Preformulation Studies on Phenytoin Agglomerates Prepared by Hot Melt

PREFORMULATION STUDIES ON PHENYTOIN AGGLOMERATES PREPARED BY HOT MELT

Dr. Maged Fawzy DEEF Pharmaceutical Industries Company, SA This work was a part of Master thesis presented in Faculty of Pharmacy, AlAzhar University, Egypt 2007

INTRODUCTION
Hot Melt
Hot melt agglomeration is one type of wet granulation techniques that may be utilized to enhance the dissolution profile of water insoluble drugs. Hot melt agglomeration is also called thermoplastic granulation and it may be defined as the process in which the granulation is achieved via utilization of granulating agent which is in the solid state at room temperature, this granulating agent has low melting point (40 -80)C. Melt agglomeration techniques: 1. In situ agglomeration, in which the whole powder bed including agglomerating agent are mixed and the bed temperature raised stepwise till reaching the melting point of the agglomerating agent. 2. Pump on melt agglomeration in which the melted agglomerating agent is added to pre-heated powder bedi . 3. Utilization high shear mixing in melt agglomeration, using mixer with impeller speed range 100 -500 rpm, as the intensive agitation raises the temperature of the powder bed enough to melt the agglomerating agent. 4. Spray congealing by atomizing a drug dispersion in a melted carrier, then allow the melted droplets to solidify.ii Advantages of Hot melt process: 1. No solvent is added. 2. Fewer processes are performed. 3. Drying step is eliminated. 4. Safe application to the environment. 5. Uniform distribution of the particles. Disadvantages of hot melt process: 1. It is a high energy input process. 2. The process could not be applied to thermolabile drugs. Rumpf iii-iv classified the mechanisms of agglomeration binding into: 1. Nucleation of primary particles (Figure 1A): as the system temperature raised to about 2/3 of the melting temperature of solids - by heat introduced to the system from external source or created during the below process by friction - diffusion of molecules from one particle to another occurs forming solid bridge. 2. Coalescence (Figure 1B) takes place following random collision of well formed nuclei. The coalescence occurs only if the particles have excess surface moisture. 3. Layering (Figure 1C) is the growth mechanism of the formed nuclei which occurs by successive addition of material on already formed nuclei. 4. Abrasion transfer (Figure 1D) involves transfer of material from one particle to another without any preference in either direction. 5. Size reduction which has indirect effect on the growth mechanisms. Size reduction may occur via attrition (Figure 2A), breakage (Figure 2B) and shatter (Figure 2C). The produced fragments redistribute onto surviving nuclei and participate in the growth processiv. The agglomerating liquid plays an important role in determining the particle size of the produced granules. A uniform liquid distribution is an important factor for controlling the growth of nuclei. The interaction between the granulating liquid and the particle surface is dependent on the ability of the liquid to wet the particle surface, this is achieved if the granulating liquid has surface activity v. Models for moist agglomerates according the state of liquid in moist agglomeratesiv: 1. Pendular state, occurs when the liquid fill part of the void space to form discrete lens-like rings at the contact points between particles forming the agglomerates (Figure 3 A). 2. Funicular state occurs when the liquid bridge containing gas and pores filled with liquid are present and pockets of air are dispersed throughout the agglomerate (Figure 3 B) 3. Capillary state is reached when all void space within the agglomerate is completely filled with liquid and the primary particles are held together only by the surface tension of the droplet (there is no interparticle capillary bonding) (Figure 3 C) 4. Droplet state in which the liquid completely envelopes the agglomerates (Figure 3 D) .The occasion of each model depends on the

relative amount of the liquid phase in the agglomerate according to the following tablev:

Table 1: Different models of agglomeration according to percent liquid saturation62 Model Pendular state Funicular state Capillary state Droplet state Liquid saturation 18.2% - 24.3% up to 25% 25 % - 80% > 80%

A

B

C

D

Figure 1: The different mechanisms of agglomeration bindingiv

A

B

C

Figure 2: Different mechanisms of size reduction of the formed agglomeratesiv

B A

C

D

Figure 3: The different models describing the liquid bonding for the state of liquid in moist agglomerates iv.

