5f solubility enhancement by preparation of nanoparticles...

RESULTS AND DISCUSSION

SPTM, SVKM’S, NMIMS, MUMBAI 204

5F Solubility Enhancement By Preparation Of Nanoparticles

5F.1 Preparation of nanoparticles with supercritical antisolvent precipitation

5F.1.1 Formulation of nanoparticles with antisolvent precipitation

Table 5F.1 Key formulation characteristics of nanoparticles with antisolvent

precipitation

Formul

-ation

code

Solvent

used

Concentrat

-ion of drug

solution

(%)

Temper-

ature

Percentage

yield

(%W/W)

SD,n=3

Particle

size(nm)

SD

n=50

Drug

content

(%)

SD, n=3

SAS 1 Acetone 2 60 2-3 - -

SAS2 Methanol 2 60 2-3 - -

SAS 3 Methanol 2 72 6 1776.4 99.072.

46

From the above mentioned systems only SAS3 is chosen for further analysis as

showing improved yield.

5F.1.2 Saturation solubility testing

Table 5F.2 Saturation solubility of nanoparticles with antisolvent precipitation

Study parameter 0.1N HCl

Phosphate buffer

pH 6.8

Water

Solubility (mg/ml) 0.014405

0.024905

0.024783

Figure 5F.1 Saturation solubility of nanoparticles with antisolvent precipitation



5F.1.3 Multimedia dissolution studies

Table 5F.3 Multimedia dissolution of nanoparticles with antisolvent precipitation

Time

(Min)

0.1 N

HCl %RSD Water %RSD

Phosphate

buffer pH

6.8

%RSD OGD

Media %RSD

5 9.287 4.624 4.733 7.991 8.941 3.790 88.173 3.364

10 11.12 3.849 5.224 6.987 12.638 2.714 92.202 3.209

15 11.69 4.226 5.818 6.068 14.808 2.288 94.030 2.690

20 12.33 4.308 7.805 4.726 15.373 2.445 94.241 3.219

30 12.63 3.843 12.068 2.778 16.630 2.273 95.631 3.667

45 10.92 4.360 16.844 1.970 17.135 2.493 96.492 2.436

60 10.88 4.626 21.394 1.599 17.897 3.202 97.889 2.187

In-vitro multimedia dissolution of nanoparticles with

antisolvent precipitation

0

20

40

60

80

100

0 5 10 15 20 30 45 60

Time point(mins)

% C

um

ula

tive r

ele

ase

0.1 N HCl Water Phosphate buffer 6.8 Phosphate buffer 6.5+ 0.35%tween 20

Figure 5F.2 Multimedia dissolution of nanoparticles with antisolvent

precipitation



5F.1.4 Physicochemical Characterization

5F.1.4.1 XRD

Figure 5F.3 X-ray diffraction spectra of nanoparticles with antisolvent

precipitation

5F.1.4.2 FTIR spectra

Figure 5F.4 FTIR spectra of nanoparticles with antisolvent precipitation



5F.1.5 Particle size distribution

Table 5F.4 Particle size distribution of nanoparticles with antisolvent

precipitation

System D(10%) D(50%) D(90%) Polydispersity

index

Zeta

potential

SAS 3 1331 1535.7 1776.4 0.083 25.40

Figure 5F.5 Intensity distribution curve of nanoparticles with antisolvent

precipitation

As micronisation was reported through the use of supercritical antisolvent

precipitation, this method was tried to enhance the saturation solubility and

dissolution rate of candesartan cilexetil. On the basis of solubility of drug and

previous experimentation, acetone was tried as solvent of choice with 2% drug

concentration but the yield was found very low .The solvent was then changed to

methanol by keeping the concentration of drug same as that of previous experiment.

At the temperature of 60◦ C, the yield was found to be less but appearance of product

was improved. After changing the temperature to 72◦ C, slight improvement in yield

was observed finally reproducible batches of batch 3 were taken and the product was

stored in desiccator till further use. The product was then evaluated for particle size,

polydispersity index and zeta potential. Particle size was found to be around 1776 nm,

considerable reduction as compared to particle size of pure drug was found.



Important observation as reported in the advantages of Antisolvent precipitation is

particle size distribution was very narrow. Polydispersity index and zeta potential

were found to be within acceptable limit around 0.083 and 25.40 mV respectively.

Improvement in saturation solubility and multimedia dissolution was observed as

compared to pure drug and marketed formulation.IR spectra showed that no change in

the properties of drug as the characteristic peak was observed at 1717 cm-1 Change of

crystallinity of drug was seen in XRD spectra. There is a scope for further work in

this area in enhancing the reduction in particle size as well as to enhance the yield by

altering the experimental conditions.



5F.2 Preparation of Nanoparticles Using Ion Gelation Technique

5F.2.1 Preparation of nanoparticles using ion gelation technique

Chitosan and sodium tripolyphosphate nanoparticles were prepared by ion gelation

method. The ratios of chitosan and STPP were decided based on literature survey. As

it is reported that release of drug is retarded with increase in chitosan concentration,

concentration of chitosan was kept below 0.1%. Total 9 systems were prepared as

shown in experimental. According to literature, with increase in drug loading particle

size also increases amount of drug added in each batch was kept 10 mg /5 ml.

Table 5F.5 Selection of nanoparticles using ion gelation technique

Concentration

of STPP

Concentration

of chitosan 0.05%W/V 0.075%W/V 0.1%W/V

0.05%W/V S1 S2 S3

0.1%W/V S4 S5 S6

0.2%W/V S7 S8 S9

Amongst all prepared systems, only systems (In bold) which were having comparative

clear appearance were chosen for further studies.

