RESULTS AND DISCUSSION

3.1 Preparation and characterization of CAS

The three methods described in the experimental section produced similar product but the

product from method 3 was much free of impurities, mainly CS. Thus, the drug was prepared

in bulk by using this method. The characterization was carried out by analytical, physical and

spectral data. The analytical results are given in Table 3.1.

A sample of copper (II) acetylsalicylate did not melt but turned brown on heating to 300ºC as

reported in literature [77]. The IR spectrum (Figure. 3.1) resembled that already reported in

the literature [76].

Table 3.1. Analytical results of CAS.

Elements
% Calculated % Found
Analyzed

C 51.25 51.75

H 3.35 3.34

O 30.34 30.39*

Cu 15.06 14.95
*
determined by difference
Transmittance (%)

Figure 3.1. FTIR spectrum of CAS.

Wave number (cm-1)
3.2 Pharmaceutical properties

Crystallanity

The powder X-ray diffractograms of CAS and ASA are shown in Figure 3.2. It appears that

crystalline character decreases on complexation of ASA with copper. Thus, the CAS is

expected to possess enhanced bioavailability and improved tabletting properties [122 - 125].

Particle size analysis

The results of particle size analysis are presented in Table 3.2. It appears that the particle size

decreases on complexation with the metal ion. This indicates that the complex will have

enhanced compactability and bioavailability. Particle size analysis indicates the free flowing

nature of CAS, as more than 50% of the material was retained up to 150 μm.

Moisture

The moisture contents of ASA and CAS are given in Table 3.3. CAS was anhydrous.

Density measurements

The bulk, tap, and true densities for the powders are listed in Table 3.3. ASA has higher bulk

and tap densities than those of CAS. The data indicate a reduced die-filling character of CAS.

Porosity

CAS had the highest porosity followed by ASA (Table 3.3). This is a positive contribution of

complexation whereby the new materials will have better compressibility.

Flow

The values for these parameters are listed in Table 3.3. These are consistent with the flow

rates determined by flow meter. The values of angle of repose and flow rate in case of CAS

indicate an improvement in flowability with respect to ASA. Percent compressibility and the

Hausner ratio methods were used for the compressibility index. The results of the test are

given in Table 3.3.

Taste

Twenty healthy volunteers completed the taste study in a blind fashion. The taste and

aftertaste were evaluated. The results indicate no bitter taste and, therefore, it could be used

orally.
 4000
CAS
3000

2000

1000

0
Intensity

20 40 60 80 100

16000
14000
14000
12000
12000 ASA
10000
8000
6000
4000
2000
0
0 20 40 60 80 100

2 Theta

Figure 3.2. PXRD spectra of CAS and ASA

Table 3.2. Particle size analysis of CAS by sieve analyzer.

Mesh Retention Mesh Retention

(μm) (%) (μm) (%)
20 0.37 180 μ 39.2
40 1.0 250 μ 36.5
60 1.13 280 μ 35.8
80 1.35 325 μ 35.04
100 1.58 400μ 1.92
150 50.5

Table 3.3. Powder properties.

Parameter ASA CAS

Moisture (% w/w) 0.05 ± 0.01 0.00

ρtrue (g mL-1) 1.97 ± 0.05 1.82 ± 0.03

ρbulk (g mL-1) 0.43 ± 0.01 0.51 ± 0.01

ρtap (g mL-1) 0.52 ± 0.01 0.65 ± 0.01

Bukliness (mL g-1) 1.99 ± 0.02 2.30 ± 0.01

ɛº (%) 69.5 ± 0.08 75.35 ± 0.06

HR 1.40 ± 0.01 1.45 ± 0.01

CI (%) 17.31 ± 0.03 21.5 ± 0.05

α(10 g, i.d. 10 mm, 20 rpm) 16˚ 15˚

Flow rate (g s-1) 0.50 0.44

3.3 Evaluation of tablets

The results of hardness, friability, disintegration and dissolution are given in Table 3.4.

Hardness, friability and disintegration results were found to be according to required standard

specifications. Similarly, the dissolution of all batches is greater than 80% up to 30 min

according to USP specifications [111]. Dissolution behaviour of these batches is also shown

in Figure 3.3.
Table 3.4. Evaluation of tablets.

