DRUG IDENTIFICATION TESTS Determination of melting …shodhganga.inflibnet.ac.in/bitstream/10603/2206/13/13_chapter 5.pdf · DRUG IDENTIFICATION TESTS Determination of melting point(s)

Preformulation studies

School of Pharmaceutical Sciences 42

DRUG IDENTIFICATION TESTS

Determination of melting point(s)

Differential scanning colorimetry (DSC) was performed to determine the

melting point of the dicyclomine and paracetamol. Accurately weighed samples (2

mg) were transferred to aluminum pans and sealed. All samples were run at a heating

rate of 20oC/min over a temperature range 40-430oC using Shimadzu DSC-60

Thermal Analyzer. The thermograms are represented in Figure 3 & 4.

100.00 200.00 300.00 400.00Temp [C]

-30.00

-20.00

-10.00

0.00

mWDSC

174.23C

323.97C

373.08C

388.44C

Thermal Analysis Result

100.00 200.00 300.00 400.00Temp [C]

-20.00

-10.00

0.00

mWDSC

175.97C

254.53C

282.62C

Thermal Analysis Result

Figure 3 DSC Thermogram of Dicyclomine

Figure 4 DSC Thermogram of Paracetamol



Determination of absorption maxima (λmax)

Dicyclomine

10 mg of dicyclomine was accurately weighed and transferred to 100 ml

volumetric flask. The drug was dissolved in 0.1 N hydrochloric acid and the volume

was made up to 100 ml to obtain a stock solution of 100 μg/ml. One ml of this stock

solution was added to 5 ml of methyl orange solution and was extracted with

chloroform (3x1.5 ml). Organic layers were separated and pooled. The volume of

organic layer was made up to 10 ml with 0.5 % sodium acetate solution (Sethi 2008).

This solution was scanned between 400 nm to 500 nm in a double beam UV/ Visible

spectrophotometer (Shimadzu 1700). The λmax of the dicyclomine is shown in

Table 8.

Paracetamol

10 mg of paracetamol was accurately weighed and transferred to 100 ml of

volumetric flask. The drug was dissolved in methanol and the volume was made up to

100 ml to obtain a stock solution of 100 µg/ml. One ml of this stock solution was

again diluted with methanol up to 10 ml to obtain a solution of 10 µg/ml

(Pharmacopoeia of India 1996). The resulting solution was scanned between 200 nm

to 400 nm in a double beam UV/ Visible spectrophotometer (Shimadzu 1700). The

λmax of the paracetamol is shown in Table 8.

Loss on Drying

Dicyclomine

Loss on drying was determined by accurately weighing 1 gm of the drug and

drying at 105οC for three hours. It lost 0.0069 gm (NMT 1.0 %) of its weight

(Pharmacopoeia of India 1996). The results are presented in Table 8.

Paracetamol

Loss on drying was determined by accurately weighing 1 gm of the drug and

drying at 105οC for three hours. It lost 0.0023 gm (NMT 0.5 %) of its weight

(Pharmacopoeia of India 1996). The results are presented in Table 8.



Table 8 Comparative values of respective parameters used to identify the

drug(s)

S.

No.

Drug(s) Melting point

(0C)

λmax

(nm)

Loss on Drying

(%)

1. Dicyclomine 174

(172-174)

420

(420)

0.69

(NMT 1%)

2. Paracetamol 176

(174-176)

249

(249)

0.23

(NMT 0.5%)

Infra Red Spectroscopy

The Infra red spectroscopy of the sample was carried out to ascertain identity

of the drugs. A pellet of approximately 1 mm diameter of each drug was prepared by

compressing 3-5 mg of the drug with 100-150 mg of potassium bromide in KBr press

(Model M-15, Techno Search Instruments). The pellet was mounted in IR

compartment and scanned between wave number 4000-1 – 600 cm-1 using a Shimadzu

Model 8400 FTIR. The FTIR spectra are represented in Figure 5 & 6 and their

interpretation is presented in Table 9.

Figure 5 FTIR Spectrum of Dicyclomine



Figure 6 FTIR Spectrum of Paracetamol

Table 9 Interpretation of FTIR spectra of drugs

S. No.

Drug Reported Peaks (cm-1)

Observed Peak (cm-1)

Inference

1250-1020 1134.07 C-N stretching

1300-1000 1193.85 C-O stretching

3000-2840 2929.67 C-H stretching

1. Dicyclomine

1725-1700 1718.45 C=O (ester) stretching

3400-3200 3326..98 O-H stretching

3500-3100 3413.77 N-H stretching

1655-1620 1654.81 C=O (amide) stretching

1570-1515 1560.30 Amide II band

1250 1259.43 C-N-H group

2. Paracetamol

850-750 837.05 Para-disubstituted aromatic ring



CALIBRATION CURVES

Calibration Curve of Dicyclomine

Preparation of Stock Solution

100 mg of dicyclomine was accurately weighed and transferred to 100 ml

volumetric flask. The drug was dissolved in 0.1 N hydrochloric acid to get a solution

of 1000 μg/ml (stock solution I). 10 ml of stock solution I was diluted to 100 ml with

0.1N HCl (Stock solution II). Further, 10 ml. of stock solution II was diluted up to 50

ml with methyl orange solution (1%w/v) and extracted with chloroform (3x15 ml).

Organic layers were separated and pooled. The volume of pooled organic layer was

made up to 100 ml with sodium acetate solution (Stoke solution III). This stock

solution III was used to prepare a series of standard dicyclomine solutions as

discussed below.

Procedure

From stock solution III aliquots of 1, 2, 3, 4, 5 6, 7 & 8 ml were transferred to

a series of 10 ml volumetric flasks. The volume was made up with 0.1 N HCl to give

10, 20, 30, 40, 50, 60, 70 & 80 μg/ml of dicyclomine. The absorbance of these

solutions was measured at 420 nm against blank. The same procedure was followed

for the preparation of standard curve of dicyclomine in phthalate buffer pH 4.5,

phosphate buffer pH 6.8, and phosphate buffer pH 7.4. The standard curve of

dicyclomine in phosphate buffer pH 6.8 with pectinex ultra-SPL was also prepared by

this method, where the drug was dissolved in mixture of 99 ml of buffer and 1 ml of

pectinex ultra-SPL for the preparation of stock solution III. The data are recorded in

Tables 10 & 11 and the curves are plotted Figure 7-Figure 11.



Table 10 Calibration curves data of Dicyclomine

Absorbance± S.D. Concentration

µg/ ml 0.1 N HCl Phthalate buffer

pH 4.5

Phosphate buffer

pH 6.8

Phosphate buffer

pH 7.4

Phosphate buffer

pH 6.8 with

Pectinex ultra SPL

10 0.135±0.006 0.139±0.049 0.134±0.015 0.129±0.010 0.089±0.016

20 0.252±0.005 0.254±0.006 0.25±0.022 0.245±0.021 0.205±0.007

30 0.358±0.003 0.361±0.015 0.361±0.001 0.365±0.002 0.325±0.025

40 0.482±0.007 0.485±0.005 0.47±0.006 0.493±0.001 0.453±0.014

50 0.622±0.012 0.610±0.015 0.601±0.014 0.612±0.018 0.572±0.007

60 0.748±0.024 0.724±0.010 0.710±0.002 0.735±0.001 0.695±0.002

70 0.868±0.005 0.825±0.004 0.83±0.004 0.845±0.003 0.805±0.025

80 0.982±0.002 0.955±0.012 0.941±0.012 0.985±0.004 0.945±0.013

* Mean±S.D. (n=3)



Figure 7 Calibration curve of dicyclomine in 0.1 N HCl

Figure 8 Calibration curve of dicyclomine in phthalate buffer pH 4.5

y = 0.0123x + 0.0031R2 = 0.9991

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100

Concentration (mcg/ml)

Abs

orba

nce

(nm

)

y = 0.0116x + 0.0201R2 = 0.9995

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100


Abs

orba

nce

(nm

)



Figure 9 Calibration curve of dicyclomine in phosphate buffer pH 6.8

Figure 10 Calibration curve of dicyclomine in phosphate buffer pH 7.4

y = 0.0116x + 0.016R2 = 0.9997

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100


Abs

orba

nce

(nm

)

y = 0.0122x + 0.0036R2 = 0.9997

0

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100


Abs

orba

nce

(nm

)



Figure 11 Calibration curve of dicyclomine in phosphate buffer pH 6.8 and

pectinex ultra SPL

y = 0.0122x - 0.0364R2 = 0.9997

0

0.15

0.3

0.45

0.6

0.75

0.9

1.05

0 20 40 60 80


Abs

orba

nce

(nm

)



Table 11 Characteristic of calibration curves of dicyclomine

Values Parameters

0.1 N HCl Phthalate buffer

pH 4.5

Phosphate buffer

pH 6.8

Phosphate buffer

pH 7.4

Phosphate buffer

pH 6.8 with

Pectinex ultra SPL

λmax (nm)

Beer’s law limit (mcg/ml)

Slope (b)

Intercept (a)

Regression equation

(y= a+bx)

Correlation coefficient

420

10-80

0.0123

0.0031

0.0123x-0.0031

0.9991

420

10-80

0.0116

0.0201

0.0116x + 0.0201

0.9995

420

10-80

0.0116

0.016

0.011x + 0.016

0.9997

420

10-80

0.0122

0.0036

0.0122x + 0.0036

0.9997

420

10-80

0.0122

0.0364

0.0122x - 0.0364

0.9997



Calibration Curve of Paracetamol

Preparation of Stock Solution

Accurately weighed 100 mg of the drug was transferred to 100 ml volumetric

flask. The drug was dissolved in 5 ml methanol. The volume was made up to the mark

with methanol (stock solution I) to make a solution of 1000 μg/ml. One ml of stock

solutions I (1000 μg) diluted to 50 ml with methanol to give a stock solution of

concentration 20 μg/ml (Stock solution II). Stock solution II was used to prepare a

series of standard drug solutions.

Procedure

From stock solution II aliquots of 1, 2, 3, 4, 5, 6, 7 & 8 ml were transferred to

a series of 10 ml volumetric flasks and the volume was made up to the mark with 0.1

N hydrochloric acid. The absorbance of standard solutions was measured at 249 nm.

