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PHARMANIAGA RESEARCH CENTRE SDN BHD

PRODUCT DEVELOPMENT DEPARTMENT


ANALYTICAL METHOD VALIDATION

ATTACHMENT 1.0

Reference Protocol No

AMVP-06/15

Characteristics

Formulation of Diclofenac Sodium 75 mg/ 3mL Injection

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Ingredients for Actimol Menstrual


Tablet

Quantity/ 3mL
mg

Diclofenac Sodium

75.00

Benzyl Alcohol

120.00

Mannitol
Sodium metabisulphate
Propylene glycol

8.58
2.01
780.00

Sodium hydroxide

q.s

Water for Injection

q.s to 3 mL

Analytical Method Validation : Linearity


Prepared by:
Date:

Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Identification for Diclofenac sodium and Benzyl Alcohol

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Blank, Placebo, Standard and Sample

Blank

Placebo
Preparation

Standard Preparation for Benzyl Alcohol

Standard Preparation for


Diclofenac Sodium

Purity, %

NA

NA

100.0%

100.0%

Weight ,mg

NA

NA

162.40

100.60

Compound

% Difference in Retention Time of Standard Preparation Compared with Sample Preparation for Benzyl Alcohol
Retention Time (min)
Preparations
Benzyl Alcohol

Standard Preparation

Sample Preparation

3.873

3.866

% Difference in Retention Time of Standard Preparation Compared with Sample Preparation for Diclofenac Sodium
Retention Time (min)
Preparations

Diclofenac Sodium

Standard Preparation

Sample Preparation

12.905

12.899

An

An
Prepared by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

Weighing printout

AMVP-06/15
Identification for Diclofenac sodium and Benzyl Alcohol
Diclofenac Sodium 75 mg /3 mL Injection

An
Prepared by:
Date:

ATTACHMENT 2.0

Sample Preparation for


Diclofenac Sodium
NA
NA (Spiked sample)

% Difference in Retention Time ( 5.0%)


0.18

% Difference in Retention Time ( 5.0%)

0.05

Analytical Method Validation : Linearity

Analytical Method Validation : Linearity


Reviewed by:
Date:

ATTACHMENT 2.0

Analytical Method Validation : Linearity


Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Specificity - Degradation Study (Diluent)
Diclofenac Sodium 75mg/ 3mL Injection

Stress Condition-Diluent Solution


Control

Solution

Resolution

NA
NA

NA
NA

NA
NA

NA

NA

NA

NA

NA

NA

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

3.069

NA

NA

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

Benzyl Alcohol
Diclofenac Sodium

Acid Degradation, 3M Hydrochloric Benzyl Alcohol


Acid
Diclofenac Sodium
Alkaline Degradation, 3M Sodium
Hydroxide
Oxidation Degradation, 30%
Hydrogen Peroxide

Thermal Degradation, 80C


Photolytic Degradation, UV 254nm

Purity

Retention time, Min

H2O2

Angle

Prepared by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Specificity - Degradation Study (Placebo)
Diclofenac Sodium 75mg/ 3mL Injection

Stress Condition-Placebo Solution

Solution

Resolution

NA
NA

NA
NA

NA
NA

Peak 1

3.115

NA

NA

Peak 2

10.651

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Peak 2

NA

NA

NA

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

Peak 1

NA

NA

NA

Peak 2

10.541

NA

NA

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

3.059

NA

NA

Benzyl Alcohol
Control

Diclofenac Sodium

Benzyl Alcohol
Acid Degradation, 3M Hydrochloric Diclofenac Sodium
Acid
Peak 1

Alkaline Degradation, 3M Sodium


Hydroxide

Oxidation Degradation, 30%


Hydrogen Peroxide

Purity

Retention time, Min

H2O2

Angle

Thermal Degradation, 80C

Photolytic Degradation, UV 254nm

Prepared by:
Date:

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

Peak 1

3.029

NA

NA

Peak 2

10.577

NA

NA

Benzyl Alcohol

NA

NA

NA

Diclofenac Sodium

NA

NA

NA

Peak 1

3.103

NA

NA

Peak 2

10.551

NA

NA

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Specificity - Degradation Study (Standard)
Diclofenac Sodium 75mg/ 3mL Injection

Standard Preparations
Compound
Weight, mg (Control)
Purity, %
Expiry Date

Diclofenac Sodium
100.6
100.0
NA

Benzyl Alcohol
162.4
100.0
NA

*Refer to Attachment 2.0 for the standard weighing printout.