Friability Disintegration Released after

Product Hardness (Kg)
(%) time (min) 30 min (%)

ASA(WG) 9.0 ± 1.0 0.22 1.6 80.0

ASA(DC) 6.5 ± 1.5 0.35 4.0 84.0

CAS(WG) 9.4 ±1.0 0.18 1.5 83.5

CAS(DC) 6.7 ± 1.5 0.25 2.6 85.0

 100

90

80

70

60
Release (%)

50

40

30

20

10

0
0 10 20 30 40 50 60 70 80 90 100 110
Time (min)

Figure 3.3. Dissolution behaviour of ASA (WG) □; CAS (WG) ×; ASA (DC) ♦;
CAS (WG)▲ tablets. DC = direct compression; WG = wet granulation.
3.4 Development and validation of assay method(s)

3.4.1 HPLC method

Three different mobile phases were used for the development of assay method on HPLC. In

the first method, mobile phase A (water, acetonitrile and 0.1 M phosphate buffer pH 2.5 in

35: 25: 40 ratio) was used along with chromatographic conditions as given in experimental

section. The resultant chromatogram is shown in Figure 3.4. By using this method, only two

peaks representing ASA and SA were identified by spiking technique. These results indicate

that CAS hydrolyzes in this mobile phase and is converted to ASA and SA. As this method

did not show any peak for CAS, so it could not be used for the assay of CAS.

In the second method, methanol-ethanol (50:50) was used as mobile phase for the assay of

CAS according to the chromatographic conditions as given in the experimental section. The

chromatogram (Figure 3.5) shows again the resolution of only ASA and SA. So, this method

also could not be used for the assay of CAS.

In the third method, mobile phase C i.e. methanol-acetic acid (20:1) was used. By using this

method, four peaks representing CAS, ASA, SA and CS were resolved. The typical

chromatogram is shown in Figure 3.6.

The system optimization was carried out by using various compositions of this mobile phase

as given in Table 3.5. It was found that the separation deteriorated on increasing or

decreasing methanol-acetic acid ratio beyond 20:1. A test run was obtained by using the

mixture of CAS, ASA, CS, and SA standards and all four components were separated. The

typical chromatogram is shown in Figure 3.6. The retention time for CAS, CS, ASA, and SA
was 2.6, 2.8, 3.0 and 3.2, respectively. In this study, the resolution was 1.0 for CS and greater

than 1 for CAS and ASA. The peaks in the mixture were verified by spiking with the

standards. The performance parameters thus obtained are given in Table 3.6.
 1
(mV × 100) 10

0
6 8 10

Time (min)

Figure 3.4. Chromatogram of CAS showing separation of ASA (1) and SA (2) in the mobile
phase A.

(mV × 100) 10
1
2

0
0 1 2 3 4 5

Time (min)
Figure 3.5. Chromatogram of CAS showing separation of ASA (1) and SA (2) in the mobile
phase B.
(mV × 100) 3
2 1
4

2
1 1

00 11 22 3 3 4 4 5 5
Time(min)
Time (min)

Figure 3.6. Chromatogram showing separation of CAS (1), CS (2),

ASA (3) and SA (4) in the mobile phase C.
 Table 3.5. Effect of mobile phase composition on performance parameters.

Mobile phase Parameter CAS CS ASA SA

Methanol-Acetic acid N 2260 4659 5153 10725

(20 : 1.0) Rs 1.97 0.89 1.69 ̶

As 1.00 0.90 1.10 1.00

K 4.20 4.80 5.10 5.50

Methanol-Acetic acid N 2205 4552 5150 10656

(20 : 0.5) Rs 0.82 0.45 0.75 ̶

As 1.80 1.00 1.50 1.30

K 8.20 9.80 10.10 10.50

Methanol-Acetic acid N 2105 4052 4150 10206

(20 : 1.5) Rs 0.65 0.45 0.75 ̶

As 2.00 1.20 1.40 1.50

K 6.20 8.80 9.00 11.50

Table 3.6. Performance parameters.

 Parameter CAS CS ASA SA

N 2260 4659 5153 1072

Rs* 1.97 1.01 1.69 ̶

As 1.00 0.90 1.10 1.00

K 4.20 4.80 5.10 5.50

* between the adjacent peaks

3.4.2 Method validation

Accuracy and precision

The accuracy ranged from 80.7 to 99.65 % in terms of recovery. The precisions were

determined within the day and between the days. The analyses were performed at three

different concentration levels, covering the entire linear range. The CV ranged from 0.03 to

0.08 and 0.05 to 0.11 for within the day and between the days, respectively. The results are

given in Table 3.7.

Linearity

Concentration and peak area were found to be linearly related for CAS, CS, ASA and SA in

concentration ranges under study. The linearity data are given in Table 3.8. The calibration

curves in mobile phase and plasma are shown in Figures 3.7 and 3.8, respectively.

Limit of detection (LOD) and limit of quantification (LOQ)

The values of LOD and LOQ are given in Table 3.7. The very low levels indicate that the

method is very sensitive for the determination of the substances under investigation in

presence of each other.

