5. PREFORMULATION STUDY

Transcription

1 5. PREFORMULATION STUDY Preformulation studies are the first step in the rational development of dosage form of a drug substance. The work was conducted by using Losartan Potassium as a model drug. The pure was subjected to Preformulation study. The drug was studied for its organoleptic properties, solubility, compatibility with other excipients. A through investigation of physicochemical properties may ultimately provide a rationale for formulation design or support the need for molecular modification or merely confirm that there are no significant barriers to compounds development. The goals of the study therefore are: To establish the necessary physicochemical characteristics of a new drug substance. To establish its compatibility with other excipients To establish its kinetic release profile 5.1 Analytical Method Development for Losartan Potassium Preparation of Phosphate Buffer ph 6.5 solution (IP-2007) According to specification described in Indian pharmacopoeia an accurately weighed quantity of 2.38 gm of di-sodium hydrogen phosphate, 0.19 gm of potassium dihydrogen phosphate and 8.0 gm of Sodium chloride was added to sufficient double distilled purified water and it was made up to a 1000 ml clear solution. The ph of the buffer was analyzed by digital ph meter (Hanna instruments phep, Model No.PHEP) Preparation of stock solution 85 Page

2 An accurately weighed quantity of 25mg of Losartan Potassium was taken in a clean, dry 250ml of volumetric flask. Then volume was made up to 250ml with phosphate buffer ph 6.5 and shaken vigorously to yield a clear solution of 100 mcg/ml concentration Determination of λ max and calibration curve: A particular concentration from the stock solution was scanned from nm wavelength range in UV-Visible Spectrophotometer [Jasco V 630 Double Beam UV- Visible Spectrophotometer]. From the scanning report it was evident that the wavelength of maximum absorbance (λ max ) of Losartan Potassium was found at 248.7nm. The appearance of a peak at 204.6nm was considered as the solvent peak so can be eliminated as the 1st peak and hence the λ max for Losartan Potassium was found to be at 248.7nm. 86 Page

3 Fig.6. UV Scan Report of LP in PBS 7.4 From the stock solution (100 mcg/ml) different amount of solution was withdrawn and transfer to the volumetric flask and volume was made up to 10ml with phosphate buffer ph 6.5. This gives a concentration ranging from 4 mcg/ml-16 mcg/ml. The absorbances of 9 different concentrations were obtained at λ max. The different concentrations and corresponding absorbance were given on in Table 3. Absorbance data (Y) were plotted against concentration(x) and Regression analysis was done. 87 Page

4 The linear equation found in the form of Y = mx + c, where m=slope, c = intercept. The corresponding standard curve generated by linear Regression analysis along with the mathematical representing the curve is given Fig. 7. Concentration (mcg/ml) Absorbance λ max Table 3: Calibration Plot of LP in PBS Page

5 CALIBRATION CURVE OF LP IN PBS y = 0.032x - 7E-05 R 2 = absorbance Concentration (mcg/ml) Fig.7. Calibration Curve of LP in PBS Preparation of 0.1M HCl According to specification described in Indian pharmacopoeia an accurately measured quantity of 8.4 ml of concentrated hydrochloric acid was added to sufficient double distilled purified water and it was made up to a 1000 ml clear solution. The ph of the buffer was analyzed by digital ph meter (Hanna instruments phep, Model No.PHEP) Preparation of stock solution An accurately weighed quantity of 25mg of Losartan Potassium was taken in a clean, dry 250ml of volumetric flask. Then volume was made up to 250ml with 0.1M HCl and shaken vigorously to yield a clear solution of 100mcg/ml concentration Determination of λ max and calibration curve: 89 Page