5F.2.2 Evaluation of nanoparticles using ion gelation technique

For measurement of entrapment efficiency, supernatant was suitably diluted with

methanol and analyzed at 254 nm and amount of unentrapped drug was calculated.

From that Drug loading efficiency and % drug entrapment was calculated. Drug

loading efficiency was found to vary between 24 to 35 %with system S1 showing

maximum drug loading efficiency of 35.85%.

Percentage yield was calculated by the formula described in experimental section

4F.2.

Table 5F.6 Evaluation of selected nanoparticle systems prepared by ion-gelation

technique

Formulat

ion code

DLE

(%W/V)

SD, n=3

Particle

size(nm)SD

n=50

Polydis-

persity

index

Zeta

Pote-

ntial

Percentage

yield

(%W/W)

SD,n=3

Drug

content

(%)

SD, n=3

S1 35.85 209.7 0.299 25.17 60 98.45

S5 24.62 280.8 0.261 29.34 48 99.76

S6 30.34 329.7 0.356 27.12 55 98.34



Particle size, polydispersity index and zeta potential of the given systems was

calculated using photon correlation spectroscopy and electrophoretic light scattering

using Delsa Nano instrument. Particle size was found to range from 209 nm-329 nm

of the selected systems. It was found that lower the concentration of chitosan lower

the size of particles.

Table 5F.7 Particle size distribution of selected nanoparticle systems prepared by

ion-gelation technique

System D(10%) D(50%) D(90%)

S1 24.6 75.8 209.7

S5 69.3 136.6 280.8

S6 12.8 64.4 329.7

Figure 5F.6 Intensity distribution curve of S1





TEM image

Figure 5F.9 TEM image of Nanoparticles prepared by ion gelation technique

TEM images revealed smooth surfaces and formation of spherical particles.

From the above mentioned systems only S1 is chosen for further analysis as showing

minimum particle size , acceptable polydispersity index and high drug loading

efficiency.

The saturation solubility testing and multimedia dissolution study of the selected

system was carried out in all media’s mentioned above.



Table 5F.8 Saturation solubility of nanoparticles prepared by ion-gelation

technique

Study parameter 0.1N HCl Phosphate

buffer pH 6.8

Water

Solubility

(mg/ml)

0.00654 0.018 0.025

Figure 5F.10 Saturation solubility of nanoparticles prepared by ion gelation

technique

During saturation solubility testing at the end of 48 h , compared to other systems ,

not much increase in solubility was found but increase in solubility as compared to

pure drug was found in all solvents. Solubility was found to increase more in case of

case of phosphate buffer pH 6.8 and water as compared to 0.1 N HCl .



Table 5F.9 Multimedia dissolution of nanoparticles prepared by ion-gelation

technique

Time

(mins)

0.1N

HCl %RSD Water %RSD

Phosphate

buffer pH

6.8

%RSD OGD

media %RSD

5 1.53 3.351 2.275 0. 6 2.586 1.7537 2.948 11.054

10 5.09 2.786 2.559 0. 4 2.629 1.3946 8.0733 13.490

15 6.83 2.4842 3.524 0. 11 2.701 1.0364 33.403 6.357

20 8.21 1.689 9.499 0. 14 2.774 2.360 55.140 9.153

30 8.37 0.752 11.77 0. 57 2.938 1.685 69.368 6.467

45 8.18 6.618 13.94 0. 20 4.400 24.75 78.303 3.772

60 8.49 3.077 14.11 0.191 5.187 21.83 89.78 2.964

120 16.4 6.723 - - 6.032 11.659 - -

240 24.5 4.124 - - 7.089 6.6099 - -

480 36.8 5.329 - - 17.871 21.794 - -

In-vitro multimedia dissolution of nanoparticles prepared by ion-

gelation technique

0

10

20

30

40

50

60

70

80

90

100

0 5 10 15 20 30 45 60 120 240 480

Time point (mins)

% C

um

ula

tive r

ele

ase

0.1N HCl Water Phosphate buffer 6.8 Phosphate buffer 6.5+0.35% Tween 20

Figure 5F.11 Multimedia dissolution of nanoparticles prepared by ion gelation

technique



During dissolution testing, it was found that dissolution was increased as compared to

pure drug. Same as that of the solubility, no marked increase was found in the

dissolution of drug. In 0.1N HCl release was found to be around 8.5% at the end of 60

minutes. Even though solubility of chitosan was found to be high in acidic pH but

solubility of drug in acidic pH is very low that might be the reason for low percentage

cumulative release. No lag time was fond in release must be because of continuous

swelling of chitosan in acidic medium. In case of water and Phosphate buffer pH 6.8

release was found to be around 14% and 5% respectively at the end of 60 minutes

reason for low release in Phosphate buffer pH 6.8 must be because of slow swelling of

chitosan in buffer media due to low solubility. To check maximum dissolution, study

was continued up to 8 hrs. At the end of 8th

hour release was found to be 17%.Lag

time of about 30 minutes was observed in buffer because of low solubility of chitosan.

In OGD media the release was found to be around 90% at the end of one hour with

not much lag time. To simulate gastrointestinal conditions and checking the effect on

release of drug in Phosphate buffer pH 6.8, dissolution of drug in acidic media after

one hour was continued further in phosphate buffer pH 6.8 for 8 hrs and aliquots were

withdrawn at the end of 2 hrs, 4 hrs and 8 hrs and content was analyzed. Maximum

release at the end of 8 hrs was found to be 36 %.