Specificity and reproducibility

The method was found to be specific for the determination of a particular analyte in dosage

forms, as the reproducibility of measurement (CV 0.0075 for CSA, 0.0025 for CS, 0.012 for

ASA, 0.005 for SA) of five different samples spiked with standard was very high (Table 3.9)
Table 3.7. Validation parameters of analytes in the mobile phase and plasma at three
different concentration levels.

Matrix Parameter CAS CS ASA SA

(Mean) (Mean) (Mean) (Mean)
In mobile Precision (CV, i. 0.03/0.7 i. 0.04/0.07 i. 0.03/0.05 i. 0.08/0.1
phase within at 0.004 at 0.09 µg at 0.03 µg at 0.02 µg
day/between µg mL-1 mL-1 mL-1 mL-1
days)
ii. 0.04/0.06 ii. 0.03/0.09 ii. 0.04/0.07 ii. 0.06/0.11
at 100 µg at 75 µg at 100 µg at 125 µg
mL-1 mL-1 mL-1 mL-1

iii. 0.04/0.07 iii. 0.05/0.08 iii. 0.03/0.06 iii. 0.07/0.09

at 200 µg at 150 µg at 200 µg at 250 µg
-1 -1 -1
mL mL mL mL-1

Accuracy (% i. 99.07 at i. 80.70 at i. 99.65 at i. 95.83 at

recovery) 0.004 µg 0.09 µg 0.03 µg 0.02 µg
mL-1 mL-1 mL-1 mL-1

ii. 99.1 at ii. 80.45 at ii. 99.55 at ii. 95.90 at

100 µg 75 µg 100 µg 125 µg
mL-1 mL-1 mL-1 mL-1

iii. 99.12 at iii. 80.55 at iii. 99.5 at iii. 95.78 at

200 µg 150µg 200 µg 250 µg
mL-1 mL-1 mL-1 mL-1

LOD (ng mL-1) 2.0 45.0 15.0 10.0

LLOQ (ng mL- 4.0 90.0 30.0 20.0

1
)
0.004-200 0.09-150 0.03-200 0.02-250
Concentration
range (µg mL-1)

Continued
Table 3.7 (Continued)
In plasma Precision (CV, i. 0.05/0.1 i. 0.08/0.14 i. 0.06/0.1 i. 0.12/0.18
within at 0.012 at 0.13 at 0.06 at 0.04
day/between µg mL-1 µg mL-1 µg mL-1 µg mL-1
days)
ii. 0.07/0.1 ii. 0.1/0.15 ii. 0.05/0.1 ii. 0.11/0.16
at 100 µg at 75 µg at 100 µg at 125 µg
mL-1 mL-1 mL-1 mL-1

iii. 0.05/0.11 iii. 0.09/0.13 iii. 0.04/0.09 iii. 0.13/0.17

at 200 µg at 150 µg at 200 µg at 250 µg
mL-1 mL-1 mL-1 mL-1

Accuracy (% i. 95.0 at i. 80.20 at i. 96.50 at i. 92.83 at

recovery) 0.012 µg 0.13 µg 0.006 µg 0.04 µg
mL-1 mL-1 mL-1 mL-1

ii. 95.1 at ii. 80.15 at ii. 96.45 at ii. 92.85 at

100 µg 75 µg 100 µg 125 µg
mL-1 mL-1 mL-1 mL-1

iii. 95.2 at iii. 80.0 at iii. 96.56 at iii. 92.90 at

200 µg 150 µg 200 µg 250 µg
mL-1 mL-1 mL-1 mL-1

LOD (ng mL-1) 6.0 65.0 30.0 20.0

LLOQ (ng mL- 12.0 130.0 60.0 40.0

1
)
0.012-200 0.13-150 0.06-200 0.04-250
Concentration
range (µg mL-1)
Table 3.8. Linearity parameters.

Parameter CAS CS ASA SA

R2 0.991 0.998 0.9914 0.994

Slope 134.2 116.4 53.6 40.3

Concentration range 0.004-200 0.004-200 0.03-200 0.02-250

(µg mL-1)

Table 3.9. Specificity data, in terms of % recovery, at 100 µgmL-1 of each analyte
in six plasmas.

Plasma CAS CS ASA SA

1 95.1 80.15 96.55 92.8

2 95.05 80.2 96.5 92.83

3 95.07 80.16 96.45 92.72

4 95.12 80.19 96.54 92.84

5 95.08 80.15 96.6 92.81

6 95.09 80.18 96.5 92.8

CV (%) 0.0075 0.0025 0.012 0.005

73
 600

500

400
Peak Area

300

200

100

0
0 50 100 150 200 250 300
-1
Concentration μg mL

Figure 3.7. Calibration curves of CAS (∆), CS (♦), ASA (□) and SA (×) in
mobile phase.