6 A particular concentration from the stock solution was scanned from nm wavelength range in UV-Visible Spectrophotometer [Jasco V 630 Double Beam UV- Visible Spectrophotometer]. From the scanning report it was evident that the wavelength of maximum absorbance (λ max ) of Losartan Potassium was found at 248.8nm. The appearance of a peak at 204.6nm was considered as the solvent peak so can be eliminated as the 1st peak and hence the λ max for Losartan Potassium was found to be at 248.7nm. From the stock solution (100mcg/ml) different amount of solution was withdrawn and transfer to the volumetric flask and volume was made up to 10ml with 0.1 M HCl. This gives a concentration ranging from 6mcg/ml-20mcg/ml. The absorbances of 9 different concentrations were obtained at λ max. The different concentrations and corresponding absorbance were given on in Table 4. Absorbance data (Y) were plotted against concentration(x) and Regression analysis was done. The linear equation found in the form of Y = mx + c, where m=slope, c = intercept. The corresponding standard curve generated by linear Regression analysis along with the mathematical representing the curve is given Fig.8 90 Page

7 Fig.8 Scan report of LP in ph 1.2 From the stock solution (100mcg/ml) different amount of solution was withdrawn and transfer to the volumetric flask and volume was made up to 10ml with 0.1M HCl ph 1.2. This gives a concentration ranging from 6mcg/ml-20mcg/ml. The absorbances of 9 different concentrations were obtained at λmax. The different concentrations and corresponding absorbance were given on in Table 4. Absorbance data (Y) were plotted against concentration(x) and Regression analysis was done. The linear equation found in the form of Y = mx + c, where m=slope, c = intercept. The 91 Page

8 corresponding standard curve generated by linear Regression analysis along with the mathematical representing the curve is given Fig.9. Concentration (mcg/ml) Absorbance λ max Table 4: Calibration plot of LP in ph 1.2 Absorbance CALIBRATION CURVE OF LP IN 0.1M HCl y = x R 2 = Concentration(mcg/ml) Fig.9. Calibration Curve of LP in ph Preparation of Losartan Potassium loaded Microspheres 92 Page

9 Various encapsulation techniques are readily available for microencapsulation of drugs and one of the most commonly employed is the solvent evaporation method. The method can be performed via various protocols and the selection for best option readily depends on the property of the compounds that are intended to be encapsulated. Particularly for water-soluble compounds, the most often employed method is the double emulsion type, in which aqueous phase containing the dissolved compounds is entrapped into water insoluble matrices. The state of art falls on the unique inter-phase formation between immiscible aqueous and organic layers. Emulsion occurs when aqueous solution containing the dissolved compounds is suspended within organic solvent containing dissolved polymer. This emulsion mixture is then dispersed into a secondary aqueous solution forming secondary emulsion; commonly known as water oil water (W 1 /O/W 2 ) double emulsion. Under such process, the first aqueous solution will be entrapped in the core of the polymer while physically the polymer is shaped into small fine spheres due to the secondary dispersion. In oil-in-water (O/W) emulsion techniques, drug is dispersed with the polymer in an organic phase, which is again dispersed into an outer aqueous phase. Upon contact, the organic solvent diffuses into the external water phase and evaporates at its surface. Consequently the polymer precipitates and entraps the drug. 93 Page

10 5.3 Design and Formulation of Losartan Potassium loaded Microspheres by Double Emulsion Solvent Evaporation Technique (W 1 /O/W 2 Method) For the formulation of Losartan potassium loaded microspheres by W 1 /O/W 2 method, 1 ml double distilled purified water containing 100 mg Losartan potassium was emulsified under vigorous ultra-sonication (350 watt, 2 min) into 10 ml methylene chloride containing 4:1 blend of different amounts of Eudragit RS:RL (400mg, 500mg, 600mg). This O/W emulsion was then added drop wise into 500 ml aqueous PVA solution (0.25% w/w) to yield the secondary emulsion (W 1 /O/W 2 ). Then the secondary emulsion was stirred continuous with a mechanical stirrer with a blade fitted with a four-blade butterfly propeller with a diameter of 50 mm (Lab Digital Stirrer, Remi) for 2 hours at 750 rpm in room temperature to allow microsphere hardening. The microspheres were separated by vacuum filtration using a Wattman filter paper and simultaneously washed three times with double distilled purified water and dried in a desiccator at room temperature for 48 h. Repeated (triplicate) batches were prepared to obtain reproducible results. It was observed that all the formulations produced a reproducible yield (Pe reza et al., 2000). The different batch specifications of LP loaded microspheres are given in Table Page