5F.3 Preparation of Nanoparticles Using Nanoencapsulation Technique

5F.3.1 Preparation of nanoparticles

Table 5F.10 Selection of nanoparticles using nanoencpsulation technique

Sr.

No. Drug:Polymer Organic phase: Aqueous phase

NE1 1:10 1:2

NE 2 1:10 1:3

NE 3 1:10 1:4

NE 4 1:20 1:2

NE 5 1:20 1:3

NE 6 1:20 1:4

NE 7 1:30 1:2

NE 8 1:30 1:3

NE 9 1:30 1:4

Amongst all prepared systems, only systems (In bold) which were having comparative

clear appearance were chosen for further studies.

5F.3.2 Evaluation of nanoparticles using nanoencapsulation technique

For measurement of entrapment efficiency, supernatant was suitably diluted with

methanol and analysed at 254 nm and amount of unentrapped drug was calculated.

Percentage drug entrapment was calculated by dissolving nanoparticles equivalent to

8 mg drug were dissolved in methanol. Drug loading efficiency was found to vary

between 18 to 23 % with system NE3 showing maximum drug loading efficiency of

23.18%.

Percentage yield was calculated by the formula described in experimental section

4F.3.

Table 5F.11 Evaluation of selected nanoparticle systems prepared by

nanoencapsulation technique

Formulat

ion code

Encapsulation

efficiency(%)

n=3

DLE(%)

, n=3

Drug

content

(%), n=3

Polydis

-persity

index

Zeta

Poten

-tial

Percentage

yield

(%W/W)

n=3

NE3 44.24 23.18 95.16 0.239 21.62 60

NE6 34.33 20.06 96.14 0.173 24.36 48

NE9 29.45 18.17 95.48 0.28 22.17 55



Particle size, polydispersity index and zeta potential of the given systems was

calculated using photon correlation spectroscopy and electrophoretic light scattering

using Delsa Nano instrument.Particle size was found to range from 209 nm-329 nm

of the selected systems It was found that lower the ratio of drug:polymer,lower the

size of particles. In the same way higher the drug present ,higher the drug loading

efficiency was seen.

Table 5F.12 Particle size distribution of selected nanoparticle systems prepared

by nanoencapsulation technique

System D(10%) D(50%) D(90%)

NE 3 37.3 139 551.4

NE 6 16 25.4 624

NE 9 63.9 288.6 1234

Figure 5F.12 Particle size distribution curve of NE 3

Figure 5F 13 Particle size distribution curve of NE 6



Figure 5F. 14 Particle size distribution curve of NE 9

From particle size distribution system NE3 was chosen for further analysis of

morphology saturation solubility and in vitro multimedia dissolution.

TEM images

(a)

(b)

Figure 5F. 15 TEM images of nanoparticles prepared by nanoencapsulation

technique (a = Clusture of particles, b=Individual particle)



TEM images revealed smooth surfaces and formation of spherical particles. Though

the particles are seen together in first image, they are well separated as shown in

second image.

The saturation solubility testing and multimedia dissolution study of the selected

system was carried out in all media’s mentioned above.

5F.3.3 Saturation solubility Testing

Table 5F.13 Saturation solubility of nanoparticles prepared by


Study parameter 0.1N HCl Phosphate

buffer pH 6.8 water OGD media

Solubility(mg/ml) 0.00416 0.00927 0.0102 -

saturation solubility of nanoparticles by

nanoencapsulation method

0

2

4

6

8

10

12

Solvents

solu

bilit

y (m

cg/m

l)

0.1N HCl Phosphate buffer 6.8 water

Figure 5F.16 Saturation solubility of nanoparticles prepared by


During saturation solubility testing at the end of 48 hours increase in solubility was

found as compared to pure drug in all solvents.



5F.3.4 Multimedia dissolution testing

Table 5F.14 Multimedia dissolution of nanoparticles prepared by


Time

(min)

0.1N

HCl %RSD Water %RSD

Phosphate

buffer pH

6.8

%RSD OGD

media %RSD

5 1.039 1.25 2.1564 0.0548 2.3569 3.2168 14.359 0.6543

10 1.985 2.18 3.4789 0.0365 3.6598 2.1567 30.265 0.9875

15 2.326 2.48 8.3548 0.9823 6.3524 2.6539 46.356 0.8563

20 2.925 1.65 13.356 0.5964 11.3562 1.5648 58.479 0.9665

30 3.176 2.79 18.598 0.6421 17.3654 1.5469 75.784 1.3256

45 3.825 1.13 20.354 1.1654 22.3659 1.4781 88.356 1.4872

60 4.219 2.97 25.369 1.3546 26.3267 1.2654 97.246 1.6324

120 25.85 2.36 - - 35.14 1.5896 - -

240 37.59 2.17 - - 41.59 2.3467 - -

480 43.82 1.58 - - 48.35 1.5437 - -

multimedia dissolution of nanoparticles prepared by


0

20

40

60

80

100

120

0 5 10 15 20 30 45 60 120 240 480

Time (minutes)

% C

umul

ativ

e re

leas

e

0.1N HCl

Water

Phosphate buffer 6.8

Phosphate buffer 6.5+0.35% Tween 20

Figure 5F.17 In vitro multimedia dissolution of nanoparticles prepared by




In vitro multimedia dissolution of prepared nanoparticles was carried out. It was seen

that in acidic pH, the release up to one hour was only 4% because of low solubility of

drug as well as polymer in that pH. In water and phosphate buffer pH 6.8, initial slow

release followed by a burst release at the end of 10 minutes was seen might be due to

solubilisation of polymer and release of drug. A total drug release of around 25% and

26% was seen in water and phosphate buffer pH 6.8 respectively. When dissolution

was continued in phosphate buffer pH 6.8, maximum release of 48% was seen. Not

much difference in release of drug was seen when dissolution in acidic media was

continued for three hours in phosphate buffer pH 6.8.