74
 600

500

400
Peak area

300

200

100

0
0 1 2 3 4 5 6
-1
Concentration μg mL

Figure 3.8. Calibration curves of CAS (∆), CS (♦), ASA (□) and SA (×) in
plasma.

75
3.5 Stability study of pharmaceutical preparations

3.5.1 Stability of CAS in the mobile phase C and in plasma

The stability study of drug in mobile phase as well as in plasma was carried out and it was

found that the chromatogram did not change significantly over the period of 6 h i.e. there was

no significant change in the peak position as well as the peak areas in mobile phase, and was

found to be stable for 72 h in plasma. Thus, the methanol-acetic acid system was found to be

suitable for this study.

3.5.2 Stability of CAS in tablets

It appears that the CAS, when is in contact with moisture, disproportionates into CS, ASA

and SA according to Scheme 5. The results of the accelerated stability study of the tablets are

shown in Figures 3.9 – 3.12.

76
 O

O
Cu2 + H2O

OCOCH 3
4
CAS

O
COOH COOH
O
Cu + + + (CH3COO)2Cu + CH3COOH

O OCOCH 3 OH
2
CS ASA SA

Scheme 5

77
 50.4

50.3

50.2

50.1
Assay (% w/v))

50

49.9

49.8

49.7

49.6

49.5
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.9. Stability curves showing the concentration of the active ingredient in
tablets vs number of days. CAS (WG) at 40 °C & 75% RH (□); CAS
(WG) at 50 °C & 75% RH (×); CAS (DC) at 40 °C & 75% RH(♦); CAS
(DC) at 50 °C & 75% RH (∆); DC = direct compression; WG =wet
granulation.

78
 4

3.5

2.5
Assay (% w/v))

1.5

0.5

0
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.10. Stability curves showing the concentration of the free SA in tablets
vs number of days. SA in CAS (WG) tablets at 40 °C & 75% RH
(□); SA in CAS (DC) tablets at 50 °C & 75% RH (×); SA in CAS
(DC) tablets at 40 °C & 75% RH (♦); SA in CAS (WG) tablets at
50 °C & 75% RH (∆).

79
 4.5

3.5

3
Assay (% w/v))

2.5

1.5

0.5

0
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.11. Stability curves showing the concentration of the free CS in tablets
vs number of days. CS in CAS (WG) tablets at 50 °C & 75% RH
(□); CS in CAS (DC) tablets at 50 °C & 75% RH (♦); CS in CAS
(WG) tablets at 40 °C & 75% RH (×); CS in CAS (DC) tablets at
50 °C & 75% RH (∆).

80
 48

47.8

47.6
Assay (%w/v))

47.4

47.2

47

46.8
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.12. Stability curves showing the concentration of the free ASA in
tablets vs number of days. ASA in CAS (WG) tablets at 40 °C &
75% RH (□); ASA in CAS (DC) tablets at 40 °C & 75% RH (♦);
ASA in CAS (WG) tablets at 50 °C & 75% RH (×); ASA in
CAS (DC) tablets at 50 °C & 75% RH (∆).

81
The stability profiles of the tablets are discussed as follows:

Wet granulation

The stability was monitored by determining the concentration of the active ingredient and

the decomposition products in the tablets in PVC securitainers placed at 40ºC and 50ºC with

75% RH at various intervals of time. At 40ºC, the concentration of CAS (the active

ingredient) dropped slowly according to the equation y = - 0.0051x + 50.232 with r2 =

0.8709. Accordingly, the concentration of SA (the decomposition product of CAS) increased

by following the equation y = 0.00102x + 1.6712 with r2 = 0.9003. At 50ºC, the

concentration of CAS dropped slowly according to the equation y = - 0.0043x + 50.134 with

r2 = 0.8521. Accordingly, the concentration of SA (the decomposition product of CAS)

increased by the following trends according to the equation y = 0.0115x+2.088 with r2 =

0.7536.

The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC with

75% RH was determined and it was found that the concentration of CS increased by

following the equations y = 0.0222x + 0.7191 (r2=0.9604) and y = 0.0259x + 0.692 (r2 =

0.9538), respectively. There is no significant difference (F < F critical) between the two

values. This indicates that the stability profile of CAS formulated by wet granulation is

similar at 40ºC and 50ºC with 75% RH.