Liquid saturation (L) may be defined as the relative amount of liquid phase in the agglomerate, as the liquid saturation increases, the effect of friction forces between particles diminish , and the strength gradually become controlled by mobile-liquid bondings. At the same moment the brittle agglomerates change into a plastic deformed state which followed by rapid granulation growth. So the mobile-liquid bondings can be utilized in explanation of the granule growth process.v Liquid saturation (L) has a direct relationship with the mean granule size (dgw)vi-vii.The liquid saturation should have a value close to 100% in order to achieve granulation, values exceeding 100% cause over wetting of the granulating mass. Lindberg stated that there is a strong relationship between porosity and liquid saturationviii. The following equation is established to calculate liquid saturation (L)iii:

L 

1   M w s  M s w

Where  is the porosity , Ms is the solid mass of the dry agglomerate, Mw is the mass of the wet agglomerate, s is the density of solid mass and w is the density of the agglomerating agent. Agglomeration process results in irregularly shaped porous particles with relatively low bulk density and strength while the granulation process results in nearly spherical particles with relatively high bulk density and strength. (100) Characterization of the pharmaceutical granules xlix: a. High compactability. b. Good flowability. c. Have promotive effect on the tablet disintegration. d. Have promotive effect on drug dissolution. e. Capability of being reworked without loss compressibility characteristics. f. Batch to batch reproducibility.

of

flow

or

Hydrophobic drugs are usually not well absorbed orally because of failure of the drug to dissolve in gastrointestinal fluidsix.

Phenytoin
Phenytoin is 5,5-diphenyl-2,4-imidazolidinedione having the following structural formula: Phenytoin was first synthesized by Biltz in 1908x. Phenytoin powder has melting point 295-298 C. It is practically insoluble in water, sparingly soluble in ethanol and acetone. Phenytoin is soluble in alkali hydroxidesxi,with dissociation const.(pKa) of 8.31xii . Phenytoin exerts antiseizure activity without causing general depression of the CNS, the most significant effect of phenytoin is its ability to modify the pattern of maximal electroshock seizuresx. Pharmacokinetic properties of phenytoin show that t1/2 varies between 7-60 hours and tmax is about 2-4 hoursxiii . Official Preparations are phenytoin tablet (USP & BP) and phenytoin suspension (USP & BP)xiv-xv. phenytoin sodium is also official in USP and BP in different dosage forms. phenytoin and phenytoin sodium are bioequivalent and tablets of phenytoin showed better dissolution propertiesxvi. Several papers were published on the problems associated with dissolution of the commercial products. Kotaki xviiclaimed that the commercial phenytoin products show wide variation in dissolution rates. Mansonxviii stated that various phenytoin products show different bioavilabilities in children. Different techniques were developed by many researchers to overcome phenytoin dissolution problem including: 1. Muhrerxix enhanced phenytoin dissolution by using dense-gas antisolvent technique. 2. Several research works were done on enhancing phenytoin dissolution by using cyclodextrins derivativesxx-xxi. 3. Trapaniliii developed quantitative structure property relationship for enhancement of dissolution using -hydroxy propyl cyclodextrin. He concluded that the dissolution of phenytoin increased more than 100 times.

4. Carbowax was utilized to enhance phenytoin dissolution by using solid dispersionxxii. 5. Povidone was utilized to enhance the dissolution rate using roll mixing, solid dispersion, spray drying 81-83 xxii,xxiii,xxiv. 6. Chowxxvstudied the effect of modification of physical properties of phenytoin by recrystallizationusing crystal defect inducing agents. 7. Bioreversible derivatization was introduced as a method for enhancing dissolution profile by many workers 84-86 xxvi,xxvii,xxviii. 8. Co-grinding with surfactants and porous silicate was utilized to enhance the dissolution ratexxix-xxx. 9. Koelemanxxxi studied the different dissolution rates of phenytoin and its coprecipitate with montomorillonite. 10. Bastamixxxii discussed different factors that affect release of phenytoin from various formulations. 11. Soft gelatin capsule was suggested by Batemannxxxiii to overcome the dissolution problem. 12. Solvent deposition was used by Johansen to solve the problemxxxiv. Preliminary experiments were performed using different thermoplastic agents and various fillers. It was concluded that gelucire could be utilized as agglomerating agent and lactose as filler to enhance dissolution profile of phenytoin tablet. Gelucirexxxv is composed of mixture of glycerides and esters of polyethylene glycol which are responsible for the hydrophobic and the hydrophilic properties. Gelucire is characterized by two numbers, the first one referring to the melting point and the second referring to the HLB value. Gelucire are extensively used in pharmaceutical industries to enhance the dissolution rate and drug delivery. Natarajin developed liquid filled nanoparticles for protein delivery by using gelucire as surfactantxxxvi. Schamp developed a liquid formulation to enhance dissolution of poorly soluble drug using gelucirexxxvii. Vilhelmsen studied the effect of process variables on the agglomerates produced by using gelucirexxxviii. Karatas improved the dissolution rate of piroxicam using gelucire and labrasolxxxix. Sevensson explained the mechanism of dissolution enhancement of poorly soluble drug by gelucirexl. Pongjanyknl developed gelucire pellets for protein deliveryxli. Itoh represented a microemulsion formulation for a model poorly water soluble drug for enhancement of oral absorptionxlii. Adamo enhanced the