Standard curves in phthalate buffer pH 4.5, phosphate buffer pH 6.8, and phosphate

buffer pH 7.4 were prepared by same method as described earlier. The standard curve

of paracetamol in phosphate buffer pH 6.8 containing pectinex ultra-SPL was also

prepared where the drug was dissolved in mixture of 99 ml of simulated intestinal

fluid of pH 6.8 and 1 ml of pectinex ultra-SPL for the preparation of stock solution II.

The calibration curves data is shown in Table 12 and 13 and the curves are plotted in

Figure 12-Figure 14.



Table 12 Calibration curves data of Paracetamol

Absorbance± S.D. Concentration

µg / ml 0.1 N HCl Phthalate buffer

pH 4.5

Phosphate buffer

pH 6.8

Phosphate buffer

pH 7.4

Phosphate buffer

pH 6.8 with

Pectinex ultra SPL

2 0.040±0.004 0.120±0.028 0.180±0.005 0.198±0.020 0.190±0.001

4 0.120±0.004 0.200±0.016 0.300±0.002 0.318±0.081 0.300±0.007

6 0.200±0.006 0.285±0.035 0.420±0.008 0.425±0.012 0.415±0.005

8 0.290±0.001 0.365±0.001 0.545±0.008 0.555±0.010 0.530±0.001

10 0.370±0.002 0.440±0.015 0.663±0.003 0.675±0.018 0.635±0.009

12 0.450±0.008 0.528±0.072 0.810±0.012 0.798±0.011 0.740±0.002

14 0.530±0.011 0.605±0.001 0.930±0.013 0.918±0.001 0.850±0.025

16 0.610±0.012 0.685±0.002 1.05±0.004 1.038±0.009 0.960±0.003

* Mean±S.D. (n=3)



Figure 12 Calibration curve of Paracetamol in 0.1N HCl

Figure 13 Calibration curve of Paracetamol in phthalate buffer pH 4.5

y = 0.0409x - 0.0418R2 = 0.9998

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 5 10 15 20


Abso

rban

ce (n

m)

y = 0.0404x + 0.0401R2 = 0.9998

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 5 10 15 20


Abs

orba

nce

(nm

)



Figure 14 Calibration curve of Paracetamol in phosphate buffer pH 6.8

Figure 15 Calibration curve of Paracetamol in phosphate buffer pH 7.4

y = 0.0627x + 0.0482R2 = 0.9995

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20


Abs

orba

nce

(nm

)

y = 0.0602x + 0.0735R2 = 0.9998

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20


Abs

orba

nce

(nm

)



Figure 16 Calibration curve of Paracetamol in phosphate buffer pH 6.8 with

pectinex ultra SPL

y = 0.0549x + 0.0836R2 = 0.9998

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20


Abs

orba

nce

(nm

)



Table 13 Characteristic of calibration curves of paracetamol

Values

Parameters

0.1 N HCl Phthalate buffer

pH 4.5

Phosphate buffer

pH 6.8

Phosphate buffer

pH 7.4

Phosphate buffer

pH 6.8 with

Pectinex ultra SPL

λmax (nm)

Beer’s law limit (mcg/ml)

Slope (b)

Intercept (a)

Regression equation

(y= a+bx)

Correlation coefficient

245

2-16

0.0409

0.0418

0.0409x-0.0418

0.9998

243

2-16

0.0404

0.0401

0.0404x + 0.0401

0.9998

246

2-16

0.0627

0.0482

0.0627x + 0.0482

0.9995

249

2-16

0.0602

0.0735

0.0602x + 0.0735

0.9998

249

2-16

0.0549

0.0836

0.0549x + 0.0836

0.9998



DETERMINATION OF SOLUBILITY

Dicyclomine

An excess of drug was dissolved in 10 ml buffer of different pH values (1.2,

4.5, 6.8 and 7.4) in conical flask and was continuously shaken for 24 hours at room

temperature with the help of conical flask shaker. After 24 hours sample was filtered

through Whatman filter paper no.1, diluted appropriately and the drug was estimated

using UV spectroscopy (Shimadzu 1700) (Bhatia and Seedher 2003). The value of

solubility studies are shown in Table 14.

Paracetamol

An excess of drug was dissolved in 10 ml buffer of different pH values (1.2,

4.5, 6.8 and 7.4) in conical flask and was shaken for 24 hours at room temperature

with the help of conical flask shaker. After 24 hours sample was filtered through

Whatman filter paper no.1, diluted appropriately and the drug was estimated using

UV spectroscopy (Shimadzu 1700) (Bhatia and Seedher 2003). The value of solubility

studies are shown in table 14.

Table 14 Solubility values of drugs

DRUG-EXCIPIENTS COMPATIBILITY STUDIES

FTIR spectra were recorded to assess the compatibility of the drugs and

excipients. Drug(s) and excipients in the ration of 1:1 were mixed thoroughly and

stored at 40 °C and 75% RH for and room temperature for 1 month (Mukherjeea et

al., 2005). The FTIR spectra are represented in Figure 17 – Figure 20.

Solvents

S. No.

Drugs

0.1 N HCl

Phthalate

buffer

pH 4.5

Phosphate

buffer

pH 6.8

Phosphate

buffer

pH 7.4

1 Dicyclomine 0.0163 gm/ml 0.0083 gm/ml 0.0069 gm/ml 0.0057 gm/ml

2 Paracetamol 0.0894 gm/ml 0.0061 gm/ml 0.0056 gm/ ml 0.0049 gm/ml



Figure 17 FTIR Spectra of drug(s), Eudragit RS100 and PVA alone and in combination kept at 40 oC and 75% RH for 1 month



Figure 18 FTIR Spectra of drug(s), Eudragit S100 and PVA alone and in

combination kept at 40 oC and 75% RH for 1 month



Figure 19 FTIR Spectra of drug(s), Eudragit RS100 and PVA alone and in

combination kept at room temperature for 1 month



Figure 20 FTIR Spectra of drug(s), Eudragit S100 and PVA alone and in

combination kept at room temperature for 1 month



RESULTS & DISCUSSION

Dicyclomine and paracetamol were identified using different methods viz.

melting point determination, determination of absorption maxima (λmax), loss on

drying, and FTIR spectroscopy.

The thermogram of differential scanning colorimetry showed sharp

endothermic peaks of dicyclomine and paracetamol at 174.23 °C and 175.97 °C,

respectively corresponding to the melting range of dicyclomine (172-174 °C) and

paracetamol (174-176 °C) in the crystalline form. Absorption maxima (λmax) of

dicyclomine and paracetamol were found to be at wavelength 420 nm and 249 nm

corresponding to the values reported in literature (dicyclomine - 420 nm and

paracetamol - 249 nm). The loss on drying for dicyclomine and paracetamol was

found to be 0.69% (limit NMT 1.0 %) and 0.23 % (limit NMT 0.5 %), respectively.

FTIR spectra of the dicyclomine, showed characteristic C-N, C-O, C-H, C=O

(ester) stretching bands at 1134.07 cm-1, 1193.85 cm-1, 2929.67 cm-1, 1718.45 cm-1,

respectively. FTIR spectra of paracetamol, showed characteristic O-H, N-H, C=O

(amide) stretching bands at 3326.98 cm-1, 3413.77 cm-1, 1654.81 cm-1, respectively.

Whereas, amide II band, C-N-H group and para-disubstituted aromatic rings at

1560.30 cm-1, 1259.43 cm-1 and 837.05 cm-1, respectively were also observed. The

observed FTIR specta of both the drugs were matched with reference spectra. The

study confirmed that the test samples were dicyclomine and paracetamol.

All the tests confirmed the identity and purity of both the drugs.

Calibration curves of both the drugs were prepared in 0.1N HCl, phthalate

buffer pH 4.5, phosphate buffer pH 6.8, phosphate buffer pH 7.4 and phosphate buffer

pH 6.8 with pectinex ultra SPL.

Calibration curve data of both the drugs were subjected to linear regression

analysis. Beer and Lambert’s law was found to be obeyed in the concentration range

of 10-80 mcg/ml and 2-16 mcg/ml for dicyclomine and paracetamol, respectively in

all the media. R-values were found to be 0. 9995, 0.9997, 0.9998, 0.9998 & 0.9998

for dicyclomine and 0. 9998, 0.9998, 0.9997, 0.9998 and 0.9998 for paracetamol in

0.1N HCl, phthalate buffer pH 4.5, phosphate buffer pH 6.8, phosphate buffer pH 7.4

and phosphate buffer pH 6.8 with pectinex ultra SPL, respectively which indicate

linearity.



The solubility of both the drugs was determined in different media. Both drugs

were found to be sparingly soluble in acidic medium and slightly soluble in basic

medium. The solubility of dicyclomine in 0.1N HCl, phthalate buffer pH 4.5,

phosphate buffer pH 6.8, phosphate buffer pH 7.4 was found to be 0.0163 gm/ml,

0.0083 gm/ml, 0.0069 gm/ml, and 0.0057 gm/ml, respectively. The solubility of

paracetamol in 0.1N HCl, phthalate buffer pH 4.5, phosphate buffer pH 6.8,

phosphate buffer pH 7.4 was found to be 0.0894 gm/ml, 0.0061 gm/ml, 0.0056

gm/ml, and 0.0049 gm/ml, respectively.

FTIR spectra were recorded to assess the compatibility of the drugs and

excipients. The compatibility of drugs with eudragit RS100, eudragit S100, and PVA

was assessed by FTIR spectroscopy of the samples kept at 40°C and 75% RH and at

room temperature for 1 month. FTIR spectra of drug (s), physical mixture of drug (s),

physical mixture of drug (s) & eudragit RS-100, physical mixture of drug (s) &

eudragit S-100, physical mixture of drug (s) & PVA, physical mixture of drug (s),

PVA & eudragit RS100, and physical mixture of drug (s), PVA & eudragit S100 were

recorded and examined. In FTIR spectra of paracetamol, characteristic N-H stretching

band at 3413.77 cm−1, O-H stretching band at 3326.98 cm−1, and carbonyl stretching

band at 1654.81 cm−1 were noted and in case of dicyclomine, characteristic C=O

stretching band was observed at 1718.45 cm-1 which are in agreement with the

reported values. Eudragit RS 100 showed an ester C=O stretching peak around

1726.17 cm−1 and eudragit S 100 showed carbonyl stretching at 1718.46 cm-1 and

bond characteristic to carboxylic group in the range 2437-3473 cm-1 as reported in the

literature. All characteristic peaks of drug(s) were observed in the FTIR spectra of

physical mixture of drug (s) & eudragit S-100, physical mixture of drug (s) & PVA,

physical mixture of drug (s), PVA & eudragit RS100, and physical mixture of drug

(s), PVA & eudragit S100. The results showed no chemical interaction and changes

took place in FTIR spectra of both the drugs and various excipients alone or in

combination exhibiting compatibility of the drugs with all excipients.