The concentration of ASA (decomposition product of CAS) at 40ºC and 50ºC with 75% RH

dropped slowly according to the equations y = - 0.0029x + 47.68 with r2 = 0.9254 and y = -

0.0031x + 47.671 with r2 =0.8987, respectively. This trend is obvious because the ASA

further decomposes into SA, which has increasing trend as shown in Figure.3.12.

82
Direct compression

The stability was monitored by determining the concentration of the active ingredient and

the decomposition products in the tablets placed at 40ºC, and 50ºC with 75% RH, at various

intervals of time. At 40ºC, the concentration of CAS (the active ingredient) dropped slowly

according to the equation y = -0.0043x + 50.259 with r2 = 0.6877. Accordingly, the

concentration of SA (the decomposition product of CAS) increased by following the

equations y = 0.005x + 1.8219 with r2 = 0.8304. At 50ºC, the concentration of CAS (the

active ingredient) dropped slowly according to the equation y = -0.0053x + 50.28 with r2 =

0.8738. Accordingly, the concentration of SA (the decomposition product of CAS) increased

by following the equation y = 0.0058x + 1.5933 with r2 = 0.8383.

The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC with

75% RH was determined and it was found that the concentration of CS increased by

following the equations y = 0.0153x + 0.7996 (r2 = 0.9006) and y = 0.0249x + 0.6842 (r2 =

0.9215), respectively. There is no significant difference (F < F critical) between the two

values. This shows that the stability profile of CAS formulated by direct compression is

similar at 40ºC and 50ºC with 75% RH.

The concentration of ASA (decomposition product of CAS) at 40ºC and 50ºC with 75% RH

dropped slowly according to the equations y = - 0.0028x + 47.665 with r 2 = 0.8782 and y = -

0.0035x + 47.69 with r2 = 0.9065, respectively. This behaviour of ASA contents has been

explained before.

83
3.5.3 Comparison of stability data of CAS and ASA tablets

Wet granulation

The stability was monitored by determining the concentration of the active ingredient and

the decomposition products in the tablets placed at 40ºC and 50ºC with 75% RH, at various

intervals of time. At 40ºC, the concentration of CAS (the active ingredient) dropped slowly

according to the equation y = 23.562e-0.0012x with r2 = 0.8222, whereas the decomposition

occurred at much higher rate (F > F critical) in case of tablets of ASA and followed the trend

as per equation y = 99.998x-0.0754 with r2 = 0.9214. Accordingly, the concentration of SA (the

common decomposition product of CAS and ASA tablets) increased by following the

equations y = 0.0085x+1.5454 (r2 = 0.9064) and y = 0.146x-0.2375 (r2 = 0.9271) for CAS and

ASA tablets, respectively. These results clearly show an enhanced stability of CAS

formulation as compared to that of ASA. At 50ºC, the concentration of CAS (the active

ingredient) dropped slowly according to the equation y = 0.0188x+23.947 with r2 = 0.7864,

whereas the decomposition occurred at much higher rate (F > F critical) in case of tablets of

ASA according to the equation y = -0.183x+93.089 with r2 = 0.9404. Accordingly, the

concentration of SA (the common decomposition product of CAS and ASA tablets) increased

by following the trends according to equations y = 0.0085x+1.5454 (r2=0.9064) and y =

0.146x-0.2375 (r2 = 0.9271). These results also show an enhanced stability of CAS

formulation as compared to that of ASA.

84
The concentration of CS (another decomposition product of CAS) at 40ºC and 50ºC with

75% RH was determined and it was found that the concentration of CS increased by

following the equation y = 0.0222x +0.7191 (r2=0.9604) and y = 0.0259x +0.692 (r2 =

0.9538), respectively. There is no significant difference (F < F critical) between the two

values. This indicates that the stability profile of CAS formulated by wet granulation is

similar at 40ºC and 50ºC with 75% RH.

Direct compression

The stability was monitored by determining the concentration of the active ingredient and

the decomposition products in the tablets placed at 40ºC and 50ºC (with 75% RH) and at

various intervals of time. At 40ºC, the concentration of CAS and ASA (the active ingredient)

dropped slowly according to the equations y = 25.041e-0.0005x with r2 = 0.73, and y = 98.867x-
0.076
with r2 = 0.7265 (F < F critical), respectively. Accordingly, the concentration of SA (the

common decomposition product of CAS and ASA tablets) increased by following the

equations y= 0.0195x+1.3391 (r2 = 0.745) and y = 0.3714Ln(x) + 0.2708 (r2 = 0.8579) for

CAS and ASA tablets, respectively. These results clearly indicate that there is no significant

difference in the stability of CAS formulation as compared to that of ASA. At 50ºC, the

concentration of CAS and ASA (the active ingredient) dropped slowly according to the

equations y = -0.0128x+25.092 with r2 = 0.6816 and y = -0.0505x+96.584 with r2 = 0.9399 (F

< F critical), respectively. Accordingly, the concentration of SA (the common decomposition

product of CAS and ASA tablets) increased by following the trends according to equations y

= 0.0124x+2.0862(r2 = 0.7874) and y = 0.0493x-0.426 (r2 = 0.9775). These results also

indicate no significant difference in the stability of CAS formulation as compared to that of

ASA.

