken in a glass syringe using 22 gage needle. Then it was added dropwise to a 3mL of cold de-ionized water under ice bathe (2-4oc) and sonicated by probe sonicator for different time periods (5, 10, 15 min) specified by box behnken design at 200w amplitude to solidify the SLNs. The tip diam of probe 8mm was dipped in liquid of 10mm leads to reflecting upwards and wave piteous downwards.DRUG CONTENT (ASS AY) DETERMINATION1 mL of solid diffusion from the formulations selected for evaluation was transferred into 10mL volumetric flask and diluted up to the mark with methanol. Then the solution was subjected to ultracentrifugation for 15 min at 10,000 rpm. Then the supernatant was aspirated, filtered, befittingly diluted and measured the absorbance at max 243nm. The results given in remandHR TEMThe surface morphology of prepared SLNs like speck size, particle shape and aggregation were analyzed by HRTEM operating at 120kv voltage. The samples were diluted properly, stained by 2% phosphotungistic acid. A drop of aqueous public exposure was placed /mounted on carbon coated 400mesh copper grids which is to be allowed for 5 min to dry the nanoparticles on grid before exam under IR lamp/AIR DRY and TEM images were recorded with mangnification range from 11500-50000x.PARTICLE POTENTIAL, SIZE AND POLYDISPERSITY INDEX(PI)For the prepared SLNs the mean particle size, zeta potential and PI were performed by dynamic light scattering (DLS) with a zetasizer Nano ZS90 (Malvern application center, banglore), model pane 3690 and equipped with 4mW, 633nm He-Ne laser. The measurements were made at the following conditionsRefractive index of forte 1.330Dielectric constant value 78.5Viscosity of strewing medium 0.8872Temperature 250cThen the samples (100l) were diluted to 1mL with de-ionized water to prevent multi scattering and transferred in to a fluid zeta cuvettes to record the particle size. The size measurement angle was 900. The PI value was used to determine particle size distribution. The same instrument was used for the determination of zeta potential using an laser Doppler electrophoresis technique. Zeta potential value was calculated from the mean mobility of electrophoresis values by henry equation. The measurements were done in triplicate.ENTRAMENT EFFICIENCY% EE of the PA was studied by find out the unentrapped drug concentration in supernatant layer(aqueou s layer). Accurately measured 5ml of (equivalent to 10mg of PA) nanoparticle dispersion was transferred to eppendorf centrifuge tubes and centrifuged at 15000 rpm for 30 min at -3oc using ultra cooling centrifuge. Then the supernatant was separated, diluted appropriately using SNES and filtered using 0.45 m membrane filter. The filtered samples were estimated by using UV spectrophotometer at max 240nm against blank. Encapsulation efficiency was determined using the equation.% EE = Quantity of drug added weight of drug in aqueous phase/ mass of drug added x 100INVITRO DIFFUSION STUDIESDialysis bag method was used to determine invitro diffusion release profile of PA and using a USP dissolution apparatus II. Before using dialysis membrane was soaked in SNES for 12 hours and the molecular cutoff of membrane was between 12000-14000. The following conditions were used to place out diffusion studies.The run speed of paddle 100 rpmDiffusion medium SNES (pH 5.5)Volume of medium 500mLTemper ature 370.5ocTime intervalsThe SLNs dispersion containing the 10mg/5mL(amount equivalent to one dose of drug) was transferred in dialysis bag and tied at both ends. Then the it was suspended in the receptor compartment containing SNES pH 5.5. An fractional of 5ml samples were removed at fixed time points from receptor medium. The same volume of fresh buffer was replaced after every time point to maintain constant buffer volume. The fresh buffer was also maintained at the same temperature(370.5oc ) similar to sample. The PA concentration was estimated using UV-Visible spectrophotometer at 240nm against blank (SNES). The final optimized sample concentration was further determined by HPLC. The selected formulation results data were shown in Table and graphical representation in figureRELEASE KINETICSThe release mechanism of drug from solid lipid nanoparticles were analyzed and determined by fitting the invitro release data to different kinetic models includes Zero, first order, higuch i and peppas-korsemayer.ReferencesUV-VISIBLEE.G.C. Clarke, Isolation and Identifeation of Drugs, volume 1 ,The Pharmaceutical Press, London, 1978, p. 270