Preparation and Characterization of Microsponge Formulations


PREPARATION OF MICROSPONGES

Microsponges preparation using Eudragit RS-100

Eudragit RS-100 based paracetamol and dicyclomine loaded microsponges

were prepared by quasi-emulsion solvent diffusion method. The internal phase

consisted of eudragit RS-100 (200mg) and triethylcitrate (1% v/v, as plasticizer)

dissolved in 5 ml dichloromethane. The drug was added to this with gradual stirring

(500 rpm). The internal phase was then poured into 0.5 % w/v polyvinyl alcohol

(PVA, molecular weight 30,000-70,000) solution in water, the external phase. After 8

hour of stirring the microsponges were formed due to removal of dichloromethane

from the system. The microsponges were filtered and dried at 40°C for 12 hours (Orlu

et al., 2006).

The same method was used for the preparation of microsponges with eudragit

S-100 except the stirring rate which was kept at 1000 rpm. The compositions of

various microsponge formulations are given in Table 15 & 16.

Table 15 Composition of Eudragit RS-100 based microsponge formulations

Formulation code/amount

Components FDRS1 FDRS2 FDRS3 FDRS4 FPRS1 FPRS2 FPRS3 FPRS4

Dicyclomine (mg) 600 1200 1800 2400 - - - -

Paracetamol (mg) - - - - 600 1200 1800 2400

Eudragit RS-100 (mg) 200 200 200 200 200 200 200 200

Triethylcitrate (%v/v) 1 1 1 1 1 1 1 1

Dichloromethane (ml) 5 5 5 5 5 5 5 5

PVA (% w/v) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5



Table 16 Composition of Eudragit S-100 based microsponge formulations

Formulation code/amount

Components

FDS1 FDS2 FDS3 FDS4 FPS1 FPS2 FPS3 FPS4

Dicyclomine (mg) 600 1200 1800 2400 - - - -

Paracetamol (mg) - - - - 600 1200 1800 2400

Eudragit S-100 (mg) 200 200 200 200 200 200 200 200

Triethylcitrate (%v/v) 1 1 1 1 1 1 1 1

Dichloromethane (ml) 5 5 5 5 5 5 5 5

PVA (% w/v) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

OPTIMIZATION OF FORMULATION

Effect of drug to polymer ratio on the size of microsponges

The drug and polymer in the ratios 3:1, 6:1, 9:1, 12:1 were taken to prepare

different microsponge formulations. In each formulation, the amounts of polymer

(200 mg), dichloromethane (5 ml), PVA (0.5% w/v) were kept constant. The

microsponge formulations were prepared using mechanical stirrer (Remi RQ1217-D)

at a stirring rate of 500 rpm for eudragit RS-100 based microsponges and 1000 rpm

for eudragit S-100 based microsponges for 8 hours.

Effect of the volume of internal phase on the production of microsponges

Two different volumes 5 and 10 ml were taken to study the effect of volume

of internal phase solvent (dichloromethane) on the microsponge formulations FDRS1,

FPRS1, FPS1 and FDS1.

Effect of stirring speed on the size of microsponges

The effect of stirring speed on the average size of microsponges was studied

using different stirring speeds (300, 400, and 500 rpm for formulations FDRS1 &

FPRS1 and 500 & 1000 rpm for formulations FDS1 & FPS1). The effect of stirring

rate on the size of microsponges is presented in Table 17 and depicted in Figure 21-

Figure 24.



Table 17 Effect of stirring speed on the size of microsponge formulations

Size (µm) Stirring Speed

(rpm) FPRS1 FDRS1 FPS1 FDS1

300 77.03±9.46 71.73±7.24 - -

400 66.64±5.71 63.91±5.21 - -

500 62.34±6.89 60.25±5.67 79.13±8.21 74.37±6.88

1000 - - 54.70±7.89 52.54±5.24

Figure 21 Photomicrographs of dicyclomine loaded microsponges (FDRS1)

prepared at different stirring rates (a) 300 rpm; (b) 400 rpm; (c) 500

rpm

Figure 22 Photomicrographs of paracetamol loaded microsponges (FPRS1)

prepared at different stirring rates (a) 300 rpm; (b) 400 rpm; (c) 500

rpm

* Mean±S.D. (n=3)



Figure 23 Photomicrographs of dicyclomine loaded microsponges (FDS1)

prepared at different stirring rates (a) 500 rpm; (b) 1000 rpm

Figure 24 Photomicrographs of paracetamol loaded microsponges (FPS1)

prepared at different stirring rates (a) 500 rpm; (b) 1000 rpm

Effect of the amount of emulsifying agent on the production yield and size of

microsponge

Two different concentrations viz. 0.5 % and 1.0 % w/v were taken to study the

effect of amount of emulsifying agent (PVA) on the microsponge formulations

(FDRS1, FPRS1, FPS1 and FDS1). The effect of emulsifying agent on microsponge

formulations is presented in Table 18.



Table 18 The effect of emulsifying agent on microsponge formulations

Formulation

Code

PVA

(% w/v)

Yield (%)

Mean Diameter

(µm ± S.D.)

FPS1 0.5 70.56±0.23 54.70±7.89

FPS1 1.0 68.07±1.21 55.40±6.73

FPRS1 0.5 72.00±0.43 62.34±6.89

FPRS1 1.0 67.35±2.56 66.12±3.15

FDS1 0.5 73.06±0.21 52.54±5.24

FDS1 1.0 64.82±0.82 63.59±5.64

FDRS1 0.5 79.01±0.57 60.25±5.67

FDRS1 1.0 61.34±3.67 71.02±4.28

CHARACTERIZATION OF MICROSPONGES

Angle of repose

Angle of repose was determined using funnel method. 5 gm microsponges

were allowed to pass through a funnel that was raised vertically until a maximum

cone height, h, was obtained. Diameter of heap, D, was measured. The repose angle,θ,

was calculated by formula (Rao and Patil 2005).

The standard value and experimental value of angle of repose are shown in Table 19

and 20.

* Mean±S.D. (n=3)



Carr’s Index and Hausners ratio

The Carr’s Index and Hausners ratio were calculated using formula:

Tapped density was determined by placing 5 gm of the microsponges in a

graduated cylinder tapping it for 100 times.

Poured density was determined by placing 5 gm of microsponges into a

graduated cylinder and measuring the volume (Rao and Patil 2005). The standard

value and experimental value of Carr’s Index and Hausners ratio are shown in

Table 19 and 20.

Table 19 Standard values of Angle of repose, Carr’s index and Hausner’s

ratio

S.No. Angle of

Repose

Carr’s

Index

Hausner’s

Ratio

Type of Flow

(Inference)

1 < 20 5-15 __ Excellent

2 20-30 12-16 < 1.25 Good

3 30-40 18-21 __ Passable

4 __ 23-35 >1.25 Poor

5 __ 33-38 1.25-1.50 Very Poor

6 >40 >40 __ Extremely Poor



Table 20 Characterization of microsponges

Evaluation Parameters Formulation

Code Angle of

Repose (θ)

(0)

Poured

density

(gm/cm3)

Tapped

Density

(gm/cm3)

Carr’s

Index (%)

Hausners

Ratio

FPRS1 23.74±2.18 0.47±0.01 0.50±0.01 6.00±0.01 1.06±0.01

FPRS2 20.14±1.22 0.45±0.01 0.50±0.02 9.67±0.63 1.11±0.28

FPRS3 21.12±2.11 0.40±0.01 0.42±0.01 8.01±0.51 1.05±0.01

FPRS4 23.01±1.21 0.35±0.01 0.38±0.01 8.63±0.34 1.08±0.25

FPS1 21.18±3.01 0.50±0.01 0.57±0.01 10.42±0.01 1.14±0.54

FPS2 22.10±1.18 0.45±0.01 0.50±0.01 10.00±0.01 1.11±0.25

FPS3 21.08±1.12 0.41±0.01 0.43±0.01 5.34±0.94 1.04±0.06

FPS4 24.12±2.12 0.35±0.01 0.38±0.01 7.82±0.09 1.08±0.26

FDRS1 23.48±1.31 0.47±0.01 0.50±0.01 6.61±0.86 1.06±0.47

FDRS2 19.46±2.16 0.45±0.08 0.52±0.05 13.46±0.01 1.15±0.33

FDRS3 25.05±1.97 0.40±0.01 0.41±0.01 2.39±0.05 1.02±0.11

FDRS4 21.12±1.14 0.38±0.01 0.41±0.01 5.00±0.01 1.07±0.16

FDS1 21.41±2.10 0.40±0.01 0.42±0.04 4.68±0.05 1.05±0.06

FDS2 18.81±3.15 0.39±0.01 0.41±0.01 4.97±0.09 1.05±0.07

FDS3 14.14±1.12 0.35±0.01 0.38±0.01 7.89±0.01 1.08±0.04

FDS4 17.12±2.23 0.35±0.01 0.38±0.01 7.90±0.28 1.08±0.11

Determination of production yield

The production yield of the microsponges was determined by calculating the

initial weight of the raw materials and the final weight of the microsponges obtained.

All the experiments were performed in triplicate and the mean of the each value was

reported (Jelvehgari et al., 2006). The results of production yield of the microsponge

are shown in Table 21.

* Mean±S.D. (n=3)



Actual drug content and encapsulation efficiency

For paracetamol microsponges

The weighed amount of drug loaded microsponges (100 mg) was suspended in

100 ml phosphate buffer pH 6.8 for 12 h and subjected to intermittent stirring. The

sample was filtered using 0.45_m membrane filter and analyzed at 249 nm against

blank using UV spectrophotometer (UV 1700, Shimadzu, Japan). All analyses were

carried out in triplicate. The results of actual drug content and encapsulation efficacy


For dicyclomine microsponges

The weighed amount of drug loaded microsponges (100 mg) was suspended in

100 ml phosphate buffer pH 6.8 for 12 h (sample-1) and subjected to intermittent

stirring. 10 ml of sample-1 was diluted with 10 ml of 0.1N HCl (sample-2). 10 ml of

sample-2 was further diluted with 50 ml of methyl orange (1%w/v) and extracted with

chloroform (3x1.5 ml). The organic phase was separate and pooled and the volume of

sample was made up to 100 ml with methylated sodium acetate. The solution was

filtered using 0.45_m membrane filter. The absorbance was taken at 420 nm against

blank using UV spectrophotometer (UV 1700, Shimadzu, Japan). The drug content

and encapsulation efficiency were calculated using the following formula.