11 Fig. 10: Schematic Representation of Preparation of Losartan potassium loaded microspheres by W 1 /O/W 2 method Method of Preparation Formulation Code D : P Amount of Drug taken (mg) Amount of Polymer taken in ratio 4:1(mg) Amount of Methylene Chloride ERS ERL (ml) F 1 1 : W 1 /O/W 2 METHOD F 2 1 : F 3 1 : Table 5: Batch specification of Losartan potassium loaded microspheres by W 1 /O/W 2 method 95 Page

12 5.4 Design and Formulation of Losartan Potassium loaded Microspheres by Emulsion Solvent Evaporation Technique (O/W Method) For the formulation of Losartan potassium loaded microspheres by O/W method, 100 mg Losartan potassium was dispersed under vigorous ultra-sonication (350 watt, 2 min) into 10 ml methylene chloride containing 4:1 blend of different amounts of Eudragit RS:RL (400mg, 500mg, 600mg). This resulting dispersion was then added drop wise into 250 ml aqueous PVA solution (0.25% w/w) to yield the emulsion (O/W). Then the primary emulsion was stirred continuous with a mechanical stirrer with a blade fitted with a four-blade butterfly propeller with a diameter of 50 mm (Lab Digital Stirrer, Remi) for 2 hours at 750 rpm in room temperature to allow microsphere hardening. The microspheres were separated by vacuum filtration using a Wattman filter paper and simultaneously washed three times with double distilled purified water and dried in a desiccator at room temperature for 48 h. Repeated (triplicate) batches were prepared to obtain reproducible results. It was observed that all the formulations produced a reproducible yield (Pe reza et al., 2000). The different batch specifications of LP loaded microspheres are given in Table Page

13 Fig. 11: Schematic Representation of Preparation of Losartan potassium loaded microspheres by O/W method Method of Preparation Formulation Code D : P Amount of Drug taken (mg) Amount of Polymer taken in ratio 4:1(mg) Amount of Methylene Chloride ERS ERL (ml) F 4 1 : O/W F 5 1 : METHOD F 6 1 : Table 6: Batch specification of Losartan potassium loaded microspheres by O/W method 97 Page

14 5.5 Process Optimization during Production of Batches of Microspheres a) Polymer concentration and Drug concentration When 1:1 (w/w) polymer ratios were used for both the Eudragit RS and RL polymers, the quality of microspheres formed was poor. These were irregularly shaped, not flowing, and presented with lots of indentation. Microspheres were only formed when the polymer ratio was increased to ratios of between 4:1. Discrete, spherical, and uniform microspheres were obtained within a ratio of 1:4 to 1:6 (w/w) drug/polymer ratios for both the manufacturing techniques as can be seen in SEM analysis. Drug concentration was optimized according to the dose of the drug. b) Choice and Amount of Organic Solvent The particle size and the drug loading efficiency of the microspheres appeared to show dependency on the type of organic solvent used. As a reference examination, the value of an interfacial tension between the solvent used and water phase was cited in literatures. The values of interfacial tension of Dichloromethane (DCM) (20.4) and Chloroform (CR) (31.4) were relatively higher than those of Acetone (AC) (0) and Ethyl Acetate (EA) (6.78). Apparently, the particle size of the microspheres increased with the interfacial tension in the water phase. The solvent with a low interfacial tension with water phase produced smaller-sized droplets during the preparation of the emulsion. Amount of organic solvent depends upon viscosity of polymeric solution. The polymeric solution should be less viscous and a clear solution completely solubilizing the polymers. 98 Page

