chapter 3 3.1 materials -...

Chapter 3 MATERIALS AND M ETH O D S

3.1 M ATERIALS AND EQUIPM EN TS

Table 3.1 Ingredients o f form ulation

Name o f Isigredieiits

Active Pharmaceutical Ingredient (API) 0610656/000039

Mycophenolic Acid (Impurity) 017623/324

Ac-di-sol T418C

Povidone K-90

MC.C PH 102

3395922400

5448C

MCC PH 102

Magnessium stearate

Starch 1500

Hydroxy Propyl Cellulose (Klucel LF)

Anhydrous lactose (DCL-21)

Sodium starch glycolate (Glycolys)

Magnesium stearate (Hyqual)

P205815360

A05682

10326638

E-0912

CO-1608

Biocon Ltd.

Biocon Ltd.

D M V , USA

Jubilant Organosys

Aqualon Hercules

Aqualon Hercules

M SU M b^dtT uSA

Colorcon, Indianapolis USA

Aqualon Hercules

D M V , USA

Rotique

S l i i i d a 'o d t J J S A

D epartm ent of Pharmaceutical Chemistry Page 52

C h a p t e r 3 MATERIALS AND METHODS

Table 3.2 Chemicals used along with flieir source

Nam e of C hem kais

Anhydrous acetate

Sodium Hydroxide__ __________

Potassium dihydrogen phosphate

Distilled Water / Milli-Q Water

Hydrochloric Acid

Acetic acid

Triethyiamine (TEA)

Orthophosphoric acid (OPA)

Sodium acetate

Acetonitrile (HPLC grade)

Methanol (HPLC grade)

Hydrogen peroxide

Source

Qualigens, Mumbai (India)

Thomas Baker, Mumbai


In - house






Merck, Mumbai (India)

Merck, Mumbai (India)

Ranbaxy, fine chemicals Ltd.


Chapter 3 MATERIALS AND METHODS

Table 3.3 Equipmemts/Instmmeiits used

HPLC Systems

PDA detector

Autotitrator

Dissolution Apparatus

Dissolution Apparatus

UV-Visible Spectrophotometer

UV-Visible Spectrophotometer

Digital Moisture Analyzer

Electronic Weighing Balance

pH Meter

Magnetic Stirrer

Oven

Sieves (ASTM standard)

Sonicator

IR

IR

PSD Apparatus

Photo stability chamber

NMR

Waters 2695 , separation module

Shimazdu 2816

w i ' i i T i i i ?

Orion 940, Thermo Electron Corporation

Electrolab, TDT-08L, USP

Distek Dissolution System, 2100C, USA

Perkin Elmer, Lambda 35,USA

Mettler Toledo HB43, Switzerland,

Mettler Toledo AB204-S, Switzerland

Thermo Electron Corporation, USA

Deepali Udyog, India

Narang Scientific Works, India

Jayant Test Sieves (Jayant Scientific, India)

Bandelin, GERMANY

Nicolet Avatar 370DTGS, Thermo Electron

Corporation

WIN-IR spectrophotometer

Mastersizer 2000, Malvern Instruments

Thermolab, India

Bruker (300MHz) spectrometer


Chapter£_^^^^ __________ __ _ MATERIALS AND METHODS

3.2 Pliysicocliemical C haracterization

3.2.1 A ppearance; [mniunosiippressant MMF is a White to off-White crystalline powder

Flow Properties of the M M F

3.2.2.a Angle of repose

Angle o f repose o f granules was determined by Funnel method, The accurately weighed

granules were taken in a funnel. The height of the funnel was adjusted in such a way that

the tip o f the funnel just touches the apex o f the heap of the granules. The granules were

allowed to flow through the funnel freely onto the surface. The diameter the powder cone

was measured and angle of repose was calculated using the following equation:

tan 0 = h/r

Where, h and r are the height and radius o f the powder cone,

3.2.2.b Bulk Density

M ethod

27.29 grams of the MMF was introduced in a 100 ml graduated cylinder. Powder level

was noted without compacting. Bulk density was calculated using the following equation

Bulk density = (M)/ Vn

Where, M = Mass o f test sample

Vo = Unsettled apparent volume

3.2.2.C T apped Density

M ethod

27.29 grams o f the MMF was introduced in a 100 ml graduated cylinder. Mechanically

the cylinder was tapped using the tapped density apparatus by raising the cylinder and

D epartm ent of Pharmaceutical Chemistry 5


allowing it to drop under its own weight tliat provides a fixed drop of I4±2 mm at a

normal rate of 250 drops per minute. The cylinder was tapped 500,750,1250 times

initially and measured the tapped volumes. Tapped density was calculated using the

following equation-

Tapped density = (M)/ Vf

Where, M = Mass of test sample

Vf= Final tapped volume

Because the interparticulate interaction that influence the bulking properties o f a powder

are also the interactions that interfere with powder flow, a comparison o f the bulk and

tapped densities can give a measure of the relative importance of these interactions in a

given powder. Such a comparison is often used as an index o f ability of the flow of

powder.

■r-r(s.

Fig 3.1 Apparatus For Measurement of Bulk and Tapped Density

3.2.2.d Powder Compressibility

The Compressibility Index and Hausner Ratio are measures o f the porosity of a powder to

be compressed. They measure the relative importance o f interparticuiate interactions. For

poorer flowing materials, there are frequently greater interparticuiate interactions and a

Departm ent of Pharmaceutical Chemistry Page 56

greater ditference between the bulk and tapped densities. These differences are reflected

in the Compressibility hidex and Haiisner Ratio. Tliey are calculated by the following

equation. Percentage compressibility o f the granules was determined by C arr’s

compressibility Index (Aulton, 1998).

