10 Chapter 2 Materials and Methods - Shodhganga : a...

Materials and Methods

Department of pharmaceutical sciences, JJTU. 15

MATERIALS AND METHODS

The materials and various equipments used in this research work was obtained from

different vendors.

List of materials obtained are:

Hydrogenated castor oil : Matrix, Hyderabad.

Iso propyl alcohol : Dr.Reddy’s Lab’s, HYD.

Opadry pink : Colorcon, Mumbai

Polysorbate 80 : Dr.Reddy’s Lab’s, HYD.

HPC-LF : Dr.Reddy’s Lab’s, HYD.

Poloxamer-188 : Basf

Sodium starch Glycolate : Dr.Reddy’s Lab’s, HYD.

Lactose : Dr.Reddy’s Lab’s, HYD

Meglumine : Merck, Mumbai.

Methanol : Merck, Mumbai.

Aceto nitrile : Merck, Mumbai.

Triethilamine : Merck, Mumbai

Metformin hydrochloride : Ramdev Chemical Pvt Ltd, India.

Batch No: PX-74

Percent purity: 99.09

Storage: Room temperature

: FINE CHEMICALS.

Acetonitrile gradient :HPLC grade (Qualigens Fine Chemicals, Mumbai.)

Potassium Dihydrogen

Milli-Q water : Nishka Laboratories, (Hyderabad, India)

Control rabbit plasma : Nishka Laboratories (Hyderabad, India)



INSTRUMENTS

Name of Instrument Manufacturing Company

Bilayered compression machine : Rimeck, India

HPLC : Shimadzu V.P.Serues

Automatic tablet dissolution apparatus USP I :

USP II

Vanket, USA

Electronic thickness measurement apparatus : Mitutoyo, Japan

Friability tester (USP) : Electro lab, Mumbai, India

Tablet hardness tester : Schleuniger, USA

Electronic LOD measurement apparatus : Sartorius, Germany

Bulk density apparatus : Electro lab, Mumbai, India

Electronic weighing balance, 6 Kg : Essac- Teraoka, Japan

Analytical weighing balance, 100-210 gms : Adventure. Essae, Teraoka.

Mechanical stirrer : Remimotor. Mumbai, India

Sifter : Neomachine, Calcutta, India

Planetary mixer, PLM, : Kenwood, Britain

Rapid mixer granulator : Tapasya, Mumbai, India

Rapid dryer : F.Kurt Retsch Gmbh and co., Germany

Multi mill : Neomachine, Calcutta, India

Blender : Vamp, India

Tablet compression machine 16 station single : Cadmach, Ahmedabad, India

Analytical sieve shaker. : Retsch, Calcutta, India.

HPLC : Shimadzu Corporation (Kyoto, Japan)

Pump : Shimadzu LC-10AT VP

Auto sampler : Shimadzu SIL-HTC

MS/MS Detector : API-3200, MDS SCIEX.

Degasser : Shimadzu FCV-10AL VP

System Controller : Shimadzu SIL-HTC ver 6.03

Data analysis software : Analyst 1.4.2.

Pipettes : 10-100, 100-1000 µL Gilson, France



Balance : Mettler Toledo AB 108

pH meter : Thermo Orion pH meter-model 420

(USA)

Cyclomixer : Heidolph Vibramax 110 (Germany)

Centrifuge : Multifuge (Heraeus 3S-R, Germany)

Evaporator : Zymark Turbo Vap LV, USA

Filtration unit : Millipore (XI5522050) (USA)

Filtration membrane : 0.45 µm, Millipore, Filter Type: HV

(USA)

Refrigerator : LG, 360 L, LG India Ltd (India)

Deep Freezer : Thermo Forma -80°C (USA)

Ultra sonicator : Branson (USA)

Method Conditions

Analytical column : Peerless Basic, C18

Mobile phase : methanol: water

Flow rate : 0.6 mL/min

ISTD : Glipizide

Retention time : Metformin:~ 1.38 min

: Glimipride ~ 1.73 min

Run time : 3min

Sample processing method : Liquid-Liquid extraction

Solvent/volume : 3 mL of dichloromethane: isoamylalcohol

(9:1v/v)

Reconstitution volume : Reconstitute MOBILE PHASE of 250 micro

liters

Volume of injection : 20 microliters.



PREFORMULATION STUDIES 54

Organoleptic studies of pure drugs (color, odour and taste)

In this studies pure drugs (metformin and glimepiride) taste, color, odor has been

checked physically.

Drug-excipients comparability studies

Excipients play an equal amount of importance and role in preparation of pharmaceutical

dosage forms. The complete and reliable formulation of effective and stable all

pharmaceutical dosage forms depends on the excipients selection for no or less problems

in manufacturing and provide to way for administration without any difficulties. The

inactive ingredients used in tablet formulation should assist in controlled release of active

pharmaceutical ingredients present in it, maintain therapeutic levels in blood-plasma and

should be prevented from degradation in all concerns.

The various excipients are selected for the studies and they were mixed in 1:1 ratio with

drugs. These mixture were taken in 5 ml glass vial in open (closed with perforated

aluminum foil) and closed condition and these were stored at room temperature in

stability chamber at various temperatures like 600C, 80%RH; 40 ºC, for specified period

of time according to standard books in this study kept for three month,

At the interval of initial, 2nd week, at end of one month, samples are collected and

checked for color difference in any change.

Determination moisture content

Karl fisher titration apparatus is commercially available and consists of one of

two automated burette, first one is titration vessel having electrodes which are covered

tightly. Second is magnetic stirrer. In the system air is kept along with P2OT anhydrous

Ca C11 OR silica gas.



First the Karl fisher (KF) factor is determined by using the substance of standard

moisture content (M.C) i.e. disodium tartarate. A specified quantity of disodium tartarate

is added in beaker containing the methanol and titrated using Karl fisher reagent (KF

reagent). The KF factor is calculated using the formula.