15 c) Speed of stirrer During processing, it was found that when the stirring speed was kept at 500 rpm, the shapes of particles were found to be irregular for all formulations because the stirring speed was not fast enough to disperse the inner phase in outer phase and a huge coalesced mass was obtained. This is due in part to inadequate agitation of the media to disperse the inner phase in discrete droplets within the bulk phase. At stirring speeds above 1000 rpm, the turbulence caused frothing and adhesion of the microspheres to the container walls and propeller blade surfaces, resulting in high shear and a smaller size of the dispersed droplets. When stirring speed was raised to 750 rpm the best spherical particles with good surface characteristics were obtained with all formulations which is exhibited in the scanning electron micrograph analysis. 5.6 Characterization Process of Losartan potassium Loaded Microspheres a) % Yield Value of Microspheres: The prepared microspheres were assessed for the yield value. The batch was weighed after total drying and the yield % was calculated using the formula give below. Each batch was formulated in triplicate batches (n=3) to get a reproducible yield (Lim et al., 2000). Results are given in Table 7 b) Flow properties of prepared microspheres: 99 Page

16 Flow properties of prepared microspheres were determined by bulk density, tapped density, Carr s index and Hausner Ratio or Packing factor (Jain et al.,2005). i) Determination of Bulk Density and Tapped Density: Accurately weighed quantities of prepared microspheres were carefully poured into the graduated cylinder (10ml). The initial volume was measured. The graduated cylinder was tapped for 100 times. After that the volume was measured. Bulk Density = W V O Tapped Density = W W F Where W = weight of the formulation V O = Bulk Volume W F = Tapped Volume Bulk and Tapped density expressed in gm/ml. The results were given in Table 8 ii) Carr s Index or Compressibility Index: Carr' s index Tapped density - bulk density = 100 Tapped density Grading of the powders for their Flow properties according to the Carr s index. CARR S INDEX (%) FLOW 5 15 Excellent 100 Page

17 12 16 Good Fair to passable Poor Very poor >40 Very very poor The results were given in Table 8. iii) Hausner Ratio: It indicates the flow properties of the microspheres and measured by the ratio of tapped density to bulk density. Tapped density Hausner Ratio= Bulk density Hausner Ratio Properties Free flowing Cohesive powder The results were given in Table 9. c) Drug Loading and Encapsulation efficiency Microspheres were thoroughly triturated in a mortar with a pestle. An equivalent accurately weighed amount of 50 mg of powdered microspheres was extracted with 50 ml of 0.1 M HCl by extensive stirring for 24 h at 150 rpm. The solution was then filtered through a 0.45 micron membrane filter (Millipore Millex-HV); a sample of 1mL was withdrawn from this solution, diluted to 10 ml with 0.1 M HCl. The resulting 101 Page

18 acidic solution containing the extracted drug was clarified by centrifugation at 2000 g for 20 min and assayed using UV spectroscopy at nm to find out the actual drug content of microspheres. The measured absorbance was converted to drug concentration using a standard curve for the known concentration of the drug in 0.1M HCl. The experiment was carried out in triplicate for each sample. The % drug loading (Yang et al., 2003) was calculated using the following equation: Study was performed in triplicate (n=3) to get reproducible results. Results are given in Table 10. Encapsulation efficiency (Haznedar et al., 2003) was calculated using the following equation: Study was performed in triplicate (n=3) to get reproducible results. Results are given in Table 10. d) Particle Size Distribution Study The size distribution study was carried out using an optical microscope, and the mean particle size was calculated by measuring 300 particles with the help of a calibrated ocular micrometer. Calibration of the optical microscope was carried out using following procedure (Martin et al., 1993): 2 divisions of stage micrometer coincided with 3 division of eye-piece micrometer 102 Page

19 Each division of stage = 0.01 mm 1 division of eye-piece will correspond to = 2/3 X 0.01 X 1000 = 6.66 µm The microspheres are mounted on a slide and placed on a mechanical stage. The microscope eye-piece is fitted with a micrometer by which the size can be determined. The field is projected on to the screen and the particles are measured along an arbitrary chosen fixed line horizontally across the center of the particles. A size-frequency distribution curve is plotted as shown in Fig Page