Stress Condition-Sample Solution

Solution

Resolution

3.873
12.948

NA
NA

0.237
0.205

Peak 1

NA

NA

NA

Peak 2

NA

NA

NA

3.882

NA

0.249

12.923

17.300

0.261

7.272

17.000

0.846

NA

NA

NA

3.871

NA

0.326

Benzyl Alcohol
Control

Diclofenac Sodium

Benzyl Alcohol
Acid Degradation, 3M Hydrochloric Diclofenac Sodium
Acid
Peak 1
Peak 2
Benzyl Alcohol
Alkaline Degradation, 3M Sodium
Hydroxide

Purity

Retention time, Min

Angle

Alkaline Degradation, 3M Sodium


Hydroxide

Oxidation Degradation, 30%


Hydrogen Peroxide

Thermal Degradation, 80C

Photolytic Degradation, UV 254nm

Prepared by:
Date:

Diclofenac Sodium

12.918

NA

0.203

Peak 1

NA

NA

NA

Peak 2

NA

NA

NA

Benzyl Alcohol

3.878

NA

0.211

Diclofenac Sodium

12.925

NA

0.320

H2O2

3.069

NA

NA

Benzyl Alcohol

3.878

NA

0.342

Diclofenac Sodium

12.930

NA

0.184

Peak 1

NA

NA

NA

Peak 2

NA

NA

NA

Benzyl Alcohol

3.882

NA

0.310

Diclofenac Sodium

12.939

NA

0.243

Peak 1

NA

NA

NA

Peak 2

NA

NA

NA

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Specificity - Degradation Study (Sample)
Diclofenac Sodium 75mg/ 3mL Injection

Sample Preparations
Compound
Weight, mg (Control)
Purity
Expiry Date

Dicllofenac Sodium
100.6
100.0
NA

Benzyl Alcohol
162.4
100.0
NA

*Refer to Attachment 2.0 for the sample weighing printout.


Stress Condition-Sample Solution

Purity

Retention time, Min

Resolution

Benzyl Alcohol
Diclofenac Sodium

3.867
12.902

NA
7.4

1.781
1.143

Peak 1

5.612

9.7

NA

Peak 2

10.349

NA

NA

Peak 3

15.056

5.0

NA

Benzyl Alcohol
Diclofenac Sodium

3.868

NA

0.355

12.906

17.300

0.286

Acid Degradation, 3M Hydrochloric Peak 1


Acid
Peak 2

NA

NA

NA

NA

NA

NA

Peak 3

NA

NA

NA

Control

Solution

Angle

Acid Degradation, 3M Hydrochloric


Acid

Alkaline Degradation, 3M Sodium


Hydroxide

Oxidation Degradation, 30%


Hydrogen Peroxide

Prepared by:
Date:

Peak 4

7.257

17.000

NA

Benzyl Alcohol
Diclofenac Sodium

3.869

NA

0.232

12.906

NA

0.177

Peak 1

NA

NA

NA

Peak 2

NA

NA

NA

Peak 3

NA

NA

NA

Benzyl Alcohol
Diclofenac Sodium

3.858

NA

0.246

12.905

NA

0.241

H2O2

3.058

NA

NA

Peak 1

NA

NA

NA

Peak 2

NA

NA

NA

Peak 3

NA

NA

NA

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Specificity - Degradation Study (Sample)

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Stress Condition-Standard Solution

Solution

Resolution

3.873

NA

0.462

12.913

7.100

0.140

Peak 1

NA

NA

NA

Peak 2

10.346

NA

NA

Peak 3

15.091

5.400

NA

Peak 4

NA

NA

NA

Peak 5

8.216

23.000

NA

Benzyl Alcohol
Diclofenac Sodium

3.871

NA

0.813

12.901

6.800

0.231

Peak 1

5.625

7.200

NA

Peak 2

10.325

NA

NA

Peak 3

15.059

5.100

NA

Benzyl Alcohol
Diclofenac Sodium
Thermal Degradation, 80C

Photolytic Degradation, UV 254nm

Purity

Retention time, Min

Angle

Prepared by:
Date:

ATTACHMENT 3.0

Purity
Threshold

Result

NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time
No peak detected at Benzyl Alcohol and
Diclofenac Sodium retention time. H2O2
peak detected at 3.069
No peak detected at Benzyl Alcohol and
Diclofenac Sodium retention time
No peak detected at Benzyl Alcohol and
Diclofenac Sodium retention time

Reviewed by:
Date:

ATTACHMENT 3.0

Purity
Threshold
NA
NA
NA
NA

Result
No peak detected at Benzyl Alcohol and
Diclofenac Sodium retention time. Two
placebo peaks were observed at 3.115 and
10.651 minutes respectively.

NA
NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
NA
NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
NA
NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time. H2O2
peak detected at 3.059

NA
NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
NA
NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA

Reviewed by:
Date:

ATTACHMENT 3.0

Purity
Threshold
1.190
1.194
NA

Result

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
1.214
1.273
1.917
NA

1 degradation peak detected appeared at


7.272 minute with resolution of 17.0 and 17.3
from Benzyl Alcohol peak and Diclofenac
Sodium peak respectively.

