Preformulation study is the first step in the rational development of dosage forms of a drug substance. It is study of physical and chemical properties of drug particles alone and combined with different exepients. The study is useful to find out any interaction between drug and exepients and also useful in developing stable and bioavailable dosage forms which can be mass produced.
The FT-IR spectrum of pure Simvastatin, Polyvinl Pyrrolidine, Physical mixture and Nanosuspension of Simvastatin.
PREPARATION OF STANDARD CURVE:
Preparation of 0.1N HCl:
8.5 ml of concentrated hydrochloric acid was taken and diluted with distilled water up to 1000 ml.
Preparation of Phosphate buffer pH 7.0:
Preparation of standard curve of Simvastatin with 0.1N HCl:
100 mg of simvastatin was accurately weighed and dissolved in a small portion of 0.1N HCl in a 100 ml volumetric flask and volume was made to 100 ml. this was primary stock solution contained 1000 µg/ml. From this primary stock solution 10 ml was pipette out and transferred to 100 ml volumetric flask and volume was made up to 100 ml with 0.1 N HCl which contained the concentration of 100 µg/ml. From the second solution 10 ml was pipetted out and diluted to 100 ml with 0.1 N HCl. From third stock solution aliquots equivalent to 2-10 µg/ml (2, 4, 6, 8 and 10 ml) were pipette out in to a series of 10 ml volumetric flask and volume was made up to 10 ml with 0.1 N HCl. The absorbance of this solution is measured at 239 nm using UV-Visible spectrophotometer. The calibration curve was plotted taking concentration µg/ml on X-axis and absorbance on Y- axis.
FORMULATION OF NANOSUSPENSION:
Nanosuspension of simvastatin was prepared by High pressure homogenization technique. Simvastatin powder (1%w/v) was dispersed in aqueous surfactant solution of different concentration under magnetic stirring. After dispersion first size reduction step was carried out using an Ultra-Turrax T25 Basic homogenizer (IKA-Werke, Staufen, Germany) at 9500 rpm for 10 min. The obtained mixture was homogenized using Micron LAB 40 (APV system, Unna, Germany). At first two cycles at 100 bar and 2 cycles at 500 bar and then finally 15 cycles at 1500 bar were run to obtain nanosuspension.
Formulation of different batches of simvastatin nanosuspension
Ingredients
FN-1
FN-2
FN-3
FN-4
FN-5
Simvastatin (%w/w), g
1
1
1
1
1
Polyvinyl pyrrolidine (%w/w), mg
500
400
300
200
100
Distilled water (ml)
98.5
98.6
98.7
98.8
98.9
PRODUCTION OF DRY NANOPARTICLES:
The lyophilization method is used to obtain nanoparticles in dried-powder state from the nanosuspension obtained after homogenization
PARTICLE SIZE ANALYSIS:
The particle size analysis was performed using Microtac Blue wave particle size analyzer. The results particle size distribution were used as a characterization parameters. Before measurement the samples had to be diluted with deionized water to obtain a suitable concentration for measurement.
SATURATION SOLUBILITY STUDIES:
Saturation solubility measurements were assayed through ultraviolet absorbance determination at 239 nm using shimadzu UV-Visible spectrophotometer. The dried powder and pure drug saturation solubility studies was performed and reported by hecq et al. Weighed amount pure drug and nanoparticles equivalent to 20 mg of drug were separately introduced in to 25 ml stoppered conical flask containing 10 ml distilled water. The sealed flasks agitated for 24hrs at 370 c and equilibrated for 2 days. An aliquot was passed through 0.1 µm membrane filter and filtrate was suitably diluted and analyzed on a UV spectrophotometer at 239 nm.
DIFFERENTIAL SCANNING CALORIMETRY:
DSC is an important evaluation technique to find any possible interaction between drug and polymer. Any such interaction leads to reduce entrapment efficiency of polymer and also efficacy of drug. Differential scanning calorimetric analysis was performed using Shimadzu DSC- 60 system. Polymeric sample of nanosuspension was sealed in aluminum cells and set in a shimadzu DSC-60 apparatus between 30°C - 300°C. Thermal analysis was performed at a heating rate maintained at a 10°C per minute in a nitrogen atmosphere. Alumina was used as the reference substance.Enthalpy changes (ΔH) were calculated from peak areas of samples and to study the polymeric changes in formulations.
ZETAPOTENTIAL ANALYSIS:
The particle charge is one of the factors determining the physical stability of emulsion and suspensions. The higher particles are equally charged, the higher is the electrostatic repulsion between the particles and the higher is the physical stability. Typically the particle charge is quantified as called zeta potential, which is measured e.g. via the electrophoretic mobility of the particles in an electrical field. Alternatively the particle charge can be quantified in surface charge per surface unit, determined by colloid titration. Zeta potential is an abbreviation for electrokinetic potential in colloidal systems. In the colloidal chemistry literature, it is usually denoted using the Greek letter zeta, hence ζ-potential. From a theoretical viewpoint, zeta potential is electric potential in the interfacial double layer (DL) at the location of the slipping plane versus a point in the bulk fluid away from the interface. Zeta potential analysis was performed to estimate the stability of the nanosuspension using Malvern Zetasizer ( Malvern instruments). The samples were diluted with deionized water with conductivity adjusted by addition of sodium chloride before measurement.
IN-VITRO DRUG RELEASE STUDIES:
The in-vitro release of simvastatin from formulation was studied by Uin from formulation was studied by USP dissolution test apparatus using paddle method at a rotation speed of 60 rpm. The dissolution medium used was freshly prepared acidic buffer (pH 1.2) and phosphate buffer (pH 7.0). 10 mg of pure drug and nanosuspension equivalent to 10 mg of drug was taken and placed in dissolution medium. The volume and temperature of dissolution medium were 900 ml and 37.0 ± 0.20c, respectively. Samples were withdrawn at fixed time and were filtered and assayed through ultraviolet absorbance determination at 239 nm using Shimadzu UV-Visible spectrophotometer. The results obtained were compared with the dissolution profile of unprocessed drug.
Table no.6: Zeta potential & Stability behavior of Nanosuspension
Zeta Potential [mV]
Stability behavior of the colloid
from 0 to ±5,
Rapid coagulation or flocculation
from ±10 to ±30
Incipient instability
from ±30 to ±40
Moderate stability
from ±40 to ±60
Good stability
more than ±61
Excellent stability