5G Optimization of Formulation

5G.1 Complexation with cyclodextrin using lyophilization technique

5G.1.1 Optimization of formula

Optimized formula

Table 5G.1 Optimized formula of F16

Ingredient Amount %

Drug complex(lyophilized) 44.601

Avicel (102) 50.40

Disintegrant(SSG) 5

5G.1.2 Evaluation

5G.1.2.1 Drug content and Weight variation

Table 5G.2 Drug content and weight variation of optimized F16 formulation

Formulation Drug content(%)±%RSD

n=3

Weight variation±%RSD,

n=20

F16 100.1433±1.065

99.5225± 0.891416

5G.1.2.2 Multimedia dissolution testing

Table 5G.3 Multimedia dissolution of optimized F16 formulation

Time Points 0.1NHCl Phosphate

buffer6.8 Water OGD media

0 0 0 0 0

5 24.18437 24.37989 89.15037 90.66809

10 27.9755 32.77144 91.59677 92.5003

15 35.61409 39.85314 93.00127 93.70947

20 38.78095 48.55875 93.97953 95.06

30 41.26329 56.17042 94.87149 96.21551

45 42.11395 64.69941 95.36336 98.41429

60 42.61535 69.20625 94.96644 99.99302



In vitro multimedia dissolution of optimized F16 formulation

0

20

40

60

80

100

120

0 5 10 15 20 30 45 60

time points (minutes)

% C

um

ula

tive r

ele

ase

0.1NHCl Phosphate buffer6.8 Water OGD medium

Figure 5G.1 Multimedia dissolution of optimized F16 formulation

5G.2 SMEDDS

5G.2.1 Optimization of formula

Table 5G.4 Optimized formula of SMEDDS

Ingredients Quantity(mg)

Drug 8

S/cos 800

Oil 88.8

Prepared formulations were filled in hard gelatin capsule No. 3.

5G.2.2 Evaluation

5G.2.2.1 Drug content and weight variation

Table 5G.5 Drug content and weight variation of optimized SMEDDS

formulation

Formulation Drug

content(%)±%RSD n=3

Weight

variation±%RSD

SMEDDS 1

100.03±1.23

896.75±0.08



5G.2.2.2 Multimedia dissolution testing

Table 5G.6 Multimedia dissolution of optimized SMEDDS formulation

Time Points 0.1NHCl Phosphate

buffer pH 6.8 Water OGD media

0 0 0 0 0

5 72.113 88.216 90.215 91.82

10 74.988 90.96 91.100 94.79

15 86.13 92.89 94.5176 96.64

20 87.0263 93. 97 96.3352 98.147

30 87.227 94.18 96.7982 99.16

45 87.505 94.99 97.2146 99.48

60 88. 012 96.58 97.5413 99.79

In vitro multimedia dissolution of optimized SMEDDS

formulation

0

20

40

60

80

100

120

0 5 10 15 20 30 45 60

Time points(minutes)

% c

um

ula

tive r

ele

ase

0.1NHCl Phosphate buffer6.8 Water OGD medium

Figure 5G.2 Multimedia dissolution of optimized SMEDDS formulation



Selected formulae were optimized for proper filling.To increase the bulk and to

enhance the property of filling,diluent was added in complexes of drug and

HPBCD(1:2).To avoid any plug formation disintegrant, sodium starch glycolate (5%)

was added in the mixture .

Drug content was found to be within the range.

No effect of disintegrant on release profile and stability of drug was seen.

No change in formula in case of SMEDDS was done.SMEDDS concentrate was

added with a pipette in the empty hard gelatin capsule.

.

5H In -Vivo studies

5H.1 Development of analytical method for analysis of Candesartan in plasma

Summary of validation

Analyte : CANDESARTAN

Analytical Technique : LC/MS/MS

Equipment used : Perkin Elmer Series 200 pump fitted

with Perkin Elmer

Series 200 autosampler

Software used : Analyst Software version 1.3

Scan Type : MRM

Column type : Restek C18 (150mm x 2.1 mm, i.d.) 5µ

Mobile Phase : 0.01 % Formic acid: Acetonitrile

(10:90)

Flow Rate : 0.2 mL / minute.

Biological Matrix : Rat Plasma

Anticoagulant used : K2EDTA

Sample Extraction : Solid Liquid Extraction

Linearity Range : 8.00ng/mL – 250.00ng/mL

Equation Type : Linear, y = ax + b

Weighting Factor : 1/x2

Validated LQC : 10.00 ng/mL for CANDESARTAN

Validated MQC : 100.00ng/mL for CANDESARTAN

Validated HQC : 200.00ng/mL for CANDESARTAN

Freeze Thaw Stability : 3 Cycles at – 20 ± 5 oC

(CANDESARTAN)

Long Term Stock : For 14 days at 2 - 8oC

Solution Stability

Long Term Stability In Matrix : For 30 days at 20±5oC

(CANDESARTAN)



(a) (b)

Figure 5H.1 Representative LC/MS/MS spectra of drug(a) and internal

standard(b)

Validation parameters for LCMS method

5H.1.1 Specificity

The specificity of the intended method was established by screening the

standard blank (without spiking with CANDESARTAN of different

batches/lots of commercially available rat blank plasma). Seven different

batches of plasma (K2 EDTA) including one haemolysed plasma were

screened and found free from endogenous significant interferences. Rat

plasma batches, free of significant interferences at the retention time of

CANDESARTAN were used to prepare calibration curve standards and

quality control samples.