1.256
No peak detected at Benzyl Alcohol and
Diclofenac Sodium retention time

1.197
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
1.206
1.184
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time. H2O2
peak detected at 3.069

1.283
1.197
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
1.252
1.244
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA

Reviewed by:
Date:

ATTACHMENT 3.0

Purity
Threshold
3.549
3.249
NA
NA
NA

Result
Total 3 peaks observed appeared at 5.612,
10.349 and15.056 minute. The resolution of
peak 1 from Benzyl Alcohol peak is 9.7.
While the resolution of peak 2 and 3 from
Diclofenac Sodium peak are 7.4 and 5.0
respectively.

1.314
1.254
NA
NA
NA

1 degradation peak detected appeared at


7.257 minute with resolution of 17.0 and 17.3
from Benzyl Alcohol peak and Diclofenac
Sodium peak respectively.

7.257 minute with resolution of 17.0 and 17.3


from Benzyl Alcohol peak and Diclofenac
Sodium peak respectively.
NA
1.162
1.172
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time

NA
NA
1.257
1.295
NA
NA

No peak detected at Benzyl Alcohol and


Diclofenac Sodium retention time. H2O2
peak detected at 3.069

NA
NA

Reviewed by:
Date:

ATTACHMENT 3.0

Purity
Threshold

Result

1.148
1.138
NA
NA
NA
NA

Total 3 peaks observed appeared at 8.216,


10.346 and15.091 minute. The resolution of
peak 5 from Benzyl Alcohol peak is 23.
While the resolution of peak 2 and 3 from
Diclofenac Sodium peak are 7.1 and 5.4
respectively.

NA
1.235
1.264
NA
NA
NA

Total 3 peaks observed appeared at 5.625,


10.325 and15.059 minute. The resolution of
peak 1 from Benzyl Alcohol peak is 7.2.
While the resolution of peak 2 and 3 from
Diclofenac Sodium peak are 6.8 and 5.1
respectively.

Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Standard Benzyl alcohol

Finished Product

Diclofenac sodium 75 mg/ 3mL

Standard and Preparation for Benzyl Alcohol


Compound

Standard for Benzyl Alcohol

Weight, mg

164.89
100%

Purity, %
Stock concentration, mg/mL

0.82

Range (%)

Conc (mg/ml)

Injection 1

Injection 2

Injection 3

Average

Std
deviation

% RSD

Ycal

5 (LOQ)

0.0021

3070.6

3151.5

3073.1

3098.4

46

1.48

3097.298613623

60

0.0247

40135.1

40490.3

40721.6

40449.0

295

0.73

39418.95992767

80

0.0330

51256.7

51063.6

51175.5

51165.3

97

0.19

52626.83676914

100

0.0412

66480.7

66293.9

66364.4

66379.7

94

0.14

65834.71361061

120

0.0495

77712.9

78276.8

78639.7

78209.8

467

0.60

79042.59045208

140

0.0577

92487.5

92840.8

93577.9

92968.7

556

0.60

92250.46729355

intercept

SUMMARY OUTPUT
Regression Statistics
Multiple R

0.9995322597

R Square

0.9990647381

Adjusted R Square

0.9988309226

Standard Error

1084.38816878

y-intercept=

100

Concentration=

Wt of std x F
200
100

area average of 100%


y-intercept= -0.31

Where F: 0.25(LOQ), 3(60%), 4(80

Observations

ANOVA
df

SS

MS

Regression

5024466140.348

5024466140.34798

Residual

4 4703590.8023886

1175897.70059714

Total

5 5029169731.1504
Coefficients

P-value

958.6599543811

-0.213496554

0.8413801327 -2866.337335 2456.996141 -2866.337334636 2456.996141146

X Variable 1

1602022.78385 24508.035284629

0.0011355596

2 39418.9599277 1030.0400723327

1.0619996084

3 52626.8367691 -1461.5701024714

-1.5069189231

544.9530560579

0.5618615699

5 79042.5904521 -832.7904520795

-0.8586298318

6 92250.4672936

Date:

Standard Residuals

1.1013863773

4 65834.7136106

Prepared by:

Residuals

718.266039783

Lower 95%

Upper 95%

Lower 95.0%

Upper 95.0%

65.367246507 3.281203679E-007 1533977.5692 1670067.998 1533977.5692483 1670067.998456

RESIDUAL OUTPUT

1 3097.29861362

4272.8769159056 3.28120E-007

t Stat

-204.67059675

Predicted Y

Significance F

Standard Error

Intercept

Observation

0.7405520169

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Standard Benzyl alcohol

Finished Product

Diclofenac sodium 75 mg/ 3mL

Linearity and Peak Response of Standard Benzyl Alcohol

100000.0

f(x) = 1602022.78385236x - 204.670596745


R = 0.9990647381
80000.0

Peak Response

60000.0

40000.0

20000.0

20000.0

0.0
0.000

0.010

0.020

0.030

-20000.0

Concentration (mg/mL)