Actual drug content (%) =Mact/Mms × 100

Encapsulation efficiency (%) =Mact/Mthe × 100

Where Mact is the actual drug content in microsponges, Mms is the total amount of the

microsponges and Mthe is the amount of drug added to the microsponges. All analyses

were carried out in triplicate. The results of actual drug content and encapsulation

efficacy are shown in Table 21.

Particle size analysis

Particle size was determined by photomicroscope (RXLr-3T, Radical). Microsponges

were suspended in glycerol, and the particle size was determined using the software,

Biowizard. The results of particle size analysis are shown in Table 21.



Table 21 Characterization of various microsponge formulation (n=3)

Mean Particle size Formulation Drug:Polymer Ratio

Production yield

(% ± S.D.)

Theoretical drug content (%)

Actual drug content

(% ± S.D.)

Encapsulation efficiency (% ± S.D.) (µm ± S.D.)

FDRS1 3:1 79.01±0.57 75.0 62.05±0.06 82.73±0.45 60.25±5.67

FDRS2 6:1 70.65±0.28 85.7 70.12±0.07 81.91±0.43 53.62±7.11

FDRS3 9:1 76.60±0.56 90.0 75.32±0.08 83.68±0.23 49.34±6.45

FDRS4 12:1 70.48±0.78 92.3 80.69±0.34 87.42±0.56 43.66±6.20

FDS1 3:1 73.06±0.21 75.0 67.12±0.04 89.49±0.01 52.54±5.24

FDS2 6:1 72.30±0.03 85.7 72.39±0.78 84.45±0.34 48.78±4.89

FDS3 9:1 68.45±0.02 90.0 78.92±0.41 87.68±0.56 41.76±5.21

FDS4 12:1 77.30±0.07 92.3 83.42±0.34 90.37±0.90 34.43±4.99

FPRS1 3:1 72.00±0.43 75.0 73.56±0.09 98.08±0.89 62.34±6.89

FPRS2 6:1 74.12±0.34 85.7 84.32±0.04 98.37±0.56 54.67±5.39

FPRS3 9:1 76.23±0.36 90.0 88.32±0.45 98.13±0.09 48.23±7.24

FPRS4 12:1 75.02±0.60 92.3 90.81±0.34 98.38±0.67 41.45±5.34

FPS1 3:1 70.56±0.23 75.0 71.82±0.20 95.76±0.66 54.70±7.89

FPS2 6:1 71.12±0.56 85.7 82.14±0.02 95.83±0.56 46.30±7.67

FPS3 9:1 74.12±0.34 90.0 86.09±0.09 95.65±0.47 41.63±6.37

FPS4 12:1 76.76±0.45 92.3 88.67±0.06 96.06±0.86 37.64±5.54



Fourier transform infrared (FTIR) analysis

FTIR spectra were recorded to assess the chemical interaction or changes

during microsponge preparation. FTIR spectra of the drug (s), physical mixture of

drug (s) with different polymers, and different microsponge formulations were

recorded in potassium bromide disc using a Shimadzu Model 8400 FTIR

spectrometer. The FTIR spectra of different microsponge formulations are shown in

Figure 25- Figure 28.

Figure 25 FTIR Spectra of dicyclomine, physical mixture of drug & Eudragit

RS-100, and microsponge formulations FDRS1–FDRS4



Figure 26 FTIR Spectra of dicyclomine, physical mixture of drug & Eudragit S-

100, and microsponge formulations FDS1–FDS4



Figure 27 FTIR Spectra of paracetamol, physical mixture of drug & Eudragit

RS-100, and microsponge formulations FPRS1–FPRS4



Figure 28 FTIR Spectra of paracetamol, physical mixture of drug & Eudragit S-

100, and microsponge formulations FPS1–FPS4



Differential scanning calorimetric (DSC) analysis

DSC provides information about the physical properties of the drugs and

demonstrates a possible interaction between drug and other compounds in

microsponges. Thermal analysis using DSC was carried out on drug (s), physical

mixture of drug (s) with different polymers and different microsponge formulations

using Shimadzu DSC-60 Thermal Analyzer. 2 mg of samples were loaded into

aluminum pans and sealed. All samples were run at a heating rate of 20oC/min. over a

temperature range 40-430oC. The DSC thermograms of different microsponge

formulations are shown in Figure 29- Figure 32. .

Figure 29 DSC thermograms of dicyclomine, physical mixture of drug &

Eudragit RS-100, and microsponge formulations FDRS1–FDRS4



Figure 30 DSC thermograms of dicyclomine, physical mixture of drug &

Eudragit S-100, and microsponge formulations FDS1–FDS4



Figure 31 DSC thermograms of Paracetamol, physical mixture of drug &

Eudragit RS-100, and microsponge formulations FPRS1–FPRS4



Figure 32 DSC thermograms of Paracetamol, physical mixture of drug &

Eudragit S-100, and microsponge formulations FPS1–FPS4

Morphology

The morphology of the microsponges was studied using scanning electron

microscopy (SEM). All the samples were coated with gold–palladium alloy under

vacuum. Coated samples were then examined using LEO 430 SEM analyzer. The

SEM micrograph of different microsponge formulations are shown in Figure 33 -

Figure 36.



Figure 33 (a-h) SEM photograph of microsponge formulations (dicyclomine:

eudragit RS-100). The photograph coded ‘A’ represents whole

image; ‘B’ represents surface photograph



Figure 34 (a-h) SEM photograph of microsponge formulations (dicyclomine:

eudragit S-100). The photograph coded ‘A’ represents whole image;

‘B’ represents surface photographs.



Figure 35 (a-h) SEM photograph of microsponge formulations (paracetamol:

eudragit RS-100). The photograph coded ‘A’ represents whole

image; ‘B’ represents surface photographs.



Figure 36 (a-h) SEM photograph of microsponge formulations (paracetamol:

eudragit S-100). The photograph coded ‘A’ represents whole image;

‘B’ represents surface photographs.



RESULTS AND DISCUSSION

Quasi-emulsion solvent diffusion method was used for preparation of

microsponges because of its simplicity and reproducibility. Moreover, it has

advantage of avoiding solvent toxicity (Orlu et al., 2006). The drug and polymer in

the ratios 3:1, 6:1, 9:1, 12:1 were taken to prepare different microsponge

formulations. In each formulation, the amounts of polymer (200 mg),

dichloromethane (5 ml), PVA (0.5% w/v) were kept constant. The microsponge

formulations were prepared using mechanical stirrer (Remi RQ1217-D) at a stirring

rate of 500 rpm for eudragit RS-100 based microsponge and 1000 rpm for eudragit S-

100 based microsponge for 8 hours. The various microsponge formulations namely

FDRS1, FDRS2, FDRS3, FDRS4 & FPRS1, FPRS2, FPRS3, FPRS4 containing

drug:eudragit RS-100 in the ratios 3:1, 6:1, 9:1, 12:1, respectively and FDS1, FDS2,

FDS 3, FDS4 and FPS1, FPS2, FPS3, FPS4 containing eudragit drug:S-100 in the

ratios 3:1, 6:1, 9:1, 12:1, respectively were prepared.

The effect of various variables like drug to polymer ratio, stirring rate, volume

of internal phase, amount of emulsifying agent on the nature of microsponges was

studied.

Effect of drug-polymer ratio on the size of microsponges

The morphology of the microsponges was studied by scanning electron

microscopy (SEM). The microsponges were observed to be spherical and uniform

with no drug crystals on the surface. It was noted that drug-polymer ratio has

considerable effect on the morphology and size of microsponges. It was observed that

as the ratio of drug to polymer was increased, the particle size decreased. The mean

particle size of formulations FDRS1-FDRS4, FPRS1-FPRS4, FDS1-FDS4, and FPS1-

FPS4 in the ratios of 3:1, 6:1, 9:1 and 12:1 were found to be between 60-44µm, 62-41

µm, 53-34 µm and 55-38 µm, respectively. This could probably be due to the fact that

in high drug to polymer ratios, the amount of polymer available per microsponge was

comparatively less. Hence less polymer surrounded the drug resulting in smaller

microsponges (Chaurasia and Jain.2004).



Effect of stirring rate on the size of microsponges

The effect of stirring rate on the size of microsponges was studied by photo

microscope (RXLr-3T, Radical, India). The formulation with the lower drug to

polymer ratio (i.e., 3:1) was chosen to investigate the effect of stirring rate on the

morphology of microsponges. The stirring rate was varied in the range of 300 to 500

rpm for eudragit RS-100 based formulations and 500 to 1000 for eudragit S-100 based

formulations. The dispersion of the drug and polymer into the aqueous phase and the

formulation of microsponge were found to be dependant on the agitation speed. As

the speed was increased, smaller spherical microsponges with uniform size were

formed (Perumal 2001). When the rate of stirring was increased 300 - 500 rpm

eudragit RS-100 based microsponges, the spherical microsponges were formed with

mean particle size of 72 µm - 60 µm and 77 µm - 62 µm for formulation FDRS1 and

FPRS1, respectively. When the rate of stirring was increased 500 - 1000 rpm for

eudragit S-100 based microsponges the spherical microsponges were formed with

mean particle size of 74 µm - 53 µm and 79 µm - 55 µm for formulation FDS1

and FPS1, respectively.

Effect of volume of internal phase on the formation of microsponges

It was observed that on increasing the volume of internal phase from 5 to 10

ml microsponges were not formed. This may be due to the decrease in viscosity of the

internal phase (Yang et al., 2003). As the amount of dichloromethane was increased,

though the finely dispersed spherical quasi-emulsion droplets were seen in solvent

under the agitation, but as the stirring was discontinued emulsion droplets adhered to

each other and coalesce. Consequently, no microsponges could be formed. The result

suggests that the amount of dichloromethane need to be controlled within an

appropriate range to effect not only the formation of quasi-emulsion droplets at the

initial stage but also the solidification of drug and polymer in the droplets.