Com pressibility Index (C a rr’s Index):-

(Tapped density - Poured density)

Cl = ---------------------------------— -------- X 100

Tapped density

H m m e r Ratio (HR):-

Chapter 3 ^ __________________________MATERIALS AND METHODS

HR =

Tapped density

Poured density

3.2.2.e Particle Size Analysis By Sieve Shaker

Tare each test sieve to the nearest 0.1 g. Place an accurately weighed quantity o f test

specimen on the top (coarsest) sieve, and replace the lid. Agitate the nest o f sieves for 5

minutes. Then carefully remove each from the nest without loss o f material. Reweigh

each sieve, and determine the weight o f material on each sieve. Determine the weight o f

material in the collecting pan in a similar manner, Reassemble the nest o f sieves, and

agitate for 5 minutes. Remove and weigh each sieve as previously described. Repeat these

steps until the endpoint criteria are met. Upon completion o f the analysis, reconcile the

D epartm ent of Pharmaceutical Chemistry Page57

weights o f material. Total losses must not exceed 5% o f the weight of the original test

specimen.

Endpoint D eterm ination~The test sieving analysis is complete when the weight on any

o f the test sieves does not change by more than 5% or 0.1 g (10% in the case o f 76-mm

sieves) o f the previous weight on that sieve. If less than 5% of the total specimen weight

is present on a given sieve, the endpoint for that sieve is increased to a weight change of

not more than 20% of the previous weight on that sieve.

If more than 50% of the total specimen weight is found on any one sieve, unless this is

indicated in the monograph, the test should be repeated, but with the addition to the sieve

nest of a more coarse sieve, intermediate between that canying the excessive weight and

the next coarsest sieve in the original nest, i.e., addition o f the ISO series sieve omitted

from the nest of sieves,

3.3 Ideiitltlcatioii of the M M F

3.3,1 By U,V. Spectral Analysis

A. Preparation of Different Buffers

L P repara tion of 0.1 N HCl (IP)

Hydrochloric acid (8.5 mi) was added to 200 ml of distilled water and volume was made

up to the 1000 ml with distilled water.

2. Preparation of 4,5 N Acetate Buffer

Anhydrous acetate (1.6g) and acetic acid (1.8ml) was added to200 ml o f distilled water

and volume was made up to the 1000 ml with distilled water.

3, Preparation of 6.8 N Phosphate Buffer

Potassium hydrogen phosphate (6,8g) and sodium hydroxide (0.89g) added to200 ml o f

distilled water and volume was made up to the 1000 ml with distilled water,

C h a p t e r 3 ^ _____ M ATERIALS AND ME^TIODS


^ MATERIALS AND METHODS

B. A bsorbance Spectra of the M M F

L P rocedure for A bsorption S pectra of M M F in OdN H C l

The stock solution was prepared by dissolving the drug (98.4mg) in sufficient amount of

water in 1000 ml o f the volumetric flask, sonicated it for 15 minutes and thereby making

up the volume to 1000ml with water. The stock solution prepared thus was o f the strength

98.4mcg/ml. 5 ml o f the stock solution was diluted to 50 ml with water (strength o f 9.9

mcg/ml) and the absorbance spectrum was obtained using UV spectrophotometer in the

region 200-400 nm.

II. Procedure for A bsorption Spcctra of M M F in pH 4,5 A cetate B uffer

The stock solution was prepared by dissolving the drug (99.2mg) in sufficient amount of


up the volume to 1000ml with water. The stock solution prepared thus was o f the strength

99.2mcg/ml. 5 ml o f the stock solution was diluted to 50 ml with water (strength o f 9.92

mcg/ml) and the absorbance spectrum was obtained using UV spectrophotometer in the

region 200- 400 nm.

III. Procedure for A bsorption Spectra of M M F in pH 6.8 P hosphate Buffer

The stock solution was prepared by dissolving the drug (99mg) in sufficient amount o f


up the volume to 1000ml with water, The stock solution prepared thus was of the strength

99 mcg/ml. 5 ml o f the stock solution was diluted to 50 mi with water (strength o f

9.9mcg/ml) and the absorbance spectra was obtained using UV spectrophotometer.

IV. P rocedure for Absorption Spectra of MMF in DM w ater

The stock solution was prepared by dissolving the drug (99.5mg) in sufficient amount o f



up the voh,ime to 1000ml with water. The stock solution prepared thus was o f the strength

99.5mcg/ml, 3 ml of the stock solution was diluted to 50 ml with water (strength of

5,97mcg/ml) and the absorbance spectrum was obtained using UV spectrophotometer.

C. S tan d ard Plots of the M M F

I. P rocedure for S tandard Plot of M M F in O.IN HCl

The stock solution (98.4mcg/mi) was diluted to obtain the concentrations o f 2, 4, 6, 8 and

10 mcg/ml and absorbance was taken using UV spectrophotometer and the calibration

curve was plotted as a function of absorbance v/s concentration

II. P rocedure for S tandard Plot of M M F in pH 4.5 A cetate Buffer

The stock solution (99,2mcg/ml) was diluted to obtain the concentrations o f 2 ,4 , 6, 8 and


curve was plotted as a function of absorbance v/s concentration

III. P rocedure for S tandard P lot of M M F In pH 6.8 Phosphate Buffer

The stock solution (99mcg/ml) was diluted to obtain the concentrations o f 2, 4, 6, 8 and


curve was plotted as a function o f absorbance v/s concentration.

33 .2 Infrared Spectral Analysis

Infrared spectroscopy o f the MMF was studied for identification purpose. Nicolet Avatar

370DTGS infrared spectroscope was used for this purpose.

M ethod: Approximately 2 g o f the MMF was triturated with 400 mg of finely powdered

and dried potassium bromide. These quantities are usually sufficient to give a disc 10-15

mm diameter and a spectrum of suitable intensity. The mixture was carefully grinded,

spread uniformly in a suitable die and submitted to a pressure o f about 800 M Pa (8 tcm '

). A background scan was performed sing KBr disc without the MMF and then the scan

Chapter 3 MATERIALS A W METHODS


for KBr disc containing the MMF in the range of 4000~400cm'' was performed, Similarly

the scan o f EP reference standard was done,

3.3.3 NMM Analysis

As part o f our ongoing research program on API characterization we have been involved

in the characterization o f Mycophenolic acid and its derivative like mycophenolate

mofetil (MMF). These compounds are used as immunosuppressant drug in various organ

transplant surgeries.