Gelucire

dissolution of Diclofenac sodium by using gelucire 50/13 solid dispersion techniquexliii.

Lactose
Anhydrous lactose is hydrophilic filler that is commonly used in solid formulation to enhance wettability and dissolution of poorly soluble drugs. Anhydrous lactose is compressed by defragmentation mechanism. Comparing to lactose monohydrate, anhydrous lactose is more compactable, soft, less elastic and undergo brittle fracture much more readily at lower stresses than monohydrate. The main disadvantage of anhydrous grade is the relatively slowing disintegration of tablets due to poor water penetration due to small pore diameter. Direct compression (D.C.) grade lactose is more flowable and more compressible than powdered lactose. Lactose D.C. is prepared of pure lactose with a small amount of amorphous lactose. The amorphous lactose improves the compression force/ hardness profile of lactose.xxxv,xliv The objective of this chapter was to formulate phenytoin agglomerates prepared by hot melt agglomeration technique. The prepared phenytoin agglomerates were subjected to the following tests: Drug content, agglomerate shape, micromertic characteristics, friability testing, NIR characterization, agglomerate bed hydrophilicity and in-vitro dissolution. The micromertic characterization includes; particle size analysis, agglomerates densities, flow properties, pore size analysis and specific area measurement.

EXPERIMENTAL nnnnnnnnbn
MATERIALS
1. 2. Phenytoin USP28 was supplied from Recordati, Italy Anhydrous lactose USP28 direct compression grade was supplied from Lactose New Zealand, New Zealand 3. Gelucire 50/13 was supplied from Gattefosse, France 4. Magnesium stearate supplied from Alba chemicals, USA 5. Methanol HPLC far UV was supplied from Merck, Germany 6. Z 7. Acetonitrile HPLC far UV was supplied from Merck, Germany 8. Triethylamine was supplied from Sisco Research Lab, India 9. Tris(hydroxymethyl)aminomethane was supplied from Fluka, Germany 10. Phosphoric acid was supplied from Merck, Germany 11. Sodium lauryl sulfate was supplied from Canadian alcolac, Canada 12. Heavy liquid paraffin was supplied from Witco, Germany

EQUIPMENT
1. Digital balance, (Sartorius, Germany) 2. Series of standard stainless steel sieve ,(Fritsch, German) 3. Mastersizer MS 2000 particle analyser, Laser diffraction particle size analyzer (Malvern, England) 4. Infra red spectrophotometer, (Schimadzu, Japan) 5. Laboratory scale Temperature controlled high shear rotary processor with the impeller fixed from its upper axis, the impeller has variable speed ranging from 550 rpm to 1050 rpm. The pot volume is 3000 ml, the equipment has no chopper. (Philips, Czech) 6. Laboratory scale High shear double jacketed granulator, (Zanchetta, Italy) with three bladed impeller of plough like shape fixed in the bottom of the pot, the impeller has variable speed from 120 rpm to 1200 rpm. Zanchetta is equipped with chopper that is fixed in the lid , chopper speed is fixed. The pot volume is 2500 ml. 7. Powder Flowmeter (Erweka, Germany), 8. Tape Density meter , (Campbell, India)

9. Helium pycnometer Ultrapycnometer 1000, (Quantachrome, USA) 10. Ultrasonic water bath, (Selecta,Spain) 11. Near Infra Red spectrometer ( Bruker, USA) 12. Vibrator ( Selecta Vibromatic -384, Spain) 13. Friability tester (Pharmatest, Germany) 14. USP Dissolution tester (Pharmatest, Germany) 15. High speed gas sorption analyzer (Nova 1000 series, Quantachrome, USA) which used nitrogen gas as adsorbate , the theory of working depends on measuring the surface area using BET sorption isotherm. The instrument is capable of measuring surface area, specific surface area, true density, pore size and pore size distribution. 16. HPLC (1100 series, Agilant, USA)