KF factor = (0.1556 amount of disodium tartarate) / titer volume.

Then the sample was titrated in he same way, as the percentage of moisture

content was determined using the formula.

Percentage moisture content = (KF factor x titer value x 100) / weight of the sample

taken.

Fourier transforms infrared spectroscopy:

Active pharmaceutical ingredients along inactive ingredients are conducted for IR

studies. By taking pure drugs metformin and glimepiride one mg each, taking equal

propionate of 1:1 ratio of metformin and glimepiride, metformin with its excipients and

glimepiride with its excipients are taken in same ratios and are properly mixed by taking

potassium bromie 100mg separately in agate mortar. The prepared powder of mixture is

pressed at a pressure around 10,000 – 15,000 psi into Potassium bromide pellet. Those

were analyzed by FTIR.

Solubility and selection of dissolution media: literature review gives details of

solubility data of metformin hydrochloride and glimepiride in different solvent mediums

represented table wise manner as follows table –2 and table – 3.



Table.2

Solubility of metformin hydrochloride in different solvents

Table-3

Solubility of glimepiride in different solvents

.

pH solubility of metformin hydrochloride by saturated solubility method: literature

review gives details of ph dependent solubility data of metformin hydrochloride and

glimepiride in different ph.d mediums determined by saturation solubility method given

in table – 4 and table – 5.

Sl.No Solvent Extent of solubility

1. Isopropyl alcohol and methylene chloride Freely soluble

2. Water Freely soluble

3. Ethanol Soluble slightly

4. Acetone Not soluble acetone.

Sl.No Solvent Extent of solubility

1. n, n-dimethyl acetonide and

acetic acid Freely soluble

2. Water Practically insoluble

3. 0.1N HCL Insoluble

4. Acetone, acetonitrile,

triethilamine Freely soluble



Table-4

pH solubility of metformin hydrochloride

Sl.No. Medium Solubility in mg/ml

1. Water 4.61

2. 4.0 pH Acetate Buffer 4.78



5. 5.8 pH Phosphate Buffer 4.15




9. 7.5 pH Sodium Phosphate Buffer 5.12

Table-5

pH solubility of glimepiride by saturated solubility method

Sl.No. Medium Solubility mg/ml

1. Water Not soluble

2. 0.1 N Hcl Not soluble

3. 3.0 pH Acid Phthalate Not soluble

4. 4.5 pH Acetate Buffer Not soluble

5. 6.8 pH Phosphate Buffer Not soluble

6. 7.4 pH Phosphate Buffer Not soluble


8. 0.5 %SLS 0.03

9. 6.8 pH + 1%SLS 0.07

10. 7.4 pH Phosphate Buffer +1%SLS 0.08

11 7.8 pH Phosphate Buffer 1.15



Characteristics of the active pharmaceutical drug:55

The characteristics of active drug like density such as bulk density, tapped

density, compression properties like compressibility index, and flow properties of

granules depend on angle of repose and loss on drying are determined as follows.

Physical properties of metformin hydrochloride and powder blend of tablet batches

Determination of loss on drying (LOD):

LOD is determined using electronic LOD determination apparatus as shown in the

Figure 5, page number 52 and 53. Approximately one gram of sample is taken on lid,

tarred for accurate weight and closed the lid. Temperature is adjusted to 105 °C and

operated the equipment. LOD value was displayed automatically after getting a constant

value.

Conducting angle of repose for powders and granules:55

Experiment for angle of repose can be done with help of automatic digital

apparatus. The apparatus works with principle of IR radiation moving vertically and

horizontally. Sample is placed in hopper and operated. The bottom lid opens and sample

is moved on to a stage where height (h), radius (r) and diameter (d) are measured

automatically and angle of repose is displayed.

θ = tan-1

In the above given equation h is height and r is radius of powder cone respectively.

Determination of densities and compression parameters:55

Bulk density: loose bulk density and tapped bulk density determined. Metformin

hydrochloride allowed passing from sieve #18 for break of clumps, if any. Accurately

weighed 50 g of the drug was placed in measuring cylinder of 100 ml and initial level of

volume reading is observed and taken. Then the cylinder allowed to tap for 500 times

initially from a distance of 14+2 mm. The tapped volume (Va) was measured. The same

h r



is repeated for another 750 times and tapped volume is measured. The same thing was

done for powder blend of the tablet. The LBD and TBD were calculated in g per ml by

give formula.

Loose bulk density = wt of taken powder/ initial loose volume occupied by powder.

Tapped bulk density= wt of taken powder /volume occupied after tapping.

Compression properties of blend or granules:

Carr indices:

Compression properties of granules and blend or powders can be calculated by formula:

CI = (VO – Vf) multiplied by 100

V0

Hauser’s ratio:

The flow properties of blend, granules or Powder are measured by ratio of TBD to

LBD.

Hauser’s Ratio = TD/BD

Distribution of granules based on their particle size: This practice was done for the

granules obtained after wet granulation to check average size of the granules. 100 gms of

the granules obtained by wet granulation method are shifted in to sieve shaker, the

machine was run for 5 minutes, all the meshes were taken out and retained granules were

collected by respective mesh and the % retention of granules by that mesh was calculated.

Average particle size was determined. A graph was plotted taking average particle size on

X – axis and percent weight undersize on Y – axis.56



Experimental methods

Preparation of buffers and reagents

PH 6.8 solution of phosphate buffer is prepared as: 250 ml of 0.2 M Potassium

dihydrogen orthophosphate and 112 ml of 0.2 M NAOH is prepared and taken into 1000

ml volumetric flask, and then volume up to 1000ml is adjusted with distilled water and

pH is adjusted at 6.8 using diluted NAOH.

Analytical methods:

Stock solution of metformin hydrochloride: 1000 micrograms/ml.