M ethod

The API for study procured from Biocon ltd. Bangalore, The 'H-NMR, '-^C-NMR,

HMQC and HMBC spectra were recorded on Bruker DPX 300 MHz spectrometer using

DMSO-df, as a NMR solvent, TMS was used as internal standard and chemical shifts

reported in parts per million (ppm).

3.4 Com patibility Studies O f The M M F W ith V arious Excipients

3.4.1 Physical Com patibility Study

A physical compatibility study was designed to determine the interaction o f the drug with

various excipients. The samples i.e. drug alone, Excipients alone and homogeneous

mixture o f drug and each excipient were kept at accelerated conditions of 60°C in sealed

glass vials, and 40°C/75% RH in open glass vials (punctured to enable exposure to RH

conditions for four weeks). These samples were then periodically examined against a

control sample kept at 4°C.

Control (2-8°C) ; ~ Sealed vials

40°C, 75% RH (open) open vials

40°C, 75% RH (Close) Sealed vials

60“C (open) open vials

60°C (Close) Sealed vials

Chapter 3 MATERIALS AND METFIODS

D epartm en to f Pharmaceutical Chemistry Page 61

The ratio for physical mixture o f drug and the excipients was selected based on the

probable concentration o f the excipients in the capsule formulation.

3,5 Forniuiatioii Developm ent

From the literature and from the patent search and from the cosTipatibility studies o f the

excipients the most favorable excipients were short-listed. All the excipients chosen are

well known for their suitability and fitness of purpose. Each excipient is controlled by

pharmacopial specification and are the same or similar to those used in the reference

innovators product. The object o f the development programme was to produce a generic

capsule having pre-determined characteristics using design o f experiments. The final list

of excipients to be used with their probable functions is in Table 4.4

Excipients used in the Capsules are given below with their possible functions


D epartm en to f Pharmaceutical Chemistry Page 62

C h a p te r 3 MATERIALS AND METHODS

Table 3.4 Prototype formula for the of the M M F

3.5.1 Selection of Average W eight

Based on market product (Avg. weight o f reference product is 300mg with streggth 250

mg), we decided to keep the average weight o f our product similar as o f the reference

product that is 303 mg.

Department of Pharmaceutical Chemistry Page 63


Table 3.5 Average weight of capsules

S trength (mg)

W eight (mg)

Innovator In-house

250 300 303

3.5.2 P reparation of capsules

Capsules were prepared using wet granulation method according to the design matrix

obtained from the design expert software for three variables. All 17 batches were

prepared and analyzed for the chosen responses. Table gives the composition o f the three

variables in all the batches prepared.



Table 3.6 Niiinerlcal value o f th ree factors iii cen tral com posite eKperinienta! design

as obtained wsieg design expert softw are 7 .13

Factor 1 Factor 2 F actor 3

Std Run A:Povidone-K90 B;M CC PH-105 Ac-di-sol

14 1 1.00 10.00 1.00

4 2 1.50 15.00 1.00

1 3 0.50 5.00 1.00

13 4 1.00 10,00 1.00

11 5 1.00 5.00_ _ _ _

5 6 0.50 — =1 10.00 0.50

7 0.50 10.00 T 5 r ~ ~ ~

17 8 1.00 10.00 1.00

T 5 ~ “ 9 1.00 10.00 1.00

6 10 1.50 10.00

2 11 1.50 5.00 1.00

12 12 1.00 15.00 1,50

16 13 1.00 10.00 1.00

9 14 1.00 5.00 0.50

3 15 0.50 15.00 1.00

8 16 1.50 10.00 1.50

10 17 1.00 15.00


3 .53 M anufacturing procedure

1. First the drug weighed and passed through # 40 mesh screen.

2. In next step MCC PH 105 and Ac-Di-Sol were weighed, passed through # 40 mesh

screen and both mixed with drug,

3. The granulating solution was prepared liy dissolving Povidone K90 in purified water

as specified in prototype formula,

4. Granules were prepared by using this granulating solution and above ingredients,

The prepared granules were subjected for drying in FBD at 60°C until LOD reached

1,5-2%.

5. Dried granules were sifted through #25 mesh screen.

6. Then Extragranular material (MCC PH 102, Ac-Di~Sol) were weighed and passed

through #40 mesh screen, This Extragranular material was mixed with granules in

double cone blender for 15 min at 10 rpm.

7. Magnesium stearate weighed and passed through # 60 mesh screen and was mixed to

double cone blender for 5 min at 10 rpm.

8. The capsules were filled using these granules,

C h ap te rs MATERIALS^yp METHODS



Table 3.7 Processing variables th a t were kept constant d u rin g form ulation o f ail the

batches

S. No Processing variables C onstan t

1 Amount of DM water used for binder solution

(Povidone kl9)

75 ml

2 Mixing time of the ingredients 10 min„ „ _ __ „

3 Binder solution adding time 3 min

Drying temperature of the granulation Ir 60° C

6 Loss on drying o f the blend Not more than 2

Mixhig time of granules and Extragranular

material in double cone blender

IS min

Mixing time of magnesium stearate to the blend in

double cone blender

5 min

The granules were filled in to capsules to a un it fill weight o f 303 mg.

D epartm ent pf Pharmaceutical Chemistry Page 67

C hapter 3 MATERIALS AND METHODS

Fig 3.2 FLOW CHART OF WET GRANULATION PROCESS AND CAPSULE

FILLING

Welgli the raw materials

Granulate the above blend using aqueous binder solution of Povidone 1^90,

r

:.vt

Dry the wet granules at an inlet air temperature of 60 °C until % Loss on

drying achieved NMT 2,0%.

Sift above dried granules through # 25 mesh ASTM.

Sift extragranular material ( MCC PH102+Ac-Di-Sol)Throu|;h # 40 mesh

ASTM, Add it to the above sifted granules



Blend for 15 minutes blender

Blending'.'