20 e) Scanning electron microscope analysis The external and internal morphology of the microspheres were studied by scanning electron microscopy (SEM). The samples for SEM were prepared by lightly sprinkling the powder on a double adhesive tape stuck to an aluminum stub. The stubs were then coated with gold to a thickness of about 300 Å under an argon atmosphere using a gold sputter module in a high-vacuum evaporator. The coated samples were then randomly scanned and photomicrographs were taken with a scanning electron microscope (Jeol JSM-1600, Tokyo, Japan). SEM images are shown in Fig (Kılıcarslan et al., 2003) f) Fourier Transform Infrared Spectroscopy (FT-IR) FT-IR spectra were taken on JASCO FT-IR (Model 4100, Japan) to confirm cross linking and to investigate chemical interactions between drug and polymer matrix. 2% of samples were crushed with KBr to get transparent pellets by applying a pressure of 6 Ton. FT-IR spectra of pure LP (Losartan potassium), pure polymers (ERS 100 & ERL 100) and Losartan potassium loaded microspheres prepared by both the techniques were scanned in the range between 500 and 4000 cm -1. FT-IR spectra are shown in Fig (Chopra et al., 2007) 104 Page

21 g) Proton NMR Spectroscopy Approximately 200 mg of the sample was introduced into the probe rotor. Solid state 1 H NMR spectra were recorded on a Bruker MSL-100 NMR spectrometer operating at MHz with a magnetic field strength of 2.35 T. A ninety degree pulse 1 H decoupling field of 15 gauss with a pulse duration of 4 ms and a spinning frequency rate of 4.2 khz was used to record the cross-polarization magic angle spinning (CP:MAS) spectra. More than 2400 scans were acquired for each resulting spectrum. All observations were made at temperatures between 20 and 22 C (Omari et al., 2004). 1 H NMR spectra are shown in Fig h) Thermal Analysis of Drug crystallinity (Differential Scanning Calorimetry) Differential scanning calorimetry (DSC) thermograms were obtained by a Mettler Toledo DSC 822e Stare 202 System (Mettler Toledo, Switzerland) equipped with a thermal analysis automatic program. Aliquots of about 5mg of each sample were placed in an aluminium pan of 40µl capacity and 0.1mm thickness, press-sealed with a perforated aluminium cover of 0.1mm thickness. An empty pan sealed in the same way was used as reference. DSC curves of pure API (LP), pure polymers (ERS & ERL) and LP loaded microspheres prepared by both the techniques were recorded. Conventional DSC measurements were performed by heating the sample from 20 to 300 O C at the rate of 5 O C/min under a nitrogen flow of 50 cm 3 /min. The starting temperature was 20 O C. Indium (99.99% purity) was used as a standard for calibrating the temperature (Yfiksel et al., 1996). DSC Thermograms are shown in Fig i) Crystallographic characterization using Powder XRD Powder XRD patterns were obtained with a Philips XRD (Model: 1730/10) with CuK α radiation (0.154 nm) at 35 kv and 20 ma over a 2θ range of Diffraction patterns for pure API (LP), pure polymers (ERS & ERL) and LP loaded microspheres 105 Page

22 prepared by both the techniques were recorded. The samples were grounded before analysis (Aso et al., 1993). X-Ray Diffraction spectra are shown in Fig j) In-Vitro Drug Release Study i) Release Kinetics Data obtained from In-vitro release studies were fitted to various kinetics models to find out the mechanism of drug release from microspheres (Li W et al., 1994). Kinetics models used for In-vitro drug release from microspheres: Zero order release kinetic model. First order release kinetic model [1897]. Hixon-Crowell Model [1931]. Higuchi Model [T.Higuchi 1963 & W.I. Higuchi 1967]. Zero order Kinetics: According to this model, under standard condition of temperature and agitation, the dissolution medium, the dissolution rate model can be described by the equation. dq/dt = K 0 or, Expressing in an integrated form q = K 0 t Where, q = amount of drug released per unit surface area. K 0 = zero order release rate constant t = time A plot of q vs. t gives a straight line. First order Release Kinetics (Noyes Whitney s Equation): 106 Page