85
The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC (with

75% RH) was determined and the concentration of CS increased by following the equation,

y= 0.0153x+0.7996 (r2 = 0.9006) and y = 0.0249x +0.6842 (r2 = 0.9215), respectively. There

is no significant difference (F < F critical) found between the two values. This indicates that

the stability profile of CAS formulated by direct compression is similar at 40ºC and 50ºC

with 75% RH. The comparisons of the stability study data of CAS and ASA tablets are

shown in Figures 3.13 – 3.16.

86
 100

95

90

85

80

75
Assay(%w/v))

70

65

60

55

50

45

40
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.13. Stability curves showing the concentration of the active ingredient in tablets
vs number of days. ASA (DC) at 40 °C & 75% RH (*); ASA (DC) at 50 °C &
75% RH (●); ASA (WG) at 40 °C & 75% RH (-); ASA (WG) at 50 °C &75%
RH (×); CAS (DC) at 40 °C & 75% RH(♦); CAS (DC) at 50 °C & 75% RH(▲);
CAS (WG) at 40 °C & 75% RH (■); CAS (WG) at 50 °C & 75% H(+); DC =
direct compression; WG = wet ganulation.

87
 18

16

14

12
Assay (%w/v))

10

0
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.14. Stability curves showing the concentration of the free SA in tablets
vs number of days. SA in ASA (DC) tablets at 40 °C & 75 % RH
(■); SA in ASA (DC) tablets at 50 °C & 75% RH (●); SA in ASA
(WG) tablets at 40 °C & 75% RH(-); SA in ASA (WG) tablets at 50
°C & 75% RH (×); SA in CAS (DC) tablets at 40 °C & 75% RH (♦);
SA in CAS (DC) tablets at 50 °C & 75% RH (*); SA in CAS (WG)
tablets at 40 °C & 75% RH (▲); SA in CAS (WG) tablets at 50 °C
& 75% RH (+).

88
 100

90

80
Assay(%w/v))

70

60

50

40
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.15. Stability curves showing the concentration of the active ingredient
in wet granulated tablets vs number of days. ASA (WG) at 40 °C
& 75% RH (■); ASA (WG) at 50 °C & 75 % RH (×); CAS (WG)
at 40 °C & 75% RH (♦); CAS (WG) at 50 °C & 75% RH (▲).

89
 18

16

14

12
Assay (%w/v))

10

0
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.16. Stability curves showing the concentration of the free SA in wet
granulated tablets vs number of days. SA in ASA (WG) tablets
at 40 °C & 75% RH (■) ;SA in ASA (WG) tablets at 50 °C &
75% RH (×); SA in CAS (WG) tablets at 40 °C & 75% RH (♦);
SA in CAS (WG) tablets at 50 °C & 75% RH (▲).

90
3.5.4 Stability comparison of CAS and ASA capsules

The stability was monitored by determining the concentration of the active ingredient and the

decomposition product in the capsules placed at 40ºC and 50ºC with 75% RH, at various

intervals of time. The results of the accelerated stability study of capsules are shown in

Figures 3.17 – 3.19.

At 40ºC, the concentration of CAS and ASA (the active ingredient) dropped slowly

according to the equation, y = 25.021e-0.0005× with r2 = 0.78 and y = 98.856x-0.076 with r2 = 0.765

(F < F critical), respectively. Accordingly, the concentration of SA (the common

decomposition product of CAS and ASA capsules) increased by following the equations y =

0.0018x+0.1287 (r2 = 0.8328) and y = 0.0013x -0.0014 (r2 = 0.9065) for CAS and ASA

capsules, respectively. These results clearly indicate no significant difference in the stability

of CAS capsules as compared to that of ASA. At 50ºC, the concentration of CAS and ASA

(the active ingredient) dropped slowly according to the equation y = -0.0122x+24.092 with r2

= 0.7816 and y = -0.0405x+98.584 with r2 = 0.94 (F < F critical), respectively. Accordingly,

the concentration of SA (the common decomposition product of CAS and ASA capsules)

increased by following the trends according to equations y = 0.0016x+ 0.121(r 2 = 0.8228) and

y = 0.0016x-0.0085 (r2 = 0.9689). These results also indicate similar stability of CAS and

ASA formulation.