Magnesium Stearate USNF (Through # 40 mesh ASTM) Add it to the above blend

and blend for 5 minutes

3.5.4 Evaluation of the capsules of the M M F

a) W eight Variation

Twenty capsules o f each formulation were weighed individually and mean weight

and percentage relative standard deviation was calculated,

Six capsules of each formulation were also examined for their thickness using

sliding Vernier caliper and the mean thickness value was calculated.

b) D isintegration Time

Six capsules of each formulation were used to determine disintegration time. Water

was used as disintegration medium and temperature was maintained at 37±2°C.

c) Dissolution profile

Dissolution was performed on capsules (n=6) using USP apparatus 2 (Paddle) 100

rpm in 900 ml of 0.1 N HCl (pH =1.2) maintained at 37± 0.5° C. 10 ml o f the

samples were withdrawn, fdtered and 0.1 nil o f the sample is diluted upto 10 ml and

analyzed spectrophotometrically at 293 nm for Mycophenolate mofetil content.


3,5.5 Stability of In-house product

Accelerated stability studies according to the ICH guidelines

The MMF was packed in HDPE bottle and kept in stability chamber maintained at 40° C

and 75 % Relative Humidity for 90 days. Samples were withdrawn at interval o f 0, 30, 60

and 90 days. The samples were analyzed for their drug content, DT, hardness and

dissolution in 0. IN HCl in USP I at 100 rpm, (As this is the official apparatus and media

in USFDA).

3.6 Dissolution M ethod Development

3.6.1 Introduction

Drug absorption from solid dosage forms after oral administration depends on the release

of the drug substance from the drug product, the dissolution or solubilization o f the drug

under physiological conditions, and the permeability across the gastrointestinal tract.

Because o f the critical nature of the first two of these steps, in vitro dissolution may be

relevant to the prediction of in vivo performance. Based on this general consideration, in

vitro dissolution tests for immediate release solid oral dosage forms are used to;

• Assess the lot-to-lot quality of a drug product;

® Assess the stability of the drug product;

® Ensure continuing product quality and performance after certain changes, such

as changes in the formulation, the manufacturing process, the site of

manufacture, and the scale-up of the manufacturing process; and

• Develop new formulations.

In formulation development, dissolution testing can aid in the selection o f excipients, help

optimize the manufacturing process, and enable formulation of the test product to match

the release of the reference product. Dissolution testing has emerged in the

pharmaceutical field as a very important tool to characterize drug product performance.


Chapter 3 ^ MATERIALS AND METHODS

The dissolution test is an analytical technique that has undergone significant equipment

modifications and improvements spanning the last decade. Dissolution has become an

important and widely utilized test receiving more emphasis worldwide fi'om regulatory

authorities during the last 15 years. The significance o f a dissolution test is based on the

feet that for a drug to be absorbed and available to the systemic circulation, it must

previously be dissolved. Therefore, dissolution tests are used not only for quality control

of finished products, but also to assess several stages o f formulation development, for

screening and proper assessment o f dift'erent formulations. Basically, the dissolution test

makes it possible to assess the dissolution properties of the drug itself and thereby to

select the most appropriate excipients and to optimize proportions among them to

obtaining the desired drug release behavior.

Moreover, when an ‘in vitro/in vivo’ correlation is available, dissolution can be used as a

test to reflect the bioavailability o f a product in humans and therefore to determine the

actual bioequivalence of different products containing the same drug at the same dosage.

During a preformulation study, preliminary testing conditions are commonly elaborated

taking into consideration the state o f the art for dissolution testing. Different official

apparatus are available and, for each, Compendia, e.g., USP, BP, and EP, report detailed

specifications in both general chapters and individual monographs on solid oral dosage

forms.

Chapter 3 MATERIALS AND M F £ H O ^



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Fig 3 3 A pplications of in v itro dissolution studies

3.6.2 Selection of a suitable appara tus

The preferred apparatus for a capsule dosage form (Mycophenolate Mofetil) is by

paddle type (USP-II) as mentioned in USFDA guidance for dissolution o f generic

drugs.

3.6.3 Selection o f a suitable Dissolution M edia

0.1 N HCL was selected as the dissolution media as mentioned in USFDA guidance

for dissolution o f generic drugs.

3.6.4 Placebo In terference

Placebo o f the capsule formulation was analysed by UV spectrometer to see if it has

any interference on the UV absorbance of the MMF.


Weighed amount of the placebo was taken and dissolved in 900 ml o f O.IN HCl and

sonicated for 15 min to facilitate maximum solubility o f the placebo in the dissolution

media. 2 ml o f this solution was pipetted out into a 50 ml volumetric flask and the

volume was made upto 50 mi with O.IN HCl. Absorbance o f this dilution was taken

by LIV spectrophotmeter at 293.0 nm.

3.6.5 F ilter V alidation

The effect o f filter interference in the UV analysis was done using the following filters:

® Wattmann filter paper

® 0.45 pm PVDF Filter

® 0,45 pm PTFE Filter

• 0.45 pm Nylon Filter

The placebo solution was passed through the above filters and their UV absorbance

taken at 250.0 nm.

3.7 Dissohitlon M ethod V alidation

Dissolution method is developed in O.IN HCL by UV Spectroscopy method. There are

different parameters which are used to validate this method.

3.7,1 Specificity and selectivity

Mycophenolate mofetil solutions (22 pg m f ') were prepared in both the selected media

along with and without common excipients (microcrystalline cellulose, magnesium

stearate, talc, HPMC, iron oxide red, titanium dioxide) separately. All the solutions

(included both dissolution mediums and placebo solution) were scanned from 400 to 200

nm at a speed of 400 nm min"' and checked for change in the absorbance at respective

wavelengths and any interference by dissolution mediums or placebo. In a separate study,

dmg concentration o f 22 pg ml~‘ was prepared independently from pure drug stock

Chapter 3_________________________________________ MATERIALS AND METHODS

D ep ar tm en to f Pharmaceutical Chemistry Page 73

MATERIALS AND MRTHOD_S

solution in selected media and analyzed (« = 6). The standard deviations were determined

in both cases.