Stock solution of metformin hydrochloride can be prepared by taking 100mg of

metformin and made it to dissolve in buffer solution of phosphate of 100ml volume in

100 volumetric flask to get 1000 micrograms per ml concentration of solution.

Procedure to determine analytical wavelength:

Metformin:

From the prepared stock solution, quantity of 0.5 ml is pipette into 100 ml

volumetric flask. Using phosphate buffer solution pH 6.8, volume is being made till

100ml. The resulting solution containing 5 µg/ml was scanned between 200 and 400 nm.

The λmax was found to be 233 nm. IP 1996 specifies λmax of 233 shown in Figure (Fig. 1)

Fig. 01: Spectrum of metformin hydrochloride (UV) in solution

pH, 6.8 (5 µg/ml) of phosphate buffer

00.10.20.30.40.50.60.70.80.9

1

200 250 300 350 400

Ab

sorb

anc

e

Wavelength (nm)

233 nm



Metformin hydrochloride calibration curve in soluti on of pH 6.8 phosphate buffer.

Accurately weighed quantity metformin hydrochloride (50 mg) was dissolved in

little quantity of phosphate buffer solution pH, 6.8 and volume was made up to 100 ml.

Appropriate aliquots were taken into different volumetric flasks and made up to 50 ml

with pH, 6.8, solution of phosphate buffer so as to get drug concentrations of 2 to 10

µg/ml. The absorbencies of final diluted aliquots are determined at λmax 233 nm. This

procedure was performed in triplicate to validate the calibration curve shown in Figure

(Fig. 02) using the data given in Table - 6.

Table - 6

Data of calibration curve of metformin in pH 6.8 phosphate buffer, at 233 nm

Sl. No

.

Concentration,

µg/ ml

* Absorbance at 233 nm

AM + SD

1 0 0.000 ± 0.000

2 2 0.159 ± 0.001

3 4 0.319 ± 0.003

4 6 0.480 ± 0.001

5 8 0.647 ± 0.004

6 10 0.802 ± 0.002

* Each value is an average of three determinations



Fig.02: Metformin calibration curve in pH, 6.8 phosphate buffer solution, at 233 nm.

Linear regression analysis for standard curve in pH 6.8:

The linear regression study analysis is done on absorbance data points. Results can

be analyzed as:

The Slope = 0.0806

The intercept = 0.0027

The correlation coefficient = 0.9999

A straight-line equation (y = mx + c) was generated to facilitate the calculation for

amount of drug. The equation is as follows.

Absorbance =0.0806 X Concentration + 0.0027

y = 0.080x - 0.002R² = 0.999

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10 12

Ab

sorb

ance

concentration (mcg/ml)



Glimepiride:

Glimepiride stock preparation (A): Weigh and transfer accurately 55 mg of glimepiride

in a 100 ml volumetric flask dissolve and make up to the volume by taking aceto nitrile.

Take out 10ml of sample into a 50 ml of pH 7.8 Phosphate buffer.

Preparation of working standard: take solution A of 2.0ml in volumetric flask of 50ml

capacity and make up to the volume with dissolution medium.

Chromatographic conditions:

Buffer : Tranfer triethylamine 7.0 ml of into 1000 ml H2O. PH Adjusted to 3.0 using

dilute orthopthosphoric acid.

Elution phase (Mobile): Degassed filtered mixture of buffer and acetonitrile ratio of 50:

50 v/v. was prepared.

Column : Hypersil BDS-C18, 150x4.6mm, 5µ.

Flow : 1.0 ml/minute

Temperature : Ambient

Load : 20 µl

Runtime : 12 minutes

In-vitro dissolution studies of bilayered tablets

A) For Metformin hydrochloride

Dissolution Parameters:

Medium: pH 6.8 USP Phosphate buffer USP-Type II (Paddle)

RPM: 75. Temperature: maintained about 37±0.50 C.

Volume of medium: dissolution medium 900 ml.



0.2M Potassium dihydrogen orthophosphate: Transfer 27.218 g of potassium di

hydrogen orthophosphate accurately weighed to a suitable container, dissolve and make

up the volume with purified with water to 1000 ml.

0.2M Sodium hydroxide: Transfer about 8.0 g of NAOH pellets to a suitable container

dissolve and make up the volume with purified with water to 1000 ml.

Phosphate buffer pH 6.8: Take 250ml of 0.2 M potassium dihydrogen orthophosphate

and 195 ml of 0.2 M NAOH in suitable container, dissolve and make up the volume with

purified with water to 1000 ml and adjust to 6.8 pH using NAOH or orthophosphroic acid

Standard preparation: weigh and transfer 56mg of metformin hydrochloride to 100ml

flask and make up the volume by taking dissolution medium. Pipette 1 ml to 100 ml in

another 100ml volumetric flask and make up volume using dissolution fluid.

Test operation: six Tablets were placed individually in flasks of dissolution with sinkers

in dissolution medium of 900ml which been maintained at thirty seven degrees. Care was

taken for preventing the formation of air bubbles from superficial area of the tablet.

Samples were collected after the specified time. Sample of aliquots are withdrawn from

middle space of dissolution vessel and rotating shaft containing blade using filter through

0.45µ membrane filtered by discarding initial five ml. Dilute one ml of the sample

collected to 100 ml with dissolution medium. Dissolution media was used as blank.

Absorbances of both standard and sample preparation were measured in a suitable UV

spectrophotometer at 233 nm using dissolution medium as blank.

Calculation: Amount of metformin HCl dissolved from each Tablet in percent on label

claim was calculated using the formula:

Sw 1 900 100 P

----------- x --------- -x -------- x ------- x --------- x LC

SA 100 100 1 1



Where,

TA = Absorbance due to test

SA = Absorbance due to standard

Sw = Weight of metformin hydrochloride standard taken in mg.