The concentration of CS, another decomposition product of CAS, at 40ºC and 50ºC with

75% RH was determined and it increased by following the equations y = 0.0155x+0.7992 (r2

= 0.9106) and y = 0.0245x +0.6642 (r2 = 0.9015), respectively. There is no significant

91
difference (F < F critical) found between the two values. This shows that the stability profile

of CAS capsules is similar at 40ºC and 50ºC with 75% RH.

92
 100

90

80
Assay(%w/v))

70

60

50

40
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.17. Stability curves showing the concentration of the active ingredient
in capsules vs number of days. CSA capsules at 40 °C & 75% RH
(■); ASA capsules at 40°C & 75% RH (●); ASA capsules at 50 °C
& 75% RH (×); CAS capsules at 50 °C & 75% RH (▲).

93
 0.8

0.7

0.6

0.5
Assay (% w/v))

0.4

0.3

0.2

0.1

0
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.18. Stability curves showing the concentration of free SA vs number

of days: in ASA capsules at 50 °C & 75 % RH (□); in ASA
capsules at 40 °C & 75% RH (♦); in CAS capsules at 50 °C &
75% RH (×); in CAS capsules at 40 °C & 75% RH (∆).

94
 4

3.5

2.5
Assay (% w/v))

1.5

0.5

0
0 20 40 60 80 100 120 140 160 180
Days

Figure 3.19. Stability curves showing the concentration of free CS vs number of

days: in CAS capsules at 50 °C & 75% RH (□); in CAS capsules at
40 °C & 75% RH (∆).

95
3.5.5 Stability comparison of CAS and ASA aqueous suspension

Stability study of CAS and ASA aqueous suspension was carried out under the same

conditions as used for tablets and capsules; however, it was observed that the suspension of

both the products were not stable. The results are shown in Figure 3.20.

3.5.6 Study of commercial CAS preparation

Samples of commercially available CAS preparation, Nuhas capsules (Sigma Herbals,

Lahore), 60 mg CAS, were analyzed for the CAS, ASA, CS and SA contents. The results are

given in Table 3.10. The data indicate excellent stability of the product over the period under

study.

96
 100

90

80

70

60
Assay(%w/v))

50

40

30

20

10

0
0 2 4 6 8 10 12 14
Hours

Figure 3.20. Stability curves showing the concentration of the active ingredient
in ASA and CAS syrup vs time (hours). ASA at 40 °C & 75% RH
(■); ASA at 50 °C & 75% RH (×); CAS at 40 °C & 75% RH (♦);
CAS at50°C&75%RH(▲).

97
Table 3.10. Three years data of commercially available 60 mg CAS capsule (Nuhas, Sigma
Herbals). The contents of various analytes are given as mg per capsule.

Age (Months)
Analyte
0 6 12 24 36

CAS
59.85 59.12 59.0 58.92 58.51

0.35 0.3 0.28 0.20 0.14

ASA

CS 0.10 0.13 0.12 0.11 0.10

SA 0.05 0.06 0.10 0.15 0.2

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3.6 Pharmacokinetic studies

The pharmacokinetic data following the single oral administration of 60 mg CAS (tablet or

capsule) is given in Tables 3.11 and 3.12. The pharmacokinetic data of both the tablets and

capsules was almost similar and the reported results are an average of both dosage forms.

The chromatograms of blank plasma and sample plasma are shown in Figure 3.21. The

plasma concentration-time curves are shown in Figure 3.22. It was noted that about 1.5% (i.e.

AUC0-∞h × 100/Dose) of unconverted CAS of the oral dose reaches systemic circulation. This

behavior was found to be similar to that of ASA, indicating similar bioavailability after oral

administration [126]. The extrapolated AUC∞ was < 10% in case of CAS and ASA (as

metabolite). The areas under the curves (AUC0-∞h) of the metabolites including ASA, SA and

CS (0.45, 0.31 and 2.35 hmgL-1, respectively) show that significant amounts of these species

remain available in plasma for longer periods of time; the concentration of CS being the

highest, whereas, after administration of 900 mg ASA, the level of ASA in plasma rises

rapidly to reach a maximum, with only small amounts remaining after 2 h [126]. The

elimination of CAS, ASA, CS and SA follows the first order kinetics with r 2 0.960, 0.930,

0.999 and 1.000, respectively. The mean pharmacokinetic data of CAS, ASA, CS and SA is

given in the Tables 3.11 and 3.12.