3.7.2 A ccuracy

To determine the accuracy o f the proposed methods, different levels o f drug

concentrations-lower concentration (LC), intermediate concentration (IC) and higher

concentration (HC) (in both media) were prepared Irom independent stock solutions and

analyzed {n = 9), Accuracy was assessed as the standard deviation, percentage R.S.D. at

each level; overall standard deviation and overall % R.S.D. and compiled %recovery was

determined. To provide an additional support to the accuracy of the developed assay

method, standard addition method was employed, which involved the addition o f

different concentrations o f pure drug (5, 11, and 22 \xg ml”' in HCl medium and in the

acetate buffer medium) to a known pre-analyzed formulation sample and the total

concentration was determined using the proposed methods (n-9) [18]. The %recovery of

the added pure drug was calculated as, %recovery = [(Ct ~Cs)/Ca]xlOO, where Ct is the

total drug concentration measured after standard addition; Cs, drug concentration in the

formulation sample; Ca, drug concentradon added to formulation.

3.7.3 Precision

Repeatability was determined by using different levels o f drug concentrations (same

concentration levels taken in accuracy study), prepared from independent stock solutions

and analyzed («=6). Inter-day, intra-day and inter-instrument variation were studied to

determine intermediate precision of the proposed analytical methods. Different levels of

drug concentrations in triplicates were prepared three different times in a day and studied

for intra-day variation. Same procedure was followed for three different days to study

inter-day variation (n = 18). One set o f different levels o f the concentrations was re

analyzed using Shimadzu UV-2450 UV-vis spectrophotometer connected to computer


with UV-PC software, by proposed methods to study inter-instrument variation [n = 3),

The percent relative standard deviation (% R.S.D.) o f the predicted concentrations from

the regression equation was taken as precision.

3.7.4 L inearity

To establish linearity o f the proposed methods, five separate series o f solutions of

mycophenolate mofetil (10 - 30 (ig niL’' in O.IN hydrochloric acid and in acetate buffer

medium) were prepared from the stock solutions and analyzed. Least square regression

analysis was done for the obtained data. One-way ANOVA test was performed based on

the absorbance values, observed for each pure drug concentration during the replicate

measurement o f the standard solutions.

3.7.5 Eobustiiess

Robustness o f the proposed method was determined by (a) changing pH o f the m edia by

±0.1 units and (b) stability o f the mycophenolate mofetil in both the selected media at

room temperature for 24 h. Three different concentrations (LC, IC and HC) were

prepared in both the media with different pH and mean %recovery was determined.

3.8 Assay M ethod developm ent and validation

3.8.1 Reagents and solvents

Pure MMF was obtained as gift sample from Biocon Ltd., (Bangalore, India) and certified

to contain 99.7% (w/w) on dried basis. Acetonitrile was of HPLC grade & purchased

from SDFine- Chem limited (Mumbai, India) and all other chemicals and reagents used

were o f analytical grade and were purchased from Merk Ltd. (Worli, Mumbai, India).

Milli-Q (MA, USA) water is used for the preparation o f buffer. Triethylamine buffer

solution was prepared and filtered through 0.22ja, filter (Millipore, USA). Formulation of

MMF used for the study was MMF tablets (label claim; 500 mg/tablet), capsules (label

Chapter 3 ___ _________^ ^ .......... JVIAT£RIALSAND METHODS


claim: 250 mg/capsiiles) and PLGA MMF nanoparticles (label claim: 10.2 mg/lOOmg of

PLGA nanoparticles) procured from Jamia

Hamdard, (New Delhi, India).

3.8.2 Iiistriiinentatioii and chroniatograpliic conditions

HPLC analysis was performed with Waters (Model 2695, Waters, USA). HPLC system

equipped with PDA detector. The chromatographic separation was performed using a

Zorbax Rx-C8 (250 mm X 4.6 mm), 5 j,im (Agilent Technologies, US), The mobile phase

consisting o f a mixture o f triethylamine buffer, pH o f which adjusted to 5.3 with diluted

Orthophosphoric acid and acetonitrile in the ratio o f 40:60 (%V/V) with the flow rate of

1.0 niL min-1 was employed. The detector wavelength was set at 250 nm (PDA detector

model waters 2996). The injection volume was 10 pL while column was maintained at

45°C,

3.8.3 C alibration curve o f M ycopiienolate niofetil

A sock solution o f MMF was prepared in diluent consisted mixture o f water and

acetonitrile in the ratio o f 20:80 (%V/V) at 5 mg mL"'. With proper dilution, standard

solutions were prepared in the concentration range 100- 600 |ig mL"'. Ten microlitres

from each standard solution was injected in the column in the above mentioned

chromatographic conditions (Section 2.2). Each concentration was injected six times in

the column. The data o f peak areas plotted against corresponding concentration were

treated by linear-square regression analysis.

3.8.4 M ethod V alidation

3.8.4.1 Specificity

The specificity o f the method was ascertained by performing interference studies. Six

injections o f standard solution (for system suitability), one injection o f diluents, one

injection o f placebo (equivalent to 200 mg mycophenolate mofetil) which has been

Chapter 3 ............ ............... MATERJALS AlW M EJJODS


prepared as sample solution preparation, one injection of sample solution and three

injections of spiked sample solution which has been spiked with known impurity (MPA)

at 0.5% level has been injected in column and analyzed. Also the peak purity o f MMF

was assessed.

3,8.4.2 Precision

3.8.4.2.1 System precision

System precision o f the system was determined by injecting six replicates o f the standard

solution (injection volume, 10 },iL) and measurement carried out o f peak areas o f the main

peak. Data was treated to calculate % RSD.

3.8.4.2.2 M ethod precision

Method precision o f method w'as determined by injecting six replicates (injection volume,

10 pL) o f the sample soludon from single batch o f capsules individually and

measurement carried out o f peak areas o f the main peak. Data was treated to calculate %

RSD.

3.5.4.2.3 In term ediate precision

Intermediate precision o f method was determined by injecting six replicates (injection

volume, 10 pL) o f the sample solution from single batch of capsules individually as per

the method by a different analyst on different instrument using different column and on a

difYerent day. Measurements carried out of peak areas of the main peak. Data was treated

to calculate % RSD.