LC = Label claim of metformin hydrochloride in mg per Tablet,

P = Percent purity of metformin hydrochloride working standard used.

B) For glimepiride:

Dissolution parameters:

Medium: Phosphate buffer pH 7.8 Apparatus: USP- Type II (Paddle)

RPM: 100. Temperature: 370±0.50C.

Volume of medium: 900 ml.

Dissolution medium preparation: sodium dihydrogen phosphate (15 g) and 78.05 g of

NAOH weighed accurately and added to 1000 ml of water and allowed it to dissolve..

100 g of sodium lauryl sulphate dissolved and mixed, pH adjusted to 7.8 using either

dilute orthophosphoric acid or sodium hydroxide solution.

Chromatographic conditions:

Buffer 7.0 ml of triethylamine was transferred into 1000 ml water, pH adjusted to 3.0

using dilute ortopthosphoric acid.

Mobile Phase: Prepare an acetonitrile and buffer mixture ratio of no gas present in it and

was filtered in 50: 50 v/v.

Column : Hypersil BDS-C18, 150x4.6mm, 5µ.

Flow : 1.0 ml/minute

Wave length : 226 nm

Temperature : Ambient

Load : 20 µl

Runtime : 12 minutes



Glimepiride stock preparation (A): Weighed and transferred accurately 55 mg of

glimepiride in 100 ml flask volumetric and allow to dissolved, diluted to volume with

acetonitrile. Pipette out specified amount of sample of 10ml into pH phosphate buffer of

7.8 in 50ml.

Preparation working standard: take 2ml of sample solution A in 50 ml volumetric

flask and make up volume using dissolution medium.

Test preparation: six Tablets were placed individually in flasks of dissolution with

sinkers in dissolution medium of 900ml which been maintained at thirty seven degrees.

Care was taken for preventing the formation of air bubbles from superficial area of the

tablet. Samples were collected after the specified time. Sample of aliquots are withdrawn

from middle space of dissolution vessel and rotating shaft containing blade using filter

through 0.45µ membrane filtered by discarding initial five ml. Dilute one ml of the

sample collected to 100 ml with dissolution medium. Dissolution media was used as

blank.

System suitability: The percentage RSD for 5 standard injections found not more

required not less than 2.0%. Tailing factor of main peak will not be more than two.

Procedure: dissolution medium is taken as blank and test and reference samples are

allowed to inject in to chromatogram an area of major peaks are recorded.

Calculation: Glimepiride amount dissolved from each tablet in percent on label claim

was calculated using the formula:

TA Sw 10 2 900

-------- Multi ---------Multi--------Multi-------Multi--------Multi with P

SA 100 50 50 1

Where,

TA = Peak area due to glimepiride in the test preparation

SA = Area of peak due to glimepiride in the reference solution.

Sw = Glimepiride weight of reference solution.

P = percentage purity glimepiride working standard used.



FORMULATION OF BILAYER TABLETS FLOW CHART:

Process of bilayered tablet preparation represented in form of flow chart given in figure

(fig. 03).

PROCESS FLOW CHART

DIRECT COMPRESSION WET GRANULATION

Metformin hydrochloride Glimepiride

Dispensing Dispensing

Sifting Sifting

Dry mixing dry mixing

Binder solution

Lubrication

Granulation

Drying

Sifting

Bilayered tablet compressed on

27 station bilayer compression Lubrication

machine

Figure 03: Process flowchart of metformin-glimepiride bilayered tablets.



FORMULATION OF METFORMIN AND GLIMEPIRIDE AS BILAYER ED

TABLETS:

Method: Direct compression and wet granulation technique and direct compression

technique were followed for the manufacture of metformin sustained release tablets from

batches 1-17, where as in case of glimepiride only wet granulation technique was

followed from batch 7-17. Metformin granules and glimepiride blend made to prepare

separately using wet granulation technology.

Manufacturing of metformin blend with hydrogenated castor oil (HCO) and

hydroxy propyl methylcellulose (HPMC K 100M) drug retarding agent:

Hydrogenated castor oil and HPMC K 100M were used in batches 1-2 and from batches

3-16 respectively.

Preparation of granulating medium: Polyvinylpyrrolidone K90 (PVPK90 D) was used

as a granulating agent. PVPK90 D (50%) was dissolved in a mixture of water and

isopropyl alcohol (IPA) (7:3).

Shifting of excipients: Metformin hydrochloride, microcrystalline cellulose 102,

hydrogenated castor oil (HCO), hydroxy propyl methylcellulose (HPMC K 100M) was

sifted through # 40 sieve. Magnesium stearate, aerosil were passed through # 60 sieve.

Mixing of excipients: In batches 1 and 2 metformin hydrochloride, hydrogenated castor

oil and in batches from 3-17 metformin hydrochloride, hydroxy propyl methylcellulose

and microcrystalline cellulose were blended thoroughly in rapid mixer granulator for 10

min by running impeller of the mixer at 45 rpm.

Granulation: Granulating medium of PVPK90 in water and IPA (7:3) was added into

the rapid mixer granulator with impeller speed of around 45 rpm for 10 minutes then

chopper was put on to break down the large lumps of wet mass.



Drying : After granulation the wet mass was dried by keeping mass in dryer like FBD till

(LOD) come in between of 0-1 % its units are weight per weight, by keeping 50

atomization and 45o C temperature on FBD, initially the wet mass was dried for about 10

minutes without any temperature and atomization to evaporate the IPA present in the wet

mass.

Dry screening: After obtaining the LOD between 0-1 % w/w. granules are dried and

these granules are made to pass from sieve # 20 sieve for obtain of equal size particles or

granules.

Blending: granules after drying and passing through desired sieve number, they are

allowed to blend for 10 minutes in double cone blender and blend for 10 minutes. Next

lubricants are added and mixing was continued to 5 minutes, then the final blend is ready

for compression.

