Identification of Prednisolone Acetate

IDENTIFICATION AND CHARACTERIZATION OF DRUG

The procured prednisolone acetate was identified and characterized based on the following parameters.

1. ORGANOLEPTIC PROPERTIES

For the selected samples (drug + excipients) the following organoleptic properties were studied using descriptive terminology. Those were nature, odour and colour

2. PARTITIONING BEHAVIOR OF PA IN DISTINCT LIPIDS:

The percent partition coefficient of Prednisolone acetate in selected lipids was analyzed as elucidated elsewhere. On the part of the study/briefly, a mixture composed of melted lipid and 10mL of de-ionized water to this 10mg of PA was added. The resultant mixture was agitated reciprocally for one hour maintained at 5⁰C above the lipid melting point. The quantity of lipid utilized was mentioned in table 1. The resultant dispersion was allowed to cool. Upon making cool both phases were separated by ultracentrifugation ( ) at 15,000 rpm and filtered. The drug content in supernatant was analyzed spectrophotometrically (240nm). The percent partition coefficient was predicted as follows:

% partition = w₁-w₂/w₁ x 100

W₁ = Amount of drug added

W₂ = Amount of drug in aqueous phase

3. DETERMINATION OF MELTING POINT

PA melting point was determined by capillary method. Theoritical value was compared with practical value.

4. LOSS ON DRYING

This method measures the weight of volatile compounds or moisture of any kind which can be driven off under the specified 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. Then the loaded bottle was in electric oven, removed stopper and left near by it in drying chamber at 105⁰c for 2 hours at atmospheric pressure. After two hours the glass container was taken out from oven and weighed it again.

The following formula was used for the calculation of LOD

% LOD = Mass of test specimen – Mass of loaded bottle after drying/ Mass of sample – Mass of empty glass bottle

5. IR SPECTROSCOPY OF DRUG

Thin pellet was prepared using potassium bromide and drug in a ratio of 100:1 respectively. The molecular state of prednisolone acetate was studied using FTIR spectrometer.

6. DSC

PA thermogram was obtained in DSC 200 F3 Maia using empty aluminum pan as reference. The accurately weighed 2mg of sample was exposed to temperature range 0^oc – 500^oc under nitrogen atmosphere (flow rate: 60ml/min) at a heating rate of 10^oc/min.

7. UV-VISIBLE SPECTROPHOTOMETER (WAVE LENGTH SELECTION)

10mg of drug was dissolved in 100mL of simulated nasal electrolyte solution (SNES) which results in 100µg/ml concentration solution. From this 25 µg/ml was prepared. A few ml of this solution is transferred into cuvette (path length of 10mm cell) and scanned in the wave length range of 200-400nm against SNES as blank in double beam UV-Vis spectrophotometer.

COMPATIBILITY STUDIES OF LIPID AND SURFACTANT MIXTURE

The selected mixtures of lipid and surfactant, lipid and cosurfactant were blended at different ratios like 1:1, 2:1, 3:1 and 4:1. The blend was mixed for 10min and mixtures were observed visually for clarity (or) absence of turbidity.

SOLUBILITY STUDIES OF PREDNISOLONE ACETATE

The solubility of drug was estimated in different buffers, lipids, surfactant mixture ratios and solvents which are essential during development of formulation.

1. IN DIFFERENT LIPIDS

Test tube method was used 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 dissolution of drug was determined.

2. IN VARIOUS RATIOS OF SURFACTANT MIXTURE

The solubility of prednisolone acetate was investigated in numerous surfactant and co-surfactant ratios range from 1:1 to 4:1(Sur: Co-sur- Sur: Co-sur). Solubility studies were analyzed by adding an excess amount of PA in a 20 mL screw capped containers consisting different ratios of 10mL of each surfactant mixture. The mixtures were vortexed on orbital shaker at 50 rpm for 2 days at 37^oc 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 µm membrane filter. Then, the diluted samples were used to quantify the PA by UV-Vis spectrophotometer at 240nm.

3. IN DIFFERENT SOLVENTS AND BUFFERS

The excess quantity of drug was added to 10ml of different solvents includes pH 6, 6.4, 7, 7.4, 8 phosphate buffer, ethanol, double distilled water and methanol in a 25ml volumetric flask. Then flasks were properly capped and agitated at 37±0.5^oc 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 0.45µm membrane filters. Then samples were analyzed at 240nm by UV spectrophotometer. ds

COMPATIBILITY STUDIES OF DRUG AND EXCIPIENTS

The 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 international manufacturers. The study of compatibility between drug and excipients was followed as.