3.5.4.3 L inearity

Linearity of the method was studied in two replicate of each level in the range of 30% -

200% o f the standard concentration. A stock solution of 5000 ppm o f MMF standard was

prepared and diluted the stock standard solution for the preparation o f solution o f

different concentrations o f MMF i.e. 120 pg mL"', 160 jag mL"', 240 f g mL"', 320 |.Lg mL‘

Chapter 3 ................. .................. MATERIALS AND METHODS


Q i a p t e r ^ ________________ _ _ MATERIAJ.S AND M jriiO D S

400 jig m l./', 480 p,g m L '', 560 j.ig rriL/’, 640 },ig n iL '’, 720 f.ig mL"' and 800 j-ig n iL '’, In

order to assess the linearity o f the method data were plotted in the form o f linearity curve

and slope, intercept and correlation coefficient o f the curve has been calculated.

3.8.4.4 A ccuracy

Accuracy studies were carried out by applying the method to placebo samples to which

known amount of MMF corresponding to 80, 100, 120 and 200% of the MMF label claim

had been added. At each level of the amount, samples prepared in triplicate and

determination was performed. Data was treated to calculate % R.S.D. at each level and

overall,

3.8.4.5 Robustness of the m ethod

Robustness was studied in six replicate at a concentration level o f 400 ppm. In this study,

five parameters (wavelength o f detection, column oven temperature, flow rate o f mobile

phase, pH o f buffer in the mobile phase and composition o f the mobile phase) were

investigated and the effects on the results were expressed as standard deviation and %

R.S.D. The wavelength of detection was varied by +5 nm (at 245 nm and 255 nm),

column oven temperature was varied by +5°C (at 40°C and 50°C).Tlie flow rate of mobile

phase and pH of the buffer in mobile phase were varied by +10% (at 0.9 mL m iif' and 1.1

mL min"') and +0.2 (at pH 5,1 and pH 5.5), respectively. In the mobile phase, the minor

component was varied by + 2% absolute or 10 % relative, whichever is less. Data were

treated to calculate % R.S.D. in each case,

3.8.4.6 L im it of detection and lim it of quantitation

In order to estimate the limit o f detection (LOD) and limit of quantitation (LOQ), single

injection o f blank and six injections o f standard solution at concentration level o f 400 |ig

mL’' were injected in column and system suitability was determined by determining %

R.S.D. o f six injections. Several lower concentrations o f MMF were prepared and


Einalyzed six times for determination of LOD and LOQ based on % 11, S. D. The LOD

was expressed as 10% R, S, D. whereas LOQ was expressed as 33% R, S. D, o f six

injections.

3.S.4.7 Stability of analytical solution

In order to determiiie the stability o f the analytical solution, standard solution and sample

solution to be analyzed initially and at different time intervals at 25'’C for around 24 hours

and / or standard solution and sample solution to be analyzed initially and at different

time intervals at 5“C for around 24 hours. For that six injections o f standard solution v/ere

injected in column for the determination o f system suitability and one injection o f each

standard solution as well as sample solution were injected at different time intervals for

around 24 hours.

3.8.4.S Forced degradation studies

To perform the forced degradation studies first injected six injections o f standard solution

(for system suit). Then the sample solution and placebo solution were treated separately

in each condition as followed:

(a) Two milliliters o f IN HCl was added and mixture was heated at 70°C for 30 minutes

and neutralized by addition o f IN NaOH solution and 10 mL o f diluent. (Acid induced

degradation)

(b) One milliliters o f IN NaOH was added and mixture placed at room temperature for 5

minutes. (Base induced degradation)

(c) Two milliliters o f 30% w/v H2O2 was added and mixture was heated at 70°C for 15

minutes. (Hydrogen peroxide H2O 2 induced degradation)

(d) Tablets, capsules and nanoparticles o f MMF were placed in UV chamber at 6500

LUX for 15 days. (UV induced degradation)

C h a p te r s^ ^ MATERIALS AND METHODS


(e) Tablets, capsules and nanoparticles o f MMF were placed in oven at 105°C for 15

days. (Thermal induced degradation) In all degradation studies, 10 |.iL o f the resultant

solutions were injected in column and chromatograms were run as described in section

2.2. The peak area o f each peak has been determined and peak purity determined in each

case. Data was treated to calculate the degradation in each case.

3.5.4.9 Analysis and stability testing of form ijlations

To determine the content o f MMF in commercial capsules (label claim; 500 mg/tablet and

250 mg/tablet) and in prepared PLGA-MMF~nanoparticles (label claim; 10.2 mg/lOOmg

of PLGA nanoparticles), the twenty tablets/capsules were weighed and their mean weight

determined. Powder (tablet/capsule) and nanoparticles equivalent to 4.0 mg o f MMF were

accurately weighed and transferred into a 10 mL volumetric flask, containing 5.0 mL o f

diluent ( mixture o f water ; acetonitrile, 20;80%v/v). To ensure complete extraction o f

drug, it was sonicated for 30 min and diluted to 10 mL with diluent. The resulting

solution was centrifuged at 2000 rpm for 10 min and supernatant was analyzed for drug

content. Ten microlitres of the filtered solution was injected into the chromatographic

conditions as mentioned in section 2.2. The analysis was repeated in three replicate and

the possibilities o f excipient interference in the analysis was studied. For stability studies,

prepared tablets, capsules and PLGA-MMF-nanoparticles (in closed high density

polyethylene containers) were stored at accelerated conditions (40°C/75%RH) and drug

content was analyzed after 1, 3, and 6 months,

3.8.4.10 System suitability

System suitability in each parameter o f validation has been determined and the

acceptance criteria for the system suit were as followed;

9 Tailing factor o f MMF peak from the standard solution should not be more than

2 ,0.

C h a p te rs ^ MATERIALS AND METHODS


® Theoretical plates of MMF peak froiTi the standard solution should not be less than

3000.

® % R.S.D. o f the area of MMF peak from the five injection of the standard solution

should not be more than 2.0

3.9 Related siibstaiices method! developm ent and validation

3.9.1 RS nietliod developm ent

3.9.1.1 P reparation of Buffer (pH 5.30):

Add 2mL of Triethylamine in 650mL of Milli-Q water. Adjust the pH to 5.30i:0.05

with orthophosphoric acid. Filter through 0.45|.im nylon membrane filter and degas.