METHODOLOGY
1. Determination of Phenytoin Standard Curve Phenytoin standard curve was adopted according to USP 28xiv method under the phenytoin tablet monograph. Standard solution was prepared by dissolving 100 mg phenytoin in 100 ml of the mobile phase(filtered and degassed mixture of water, methanol, acetonitrile, Triethylamine solution, and acetic acid (500:270:230:5:1). Serial dilutions were prepared to obtain the following drug concentrations; 0.25, 0.50, 0.75, 1.0 , 1.25 mg/ml. The liquid chromatograph is equipped with a 254-nm detector and a 4.6mm × 25-cm column that contains packing L1. The flow rate is about 1.5 ml per minute. The column efficiency should be not less than 6500 theoretical plates, the tailing factor should be not more than 1.5, and the relative standard deviation for replicate injections must be not more than 2.0%. The standard curve of phenytoin was constructed by plotting the measured AUC against the corresponding concentration. 2. Estimation of possible interaction between phenytoin and added excipients The absence of interaction between phenytoin and the added excipients was tested by utilization infra red spectroscopy. Aliquot compressed samples of the following items were scanned using IR spectroscopy: The pure API Physical mixture of API with the added excipients. Potassium bromide was used as diluent and spectra recorded in the range 4000 –850 cm-1. 3. Melt agglomeration of phenytoin Phenytoin agglomerates were prepared by using hot melt pour on technique using high shear processor according to the following procedure: a. The pot was loaded with 30 gm gelucire 50/13 (15%), the temperature of the pot was raised to 60C, the gelucire was melted completely at that temperature. b. After that 40 gm phenytoin (20%), and 130 gm anhydrous lactose (65%) were added

c. The agglomeration process was carried out in the granulator at constant temperature (60C) and for pre-set agglomeration time (10 minutes). The granulator speed was constant (550 rpm). d. Once completing agglomeration process, the hot agglomerates were sieved through 450 m stainless steel sieve. e. The yielded agglomerates were weighed to calculate the product yield. 4. Characterization of agglomerates

a. Drug content Drug content was estimated using the method described in USP28 under the monograph of phenytoin tablets.xiv. "Weigh and finely powder not less than 5 gm. Transfer an accurately weighed portion of the powder equivalent to about 250 mg of phenytoin, to a 500-ml volumetric flask, dissolve in and dilute with mobile phase to volume, and mix" after that the samples were assayed using the official USP28 HPLC method, taking in consideration that each test was performed in triplicate and relative standard deviation (RSD) was calculated.

b. Agglomerate shape Agglomerates shape was examined by light microscope fitted with digital camera and micrographs of these agglomerates were captured. c. Micromertic properties

i. Particle size analysis The yielded agglomerates were analyzed using Malvern mastersizer 2000. the basic simplest theory is the Fraunhofer model. This model can predict the scattering pattern that is created when a solid opaque disc of a known size is passed through a laser beam, Mie theory was developed to predict the light scattering behaviour of all materials under all conditionsxlv,li Wet method is utilized for particle size analysis using liquid paraffin as dispersion medium. A sample of 200 mg weight was levigated gently with liquid paraffin then the sample was poured into the measurement tank. The obscuration value should be in the range (3 – 15) % and the laser intensity should be more than 65%.

ii. Agglomerate densities The different types of densities were measured to assess different flow properties, porosity (intragranular porosity) and voidage (intergranular porosity)xlvi . Bulk density is the mass of the agglomerates divided by the packing volume while the true density is the mass of the particles divided by the volume of particles excluding open and closed pores. Apparent particle density is the same but including the closed pores. Effective particle density differs from apparent density in inclusion of open and closed pores.xlvi above Bulk and tapped densities The bulk and tapped densities and the difference between the two measures affect packing propertiesv . The bulk (DB) and tapped (DT)densities were determined using measuring cylinder tapping procedure. A sample of 100 gm of the agglomerates was poured in a measuring cylinder and the weight of the poured volume was recorded . Tapping was performed till a constant volume was reached, the final volume was measured. Effective granule density Effective granule density was measured by using pycnometer using low surface tension non solving liquid (benzene) as an intrusion fluid.xlvii a sample of about 2 gm agglomerates were poured in pycnometer of known volume (10.525 ml). After the pycnometer was filled with benzene and weighed. The volume occupied by the agglomerates was calculated by knowing the density of benzene, weight of benzene displaced by the agglomerates. The following equation was utilized to calculate the density:

g

G C  B/F

Where g is the effective particle density, G is the weight of granules (gm), C the capacity of pycnometer (ml), B the weight of intrusion liquid (benzene) (ml) and F the specific gravity of benzene( 0.887 gm/ml). Apparent granule density Apparent granule density was measured by the method described by Swarbrick by a pycnometer using mercury as an intrusion fluid.xlvii

The volume occupied by the agglomerates was calculated by knowing the density of mercury(13.534 gm/ml), weight of mercury displaced by the agglomerates. True granule density True density is measured using helium pycnometer., the theoryxlviii depends on Archimedes'priciple of fluid dsiplacement . the displaced fluid should be an inert gas that can penetrate all the pores, for this reason, helium is used since its small atomic dimensions assure penetration into pores approaching 0.25 nm in diameter. Aliquot sample of about 1.5 gm was put in the measuring cell, after that, helium gas fills the sample chamber and pressure is measured. The helium then enters another empty chamber and the pressure in both chambers is measured. The sample volume is calculated based on these pressures and then with the known weight of the sample, the true density is then calculated. iii. Flow properties of agglomerates Many factors affect the powder flowability including powder cohesiveness, particle size and size distribution. The flow properties of the yield agglomerates were assessed by using the following parameters: flow rate, angle of repose and compressibility index. The value of angle of repose of fine particles is dependent on the surface property while the flow of coarse particles is related to packing densities& the mechanical arrangement of particles so it is useful to combine more than one method to describe the flowability.xlvi c.1. Direct method Agglomerate flow rate was assessed using Erweka flowmeter. A sample of 50 gm of the agglomerates was poured into the funnel of the Flowmeter which has the following dimensions; 6.5 cm the diameter at top and 0.9 cm the diameter of the efflux tube and the flow of the powder through the funnel was determined as gm/sec. after that conversion of the flow rate units into ml/sec was performed using bulk density results.

c.2. Indirect method

Agglomerate flow rate was determined by indirect method using angle of repose. Several techniques are used to measure angle of repose including : Fixed height cone, fixed base cone, tilting table, rotating cylinder, ledge, crater and platform.xlvi The different methods may produce different values for the same powder but generally powders with angles greater than 50 have unsatisfactory flow properties, whereas minimum angles close to 25 correspond to very good flow properties. Fixed height cone was utilized as a technique for measuring angle of repose. The technique is suitable for particles of size larger than 150 microns where the cohesive forces are minimalxlvi. From measurement of the flow rate using Erweka flowmeter the diameter (D) and the height (h) of the formed heap were measured and utilized in calculation of the angle of repose according to the following equationxlix:

tan  

2h D

Carr described the flow rate by indirect method via the following equation:
C DT  DB 100 Dt

The percentage compressibility (C) is a direct measure of the potential powder arch or bridge strength. Carr categorized the powders according to the percentage compressibility into 7 categories showed in the following table: Table 2: Carr's index classification of the materials according to percentage compressibility % compressibility range 5 –15 12 –16 18 -21 23-28 28-35 35-38 >40 Flow description Excellent (free flowing granules) Good (Free flowing powdered granules) Fair( powdered granules) Poor( very fluid powders) Poor(fluid cohesive powder) Very poor(Fluid cohesive powder) Extremely poor(cohesive powder)

Hausner ratio (H), was calculated from bulk density (DB) and tapped density (Dt) results using the following equation: H=Dt/DB iv. Pore size characterization The pore sizel may be characterized according to the opening size; macropores (pores with openings exceeding 500Ao), micropore (pores with openings not exceeding 20Ao) and mesopores (pores with intermediate size openings ). Gas sorption analyzer was utilized to characterize the pore size and pore distribution. Pore size distribution is the distribution of pore volume with respect to pore size. Desorption isotherm is more appropriate than the adsorption isotherm in calculating pore size and pore size distribution since the desorption isotherm is more closer to true thermodynamic stability since it has lower free energy at lower relative pressure. Pore size can be calculated from Kelvin equation: rk  2Vm RT ln(P / Po )