1. DSC METHOD :

Differential scanning calorimetry was carried out using DSC F3 Maia instrument to analyze the melting transitions and heat capacity changes of drug alone and physical mixtures. The physical mixtures were prepared by triturating the PA and excipients in the ratio of 1:10 in a mortar 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 10^oc/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 as

S.NO.	Sample name
1	Prednisolone acetate
2	Prednisolone acetate+ Glyceryl monostearate
3	Prednisolone acetate+Stearic acid
4	Prednisolone acetate+ Palmitic acid
5	Prednisolone acetate+ Cetyl palmitate
6	Prednisolone acetate+ Tristearin
7	Prednisolone acetate+ Tripalmitin
8	Prednisolone acetate+ Tween 80
9	Prednisolone acetate+ Tween 60
10	Prednisolone acetate+ Tween 20
11	Prednisolone acetate+ Cremophor RH40
12	Prednisolone acetate+ Ethanol
13	Prednisolone acetate+ PEG400
14	Optimized formulation(Included drug + Cremophor RH40-ethanol( )+Tristearin( ) +water)

2. The above physical mixtures compatibility was also found by adding desired 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.
   1. FTIR

API was mixed with different excipients separately and spectral analysis was carried out using FTIR to study the placebo interference. The pressed 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 1:100. The mixture 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 4000-1000 cm^-1 using Bruker FTIR spectrophotometer. The obtained IR spectra’s were evaluated to determine interactions. The data mentioned in table . Graphical representation shown/given in figure .

METHOD DEVELOPMENT OF PREDNISOLONE ACETATE

A 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 work focused on development of UV spectroscopic method for dissolution 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 METHOD

Stock solution prepartion

100mg 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 (p^H5.5) to get 1000µg/ml concentration stock solution.

Prepartion of Linearity plot

From 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 µg/ml concentrations respectively. The absorbance of each concentration was recorded in 1cm cell with SNES as blank at 240nm using Shimadzu, UV spectrophotometer. This procedure was done over for 3 times. With concentration on x-axis and absorbance on y-axis a calibration graph was constructed. Inter day and intraday studies were carried out for the determination of accuracy and precision using 1,5,10 µg/ml standard concentration solutions. The readings were tabulated in table and figure .

HPLC METHOD

PSEUDOTERNARY PHASE DIAGRAM CNSTRUCTION

To construct the phase diagrams (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 1:1, 2:1, 3:1, 4:1. Solid lipids were tristearin, tripalmitin, GMS, stearic acid, Palmitic acid, cetyl palmitate. Lipid was melted at 65^oc to this required quantity of hot surfactant mixture was added followed by adding water drop by drop to mixture under stirring with magnetic stirrer until turbidity formed.

DEVELOPMENT OF DRUG LOADED MICROEMULSIONS

From 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 formulations were taken at required component weight ratios. The procedure of desired microemulsion was as follows. Required amount of lipid heated at 65^oc 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 NANOPARTICLES PREPAR TION USING PROBE SONICATOR

PA 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 gauge needle. Then it was added dropwise to a 3mL of cold de-ionized water under ice bath (2-4^oc) 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 diameter of probe 8mm was dipped in liquid of 10mm leads to reflecting upwards and wave moving downwards.

DRUG CONTENT (ASSAY) DETERMINATION

1 mL of solid dispersion 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, suitably diluted and measured the absorbance at λmax 243nm. The results given in Table

HR TEM

The surface morphology of prepared SLNs like particle 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 dispersion was placed /mounted on carbon coated 400mesh copper grids which is to be allowed for 5 min to dry the nanoparticles on grid before examination 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 ZEN 3690 and equipped with 4mW, 633nm He-Ne laser. The measurements were made at the following conditions:

Refractive index of medium: 1.330

Dielectric constant value: 78.5

Viscosity of dispersion medium: 0.8872

Temperature: 25⁰c

Then the samples (100µl) were diluted to 1mL with de-ionized water to prevent multi scattering and transferred in to a disposable 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 determining the unentrapped drug concentration in supernatant layer(aqueous 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 -3^oc using ultra cooling centrifuge. Then the supernatant was separated, diluted appropriately using SNES and filtered using 0.45 µm membrane filter. The filtered samples were estimated by using UV spectrophotometer at λ_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 100

INVITRO DIFFUSION STUDIES

Dialysis 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 carry out diffusion studies.

The run speed of paddle: 100 rpm

Diffusion medium: SNES (p^H 5.5)

Volume of medium: 500mL

Temperature: 37±0.5^oc

Time intervals:

The 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 aliquot 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(37±0.5^oc ) 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 figure

RELEASE KINETICS

The 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, higuchi and peppas-korsemayer.

References

UV-VISIBLE

E.G.C. Clarke, Isolation and Identifeation of Drugs, volume 1 ,

The Pharmaceutical Press, London, 1978, p. 270