Microsponges were formed when 3 to 5 ml of dichloromethane was used.

Effect of amount of emulsifying agent on the production yield and size of

microsponges

An increase in amount of polyvinyl alcohol (emulsifying agent) from 0.5 % to

1.0 % w/v resulted in decreased production yield and increased mean particle size.



The amount of emulsifying agent significantly effected the production yield and mean

particle size. Due to non-ionic nature of the emulsifier some hydrophobic region

might have formed which dissolved some of the drug and polymer resulting in lower

production yield3. An increased amount of emulsifying agent decreased the

production yield from 79% to 61%, 72% to 67%, 73% to 65%, 71% to 68% for the

formulations FDRS1, FPRS1, FDS1, FPS1, respectively The increase in the amount

of emulsifying agent resulted in larger microsponges, probably due to increased

viscosity, wherein larger emulsion droplets formed resulting in larger microsponges

(Devrim and Canefe 2006). An increased amount of emulsifying agent increased the

mean particle size from 60 µm to 71 µm. 62 µm to 66 µm, 53 µm to 64 µm, 55 µm to

56 µm for the formulations FDRS1, FPRS1, FDS1, FPS1, respectively.

The production yield was found to be between 72-76% for FPRS1-FPRS4, 71-

77% for FPS1-FPS4, 70-79% for FDRS1-FDRS4, and 68-77% for FDS1-FDS4. The

actual drug content was found to be between 74-91% for FPRS1-FPRS4, 72-89% for

FPS1-FPS4, 62-81% for FDRS1-FDRS4, and 67-83% for FDS1-FDS4. The

encapsulation efficiency ranged from 82-98%. The mean particle size was found to be

between 62-41 µm for FPRS1-FPRS4, 55-38 µm for FPS1-FPS4, 60-44 µm for

FDRS1-FDRS4, and 53-34 µm for FDS1-FDS4. The data obtained for various

formulations in respect to production yield, actual drug content, and encapsulation

efficiency were subjected to t-test at 95% level of significance. No significant

difference in relation to these parameters was observed amongst various formulations

at p <0.05.

Characterization of microsponges

DSC studies were carried out to confirm that no interaction took place

between drug and other compounds in microsponges (Ceschel et al., 2003).

According to the thermograms, drugs showed sharp endothermic peaks (dicyclomine

and paracetamol at 175.97°C and 174.23°C, respectively) which corresponds to the

melting point of drug in the crystalline form. In the DSC curve of physical mixture,

FPRS1-FPRS4, FPS1-FPS4, FDRS1-FDRS4, and FDS1-FDS4 the characteristic

peaks of drug(s) were seen. The result showed that drugs were compatible with

polymers. It could also be conferred that microsponge preparation processes did not

change the nature of drugs in microsponges.



FTIR spectra were recorded to assess the chemical interaction or changes

during microsponge preparation (Mukherjeea et al., 2005). FTIR spectra of drug (s),

physical mixture of drug (s) & eudragit RS-100, physical mixture of drug (s) &

eudragit S-100 and formulations FPRS1-FPRS4, FPS1-FPS4, FDRS1-FDRS4, and

FDS1-FDS4 were recorded and examined. In FTIR spectra of paracetamol,

characteristic N-H stretching band at 3413.77 cm−1, O-H stretching band at 3326.98

cm−1, and carbonyl stretching band at 1654.81 cm−1 were seen and in case of

dicyclomine, characteristic C=O stretching band was observed at 1718.45 cm-1 which

are in agreement with the reported values. All characteristic peaks of drug(s) were

observed in the FTIR spectra of different microsponge formulations namely FPRS1-

FPRS4, FPS1-FPS4, FDRS1-FDRS4, and FDS1-FDS4. These results indicated that

no chemical interaction or changes took place during microsponge preparation. The

drug was compatible with all excipients used for microsponge preparation.

In-vitro drug release studies of Microsponge Formulations


EXPERIMENTAL WORK IN-VITRO DRUG RELEASE STUDIES

In-vitro drug release studies of paracetamol loaded microsponges

In-vitro release studies were carried out in USP basket apparatus with stirring

rate 50 rpm at 37±0.5 oC. Initial drug release was carried out in 900 ml of 0.1N

hydrochloric acid for 2 hours followed by phosphate buffer pH 6.8 for next 6 hour.

Samples were withdrawn at regular intervals and analyzed spectrophotometrically at

249 nm (Orlu et al., 2006). All the readings were taken in triplicate.

The same procedure was followed for in-vitro release studies of dicyclomine

loaded microsponges. The samples were analyzed at 420 nm. The in-vitro release data

of paracetamol loaded microsponges are given in Table 22 - Table 29 and

dicyclomine loaded microsponges are given in Table 30 - Table 37.

KINETICS OF RELEASE

To determine the drug release mechanism and to compare the release profile

amongst various microsponge formulations, the in-vitro release data was fitted to

various kinetic equations. The kinetic models included zero order, first order, Higuchi

model, and Korsmeyer-Peppas model (Nokhodchi et al., 2007). The plots were drawn

as per the following details.

• Cumulative percent drug released as a function of time (zero order kinetic plots).

• Log cumulative percent drug retained as a function of time (first order kinetics plots).

• Log cumulative percent drug released as a function of log time (Korsemeyer plots).

• Cumulative percent drug released versus square root of time (Higuchi plots).

The in-vitro release data of different kinetic models are shown in Table 38 and

presented in Figure 37 – Figure 44 for paracetamol microsponges and Figure 45 –

Figure 52 for dicyclomine microsponges.



Table 22 In-vitro drug release data for formulation FPRS1

Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 15.45±0.020 1.1889 84.55 1.9271

2 1.4142 0.3010 18.23±0.010 1.2607 81.77 1.9126

3 1.7321 0.4771 39.83±0.020 1.6002 60.17 1.7793

4 2.000 0.6021 49.79±0.007 1.6971 50.21 1.7008

5 2.2361 0.6990 53.36±0.005 1.7272 46.64 1.6688

6 2.4494 0.7782 57.34±0.003 1.7585 42.66 1.6300

7 2.6458 0.8451 59.75±0.006 1.7763 40.25 1.6048

8 2.8284 0.9031 60.76±0.027 1.7836 39.24 1.5937


Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 21.49±0.008 1.3322 78.51 1.8949

2 1.4142 0.3010 24.45±0.002 1.3883 75.55 1.8782

3 1.7321 0.4771 46.14±0.012 1.6641 53.86 1.7313

4 2.000 0.6021 55.85±0.011 1.7470 44.15 1.6449

5 2.2361 0.6990 61.45±0.010 1.7885 38.55 1.5860

6 2.4494 0.7782 63.23±0.014 1.8009 36.77 1.5655

7 2.6458 0.8451 66.13±0.013 1.8204 33.87 1.5298

8 2.8284 0.9031 68.98±0.002 1.8387 31.02 1.4916




Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 27.10±0.018 1.4330 72.9 1.8627

2 1.4142 0.3010 31.79±0.034 1.5023 68.21 1.8338

3 1.7321 0.4771 51.54±0.056 1.7121 48.46 1.6854

4 2.000 0.6021 58.13±0.004 1.7644 41.87 1.6219

5 2.2361 0.6990 66.13±0.009 1.8204 33.87 1.5298

6 2.4494 0.7782 66.14±0.004 1.8205 33.86 1.5297

7 2.6458 0.8451 79.13±0.001 1.8983 20.87 1.3195

8 2.8284 0.9031 81.85±0.002 1.9130 18.15 1.2589


Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 30.33±0.004 1.4819 69.67 1.8430

2 1.4142 0.3010 36.35±0.006 1.5605 63.65 1.8038

3 1.7321 0.4771 53.14±0.004 1.7254 46.86 1.6708

4 2.000 0.6021 64.13±0.003 1.8071 35.87 1.5547

5 2.2361 0.6990 75.40±0.045 1.8774 24.60 1.3909

6 2.4494 0.7782 85.50±0.078 1.9320 14.50 1.1614

7 2.6458 0.8451 91.20±0.048 1.9599 8.80 0.9445

8 2.8284 0.9031 93.64±0.001 1.9715 6.36 0.8035



Table 26 In-vitro drug release data for formulation FPS1

Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 18.72±0.020 1.2723 81.28 1.9099

2 1.4142 0.3010 20.04±0.002 1.3019 79.96 1.9029

3 1.7321 0.4771 35.45±0.003 1.5496 64.55 1.8099

4 2.000 0.6021 44.14±0.004 1.6448 55.86 1.7471

5 2.2361 0.6990 50.45±0.040 1.7029 49.55 1.6950

6 2.4494 0.7782 54.18±0.067 1.7338 45.82 1.6611

7 2.6458 0.8451 56.94±0.004 1.7554 43.06 1.6341

8 2.8284 0.9031 57.98±0.002 1.7633 42.02 1.6235


Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 20.25±0.078 1.3064 79.75 1.9017

2 1.4142 0.3010 25.21±0.034 1.4016 74.79 1.8738

3 1.7321 0.4771 37.80±0.056 1.5775 62.20 1.7938

4 2.000 0.6021 47.70±0.004 1.6785 52.30 1.7185

5 2.2361 0.6990 57.86±0.009 1.7624 42.14 1.6247

6 2.4494 0.7782 61.40±0.004 1.7882 38.60 1.5866

7 2.6458 0.8451 62.46±0.010 1.7956 37.54 1.5745

8 2.8284 0.9031 64.09±0.002 1.8068 35.91 1.5552




Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 23.26±0.034 1.3666 76.74 1.8850

2 1.4142 0.3010 31.28±0.008 1.4953 68.72 1.8371

3 1.7321 0.4771 45.90±0.056 1.6618 54.10 1.7332

4 2.000 0.6021 52.18±0.067 1.7175 47.82 1.6796

5 2.2361 0.6990 64.22±0.056 1.8077 35.78 1.5536

6 2.4494 0.7782 71.24±0.034 1.8527 28.76 1.4588

7 2.6458 0.8451 73.18±0.022 1.8644 26.82 1.4285

8 2.8284 0.9031 74.11±0.001 1.8699 25.89 1.4131


Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 28.62±0.008 1.4567 71.38 1.8536