Where :  is the surface tension of adsorbate (nitrogen) at its boiling point (8.85 ergs/cm2 at 77K) Vm is the molar volume of liquid nitrogen (34.7 cm3/mol) R is the gas constant (8.314 x 107 ergs/deg/mol) T is boiling point of nitrogen (77 K) P/Po is relative pressure of nitrogen rk is the Kelvin radius of the pore (Ao). `So the equation may be reduced to: rk  4.15 log( P / Po )

rk is the radius of the pore in which condensation takes place at relative pressure P/Po, but some adsorption occurs on the pore walls before condensation where in the desorption phase; an adsorbed layer remains when evaporation occurs, so the actual pore radius rp is:

r p  rk  t

Where t is the thickness of the adsorbed layer which equals 3.54 Ao (the thickness of one nitrogen molecular layer) Pore size distribution is computed according to Barrett, Joyner and Halenda (BJH) method which depends on the theory that the volume of pore Vk is directly proportional to the inner capillary volume Vp1 An aliquot sample (about 300 mg) is put in the measuring cell of the high speed gas sorption analyzer, the loaded cell was fixed to the degassing station to perform degassing process at 35oC for 120 minutes, after that the cell was transferred to the measurement station to start cycles of adsorption and desorption of nitrogen gas to calculate different parameters utilized in calculating specific surface area, pore volume and pore size distribution.

v. Specific surface area Specific surface area was utilized to characterize the agglomerates. Specific surface area is utilized for comparison purpose between the different agglomerates. Specific surface area was calculated using 6 point BET isotherm method. Brunauer, Emmett & Teller (BET) lequation states that
P 1 C  P 1    V CP V(p P V C  m  o o ) m

Where: P& Po are partial pressure and saturated pressure of adsorbate. V is the total volume of adsorbed gas on the surface of adsorbate under standard temperature and pressure. Vm is the volume of adsorbed gas if entire surface of adsorbate is covered with monolayer thickness. C is a constant Plotting V versus P/Po results in a straight line where the slop equals
C V  1 m C

and the intercept equals

1 VmC

Since one mole of gas occupy 22.412 x 103 cm3 at STP, the volume of monolayer can be converted into moles. The number of molecules in the monolayer can be computed from Avogadro's number (one mole of gas at STP contains 6.023 x 1023 molecules) The cross sectional area for nitrogen is 16.2 x 10-20 which is used to further calculating the area occupied by monolayer.

The specific surface area was calculated from obtained results The pore size (t) is estimated using the following equation:

  5 t  3.54   2.303 log( P0 / P )  

1/ 3

d. Friability of agglomerates The agglomerate friability (f) was determined by the method described by Fonnerv , in which about 10 grams(Wa) of agglomerate size (200 320) m is shaken in a glass bottle for 10 minutes, subsequently the agglomerates are passed through 200 m sieve after that the friability is assessed by reweighing the retained agglomerates (Wb) according to the following equation: f  (1  Wa ) 100 Wb

e. Characterization of agglomerates using near infra red spectroscopy The yield agglomerates and phenytoin powder were scanned using NIR spectroscopy. a sample of 50 gm powder in clean dry beaker was scanned using NIR solid probe. f. Powder bed hydrophilicity Powder bed hydrophilicity was assessed using the method described by Lefebvre.(112) a sample of 2 gm of granules are put in sintered glass funnel partially plunged(1 mm) into water and the time required for water capillary to rise up to the powder bed was measured. Some methylene blue crystals at the top of powder bed help in accurate measure of this critical time.(112) g. In-vitro dissolution The dissolution procedure adopted to the monograph of phenytoin tablet in the USP28 was usedxiv; USP apparatus 2, 100 rpm, 900 ml 0.05M tris buffer was used as dissolution medium. Samples (5 ml) were withdrawn and filtered using 0.45m syringe filter after 10; 20; 30 ;60 and 120 minutes to evaluate the drug dissolution profile. The withdrawn samples were retrieved by 5ml fresh dissolution medium to keep the volume of

the dissolution medium constant. The amount of phenytoin dissolved was determined using the HPLC method described in the assay method. The dissolution test was applied to the following; a. Phenytoin agglomerates (size