Table 5H.1 Specificity for Candesartan by LCMS

Date

Curve BLANK 8.00 ng/ml 250.00 ng/ml

Code Area Ratio

Conc Area Ratio Conc Area Ratio Conc

02/08/10 SPC

0.00 0.00 0.10 70.91 3.10 2017.14

0.00 0.00 0.11 72.92 3.23 2102.72

0.00 0.00 0.12 82.00 3.43 2231.61

0.00 0.00 0.10 65.92 3.31 2157.25

0.00 0.00 0.12 81.68 3.16 2056.72

0.00 0.00 0.10 66.66 3.38 2199.30

0.00 0.00 0.10 66.76 3.49 2273.35

Mean 0.0 0.0 0.11 72.408 3.3 2148.30

S.D. 0.00 0.00 0.01 6.9205 0.14 93.92

% C.V. 0.00 0.00 10.06 9.56 4.38 4.37

Criteria % C.V. ≤ 15

5H.1.2 Plasma Linearity (Calibrant samples)

The linearity of the method was determined by using a 1/x2

weighted

least square regression analysis of standard plots associated with and

seven-point standard curve. All the three calibration curves each for

CANDESARTAN was analyzed during the course of validation were

linear for the standards ranging from 8.00ng/mL to 250.00 ng/mL

respectively. A straight-line fit is made through the data points by least

square regression analysis and a constant proportionality is observed.

The mean correlation Coefficient (r) observed was 0.9932 for

CANDESARTAN during the course of validation. The mean accuracy

and precision observed for the CC standards for CANDESARTAN

ranged from 92.69 % to 105.51 %, which are within the acceptance

limits of 85% to 115% for all CC standards except of LLOQ standard,

and for LLOQ level it is 104.95 % for CANDESARTAN. This is

within the acceptance limits of 80% to 120%.



Table 5H.2 Calibrant samples for candesartan

Curve 8.00 15.00 25.00 50.00 75.00 125.00 250.00

Date Code ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml

02/08/10 DAY- 1 MVLN1 8.99 15.86 23.74 50.24 65.95 113.32 269.91

03/08/10 DAY- 2 MVLN2 7.91 15.48 21.79 55.93 77.38 119.76 249.75

04/08/10 DAY- 3 MVLN3 9.02 14.60 26.16 49.34 69.00 106.75 273.13

05/08/10 DAY- 4 MVLN4 7.67 13.24 24.75 55.51 83.17 123.63 233.41

Mean 8.396 14.793 24.111 52.753 73.875 115.866 256.551

S.D. 0.7046 1.1614 1.8381 3.4453 7.8590 7.4158 18.5751

% C.V. 8.39 7.85 7.62 6.53 10.64 6.40 7.24

% Nominal 104.95 98.62 96.45 105.51 98.50 92.69 102.62

Criteria For % Nominal For CC standards other than LLOQ 85% - 115 %

Table 5H.3 Curve parameter summary for Candesartan (Equation Y=ax+b)

Curve Slope y-Intercept Coefficient of

code (a) (b) Determination (r2)

02/08/10 MVLN1 0.0015 -0.0056 0.9914

03/08/10 MVLN2 0.0014 0.0007 0.9982

04/08/10 MVLN3 0.0014 -0.0006 0.9864

05/08/10 MVLN4 0.0013 -0.0007 0.9969

Mean 0.0014 0.9932

S.D. 0.0001 Not applicable 0.0054

% C.V. 6.5985 0.5466

Criteria For ( r ) ≥ 0.9800

.



5H.1.3 Precision and Accuracy

The precision of the CANDESARTAN assay was measured by the percent coefficient

of variation and % Nominal over the concentration range of LQC, MQC and HQC

samples during the course of validation.

5H.1.3.1 Between Batch Precision

The between batch accuracy for all the low, middle and high quality control samples

of CANDESARTAN ranged from 90.08 % to 103.14 %, which are within the

acceptance limit of 85% to 115%. And between batch precision for all the low, middle

and high quality control samples of CANDESARTAN ranged from 2.90 % to 7.28%

respectively, which are within the acceptance limit of 15%.

Table 5H.4 Between run precision and accuracy for candesartan

Curve Code

LQC MQC HQC

10.00 100.00 200.00

ng/ml ng/ml ng/ml

MVLN2 10.34 94.83 170.49

MVLN2 9.36 101.59 177.80

MVLN2 9.44 94.09 176.81

MVLN2 8.68 104.15 175.45

MVLN2 9.49 103.08 172.55

MVLN3 10.71 107.88 180.16

MVLN3 10.45 113.51 183.77

MVLN3 10.31 98.54 182.88

MVLN3 10.99 107.19 185.73

MVLN4 10.81 100.76 186.11

MVLN4 10.50 108.44 180.20

MVLN4 11.11 105.99 186.26

MVLN4 10.75 100.73 183.86

Mean 10.226 103.137 180.160

S.D. 0.7446 5.5499 5.2297

C.V.(%) 7.28 5.38 2.90

% Nominal 102.26 103.14 90.08

Criteria For 85% - 115 %

% Nominal

Criteria For % CV ≤ 15 %



5H.1.3.2 Within Batch Precision

The within batch accuracy for all the low, middle and high quality control samples of

CANDESARTAN ranged from 87.31 % to 99.55 %, which are within the acceptance

limit of 85% to 115%. And within batch precision for all the low, middle and high

quality control samples of CANDESARTAN ranged from 1.74 % to 6.23 %, which

are within the acceptance limit of 15%.