3.9.1.2 P reparation of M obile Phase:

Prepare a suitable quantity of mixture of buffer (pH 5.30) and acetonitrile in the ratio

o f 65:35. Mix well and degas.

3.9.1.3 Preparation of Diluent:

Prepare a suitable quantity of a mixture of water and acetonitrile in the ratio o f 20:

80.

3.9.1.4 C lirom atograpM c Conditions:

Column; Zorbax SB C-8 (250mm X 4.6mm), 5|am

Flow rate: 1.5 mL / min

Injection Volume: 10 |iL

Wavelength: 250 nm

Column Temperature: 45°C

Run Time: 70 min

3.9.1.5 Preparation of System Suitability Solution;

Accurately weigh and transfer about 40mg o f MMF working standard into a lOOmL

volumetric flask. Add about lOmL o f diluent and sonicate to dissolve. Make up the

Chapter 3 ^ ___ MATERIALS AND METHODS


volume with diluent and mix. Dilute 5mL o f this solution to lOOmL with diluent and mix.

Filter through 0.45j,im nylon membrane filter.

3.9.1.6 P reparation of sam ple solution:

Determine the Average weight of not less than 10 capsules and crush them to a fine

powder. Accurately weigh and transfer powdered sample equivalent to about 200mg

of MMF into a lOOmL volumetric flask. Add about 60mL of diluent and sonicate for

about 30 min, with intermittent shaking. Make up the volume with diluent and mix.

Filter through 0.45(,im nylon membrane filter.

3.9.1.7 P repara tion of Placebo sotatloii:

Weigh and transfer the amount of placebo powder equivalent to 200mg of MMF into

a lOOmL volumetric flask. Add about 60mL of diluent and sonicate for 30 min. with

intermittent shaking. Make up the volume with diluent and mix. Filter through

0.45.Lim nylon membrane filter.

3.9.1.8 Evaluation of System Suitability:

Inject the standard solution into the chromatograph and record the chromatograms.

The system is suitable for analysis if and only if;

1. The tailing factor of MMF peak is not more than 2.0.

2. Column elficiency determined from MMF peak is not less than 7000 theoretical

plates.

3. The Relative standard deviation o f the area counts for six replicate in.jections o f

standard is not more than 5.0%,

Make necessary adjustments, i f needed, to meet the system suitability criteria.

3.9.1.9 Procedure:

Chapter3_ ______ MATERIALS AND_METHODS



Inject the placebo and sample sohition into the chromatograph and record the

chromatograms. Disregard the peaks observed in the sample chromatogram, which

are observed at the same retention time in the placebo chromatogram. The retention

time o f MMF peak is about 21 min.

The relative retention time (RRT) o f Imp_Acid impurity is as follows:

Table 3.8 MRT of Im p A d d (MPA)

S. No. Name I RRT

1 MPA — i 0.27

2 MMF ] 1.00

3.9.1.10 Calculations;

The % o f Impurities can be calculated as given below:

1. Any Individual Known Impurity

(%w/w)

AT DS P A

= — - X — — X — X 100 X-----

AS DT 100 C

2. Any Individual Unknown Impurity

ATI DS P A

(%w/w) = -....... X ..........X — — X 100 X

AS DT 100 C

3. Total Impurity = (1+2)


Where

AT = Area counts o f any individual known impurity pealc in the chromatogram o f the

sample solution.

ATI = Area counts o f any individual unknown impurity peak in the chromatogram of the

sample solution.

AS = Average area counts of ID peak in the chromatograms o f the standard solution as

obtained under system suitability.

DS = Dilution factor for standard solution.

DT = Dilution factor for sample solution.

C = Label claim of ID per tablet, in mg.

P = Percent potency of ID working standard, on as is basis.

A = Average weight of the tablet in mg.

Chapter 3 ______ _ __________________MATERIALS AND METHODS



3.9.2 RS method validation protocol and acceptance crlte iia ;-

3,9,2.1 Specificity

A. Interference Studies

Injection o f blank (diluent used in the method)

hijection o f placebo solution

Injection of sample solution

Analysis of sample spiked with known related

impurity (Imp Acid i,e MPA) at 0.5% level

A cceptance C riterion

There shall not be any peak from the

placebo solution at the RT o f MMF

and known impurity,

Peak purity o f MMF shall pass.

Peak purity o f MMF and spiked

impurity shall pass,

Sample preparation, in triplicate, and single

injection of specified degradation impurity (Imp

Acid) at sample concentration i,e.: 2000 (ig/ml

Note: If the peak saturates, the concentration shall

be adjusted, accordingly.



B. Forced D egradation Studies

Preparation and analysis of sample and

placebo by treating with Hydrochloric acid


placebo by treating with Sodium hydroxide


placebo by treating with Hydrogen peroxide


placebo by thermal degradation


placebo by photolytic degradation

(Method Precision data can be taken as

control sample)

Note: I f at 1000 pg/nil, the peak saturates, the

concentration shall be adjusted, accordingly.

A cceptance C rite ria

Peak purity o f MMF shall pass

Note : D egradation N M T 30%



3 3 2 2 Precision

A. System Precision

Experiineiit'


Six replicate injections oi' standard solution % RSD shall not be more than 5.0

B. M ethod Precision

Experim ent A cceptance C rite ria

Preparation and analysis of one batch o f 500 % RSD should not be more than

mg / tablet strength, six times as per the 10.0 o f individual and total

method impurities

In case no known impurity is present or all

impurities are below LOQ in the sample,

method precision is to be demonstrated on the

sample spiked with Imp Acid (MPA) at 0.5%

level.



C. Interm ediate Precision

Experiiiieiit

Preparation and analysis o f one batch (same

batch as used for Metliod precision) o f 250

mg / capsule strength, six times as per the

method by a different analyst on different

instrument using different column and on a

different day.