Manufacturing of glimepiride granules:

Preparation of granulating medium: hydroxy propyl cellulose (HPC-LF) (low grade

viscosity) was used as granulating agent along with meglumine, poloxomer-188 and

glimepiride was added to the granulating medium.

Meglumine was dissolved in the little quantity of water with continuous stirring then

glimepiride was added slowly dissolved in water with 3-5 drops of IPA. Then add slowly

poloxomer-188 to the above solution with continuous stirring. The solution was kept

aside till the foam gets settle down. Then HPC-LF was added slowly with continuous

stirring until it gets dissolved to form a gel like mass with out any air bobbles.

Shifting of excipients: Mannitol, MCC 114, crosspovidone and S.S.G were shifted

through # 40 sieve. Lake of sunset yellow was passed through # 100 sieve and # 80 sieve

used to pass magnesium stearate.

Mixing of excipients: Mannitol, MCC PH 114 and half the quantities of crosspovidone,

sodium starch glycolate were taken in the rapid mixer granulator and were dry mix for

about 10 minutes by running the impeller of the RMG at a speed of about 45 rpm.



Granulation: The above prepared granulating medium (glimepiride, meglumine, S.S.G.

and HPC-LF) was added to the dry mix powder in the RMG with in one minute with 45

rpm of impeller speed.

Drying : After granulation the wet mass was transferred to fluid bed dryer for drying 2-

3% LOD was attained by keeping 5 atomization and 45o C temperature on FBD, initially

the wet mass is dried for about 10 minutes with out any temperature and atomization.

Dry screening: After obtaining the LOD between 2-3 % w/w. granules are allowed to

pass # 30 sieve for uniform particle size of granules.

Blending: Dried granules were loaded in the double cone blender along with other half

quantity of povidone; S.S.G, MCC PH114, lake of sunset yellow and were blended for 20

minutes. Finally post granulating ingredients are added and blending was continued to 5

minutes, then final blend is ready for compression.

Evaluation of bilayered tablets:

Quality control tests for in process and finished tablets were done, they are thickness of

tablet, weight variation of tablets, friability of the tablets, assay etc.

Weight variation test: it a in process quality control test conducted for tablets to monitor

any fluctuations in weight of the tablets, if any major fluctuations are observed

adjustment of compression parameters are made. To conduct or check uniformity of

weight twenty tablets are randomly picked from the batch during compression and their

individual weights are measured and total weight of all twenty tablets were calculated.

Total weight is divided with twenty tablets to get average weight. Now individual weight

of each tablet is compared with average of total twenty tablets. Each tablet should not

deviate not more or less than five percent of the average value. The percent deviation was

calculated using the following formula.



Individual weight – average weight

% Deviation = x 100

Average weight

Test for hardness of tablets: it is a diametrical crushing strength and force which is

required to break the tablets. Strength of any tablet dosage form indicates its mechanical

and tensile strength. The hardness of tablet may be change depending on the inactive

ingredients used type of tablet. Hardness measured in units of kilopascals and tested by

Schleuniger tester. “Hardness factor”.

Friability test : Friability is the process of quality control tests conducted to check the

amount of tablets lose their weight when they are allowed for friability. Tablets to wit h

stand adhesions, shock when they are allowed to and during transportation when packed

in container or other suitable packaging materials. Process of friability is done by Roche

friabilator. It was rotated at a rate of 25 rpm. Ten tablets were weighed collectively and

placed in the chamber of the friabilator. It consists of rotating drum where tablets are

placed and allowed to rotate and make tablets to fall from 6 inches height in the chamber

for 4 minutes where 100 rotations made. Initial weight and final weight of the tablets

were noted and percent of weight lose was determined. Maximum percent weight loss

allowed is up to 1% and calculated using the formula.

(W1 – W2)

Friability = x 100

W1

W1 = tablets weight before test initial weight of tablets.

W2 = tablets weight after test final weight of tablets.

Active content ingredients (assay): The amount of active ingredient(s) was determined

and compared with standards stated in the monograph. Twenty tablets were used for

assay. All the batches should fall with in the limit of 95 – 105 %.



Uniformity of content (Metformin Hydrochloride and Glimepiride): Twenty tablets are

crushed in mortar after randomly selected from batch. Then, powder is placed in twenty

flasks volumetric and volume is made up with pH 6.8 phosphate buffer. The content was

shaken well for some time and kept for 30 minutes for dissolving of drug completely. The

mixtures were filtered and appropriate dilutions were made. Drug content is determined

at λmax 233 nm against blank reference in case of Metformin Hydrochloride and similarly

for Glimepiride, twenty tablets randomly selected, crushed and powder is weighed and

placed in twenty flask of volumetric and make up the volume with pH 7.8 phosphate

buffer. Amount of drug is determined at λmax 226 nm and reported.

Drug release studies: release of drug in-vitro conditions was carried out in

dissolution apparatus of USP type two a paddle one. Medium of dissolution was taken is

purified 900ml of water. Dissolution flak is kept in water bath maintained at body

temperature around thirty seven degrees centigrade. Rotation of shaft was kept for 75 rpm

and 100 rpm for metformin and glimepiride respectively. Tablets are place in basket of

dissolution apparatus. and allowed to run for 12 hrs for metformin and 45min for

glimepiride. aliquots are collected of 10ml for every specified time intervals like 2, 4, 8

and 12 hours using auto sampler for metformin and aliquots are collected of 10ml for

every specified time intervals like 10,15,30 and 45 minutes for Glimepiride. Each

collected sample is filtered through 10 micrometer filter. A fresh medium of dissolution

is replaced after taking out each sample, the collected samples are made to dilute to

required concentrations and analyzed for drug content at each time intervals using U.V

spectroscopy and for glimepiride samples are tested in and analyzed for drug in High

Performance liquid Chromatography (HPLC). Amount of drug releases is calculated.