Table 5H.5 Within run precision and accuracy for candesartan

LQC MQC HQC

Curve Code 10.00 100.00 200.00

ng/ml ng/ml ng/ml

MVLN2 10.34 94.83 170.49

MVLN2 9.36 101.59 177.80

MVLN2 9.44 94.09 176.81

MVLN2 8.68 104.15 175.45

MVLN2 9.49 103.08 172.55

Mean 9.462 99.549 174.621

S.D. 0.590 4.742 3.041

C.V.(%) 6.23 4.76 1.74

% Nominal 94.62 99.55 87.31

Criteria For 85% - 115 %

% Nominal

Criteria For % CV ≤ 15 %

5H.1.4 Percentage Extraction Yield

5H.1.4.1Recovery CANDESARTAN

The percentage mean recoveries were determined by measuring the response of the

extracted plasma quality control samples at LQC and HQC against aqueous extracted

quality control samples at LQC and HQC. The percentages mean recovery found in

LQC and HQC for CANDESARTAN was 107.60% and 101.23% respectively.



Table 5H.6 Percentage extraction yield

Curve L.Q.C. (10.00 ng/ml) H.Q.C. (200.00 ng/ml)

Code Extracted Aq.

extracted Extracted Aq. extracted

03/08/10

PEY01

9.37 8.41 172.24 173.88

9.13 8.16 175.71 171.80

8.99 8.97 174.85 170.79

Mean 9.16 8.52 174.27 172.16

S.D. 0.19 0.41 1.81 1.57

C.O.V. 2.11 4.84 1.04 0.91

% extraction 107.60 101.23

5H.1.5 Stock Solution Stability

5H.1.5.1Long Term Stock Solution Stability

Long term stock solution stability for CANDESARTAN at concentration 100.00

ng/mL and was determined by using aqueous standard after the storage for 15 days

and 30 days at 2 - 8oC. Stability was assessed by comparing against the initially

injected CANDESARTAN standard stock solution of concentration 100.00 ng/ml.

The % difference for 15 days was found as 0.71 % and for 30 days were found as 1.58

% and for CANDESARTAN standard stock solution, which is within the acceptance

limits of 2 %.



5H.1.6 Stability of Analytes in Plasma

Stability studies in plasma were conducted in the various conditions using three

replicates of LQC and HQC samples as described below:

Freeze thaw stability of the spiked quality control samples was determined during

three freeze thaw cycles stored at below -20 ± 5°C. Stability was assessed by

comparing against the freshly spiked quality control samples. The percentage

difference for LQC and HQC of CANDESARTAN was 9.71%, 9.33% and 6.75% to

3.76%, 5.08% and 8.05% respectively, which are within the acceptance limits of 10%.

Table 5H.7 Long term stock solution stability for Candesartan

Standard 100.00 ng/ml

Date Curve Initial Stability Stability

Code Fresh 15 Days 30 Days

02/08/10 SST01 1.73 1.72 1.79

17/8/2010 SST02 1.72 1.77 1.79

07/09/10 SST03 1.78 1.78 1.74

Mean 1.74 1.76 1.77

S.D. 0.033 0.029 0.029

% C.V. 1.89 1.63 1.66

% Difference 0.71 1.58

Criteria For % CV

5 %

Criteria For % Difference ≤ 5%



5H.1.6.1 Freeze Thaw Stability

Table 5H.8 Freeze thaw stability for candesartan

L.Q.C. 10.00 ng/ml H.Q.C. 200.00 ng/ml

Date Curve Initial Stability Stability Stability Initial Stability Stability Stability

Code 0 hrs 24 hrs 36 hrs 48 hrs 0 hrs 24 hrs 36 hrs 48 hrs

03/08/10 MVLN2 9.37 9.76 10.07 10.09 172.24 182.88 186.97 188.14

04/08/10 MVLN3 9.13 10.60 10.25 10.37 175.71 174.02 179.41 191.32

05/08/10 MVLN4 8.99 9.79 9.74 8.88 174.85 185.55 182.99 185.44

Mean 9.163 10.052 10.018 9.781 174.268 180.819 183.124 188.298

S.D. 0.1935 0.4778 0.2601 0.7921 1.8076 6.0346 3.7792 2.9448

% C.V. 2.11 4.75 2.60 8.10 1.04 3.34 2.06 1.56

% Nominal 91.63 100.52 100.18 97.81 87.13 90.41 91.56 94.15

% Difference 9.71 9.33 6.75 3.76 5.08 8.05

Criteria For % CV

≤ 15 %

Criteria For % Nominal 85% - 115%




5H.1.6.2Long Term Stability in Matrix

Long term stability of the spiked quality control samples in matrix was determined for

15 days and 30 days for CANDESARTAN which was stored at -20 ± 50C

temperature. Stability was assessed by comparing against the freshly thawed quality

control samples. The percentage difference for LQC and HQC of CANDESARTAN

for 15 days and 30days was 7.33% to 8.12% and 0.07% to 0.72 respectively which are

within the acceptance limits of 10%.