Individual and overall % RSD

should not be more than 10.0 of

individual and total impurities

3.9.23 L O D /L O Q

Experiment

Several lower concentration o f Imp Acid

(MPA) and MMF shall be prepared and

analyzed six times for the determination o f

LOD / LOQ based on % RSD

A cceptance C riteria

1. LOQ : % RSD should not be

more than 10

2. LOD : % RSD should not be

more than 33



3.9.2.4 L m earity

Experim ent

Linearity o f Imp Acid (MPA) and MMF to be

performed in the range o f about LOQ - 120 %

of Specification level.

For MMF Linearity range - LOQ to 2,4 ng /

mL

For Imp Acid (specification limit - 0.50 %

w/w) Linearity range ■■ LOQ to 12 |ig / mL

Correlation coefficient should not

be less than 0.980



3.9.2.S A ccuracy

Experim ent A cceptance C rite ria ~1

Placebo spiked with Imp Acid at LOQ level, in 1, % Recovery shall be in the

triplicate range o f 80 - 120.

Sample spiked with Imp Acid at about 50 % o f

specification level, in triplicate 2. Individual and overall % RSD

Sample spiked with Imp Acid at about 100 % o f % Recovery shall not be more

of specification level, in triplicate than 10.0

Sample spiked with Imp Acid at about 120 %

of specification level, in triplicate

(Mean o f Method Precision data can be

considered as Control)



2321} Robiistness


Standard solution to be prepared and analyzed under

each of the following variable conditions

Sample solution spiked with Imp Acid at 0.5% level

System suitability should

pass.

1. By changing the wavelength o f detection by +5 nm

(At 245 nm and 255 nm)

2. By changing the column oven temperature by +5"C

(At 40°C and 50°C)

3. By changing minor component of the mobile phase

by + 2% absolute or 10 % relative, whichever is less

4. By changing the flow rate by ±10% (At 1,35 ml/min

and 1.65 ml/min)

5. By changing the pH by +0.2 (At pH 5.1 and pH

5.5)

Note: RRT o f known impurity

shall be calculated in all the

variable conditions



3.9.2.7 Stability of analytical solution


to be analyzed initially and at different time

intervals at 25”C for around 24 hours

And/or


to be analyzed initially and at different time

intervals at 5°C for around 24 hours

Acceptance C rite ria

Cumulative % RSD for each

impurity should not be more than

10.0

3,9.2.8 System Suitability


3.10 H PTLC iiietliod for assay

3.10.1 P repara tion of C alibration S tandards and Q uality- C ontro l (Q C) Sam ples

A stock solution o f MMF was prepared by dissolving 200 mg MMF in 10 ml acetonitrile.

Working solutions containing from 1 to 12 mg m l'' were prepared by appropriate dilution

o f the stock solution with acetonitrile. Each working solution o f MMF (10 (.il) was used to

spike plasma (1 ml) to furnish calibration standards ranging from 10 to 120 f.ig m l'’. QC

sample solutions were prepared at concentrations o f 20, 60, and 100 )ug m l''. Each

solution was divided into 1.0 ml volumes which were immediately frozen at -20°C,

3.10.2 C hrom atography

Chromatography was performed on 20 cm x 10 cm aluminium plates coated with 200-pm

layers o f silica gel 60F254 (E. Merck, Germany). Samples were applied to the plates as 5

mm wide bands, 5 mm apart, by means of a Camag (Muttenz, Switzerland) Linomat V

sample applicator fitted with a 100 pL syringe, A constant rate o f application o f 150 nl s-

1 was used. Linear ascending development o f the plates to a

distance o f 80 mm was performed with triethylamine buffer (2 ml o f Triethyl amine was

added in 650 ml o f Milli-Q water. The pH o f the mixture was adjusted to 5.3 with

orthophosphoric acid. The buffer was filtered through 0.45 pm nylone membrane) and

acetonitrile, 20:80 (% v/v), as mobile phase in a 20 c m x 10 cm twin-trough glass

chamber(Camag), previously saturated with mobile phase vapor for 15 min at room

temperature (25 ± 2°C) and relative humidity 60 ± 5%. After development the dried

plates were scanned at 250 nm by means o f Camag TLC Scanner III in absorbance mode

operated by WinCATS software (Version 1.2.0). The source o f radiation was a deuterium

lamp emitting a continuous UV spectrum in the range 190-400 nm. The slit dimensions

were 5 mm x 0.45 ram and the scanning speed was 20 mm s'*.

Chapter 3 _________ MATERIALS AND METHODS


Sample P reparation

Before analysis, plasma samples were thawed at room temperature for approximately 10

mill. Plasma calibration standards and QC samples (1 ml) were transferred to vials and

mixed with 1.0 ml acetonitrile. After vortexing for 1 min, the samples were centrifuged (5

min, >2000g). The supernatant was transferred to new tubes and the solvent was

evaporated at 37°C under a stream of nitrogen. The residues were dissolved in 100 |.i!

acetonitrile and 1 (.il o f each sample was applied to the TLC plate to furnish a final

calibration range o f 100 to 1200 ng per zone. QC samples at final concentrations o f 200,

600, and 1000 ng per zone were obtained after application. Each concentration was

applied six times to the TLC plate.

S tability Study

QC Samples

For determining the stability, QC samples were taken at three concentration level. Plasma

was spiked with MMF and then stored at 4°C or 20°C, Then stability was assessed at 0.5,

1, 2, 4, and 8 hr interval. The stability of the drug in frozen samples (~20°C) was

detemiined for a period o f two months. The degradation rate constant (kobs), half-life

(tl/2), and shelf life (t90) o f the drug in plasma sample were also determined at 4°C and

at 20°C.

Freeze-Thaw Stability

To determine freeze-thaw stability nine aliquots of each QC sample were stored at -20°C

for 24 hrs. Then the samples were left at room temperature for thawing process. Three

aliquots o f each QC sample were analyzed after extraction. The other aliquots were again

stored at -20°C for next 24 hrs. This cycle was repeated three times.

Chapter 3 _______________ MATERIALS AND METHODS



Stock Solution Stability

The stability o f the drug in acetonitriie (stock solution) was assessed at 4°C and 20°C. At

room temperature, runtime stability o f processed samples after extraction was detennined

for QC samples. To test the stability, the samples were analyzed immediately after

preparation (control) and after a stipulated time period.


chapter 3 3.1 materials -...

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