The Cmax was found to be 0.38 mgL-1 at a tmax 0.72 h, which is about 0.6% of the administered

dose. In case of aspirin (ASA), normally, it is about 4% of the dose between 14-15 min of

administration of 900 mg [126]. The t1/2 was 8.67 h, which is ideal for once a day dosing. The

Vd and Cl values for CAS were 829 Lkg -1 and 66.30 Lh-1, respectively. The large Vd may be

due to uptake by a specific tissue or membrane, as highly lipophilic compounds are known to

distribute into lipids in cell membranes and fat stores; these effectively form slow release

99
depots of the drug and prolong the plasma levels [127]. The relatively high clearance may

lead to low exposure and low plasma average concentrations during chronic dosing. Theses

findings offer an understanding of the enhanced anti-inflammatory activity of CAS as

compared to that of ASA alone [126].

The plasma copper level was determined by atomic absorption spectrophotometer at 0, 0.25,

0.5, 1.0, 2.0, 4.0, 8.0 and 12 h after administration of a single dose of 60 mg CAS. The

results are given in Table 3.13 and the trend is shown in Figure. 3.23. It can be seen that the

plasma copper level raised about two times the normal plasma copper level, which remained

constant over a period of 12 h.

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Table 3.11. Pharmacokinetic data after a single oral dose of 60 mg CAS and 900 mg
aspirin (ASA).
Parameter CAS ASA [126]
tmax, h 0.72 0.24
-1
Cmax, mgL 0.38 36.62
t1/2, h 8.67 0.22
-1
AUC0- ∞h, hmgL 0.91 15.32
Vd, Lkg-1 829 150-200[127]
-1
Cl (Lh ) 66.30 13.33[127]
Vd: volume of distribution ; Cl: total body clearance for extra- vascular administration.

Table 3.12. Pharmacokinetic data of CAS metabolites after a single oral dose of 60 mg.

Parameter ASA SA CS
tmax, h 1.41 2.13 3.02
Cmax, µg mL-1 0.178 0.022 0.13
AUC0- ∞h, h. µg mL-1 0.45 0.31 2.35

101
 Figure 3.21. Chromatograms of blank and test plasma showing
separation of CAS(1), CS(2), ASA(3) and SA(4).
Plasma concentration (mgL-1)

1
CS
SA
0.1 ASA
CAS

0.01

Figure. 3.22. Plasma concentration-time curve of CAS, ASA, SA and CS.

0.001

0.0001
0 2 4 1026 8 10 12

Time (h)
Table 3.13. Plasma copper concentration data.

Time (h) (Concentration (µg mL-1 Time (h) (Concentration (µg mL-1

0.25 0.887522 ±0.00118 4.00 0.714411 ±0.00116

0.50 0.740773 ±0.00117 8.00 0.819859 ± 0.00118

1.00 0.670475 ±0.00118 12.00 0.688040 ± 0.00115

2.00 0.793497 ±0.00119 ̶ ̶

103
 2

1.5
Plasma copper ( µg mL )
-1

0.5

0
0 2 4 6 8 10 12 14
Time (h)

Figure 3.23. Copper plasma level-time curve.

104
 CONCLUSIONS

This work was aimed at the study of some of the important pharmaceutical and

pharmacological properties of copper (II)-acetylsalicylate, a potential anti-inflammatory

drug, with regard to its development as a drug. The work was divided into three parts. In

the first part, pharmaceutical properties of copper (II)-acetylsalicylate including

crystallanity, particle size distribution, porosity, flow, compressibility, moisture uptake

and taste were studied and compared with those of acetylsalicylic acid. The results

indicated that copper (II)-acetylsalicylate possesses reduced crystalline character, particle

size and density, better flow, no taste, and enhanced porosity. The complex was found to

be hydrophobic.

In the second part, copper (II)-acetylsalicylate was converted into various dosage forms

including tablets (through wet granulation and direct compression methods), capsules and

aqueous suspension. These dosage forms were subjected to accelerated stability studies

and compared with the profile of acetylsalicylic acid.

The third part consists of development and validation of new HPLC method for

simultaneous determination of copper (II)-acetylsalicylate and its decomposition products

or metabolites in dosage forms and plasma samples. The method was found to be suitable

for stability and pharmacokinetic studies. This method was successfully applied to

determine the various pharmacokinetic parameters in humans. The results showed that

copper (II)-acetylsalicylate possessed higher values of the time to peak drug

concentration, the half life of the drug, volume of distribution, and clearance. The data

obtained through these studies clearly demonstrate that copper (II)-acetylsalicylate

105
possesses better properties for formulation into the dosage forms studied. The results also

further our understanding regarding the enhanced anti-inflammatory effect of copper (II)-

acetylsalicylate as compared to acetylsalicylic acid.

106