23 According to Noyes Whiteny, under standard condition of agitation and temperature, the dissolution rate process for solids can be described by the equation. dq/dt = K 1 (C s C t ) Under Sink condition, i.e. when C t < 0.15 C s, the equation becomes, dq/dt = K 1 C s or in an integrated form ln q 0 /q = K 1 t Where, q=amount of drug released per unit surface area. K 1 =First order release rate constant q 0 = initial amount. C s = Saturation solubility C t = Concentration at time t. A plot of log % amount remaining to be released VS time gives a straight line with a negative slope. Hixon-Crowell Model Kinetics: As solid dissolved the surface area S changes with time. The Hixon-Crowell cube root equation for dissolution kinetics is based on the assumption that: Dissolution occurs normal to the surface of the soluble particles. Agitation is uniform on the overall exposed surface and there is no stagnation. The particles of solute retain its geometric shape. For a non-dispersed powder with spherical particles a bit mathematical derivation leads to the Kinetics equation. 107 Page

24 1/3 1/3 W0 w = K HC t Where, W 0 = Initial weight of the particles. W = Weight of the particles at t. K HC = Hixon-Crowell Release Rate constant. t = time A plot of W 1/3 1/3 0 w Vs time gives a straight line with a negative slope since W increases with time. Higuchi Model Kinetics: For coated or matrix type dosage form, the dissolution medium enters the dosage form in order for the drug to be released and diffused into the bulk solution. In such cases, often the dissolution follows the equation proposed by Higuchi. C s Q= DE ( 2A ECs) t 0.5 or, Q = K HG t 0.5 Where, Q= Amount of drug released per unit surface area of the dosage form D = Diffusion Co-efficient of the drug. E = Porosity of the matrix. T = Tortuosity of the matrix. C s = Saturation solubility of the drug in the surrounding liquid. K HG = Higuchi Release Rate constant. 108 Page

25 t = time Fitness of the data into various kinetics models were assessed by determining the correlation co-efficient, the rate constants, for respective models were also calculated from slope. ii) Experimental Procedure for In vitro drug release study The release studies of pure drug and prepared microspheres were carried out in accordance to compendia specification given in USP-XXIV / NF XVIV. For this purpose USP Apparatus 1 (Basket type) method has been used while the instrument used is an 8 basket dissolution apparatus (Electrolab Tablet Dissolution Tester, Mumbai Model NO. TDT-06P, Sr. No ). The dissolution profiles of pure drug and the drug release rate from the microspheres were studied at ph 1.2 (0.1 M HCl) and 7.4 (PBS) under sink conditions. Accurately weighed samples of microspheres equivalent to 10mg of Losartan potassium were added to 900 ml of dissolution medium kept at 37 ± 0.5 O C. The rotational speed of the Basket was fixed at 100 rpm throughout the study. The basket was covered with a nylon screen to prevent the coming out of particles during the progress of dissolution. Sample aliquots of 5 ml were withdrawn at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8 upto 12 h. A volume of dissolution medium equal to what had been removed for analysis was replaced to maintain sink condition. Samples for analysis were monitored spectrophotometrically using UV Visible Spectrophotometer (Jasco-V-630) at nm. In order to understand the mechanism and kinetics of drug release the results of in-vitro dissolution studies were fitted to various kinetics equation like zero order, first order, Higuchi and Hixon-Crowell Model. Correlation co-efficient values were calculated for linear curves obtained by regression analysis of above plots. The experiment was carried out in triplicate for each sample (n=3) to get reproducible results (Pe reza et al., 2000 & Hazendar et al., 2003). 109 Page