Effect and assistance of different drug release parameters:

The following tests were carried out to check drug release.

Metformin in different pH conditions: It is necessary to check different environment of

in release of drug from tablets Drug release study of the tablet was conducted by pH

change (gastro-intestinal condition) method using different pH dissolution medium.

Medium selected was 1.2 pH hydrochloric acid solution for initial two hrs and pH 6.8 for

last twelve hrs. dissolution medium is collected of 5ml at specified time intervals an

filtered by 10 microns filter and the filtrate is allowed to make dilute and estimated for

drug content.

Hardness effect on tablets: drug release is effected by the hardness of the tablets higher

the hardness release rate of the drug from the tablets is retarded accordingly. In present

work to see effect of hardness three different hardness containing tablets are taken like 8-

10kp (standard), less than 6-10 kp (less hardness) and more than 10 -12 kp (high

hardness).

Release study conducted in dissolution apparatus (paddle type) at rotational

speeds of 70 rpm and purified water as dissolution medium for all three types of tablets

varying in hardness. The samples were withdrawn at 2, 4, 8 and 12 hours and passed

through 10 µm filters. And samples are diluted up to desired concentrations and are

analyzed at 233 nm for drug content. The amount of drug release was analyzed

cumulatively.



Data Analysis: 57,58

Various release kinetics models are as:

To analyze the mechanism of the drug release rate kinetics of the dosage form, the

data obtained were graphed as:

1) Percent cumulative drug release verses time in minutes or hrs (In-Vitro drug release

plots)

2) Percent cumulative drug release verses time square root (Higuchi’s plots)

3) Percent Log cumulative drug remaining versed time in minutes or hrs ( concentration

dependent known as first order reaction plots)

4) Percent Log drug release verses time in log (Peppas plots)

Specific study for the hydrogels (matrix tablets)

Swelling behavior and water uptake study:

These studies are conducted in deionized water. Place metformin tablet on 20 mesh

screen which is placed in dissolution flak and allow the dissolution apparatus to run at 50

rpm for specified period of time. At specified time intervals tablet is removed and

weighed. The swelling of tablets can be calculated from following equation

100 (wet weight - dry weight)

Percent water uptake (weight gain) =

Dry weight

100 (swollen thickness-original thickness)

% swelling =

Original thickness



erosion (%) is weight of matrix tablet initial minus weight of matrix tablets after erosion

at time t divided by weight of matrix tablet initial. Value is determined is of three mean

tablets data.

Accelerated stability studies

Bilayered tablets (batch) were packed in 90 gsm thick aluminum foil strips

laminated with PVC. Tablets are exposed to different environmental conditions of

temperatures and humidities for one month by placing in stability chamber, the conditions

are 40oC and 75 % RH and 60o C during one month period. Tablets are collected after

one month and drug content is determined. 59



INVIVO STUDIES OF GLIMIPRIDE AND METFORMIN HYDROCHL ORIDE

BILAYERED TABLETS

Work study for in vivo:

Rabbits of mail gender was selected for study, their body weight is maintained from two

to two and half kilograms. Rabbits are divided in to two groups of six in each group. one

received test formulation and another received marketed formulation. Twelve hours

before drug administration, food was withdrawn from the rabbits until 24 hr post-dosing,

while, water was available for rabbits throughout the study. The tablets were

administered to rabbits using a balling gun. Blood of 1ml is collected from marginal with

specified time intervals after administration. For each animal the total number of blood

samples drawn during the study was 17. EDTA disodium salt was used as an

anticoagulant. Blood substances are removed by centrifugation. The resulting plasma

sample from each blood sample was divided into two lots, kept in cool place suitably

labeled polypropylene bags – 200C.

EXPERIMENTAL: METHOD DEVELOPMENT

Different Materials:

Metformin, glimepiride, glipizide acquired from medwin pharmaceuticals. Acetonitrile,

methanol and ethylacetate brought from MSN laboratories, Hyderabad. Potassium

Dihydrogen Phosphate and NAOH was purchased from medchem, hydrabad.

Maintaining of conditions in chromatogram.

It will have 2954 with UV detector to determine drug concentration at specified lambda

max at. C18 column is used to separation of drug from sample solution using pH 4

phosphate buffer in sixty forty ratio. The mobile phase in this study used is acetonitrile

injectedwith flow rate of 0.99ml/minute. Collected mobile phase is filtered by nylon milli

pore of 0.2 microns. Study is carried at room temperature



Stock solution Preparation:

Different stock solutions are prepared first metformin of 500, losartan of 1000 and

glimepiride of 500 micro grams per millliters and further dilutions are made by methanol

as internal standard.

Stock solution for calibration;

Stock solutions are prepared by removing drugs from blood plasma. The freshly collected

solutions are diluted accordingly to make concentrations like 0.3, 0.6, 2, 3, 5, 10, 15, 20

µg/mL for glimepiride and 0.78, 1.56, 3.12, 6.25, 12.5, 25, 50 and 100µg/mL for

metformin and are place at-200c. Detailed procedure for preparation is shown in table-07.

Table-07

Preparation of glimepiride and metformin calibration standards in Plasma

Sample ID

Concentrations

of metformin

(µg/mL)

Concentrations

of glimepiride

(µg/mL)

Drug stock

solution (µL)

Blank plasma

(µL)

OP CS1 1.56 0.3 3.72 996.28

OP CS2 3.12 0.6 7.45 992.55

OP CS3 6.25 2 16.5 983.5

OP CS4 12.5 3 31 969

OP CS5 20 5 50 950

OP CS6 40 10 100 900

OP CS7 80 15 190 810

OP CS8 100 20 240 760

Standard quality control stock solution preparations:

They are prepared at three levels namely LQC, MQC and HQC. Stock solutions are

prepared by removing drugs from blood plasma. And are diluted to get concentration of

0.9, 10, 18micrograms per liters for metformin and 2, 40 and 80 microliters for

glimepiride. Placed at -200c to get analysed. Detailed procedure for preparation is shown

in table-08.