2 1.4142 0.3010 33.14±0.004 1.5206 66.86 1.8252

3 1.7321 0.4771 39.53±0.045 1.5969 60.47 1.7815

4 2.000 0.6021 56.52±0.067 1.7522 43.48 1.6383

5 2.2361 0.6990 57.18±0.098 1.7572 42.82 1.6316

6 2.4494 0.7782 60.22±0.010 1.7797 39.78 1.5997

7 2.6458 0.8451 72.06±0.020 1.8577 27.94 1.4462

8 2.8284 0.9031 82.62±0.034 1.9171 17.38 1.2401



Table 30 In-vitro drug release data for formulation FDRS1

Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 16.43±0.078 1.2156 83.57 1.9221

2 1.4142 0.3010 19.68±0.034 1.2940 80.32 1.9048

3 1.7321 0.4771 37.45±0.056 1.5735 62.55 1.7962

4 2.0000 0.6021 46.56±0.004 1.6680 53.44 1.7279

5 2.2361 0.6990 51.21±0.009 1.7094 48.79 1.6883

6 2.4494 0.7782 56.56±0.004 1.7525 43.44 1.6379

7 2.6458 0.8451 57.90±0.008 1.7627 42.10 1.6243

8 2.8284 0.9031 58.91±0.002 1.7702 41.09 1.6137


Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 18.24±0.008 1.2610 81.76 1.9125

2 1.4142 0.3010 22.69±0.004 1.3558 77.31 1.8882

3 1.7321 0.4771 40.01±0.043 1.6022 59.99 1.7781

4 2.0000 0.6021 53.41±0.067 1.7276 46.59 1.6683

5 2.2361 0.6990 62.14±0.098 1.7934 37.86 1.5782

6 2.4494 0.7782 63.21±0.010 1.8008 36.79 1.5657

7 2.6458 0.8451 65.43±0.056 1.8158 34.57 1.5388

8 2.8284 0.9031 67.62±0.034 1.8301 32.38 1.5103




Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 24.48±0.020 1.3888 75.52 1.8787

2 1.4142 0.3010 27.88±0.002 1.4453 72.12 1.8581

3 1.7321 0.4771 47.14±0.003 1.6734 52.86 1.7231

4 2.0000 0.6021 57.59±0.004 1.7603 42.41 1.6275

5 2.2361 0.6990 66.14±0.040 1.8205 33.86 1.5297

6 2.4494 0.7782 70.26±0.007 1.8467 29.74 1.4733

7 2.6458 0.8451 73.29±0.004 1.8650 26.71 1.4267

8 2.8284 0.9031 74.01±0.020 1.8692 25.99 1.4148


Time

in hrs

T

√T

Log T

Cum. %

Drug

Released

Log Cum

% Drug

Released

Cum. %

Drug

Remained

Log Cum.

% Drug

Remained

0 0 - 0 - 100 2

1 1 0 30.41±0.045 1.4830 69.59 1.8425

2 1.4142 0.3010 34.08±0.089 1.5325 65.92 1.8190

3 1.7321 0.4771 47.50±0.043 1.6767 52.50 1.7201

4 2.0000 0.6021 56.19±0.056 1.7497 43.81 1.6416

5 2.2361 0.6990 67.59±0.067 1.8299 32.41 1.5107

6 2.4494 0.7782 79.59±0.043 1.9009 20.41 1.3099

7 2.6458 0.8451 85.71±0.012 1.9330 14.29 1.1550

8 2.8284 0.9031 86.01±0.014 1.9345 13.99 1.1458



Table 34 In-vitro drug release data for formulation FDS1

Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 16.55±0.002 1.2188 83.45 1.9214

2 1.4142 0.3010 18.97±0.001 1.2780 81.03 1.9086

3 1.7321 0.4771 32.98±0.012 1.5183 67.02 1.8262

4 2.0000 0.6021 38.08±0.010 1.5807 61.92 1.7919

5 2.2361 0.6990 45.48±0.019 1.6578 54.52 1.7366

6 2.4494 0.7782 49.67±0.012 1.6961 50.33 1.7018

7 2.6458 0.8451 52.45±0.001 1.7198 47.55 1.6772

8 2.8284 0.9031 53.45±0.006 1.7279 46.55 1.6679


Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 19.66±0.023 1.2936 80.34 1.9049

2 1.4142 0.3010 24.56±0.034 1.3902 75.44 1.8776

3 1.7321 0.4771 40.89±0.003 1.6116 59.11 1.7717

4 2.0000 0.6021 50.98±0.056 1.7074 49.02 1.6903

5 2.2361 0.6990 60.54±0.078 1.7820 39.46 1.5966

6 2.4494 0.7782 64.16±0.011 1.8073 35.84 1.5544

7 2.6458 0.8451 66.57±0.012 1.8233 33.43 1.5241

8 2.8284 0.9031 66.67±0.005 1.8239 33.33 1.5229




Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 27.14±0.009 1.4336 72.86 1.8625

2 1.4142 0.3010 30.42±0.007 1.4832 69.58 1.8425

3 1.7321 0.4771 41.44±0.001 1.6174 58.56 1.7676

4 2.0000 0.6021 51.67±0.010 1.7132 48.33 1.6842

5 2.2361 0.6990 59.59±0.005 1.7752 40.41 1.6065

6 2.4494 0.7782 63.60±0.000 1.8034 36.4 1.5611

7 2.6458 0.8451 68.14±0.001 1.8334 31.86 1.5032

8 2.8284 0.9031 71.69±0.020 1.8555 28.31 1.4519


Time

in hrs

T

√T

Log T

Cum. %

drug

released

Log Cum

% drug

released

Cum. %

drug

remained

Log Cum.

% drug

remained

0 0 - 0 - 100 2

1 1 0 30.43±0.001 1.4833 69.57 1.8424

2 1.4142 0.3010 36.75±0.009 1.5653 63.25 1.8011

3 1.7321 0.4771 41.49±0.098 1.6179 58.51 1.7672

4 2.0000 0.6021 53.58±0.086 1.7290 46.42 1.6667

5 2.2361 0.6990 61.40±0.077 1.7881 38.60 1.5866

6 2.4494 0.7782 69.45±0.045 1.8417 30.55 1.4850

7 2.6458 0.8451 77.06±0.034 1.8869 22.94 1.3606

8 2.8284 0.9031 82.62±0.023 1.9171 17.38 1.2400



Figure 37 Zero order plots of different formulations of Paracetamol and

Eudragit RS-100

Figure 38 First order plots of different formulations of Paracetamol and

Eudragit RS-100



Figure 39 Higuchi Model plots of different formulations of Paracetamol

and Eudragit RS-100

Figure 40 Korsemeyer Peppas plots of different formulations of Paracetamol and

Eudragit RS-100



Figure 41 Zero order plots of different formulations of Paracetamol and

Eudragit S-100

Figure 42 First order plots of different formulations of Paracetamol and

Eudragit S-100



Figure 43 Higuchi model plots of different formulations of Paracetamol and

Eudragit S-100

Figure 44 Korsemeyer Peppas plots of different formulations of Paracetamol

and Eudragit S-100



Figure 45 Zero order plots of different formulations of Dicyclomine and

Eudragit RS-100

Figure 46 First order plots of different formulations of Dicyclomine and

Eudragit RS-100



Figure 47 Higuchi model plots of different formulations of Dicyclomine and

Eudragit RS-100

Figure 48 Korsemeyer Peppas plots of different formulations of

Dicyclomine and Eudragit RS-100



Figure 49 Zero order plots of different formulations of Dicyclomine and

Eudragit S-100

Figure 50 First order plots of different formulations of Dicyclomine and

Eudragit S-100



Figure 51 Higuchi model plots of different formulations of Dicyclomine and

Eudragit S-100

Figure 52 Korsemeyer Peppas plots of different formulations of Dicyclomine and

Eudragit S-100



Table 38 In-vitro drug release models for different microsponges formulations

Zero Order First order Higuchi model Korsmeyer-

Peppas Model

Formulation

Code R K

(mg/hr)

R K

(hr-1)

R K

(mg/hr-

1/2)

R ‘n’

FPRS1 0.9729 7.7948 0.9846 0.1255 0.9848 24.347 0.9558 0.7939

FPRS2 0.9710 8.3785 0.9869 0.1497 0.9895 26.535 0.9622 0.6829

FPRS3 0.9805 9.4463 0.9941 0.2057 0.9954 29.678 0.9849 0.6323

FPRS4 0.9865 11.296 0.9934 0.3473 0.9957 35.083 0.9951 0.6908

FPS1 0.9788 7.1643 0.9896 0.1126 0.9915 22.410 0.9773 0.7375

FPS2 0.9789 7.9238 0.9897 0.1345 0.9928 24.849 0.9848 0.6817

FPS3 0.9809 9.0740 0.9942 0.1787 0.9954 28.480 0.9902 0.6347

FPS4 0.9834 8.8768 0.9834 0.1865 0.9922 27.541 0.989 0.6358

FDRS1 0.9538 7.4595 0.9748 0.1181 0.9783 23.322 0.9783 0.7476

FDRS2 0.9529 8.587 0.9745 0.1506 0.9755 26.796 0.9436 0.7596

FDRS3 0.952 9.1038 0.9835 0.1787 0.9847 28.706 0.954 0.6825

FDRS4 0.9733 10.351 0.9854 0.2575 0.9884 32.043 0.9932 0.6989

FDS1 0.9647 06.590 0.9832 0.0983 0.9863 20.538 0.9657 0.7231

FDS2 0.9567 8.4377 0.979 0.1488 0.9821 26.407 0.9578 0.7135

FDS3 0.9607 8.2378 0.9927 0.1534 0.9944 25.994 0.9902 0.6201

FDS4 0.972 9.2613 0.989 0.2034 0.9921 28.816 0.992 0.6199




The different microsponge formulations of dicyclomine and paracetamol were

subjected to in-vitro release studies using USP XX1V dissolution assembly. It was

observed that for each formulation the drug release decreased with increase in the

amount of polymer. This may be due to the fact that the release of drug from the

polymer matrix takes place after complete swelling of the polymer and as the amount

of polymer in the formulation increases the time required to swell also increases. The

release showed a bi-phasic pattern with initial burst effect. In the first hour drug

release of different microsponge formulations FPRS1-FPRS4, FPS1-FPS4, FDRS1-

FDRS4, and FDS1-FDS4 was noted to be 16-30%, 17-30%. 15-30% and 19-29 %,

respectively. This may be attributed to the drug present in the pores of the

microsponges. The overall cumulative percent release for different microsponge

formulations FPRS1-FPRS4, FPS1-FPS4, FDRS1-FDRS4, and FDS1-FDS4 at the

end of eight hours was found to be 59-86 %, 53-83 %, 61-94 %, and 56-86 %,

respectively.