26 In vitro dissolution results are shown in Table 13(a) & (b) for PD and Plots are shown in Fig. 45 (PD), Fig for dissolution of optimized formulation in 0.1M HCl and Fig for PBS 7.4. Correlation coefficients are shown in Table Physical Characterizations of API for tablet preparation Organoleptic Evaluation Organoleptic characters like colour, order and taste of drug were observed and recorded using descriptive terminology Solubility Study The solubility of the Losartan Potassium was evaluated in various media by quantitatively assessing the amount of drug, which is solubilized in the respective media. 110 Page

27 5.6.3 Bulk Density Bulk Density and tapped density were measured using 100 ml graduated measuring cylinder as measure of packability of powders by tapping method. Bulk density and tapped density were calculated by following formula. Bulk density = weight of powder/ bulk volume Tapped density = weight of powder/tapped volume Carr s compressibility Index Compressibility is a parameter used to evaluate the flow property of powder by comparing the bulk density and tapped density. High density powders tend to posses free flowing properties. Carr s index was calculated by using following formula. Carr s index = (Tapped density Bulk density/ Tapped density) X Hausner Ratio Huasner s ratio provides an indication of the degree of densification which could result from variation of the feed hopper. A lower value of indicates better flow and vice versa. Hausner Ratio = Tapped density/ Bulk density Angle of Repose Angle of repose is usually determined by Fixed Funnel Method and is the measure of the flowability of powder/granules. Angle of repose was calculated from radius of the base using the following formula. θ = tan -1 (h/r) = tan -1 (Height of pile/ 0.5 base) 111 Page

28 (All the results are given in table No. 15 & 16) 112 Page

29 M i c r o m e r i t i c s t u d y Preformulation before compaction of tablet Parameters LP SSG MCC Mag.St PVP MS Bulk Density(gm/ml) Tapped Density(gm/ml) Carr s Index (%) Hausner s Ratio Angle of Repose Moisture content Moisture content of LP was carried out in a vacuum oven (Alpha Scientific and Lab. Equipment,ASLE/OO). In this method first the temperature of the oven was set i.e C and the crucible was kept to be dried completely. Then vaccume was applied at less than 5mm Hg and 1 gm of sample was taken in the crucible and the oven was closed. After 30 min. dried samples were removed from the oven and kept in dessicator. Then the weight of the crucible was taken Loss on Drying (LOD) = W 2 -W 3 /W 2 -W 1 Where, W 1 = weight of the empty crucible W 2 = weight of the empty crucible along with sample W 3 = weight of the empty crucible along with sample after drying 113 Page

30 5.6.8 Loss on Drying Lp and other excipents 1-2 gm were taken and analyzed in an electronic moisture balance (Sartorius MA 150) at C for five min. Sl. No INGREDIENTS LOD (%W/W) at C 1 LP SSG MCC Mag.St PVP MS Page

31 5.7 Formulation and Development of Bi-layer Solid Dosage Form of Losartan Potassium Containing an Immediate Release Layer and a Slow Release Layer The bi-layered tablets of losartan potassium were prepared by the direct compression method. The drug, polymers and other excipients (batch size 50 tablets) used for both immediate (IR) and controlled release (CR) layers were passed through sieve # 80 and used as per formulation Table. 6.i Stage I: The Sustained release layer containing microspheres, diluents, binder and lubricants were mixed uniformly and passed through sieve # 80, then compressed on single station tablet machine using 8 mm round and flat punches with hardness between 4-5 kg/cm. Stage II: IR layer containing drug, super disintegrating agent, diluents and lubricant were mixed uniformly and compressed over CR layered tablet with hardness between 5-7 kg cm -2 to obtain bi-layer tablet. For. Ingredients of IR layer (mg) Ingredients of SR layer (mg) code Drug SSG MCC Mag.St MS MCC PVP Talc Mg.St Total Wt. B B B B B B Page