Table5H.9 Long term stability in matrix of candesartan

L.Q.C 10.00 ng/ml H.Q.C 200.00 ng/ml

Date Curve Initial

Middle Final Initial

Middle Final

Code 15 day 30 day 15 day 30 day

02/08/10 MVLN1 9.37 9.74 10.10 172.24 175.42 174.71

17/08/10 LIN01 9.13 9.91 9.74 175.71 174.78 176.99

01/09/10 LIN02 8.99 9.85 9.88 174.85 172.95 174.88

Mean 9.163 9.834 9.906 174.268 174.382 175.527

S.D. 0.1935 0.0853 0.1826 1.8076 1.2827 1.2723

% C.V. 2.11 0.87 1.84 1.04 0.74 0.72

% Nominal 91.63 98.34 99.06 87.13 87.19 87.76

% Difference 7.33 8.12 0.07 0.72

Criteria For % CV

≤ 15 %

Criteria For % Nominal 85`% - 115%


5H.1.7 Ruggedness

Ruggedness was performed by using three Quality control batches. One batch was

analyzed by using different column, second batch was analyzed by different analyst,

third batch was analyzed by using different analysis. During all the cases the %

difference for CANDESARTAN for LQC were 1.54, 1.05 and 1.06 respectively, and

for HQC were 4.10, 5.05and 3.74 respectively



Table 5H.10 Variation due to change in column

Date

Curve Injection LQC10.00 ng/ml

HQC200.00 ng/ml

Code Number COL 01 COL 02

COL 01 COL 02

04/08/10 RC

1 9.37 9.11 172.24 175.76

2 9.13 9.31 175.71 185.38

3 8.99 9.50 174.85 183.10

Mean 9.16 9.30 174.27 181.42

S.D 0.19 0.20 1.81 5.03

%C.V 2.11 2.11 1.04 2.77

% Diff -- 1.54 -- 4.10

Criteria For %C.V.

≤ 15 %

Criteria For %Difference

≤ 10%

Table 5H.11 Variation due to change in analyst

Date


HQC200.00 ng/ml

Code Number ANA 01 ANA 02

ANA 01 ANA 02

04/08/10 RA

1 9.37 9.62 172.24 182.59

2 9.13 9.10 175.71 179.40

3 8.99 9.05 174.85 187.22

Mean 9.16 9.26 174.27 183.07

S.D 0.19 0.32 1.81 3.93

%C.V 2.11 3.43 1.04 2.15

% Diff -- 1.05 -- 5.05

Criteria For %C.V.

≤ 15 %


≤ 10%



Table 5H.12 Variation in days for analysis (Interday variation)

Date


HQC200.00 ng/ml

Code Number DAY 01 DAY 02 DAY 01 DAY 02

04/08/10 RD

1 9.37 9.49 172.24 180.78

2 9.13 8.80 175.71 177.56

3 8.99 9.49 174.85 184.03

Mean 9.16 9.26 174.27 180.79

S.D 0.19 0.40 1.81 3.24

%C.V 2.11 4.28 1.04 1.79

% Diff -- 1.06 -- 3.74

Criteria For %C.V. ≤ 15 %


≤ 10%

Discussion

Chromatographic Method

Based on the experiments done during the course of validation, it can be concluded

that the intended method is validated for the estimation of CANDESARTAN in

Rabbit plasma over the concentration range of 8.00to 250.00 ng/mL respectively. The

precision and accuracy are within the acceptance limits. Consistent recoveries are

observed for LQC and HQC. The method is specific enough in the presence of

different matrices collected from different sources.

This method can be used for simultaneous quantification of CANDESARTAN in

Rabbit plasma for Pharmacokinetics studies.

Stability of Analytes

Based on the stability experiments carried out during the course of validation, it can

be concluded that stock solution is stable up to 12 hours at 2 - 8 oC for short term

stock solution stability and up to 14 days at 2 - 8 oC for long term stock solution

stability. Analytes are stable up to 24 hours at 4 oC for Auto sampler stability.

The intended analytes are stable for 4 hours during short-term ambient temperature

stability (Bench top) and three freeze thaw cycles at -20 ± 5oC. The

CANDESARTAN in matrix was stable at -20 ± 5oC for 30 days.



Ruggedness of Method

This method was proved to be rugged for different Column, different Analyst and

different Days.

5H.2 In Vivo bioavailability of selected formulations in rabbits

Table 5H.13 Descriptive statistics of the pharmacokinetic parameters of

candesartan (marketed formulation)


candesartan (F16 formulation)




candesartan (SMEDDS formulation)

Figure 5H.2 Plasma concentration v/s time curve for marketed formulation

Figure 5H.3 Plasma concentration v/s time curve for FD 16 formulation



Figure 5H.4 Plasma concentration v/s time curve for SMEDDS formulation

Figure 5H.5 Comparative data for Cmax of candesartan

Figure 5H.6 Comparative data for Tmax of candesartan



Figure 5H.7 Comparative data for AUC (0-t) of candesartan

The samples of core F16 and SMEDDS were given for in vivo study in rabbits, as no

lethal effects were reported with high doses of Candesartan and as bioavailability of

drug was very low, a dose equivalent to 8 mg was given orally to each rabbit

weighing 1.8-2.2 kg. All the samples were analysed by a validated LCMS method for

analysis of candesartan cilexetil .A considerable increase in Cmax and AUC was seen