The correlation coefficient and release rate constant values for zero order, first

order, Higuchi and Korsemeyer models were computed. The correlation coefficient

values of different microsponge formulations namely FPRS1-FPRS4, FPS1-FPS4,

FDRS1-FDRS4, and FDS1-FDS4 was found to be between 0.9729-0.9865, 0.9788-

0.9834, 0.9538-0.9733, and 0.9647-0.9720, respectively for zero order model; 0.9846-

0.9934, 0.9896-0.9834, 0.9748-0.9854, and 0.9832-0.9890, respectively for first order

model; 0.9848-0.9957, 0.9915-0.9922, 0.9783-0.9884, and 0.9863-0.9921,

respectively for Higuchi model. The R values were much closer to one for the

Higuchi kinetics. From the correlation coefficient values it is concluded that the drug

release from different microsponge formulations follow Higuchi model. Higuchi

model explained the matrix diffusion mechanism of drug release. The correlation

coefficient values for Higuchi model confirmed that drug release followed matrix

diffusion mechanism or Higuchi pattern release. The mechanism of drug release of

the all microsponge formulations was studied by fitting the release data to

Korsemeyer equation. The n values for formulations FPRS1-FPRS4, FPS1-FPS4,

FDRS1-FDRS4, and FDS1-FDS4 was found to be between 0.7939-0.6908, 0.7375-

0.6358, 0.7476-0.6989, and 0.7231-0.6199, respectively. The n value for Korsemeyer-

Peppas model was found to be between 0.5-1 indicative of non-fickian diffusion.



The in-vitro dissolution data was subjected to statistical analysis using

ANOVA. The p value was found to be 0.5754, 0.5447, 0.5930, and 0.5207 for the

formulations FPRS1-FPRS-4, FPS1-FPS-4, FDRS1-FDRS4, and FDS1-FDS4,

respectively indicating significant difference in the release behaviour (p>0.05).

Preparation and Characterization of Colon Specific Formulations


EXPERIMENTAL WORK

PREPARATION OF COLON SPECIFIC TABLET FORMULATIONS

The core tablets consisting of microsponges containing 40 mg dicyclomine

and 250 mg paracetamol, Na-CMC and magnesium stearate were prepared by direct

compression method (Vandamme et al., 2002). All tablet constituents were weighed

and mixed for 15 min and compressed using 12 mm round flat punches on an eight

station tablet press (Kambert Machinery, D-8). Core tablet compositions of drug(s)

with eudragit RS-100 and eudragit S-100 is given in Table 39 and Table 40,

respectively.

200 mg mixture of pectin:HPMC in the ratio 80:20 was used as outer coat for

compression coating of the tablets. Fifty percent of coating material was placed in the

die cavity and the core tablet was placed in centre followed by addition of the

remainder of the coating material. The coating material was compressed around the

core tablet using 16 mm round flat punches on the same tableting machine (Orlu et

al., 2006; Omo˘glu et al., 2003; Kawashima et al., 1992).

.



Table 39 Core tablet composition for Eudragit RS100 based microsponges

Microsponges formulations (mg) in each tablet Core tablet

formulation

codes

FPRS1 FDRS1 FPRS2 FDRS2 FPRS3 FDRS3 FPRS4 FDRS4

Na-CMC

(mg)

Magnesium

stearate

(mg)

CPDRS1 350.0 60.0 - - - - - - 32 8

CPDRS2 - - 300.0 55.0 - - - - 87 8

CPDRS3 - - - - 290.0 50.0 - - 102 8

CPDRS4 - - - - - - 280.0 50.0 112 8

No. of tablets: 50 for each formulation

Table 40 Core tablet composition for Eudragit S100 based microsponges

Microsponges formulations (mg) in each tablet Core tablet

formulation

codes

FPS1 FDS1 FPS2 FDS2 FPS3 FDS3 FPS4 FDS4

Na-

CMC

(mg)

Magnesium

stearate

(mg)

CPDS1 340.0 65.0 - - - - - - 37 8

CPDS2 - - 300 60.0 - - - - 82 8

CPDS3 - - - - 285.0 55.0 - - 102 8

CPDS4 - - - - - - 275.0 50.0 117 8

No. of tablets: 50 for each formulation



EVALUATION OF CORE AND COATED TABLETS

Weight Variation

The weight variation test was performed by weighing 20 tablets individually

and collectively, calculating the average weight, and comparing the individual tablet

weight to the average (Pharmacopoeia of India 1996). The data of core and coated

tablets are shown in Table 41 and 42.

Thickness

Thickness of the tablets was determined using verniar calipers. For this

purpose thickness of 10 tablets was individually measured (Lachman 1991). The data

of core and coated tablets are shown in Table 41 and 42.

Hardness

Hardness of the tablet was determined with the help of Monsanto hardness

tester. The tester consists of a barrel containing a compressible spring held between

two plungers. The lower plunger is placed in contact with the tablet, and a zero

reading is taken. The upper plunger is then forced against a spring by turning a

threaded bolt until the tablet fractured. The force of fracture was recorded by

monitoring a pointer which rides along a guage in the barrel to indicate the force. The

values of hardness of core and coated tablets are shown in Table 41 and 42.

Friability

The friability of the tablets was determined using Roche friabilator. It was

expressed in percentage (%). 10 tablets were weighed and transferred to the

friabilator. The friabilator was operated at 25 rpm for four minutes. After four minutes

the tablets were weighed again. The % friability was then calculated using the

formula;

The values of friability of core and coated tablets are shown in Table 41 and

42.



Drug content

The drug content of the tablets was measured spectrophotometrically. For this

purpose 5 tablets were collectively weighed, and crushed. The weighed amount of

powder containing equivalent to 250 mg of paracetamol and 40 mg of dicyclomine

was suspended in 100 ml simulated intestinal fluid (SIF) pH 6.8 for 12 h (sample I)

with occasional stirring and filtered using 0.45_m membrane filter.

For paracetamol, 1 ml of sample I was diluted with 10 ml SIF pH 6.8 (sample

II). Further, one ml of sample II was diluted up to 10 ml SIF pH 6.8 and analyzed at

243 nm against blank using UV spectrophotometer (UV 1700, Shimadzu, Japan).

For dicyclomine, 5 ml of sample I was diluted with 5 ml of 0.1N HCl (sample-

2). Further, 5 ml of sample II was diluted with 25 ml of methyl orange (1%w/v) and

extracted with chloroform (3x7.5 ml), then the volume of sample was made up to 50

ml with sodium acetate solution. The solution was filtered using 0.45_m membrane

filter. The absorbance was taken at 420 nm against blank UV spectrophotometer (UV

1700, Shimadzu, Japan).

The drug content was calculated using the following formula.

Actual drug content (%) =Mact/Mms × 100

Where Mact is the actual drug content of the tablet, Mms is the total amount of drug.

All analyses were carried out in triplicate. The values of drug content of core tablets




Table 41 Characterization of core tablets

Evaluation Parameters

Drug Content

% ± SD (n=5)

Core

Tablets

AAvveerraaggee

WWeeiigghhtt

mmgg ±± SSDD

((nn==2200))

AAvveerraaggee

TThhiicckknneessss

mmmm ±± SSDD

((nn==1100))

Hardness

Kg/cm2 ±

SD (n=5)

Friability

% ± SD

(n=10)

Dicyclomine Paracetamol

CPDRS1 452.21±0.13 2.8±0.2 4.8±0.2 0.24±0.06 101.17±0.15 98.32±0.23

CPDRS2 439.84±0.12 2.7±0.4 4.2±0.2 0.38±0.10 102.29±0.35 99.09±0.29

CPDRS3 437.61±0.19 2.6±0.1 4.4±0.1 0.47±0.13 99.96±0.25 102.28±0.31

CPDRS4 469.84±0.25 3.1±0.2 4.5±0.2 0.49±0.07 98.34±0.20 96.46±0.38

CPDS1 448.68±0.41 2.8±0.4 4.8±0.3 0.43±0.17 96.45±0.45 100.34±0.19

CPDS2 457.16±0.19 2.9±0.1 4.1±0.2 0.47±0.12 97.44±0.23 97.78±0.27

CPDS3 461.21±0.32 2.4±0.3 4.6±0.3 0.79±0.15 98.20±0.39 99.98±0.41

CPDS4 442.21±0.73 2.8±0.2 4.4±0.5 0.23.±0.11 97.98±0.18 98.02±0.16

Table 42 Characterization of coated tablets

Evaluation Parameters Coated

Tablets

AAvveerraaggee WWeeiigghhtt

mmgg ±± SSDD ((nn==2200))

AAvveerraaggee TThhiicckknneessss

mmmm ±± SSDD ((nn==1100))

Hardness

Kg/cm2 ± SD (n=5)

Friability

% ± SD (n=10)

CPDRS1 652.26±0.21 3.6±0.3 6.2±0.5 0.56±0.22

CPDRS2 639.56±0.10 3.4±0.3 6.1±0.3 0.78±0.31

CPDRS3 636.67±0.13 3.4±0.2 5.9±0.2 0.79±0.24

CPDRS4 670.46±0.35 3.6±0.3 6.6±0.3 0.73±0.19

CPDS1 648.32±0.31 3.8±0.2 6.5±0.3 0.53±0.27

CPDS2 653.79±0.24 3.6±0.1 6.2±0.1 0.72±0.39

CPDS3 663.28±0.29 3.7±0.4 6.8±0.1 0.41±0.23

CPDS4 649.45±0.14 3.49±0.01 5.8±0.4 0.78.±0.19




The core tablets consisting of microsponges containing 40 mg dicyclomine

and 250 mg paracetamol, Na-CMC and magnesium stearate were prepared by direct

compression method. The core tablets were evaluated for weight variation, thickness,

hardness, friability, and drug content.