32 Table 6.i: Batch specification of Losartan potassium bi-layer tablet by direct compression method 5.8 Evaluation of Tablets a) Weight Variation Test Twenty tablets were weighed and the average weight was calculated. The individual weight was compared with the average weight. The tablets pass the test if not more than two tablets are outside the percentage limit and if no tablet differs by more than two times the percentage limit. The following percentage deviation in weight variation is allowed according to USP. Weight Variation allowed as USPXX-NF XV. Average weight of tablet Percentage weight variation 130 mg or less 10 % More than 130 mg and less than 324 mg 7.5 % 324 mg or more 5 % b) Length, Width and Thickness Test 116 Page

33 Ten tablets were picked from formulations randomly and length, width and thickness were measured indivisually using Vernier-caliper (Mitutoyo Digimatic, CD-8 CDX).It is expressed in millimetre and average was calculated. c) Hardness Test Hardness indicates the ability of a tablet to withstand mechanical shocks while handling. The hardness of the tablet was determined using (Dr. Schleuniger Hardness Tester, 8M). It was expressed in Kilopound (Kp). Ten tablets were randomly picked from each formulations and hardness of the tablets were determined. The average value was also calculated. d) Friability Test The friability of tabets was determined using Roche Friabilator (Electrolab,EF-1W). It is expressed in percentage (%). Ten tablets were initially weighed (W initial) and transferred into friabilator. The fribilator was operated at 25 rpm for 4 minutes. % friability of tablets less than 1% are considered acceptable. The tablets were weighed again (W final). The % friability was calculated by, % Friability= ( Initial Wt- Final Wt)/Initial Wt X 100 % Friability of tablets less than1% are considered acceptable. e) Disintegration Time It was determined by using USP device (Electrolab, ED-2 SAPO) which consists of 6 glass tubes that are 3 inches long. To perform disintegration test, one tablet was placed in each tube and the basket arch was positioned in a 900 ml beaker of water at 37C=+-2C.A standard motor driven device was used to move the basket assembly 117 Page

34 up and down. To be in compliance with the USP standard, all tablets must disintegrate and all particles must pass through the 10 mesh in the time specified. f) Drug excipients compatibility study API, MS and excipients were thoroughly mixed in predetermined ratio given in table No.18 and passed through the sieve # 40. The blend was filled in glass vials and closed with gray rubber stoppers and sealed with aluminium seal and charged in to stress conditions like at 25 0 C/60 % RH, 40 0 C/75 %RH, and control (2-8 0 C). The samples were observed in different time interval for Physical observation and Chemical observation were carried out by FTIR spectroscopy. g) Dissolution study The release studies of pure drug and prepared microspheres were carried out in accordance to compendia specification given in USP-XXIV / NF XVIV. For this purpose USP Apparatus 1 (Basket type) method has been used while the instrument used is an 8 basket dissolution apparatus (Electrolab Tablet Dissolution Tester, Mumbai Model NO. TDT-06P, Sr. No ). The dissolution profiles of pure drug and the drug release rate from the bi-layer tablets were studied at ph 1.2 (0.1 M HCl) and 7.4 (PBS) under sink conditions as mention in the microspheres. Samples for analysis were monitored spectrophotometrically using UV Visible Spectrophotometer (Jasco-V-630) at nm. In order to understand the mechanism and kinetics of drug release the results of in-vitro dissolution studies were fitted to various kinetics equation like zero order, first order, Higuchi and Hixon- Crowell Model. Correlation co-efficient values were calculated for linear curves obtained by regression analysis of above plots. The experiment was carried out in triplicate for each sample (n=3) to get reproducible results. h) Stability study 118 Page

35 Stability study was carried out by exposing the formulation to different conditions including stress conditions of temperature and pressure as per ICH guidelines. Generally stability study was done at initial, 30 0 C/65 % RH (for 1,2,3, 6 months). After the study the samples were checked for physical and chemical properties for any changes and to be within the limit. 119 Page