Table 08:

Preparation of Glimepiride quality control standards in Plasma

Sample ID Concentrations

of metformin

(µg/mL)

Concentrations of

glimepiride

(µg/mL)

Drug stock

solution (µL)

Blank

plasma (µL)

OP LQC 2 0.9 5.8 994.2

OP MQC 40 10 100 900

OP HQC 80 18 196 804

Sample preparation method

Drug free rabbit plasma (250 µl) is spiked with appropriate volume of drug stock. To the

above prepared sample, losartan 50 microgram per milliliters, 100 microliters of sodium

hydroxide of 0.05 moles. Next each sample of aliquot is allowed for mixing to four

minutes. Then drugs are removed by addition of ethyl acetate and are placed in centrifuge

for 2000 revolution per minute for 20 minutes at controlled conditions of around four

degrees. Ethyl acetate phase containing drug is separated and added with mobile phase

300 micro liters and analyzed for drug content.



EXPERIMENTAL: METHOD VALIDATION:

VALIDATION PARAMETERS:

Specifications for experimental conditions:

Concentrations of solutions containing glimepiride and metformin is prepared having

concentrations of 0.3 micrograms per milliliters and 1.56 micrograms per milliliters.

These solutions are allowed to inject into chromatographic column for separation of two

drugs from blood-plasma. Any interference is cross identified and checked from plasma.

Determination of linearity based on range:

Least square regression is conducted for the values obtained for removed concentrations

from blood with respective area of peak against internal standard when assayed.

Calibration curves are established by using solutions of concentrations present from CS1

to CS8. After examining the deviation of percent a required model has been selected.

Limit of quantification and detection: quantification represent to minimum amount of

drug in given sample which can be estimated quantitatively with accuracy and precision.

Signal to noise type determination method is easy and accuracy where levels on

concentration of drug generates 9 to 10 than it is said as limit of quantification, where as

same if its noise is three than it is side to be limit of detection.

Accuracy and precision:

Accuracy and precision was estimated using standards quality control and (CS1) for 5

points per day, Accuracy and precision was estimated by standards of quality control like

(LQC, MQC, HQC) and (CS1) for five days each per day.

Ruggedness

Ruggedness of method is estimated by analysing spiked control samples (n=6) of medium

concentration i.e 10µg/mL using two different columns.



Stability studies

Out of different stability study methods freeze-thaw study has been selected, in this

method samples of blood of different concentration of specified time intervals are kept

for freeze cycle and thaw cycle. Samples before study, after study were analysed by

developed method. Similarly stock solution stability study (Stability after 6 Hours),

bench top stability study (Stability after 10 Hours) and Inter injection stability study were

carried out by subjecting samples to study conditions.

Subject Analysis:

A validated HPLC-UV method has been prescribed and developed to estimate metformin

and glimepiride from blood plasma samples at regular time intervals.

Estimation of metformin and glimepiride from samples of frozen plasma are heated at

room temperature initially. Next 50 and 100 micro liters of losartan and sodium hydroxide

are added to 250 micro liters of sample extracted from blood in this sodium hydroxide

acts as internal standard. Then samples are allowed for mixing to 4 minutes and drugs

samples are extracted on addition of ethyl acetate of 2.5ml. and again are mixed for same

time followed by centrifugation in cooling centrifuge at 2500 revolution per minute for 16

minutes at four degree temperature. The ethyl acetate phase is separated and analytes were

obtained as dried residues after drying using lyophiliser. The analytes residue obtained

was reconstituted with 300µl of mobile phase and analysed using HPLC system according

to parameters optimized. All the test and reference plasma samples were analysed under

the construction of standard calibration curve of metformin and glimepiride in rabbit’s

plasma. The metformin and glimepiride concentrations in the rabbit plasma samples was

calculated using regression line of peak area ratios of calibration curve

(metformin/losartan & glimepiride/losartan ) versus the concentration of metformin and

glimepiride.



Determination of pharmacokinetic parameters:

Various pharmacokinetic parameters of Metformin hydrochloride and Glimepiride are

determined by various points data collected from plasma concentration time profile

graph.

Pharmacokinetic parameters are as:

Area under curve: from zero to forty eight hours calculated by using trapezoidal rule.

AUC0-t : calculated from linear trapezoidal method.

AUC0-∞ : determined by adding the values obtain from area under curve from zero to

time T and maximum extent measurable concentrations.

C (max) : it is a maximum concentration obtained after administration of tablet

T (max) : it is time required to attain maximum concentrations.

K (el ) : graph of semi log plot of the plasma concentration versus time curve is used to

calculate elimination rate constant.

The three preparations were considered to be bioequivalent when the limits of the 90%

CL a confidence interval ratios from means of Cmax, & AUC fall within 80 –100%.

Statistical Analysis

Statistical analyses were performed on plasma Metformin hydrochloride and Glimepiride

using the Winnolin (Pharsight Version 5.0.1or higher. The analysis was including data

from 6 animals.



LCMS/MS ESTIMATION OF DRUGS:

Suitable analytical method is developed and validated for estimation and determination of

metformin hydrochlorid and glimpiride from bilayered tablets.

The dose is calculated according to the following

Dose calculation per Rabbit:

Dose of Glimipride and Metformin Hydrochloride drug to be administered in rabbits was

calculated,

Rabbit weight: 4.2 Kg

Tablet dose: 500mg

Approximate dose for metformin drug: 120 mg/kg dose.

Approximate dose for Glimepride drug: 1.2 mg/kg dose.