The average weight of the core tablet formulations CPDRS1-CPDRS4 and

CPDS1-CPDS4 were found to be between 438 to 470 and 442 to 461 mg,

respectively. The variation in weight was within the range of < 5% complying the

pharmacopoeial specifications (Pharmacopoeia of India 1996). The hardness was

found to between 4.1 kg/cm2 to 4.8 kg/cm2 indicating satisfactory mechanical

strength. The friability of the core tablet formulations were found to be between 0.23

% to 0.79 %. The friability was below 1% which indicated good mechanical

resistance. The thickness was found to between 2.76-3.01 mm.

The coated tablets were prepared by compression coating the tablets with

Pectin:HPMC (80:20) mixture as outer shell. The coated tablet formulations were also

evaluated for weight variation, thickness, hardness, and friability.

The average weights of the coated tablet formulations CPDRS1-CPDRS4 and

CPDS1-CPDS4 were found to be between 637 to 670 and 648 to 663 mg,

respectively. The variation in weight was within the range of < 5% complying the

pharmacopoeial specifications. The hardness was found to between 5.8 kg/cm2 to 6.8

kg/cm2 indicating satisfactory mechanical strength. The friability of the core tablet

formulations were found to be between 0.41 % to 0.79 %. The friability was below

1% which indicated good mechanical resistance. The thickness was found to between

3.40 to 3.67 mm.

In-vitro drug release and stability studies of colon specific Formulations


EXPERIMENTAL WORK

In-vitro drug release studies of colon specific formulations

In-vitro drug release studies of the developed formulations were carried out by

Souder and Ellenbogen extraction technique using USP basket apparatus with stirring

rate 50 rpm at 37±0.5 oC.

Different dissolution media were used as per the following scheme.

At first hour 0.1N hydrochloric acid, second and third hour phthalate buffer

pH 4.5, fourth and fifth hour phosphate buffer pH 6.8, sixth hour phosphate buffer pH

7.4 and after 6th hour mixture of phosphate buffer pH 6.8 and pectinex Ultra-SPL

(1% v/v) in order to simulate the enzymatic action of the colonic bacteria were used.

Samples were withdrawn periodically and replaced with an equal amount of fresh

dissolution medium (Chourasia et al., 2004). The samples were analyzed

spectrophotometrically at wavelengths 249 nm and 420 nm for paracetamol and

dicyclomine, respectively. The release profiles obtained for the formulations

CPDRS1-CPDRS4 and CPDS1-CPDS4 are presented in Table 43 and Table 44,

respectively. The release plot obtained for the formulations CPDRS1-CPDRS4 and

CPDS1-CPDS4 are shown in Figure 53 - Figure 56 and Figure 57 - Figure 60,

respectively.



Table 43 In-vitro drug release data for Eudragit RS-100 based colon specific formulations

Cumulative % Drug Release ± S.D.

CPDRS1 CPDRS2 CPDRS3 CPDRS4

Time

(Hrs.)

PCM DCM PCM DCM PCM DCM PCM DCM

6 1.5±0.0150 1.24±0.012 1.8±0.001 1.56±0.012 1.2±0.006 1.39±0.001 1.65±0.025 1.89±0.001

7 14.76±0.130 14.28±0.010 23.12±0.012 19.45±0.020 25.23±0.002 25.78±0.005 30.34±0.038 29.23±0.002

8 22.12±0.150 34.24±0.150 35.65±0.015 37.63±0.025 50.12±0.001 49.63±0.010 57.42±0.010 56.64±0.080

9 28.45±0.012 49.48±0.016 43.12±0.012 56.30±0.300 60.34±0.010 71.67±0.025 70.34±0.007 79.26±0.004

10 35.12±0.125 57.21±0.011 54.89±0.015 76.80±0.015 70.15±0.150 87.26±0.010 84.24±0.100 88.60±0.030

11 43.15±0.010 66.12±0.120 65.67±0.010 89.06±0.500 79.62±0.130 94.77±0.010 91.56±0.010 97.35±0.035

12 55.56±0.015 71.56±0.152 77.45±0.010 94.82±0.086 88.67±0.250 96.36±0.080 95.76±0.020 99.41±0.030

13 65.34±0.012 74.90±0.015 85.34±0.001 97.52±0.015 92.45±0.025 98.83±0.008 - -

14 68.65±0.020 76.35±0.100 88.23±0.002 99.05±0.010 - - - -

* Mean±S.D. (n=3)



Table 44 In-vitro drug release data for Eudragit S100 based colon specific formulations

Cumulative % Drug Release ± S.D.

CPDS1 CPDS2 CPDS3 CPDS4

Time

(Hrs.)

PCM DCM PCM DCM PCM DCM PCM DCM

6 1.24±0.001 2.03±0.130 2.08±0.001 1.67±0.010 1.78±0.007 1.59±0.001 1.97±0.025 1.780.01

7 19.32±0.040 15.89±0.110 18.58±0.060 18.06±0.015 23.66±0.003 24.01±0.020 29.67±0.065 36.77±0.015

8 27.45±0.060 27.56±0.012 31.97±0.001 33.87±0.025 51.89±0.020 43.82±0.001 55.75±0.250 54.88±0.010

9 43.67±0.002 41.64±0.015 51.08±0.004 51.54±0.010 77.84±0.090 65.29±0.050 79.40±0.028 74.58±0.010

10 57.43±0.009 52.56±0.062 72.34±0.002 66.72±0.100 87.10±0.001 79.06±0.003 92.07±0.012 83.46±0.001

11 67.16±0.001 59.27±0.010 84.90±0.020 77.80±0.150 94.07±0.015 87.50±0.002 97.57±0.013 93.73±0.001

12 71.69±0.001 65.83±0.101 91.32±0.060 85.44±0.121 97.37±0.065 93.22±0.001 99.45±0.005 97.59±0.004

13 74.90±0.008 69.08±0.011 94.30±0.010 89.91±0.010 99.04±0.071 97.01±0.010 - -

14 76.08±0.080 72.39±0.010 97.43±0.007 91.36±0.001 - - - -

* Mean±S.D. (n=3)



Figure 53 In-vitro drug release plots of CPDRS1


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Figure 57 In-vitro drug release plots of CPDS1


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Stability Studies

Stability studies of the developed formulations were carried out according to ICH and

WHO guidelines. The formulations CPDRS1-CPDRS4 and CPDS1-CPDS4 sealed in

aluminum foils were kept in the stability chamber (REMI) maintained at 400 C ± 20 C and 75

% ± 5% RH for 3 months. The samples were analyzed for the drug content at three time

intervals “i.e.” 30, 60 and 90 days (Edward 2005). Stability data is presented in Table 45 and

are graphically shown in Figure 61.

Table 45 Stability studies of different formulations

Drug Content (%)

After 30 days After 60 days After 90 days

Formulation

Code Dicyclomine Paracetamol Dicyclomine Paracetamol Dicyclomine Paracetamol

CPDRS1 100.78±0.23 97.10±0.56 99.27±0.94 96.76±0.32 98.10±0.55 95.32±0.39

CPDRS2 101.19±0.46 98.19±0.34 100.51±0.52 97.10±0.45 99.49±0.56 96.10±0.50

CPDRS3 98.26±0.25 100.98±0.45 97.16±0.25 100.08±0.11 96.46±0.25 99.18±0.01

CPDRS4 97.74±0.19 95.86±0.56 96.44±0.05 94.36±0.18 95.14±0.23 93.86±0.78

CPDS1 95.85±0.67 100.04±0.29 95.05±0.17 99.12±0.19 94.55±0.20 98.23±0.02

CPDS2 96.64±0.21 97.08±0.20 95.94±0.13 96.18±0.67 94.14±0.54 95.48±0.23

CPDS3 97.10±0.56 98.88±0.31 96.70±0.23 97.68±0.08 95.30±0.19 96.92±0.21

CPDS4 96.08±0.28 97.42±0.86 95.35±0.08 96.12±0.19 94.88±0.23 95.42±0.31

* Mean±S.D. (n=5)



90

92

94

96

98

100

102

DCM PCM DCM PCM DCM PCM

After 30days

After 60days

After 90days

Time (in days)

Drug

Con

tent

(%)

CPDRS1CPDRS2CPDRS3CPDRS4CPDS1CPDS2CPDS3CPDS4

Figure 61 Stability studies of different formulations at 400±20 C & 75±5% RH




The developed tablet formulations (CPDRS1-CPDRS4 and CPDS1-CPDS4) were

subjected to in-vitro drug release studies using USP XX1V dissolution assembly at the

stirring rate at 50 rpm and temperature at 37±0.5 oC. The dissolution studies were carried out

at first hour in 0.1N HCl, second and third hour in phthalate buffer pH 4.5, fourth and fifth

hour in phosphate buffer pH 6.8, sixth hour in phosphate buffer pH 7.4 and after 6th hour

mixture of phosphate buffer pH 6.8 and pectinex Ultra-SPL (1% v/v) in order to simulate the

enzymatic action of the colonic bacteria were used.

It was observed that no drug was released in the first six hours. After the lag time of 6

hours, the drug started releasing at 7th hour due to the presence of the pectinex Ultra-SPL.

Formulation CPDRS1, CPDRS2, CPDRS3, CPDRS4 released 69%, 88%, 92%, 96% of

paracetamol and 76%, 99%, 98%, 99% of dicyclomine, respectively at the end of 12-14 h.

Formulation CPDS1, CPDS2, CPDS3, CPDS4 released 76%, 97%, 99%, 99% of

paracetamol and 72%, 99%, 97%, 93% of dicyclomine, respectively at the end of 12-14 h.

The results of in-vitro drug release showed that Pectin: HPMC (80:20) coat could

protect the core for 6 hours which correspond to the time to reach the colon and then under

the influence of the enzyme, the system degraded faster and delivered the drug to the

proximal colon, a main site for bacterial carbohydrate metabolism (Orlu et al., 2006;

Omo˘glu et al., 2003)

Stability study was carried out at 40 0C ± 2 0C and 75 % ± 5% RH for 3 months. The

changes in drug content of different formulations were noted. The obtained data was

subjected to t-test at 95% level of significance. No significant difference in relation to drug

content was observed amongst various formulation at p <0.05.

The results of the In-vitro drug release and stability studies indicated that developed

formulations CPDRS1 and CPDS1 are potential formulation for targeting the dicyclomine

and paracetamol to the colon.

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