in both cases. As shown in section 6H of result and discussion , Cmax in case of

marketed formulation was found to be 781.99 ng/ml while that of SMEDDS

formulation was found to be 1905.17 ng/ml and for F16 was 3215.17 ng/ml.The

AUC 0-∞ was 8506 ng/ml*hr,12916 ng/ml*hr,and 12725 ng/ml*hr for

marketed,SMEDDS and F16 formulations.AUC 0-t was found to be 8165

ng/ml*hr,12892 ng/ml*hr and 12681 ng/ml*hr for marketed,SMEDDS and F16

formulations. Tmax in case of marketed was found to achieve after 12 hours,for

SMEDDS formulation it was 2 hours and for F16 it was 1 hour. From all above

results it was concluded that there is 3-5 fold increase in Cmax and around 2 fold

increase in AUC of experimental formulation over marketed. Eventhough Cmax of

F16 formulation was high than SMEDDS but AUC in both cases was similar this

must be because of immediate solubilisation of F16 and slow emulsification of

SMEDDS, this difference might will be eliminated with in vivo study in humans

because of large volume of fluid available for emulsification.From this data it can be

concluded that the Freeze drying and SMEDDS are effective tool for enhancing

bioavailability of Candesartan cilexetil within safe region.



5I Stability studies

5I.1 Stability studies of lyophilized drug HPBCD complex

5I.1.1 Drug content

Analysis of drug content in real time studies was done up till 6 months with

sampling points after each 1 month, similarly for accelerated stability analysis

was done uptill 3 months with sampling points after each 1 month.

Table 5I.1 Drug content of stability batches of F16

Sr.No. Time point

Drug content

Real time

studies

Accelerated

stability studies

Initial 0 Month 101.6 100.9

1 1 Month 101.1 98.42

2 2 Month 100.4 97.31

3 3 Month 99.97 95.14

4 4 Month 99.8 -

5 5 Month 98.15 -

6 6 Month 97.79 -



5I.1.2 Dissolution studies of lyophilized drug HPBCD complex

Table 5I.2 Dissolution of stability batches of F16

Real

time

Time

points

5

Min

10

Min

15

Min

20

Min

30

Min

45

Min

60

Min

0 Month

91.42

92.64

93.54

94.85

95.43

97.14

100.2

1 Month 91.81 92.98 94.01 95.27 95.93 98.17 99.76

2 Month

90.62

92.63

93.76

94.54

95.71

95.43

99.21

3 Month

90.72

91.97

93.02

95.11

95.54

96.35

98.81

4 Month

91.07

92.29

92.73

94.57

95.16

95.84

97.72

5 Month

90.64

91.23

93.48

94.72

95.15

95.75

97.14

6 Month

90.53

91.12

92.07

92.38

93.38

94.09

95.28

Acce

lerat

ed

1Month

91.70

92.47

93.64

93.96

94.54

95.43

96.87

2Month

90.44

91.39

91.89

92.23

93. 02

93.61

95.86

3Month

90.31

90.76

91.67

92.05

92.1 3

93.28

94.22



Figure 5I.1 Dissolution of stability batches (real time) of F16

Figure 5I.2 Dissolution of stability batches (accelerated) of F16



5I.2 Stability studies of SMEDDS

5I.2.1 Drug content

Table 5I.3 Drug content of stability batches of SMEDDS

Sr.No. Time point

Drug content

Real time studies Accelerated

stability studies

1 0 Month 101.2 101.2

2 1 Month 100.82 99.54

3 2 Month 100.12 97.21

4 3 Month 99.6 95.05

5 4 Month 98.42 -

6 5 Month 98.06 -

7 6 Month 97.18 -



5I.2.2 Dissolution studies of SMEDDS

Table 5I.4 Dissolution of stability batches of SMEDDS

Real

time

Time

points 5 Min

10

Min

15

Min

20

Min

30

Min

45

Min

60

Min

0 Month 90.14 92.52 93.14 94.56 94.98 97.52 99.87

1Month 90.03 92.35 92.93 94.72 95.76 97.63 99.26

2Month 90.12 91.36 92.76 93.65 95.17 96.88 99.45

3Month 89.65 90.43 91.88 93.06 94.31 96.18 99.10

4Month 89.90 90.97 91.56 93.42 95.13 96.77 98.89

5Month 89.62 89.99 91.46 93.16 95.17 96.44 98.97

6Month 89.43 89.78 91.22 93.22 94.76 96.18 98.52

Acceler

ated

1Month 91.07 92.11 94.14 95.87 97.65 98.24 100.13

2Month 90.88 91.74 93.97 95.39 97.15 98.09 99.68

3Month 90.16 91.32 92.87 94.64 96.33 96.98 98.23

Dissolution of stability batches(realtime) of SMEDDS

0

20

40

60

80

100

0 5 Minutes 10 15 20 30 45 60

Time point(Mins)

% C

um

mm

ula

tive r

ele

ase

0 Month 1Month 2Month 3Month 4Month 5Month 6Month

Figure5I.3 Dissolution of stability batches (real time) of SMEDDS



Dissolution of stability batches(accelarated) of SMEDDS

0

20

40

60

80

100

0 5 Minutes 10 15 20 30 45 60

Time point(min)

% C

um

mu

lati

ve r

ele

ase

0 Month 1M(accelarated) 2M(accelarated) 3M(accelarated)

Figure 5I.4 Dissolution of stability batches (accelerated) of SMEDDS

No significant change in dissolution and drug content of SMEDDS formulations was

seen in real time as well as accelerated stability testing upto 6 months and 3 months

respectively.

For Freeze dried formulations reduction in amount of drug content and drug release

was seen in real time as well as accelerated stability testing. This might be due to

partial change of drug in amorphous form as seen in section 5C.

From the values of drug content and dissolution of both formulations it was concluded

that drug products are safe in accelerated and real time studies.

5f solubility enhancement by preparation of nanoparticles...

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