Calibtation Curve for Glimipride and Metformin Hydr ochloride

5mg of drug is dissolved in 5 ml of Volumertic flask. Add few millilitres of DMSO and

dissolved the compound at room temperature. Then volume was made up with dimethy

sulfoxide. One milliliter is taken in to volumetric flask of 10ml and make up the volume

with methanol to get hundred micro grams per milliliter. This process is repeated to get

final solution concentration of one microgram per milliliter. Now two nano grams to five

thousand nano grams concentration was prepared from above stock solution.

Following table –09 gives the different theoretically (in-vitro) prepared concentrations

used to get calibratrion curve of glimepiride and metformin hydrochloride which is

shown in figure (fig. 04 and 06). Whereas table –10 and table –11 represents the data of

accuracy results compare to that of theoretically given concentrations. In-vivo calibration

of glimepiride and metformin and with their accuracy is given in chromatograms in

figures fig. (05 and –fig-07)



Table –09

(in-vitro) prepared concentrations used to get calibratrion curve

Calibration

Standard

Vol. spiked

Stock (µL)

Plasma

Used (µL)

Concentration of Sample

(ng/mL)

CS10 10 90 5000

CS9 10 90 2000

CS8 10 90 1000

CS7 10 90 500

CS6 10 90 200

CS5 10 90 100

CS4 10 90 50

CS3 10 90 20

CS2 10 90 10

CS1 10 90 5

CS0 10 90 2



Fig: 04. Calibration Curve for Glimipride



Table - 10 Calibration Curve Data for Glimipride



Fig. 05 chromatograph showing in vivo-calibration of glimepiride in rabbits.



Fig: 06. Calibration Curve for Metformin Hydrochlor ide



Table - 11. Calibration Curve Data for Metformin Hydrochloride



Fig. 07 chromatograph showing in vivo-calibration of metformin in rabbits.



In-vivo bioavailability studies of Glimipride and M etformin Hydrochloride

Bilayered Tablets

Male white Newzealand Rabbits (weighing about 4.2 kg) were selected as the

animal model. The age of the rabbits was 8 - 12 weeks. The Rabbits selected for the

study should not administered any food or medication for at least before two weeks

study get start. Rabbits are made in to five categories one in each category or each

group. Of which Group – I:- Control, Group – II:- Marketed, Group – III:- Standard A

(Metformin Hydrochloride 500mg), Group – IV:- Standard B (Glimepiride 2mg),

Group – V:- Test A (Metformin Hydrochloride 500mg + Glimipride 2mg). The drug

was given to the rabbits through oral gavage

From the rabbits 600 µl of blood sample was collected from ear vein at the time

interval of 0, 15mins, 30mins, 1h, 2h, 6h, 8h, 10h, 12h and 24h for all the five groups.

The blood samples were allowed to coagulate and whole sample of plasma-blood and

allowed to centrifuge around 13000 revolution per minute at RT where serum is

segregatedand kept at -200C± 2 °C till it get analysed. Bioavailability for drugs from

Bilayer tablets with metformin 500mg and glimepiride 2mg has compared with that of

Marketed Bilayered Tablet of Metformin Hydrochloride and Glimipride of Same

strengths.

Analysis of Serum samples by LC/MS/MS method103, 104

Preparation of sample aliquots:

test samples are prepared by collecting the blood-plasma of rabbit which is

extracted from rabbit marginal ear vein. Then internal standard solution is added of

concentration of five micro liters in addition of tris buffer of fifty micro litersand

mixing for one minute vertoxing. Then samples were extracted using dichloro

methane and iso amyl alcohol in 9:1 ratio and it is vortxed for thee minutes with

centrifuge at 5000revolution per minute for 15 minutes. Next dichloro methane and

iso amyl alcohol phase is separated and dried at 40 degrees with nitrogen. Final

residue mixed with mobile phase of two hundred and fifty micro liters of which

twenty micro liters was injected in to liquid chromatography-mass spectroscopy-

system.

The quantitative determination of Metformin Hydrochloride and Glimipride in

rabbit serum done in liquid chromatography-mass spectroscopy-MS method. 100µl of



serum was taken into a glass tube, 5 micro liter working solution of internal standard

approximately 50 nano grams per milliliter is added along with 100 micro liters of

sodium hydroxide zero point one percent solution and keep for vortex mixing to thirty

seconds. From this 5ml of aliquot is taken out and to this 7:3 ratio of di-ethyl-ether

dichloromethane is added and again it is vortexed for 3 minutes. Solution of Di-ethyl-

ether dichloromethane is separated of 4 micro liters and placed in glass tube to

evaporated at 40 degrees using nitrogen stream. The dried extract is mixed with

mobile phase of 250 micro liters, from which 10 micro liters of aliquot is injected into

the system.

The chromatographic system of HPLC is well consists of binary pump of LCAD, de-

gasser of FCV-10AL with auto sampler of thermostated column. Study was

performed by using peerless basic C18 by maintaining system conditions at 30 degree

centigrade. The elution phase consist of water methanol in which 0.5 % of formic acid

is mixed, which is allowed to run at the rate of 0.4 to 0.6 micro liter per minute with

split ratio of 20:80.

Mass spectrometry conditions104

Parameters used for these chromatographic conditions are of curtain gas next

gas one and nitrogen gas act as gas two. Their quantity used are as fourty fourty and

sixty units. In this time to dwell will be around 200ms and temperature maintained is

around 500 degree centigrade of voltage in 5500volts and finally mass unit resolution

has set for both mass-resolving quadrupole Q1 and Q3.

Analyst 1.4.2 software package (MDS Sciex). Is used to process and interpret

the data collected.



Metformin:

Q1: 130.1

Q3: 60.1

Glimipride:

Q1: 491.00

Q3: 126.10

Common parameters:

CAD: 6

CUR gas: 20

IS: 5500

Temp: 500° C

The results obtained and the discussion there upon was described in the next chapter “Results and Discussion”.

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