Prepared by:
Date:

0.040

0.050

0.060

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Sample Benzyl Alcohol

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Sample and Preparation for Benzyl Alcohol


Compound

Standard for Benzyl Alcohol

Weight, mg

164.89

Purity, %
Stock concentration, mg/mL

100%

Range (%)

0.82

Conc (mg/ml)

Injection 1

Injection 2

Injection 3

Average

Std deviation

5 (LOQ)

0.002

2979.0

3031.0

2931.40

2980.5

50

60

0.025

40904.5

40267.5

40334.40

40502.1

350

80

0.033

53277.8

53004.1

52954.10

53078.7

174

100

0.041

66259.0

66571.9

66623.30

66484.7

197

120

0.049

80514.3

80066.0

80530.00

80370.1

263

140

0.058

91725.4

91232.6

91860.90

91606.3

331

% RSD

Ycal
1.67 3534.688969259
0.86 39871.07768535
0.33 53084.30994575
0.30 66297.54220615
0.33 79510.77446655
0.36 92724.00672694

SUMMARY OUTPUT

Regression Statistics
Multiple R

0.9997288414

R Square

0.9994577562

Adjusted R Square

0.9993221953

y-intercept=

intercept

100

Concentration=

Wt of std x F
200
100

area average of 100%


y-intercept= 0.35

Where F: 0.25(LOQ), 3(60%), 4(80

Standard Error

825.85934882

Observations

ANOVA
df

SS

MS

Regression

5028541523.035

5028541523.03498

Residual

4 2728174.6561358

682043.664033952

Total

5 5031269697.6911
Coefficients

P-value

730.1059790736

0.3169141341

0.7671600202 -1795.718268 2258.480076 -1795.718267715 2258.480076034

X Variable 1

1602672.35859 18665.078284412

Residuals

1 3534.68896926 -554.2223025919

Date:

Lower 95%

Upper 95%

Lower 95.0%

Upper 95.0%

85.8647541773 1.102805508E-007 1550849.7933 1654494.924 1550849.7933494 1654494.923831

RESIDUAL OUTPUT

Prepared by:

7372.7560099212 1.10281E-007

t Stat

231.380904159

Predicted Y

Significance F

Standard Error

Intercept

Observation

Standard Residuals
-0.7502964912

2 39871.0776854

631.0556479807

0.8543121347

3 53084.3099458

-5.6432790838

-0.0076397728

4 66297.5422061

187.1911271851

0.2534160846

5 79510.7744665

859.3255334539

1.1633399261

6 92724.0067269 -1117.7067269439

-1.5131318813

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Sample Benzyl Alcohol

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Linearity and Peak Response of Sample Benzyl Alcohol

93000

f(x) = 1602672.35859031x + 231.3809041591


R = 0.9994577562

Peak Response
73000

53000

Peak r

Linear

Benzy
33000

Linear

13000

0.000
-7000

0.010

0.020

0.030

Concentration (mg/mL)

Prepared by:
Date:

0.040

0.050

0.0

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Linearity of Sample Benzyl Alcohol
Diclofenac Sodium 75 mg /3 mL Injection

Prepared by:
Date:

ATTACHMENT 4.0

Y-Ycal

Peak response

Ratio peak
response

61968

0.950

1030

67415

1.034

-1462

63957

0.981

545

66380

1.018

-833

65175

0.999

718

66406

1.018

Mean

65217

1.000

St Dev

1987

0.03047

% RSD

3.05

3.05

Wt of std x F
200
100
0.25(LOQ), 3(60%), 4(80), 5(100%), 6(120%) and 7(140%)

Reviewed by:
Date:

ATTACHMENT 4.0

ndard Benzyl Alcohol

Benzyl Alcohol
Linear (Benzyl Alcohol)
Linear (Benzyl Alcohol)
Linear (Benzyl Alcohol)
Linear (Benzyl Alcohol)
Linear (Benzyl Alcohol)
Linear (Benzyl Alcohol)

0.040

0.050

0.060

0.070

Reviewed by:
Date:

ATTACHMENT 4.0

Y-Ycal

Peak response

Ratio peak
response

-554

59609

0.912

631

67504

1.032

-6

66348

1.015

187

66485

1.017

859

66975

1.024

-1118

65433

1.001

Mean

65392

1.000

St Dev

2916

0.04459

% RSD

4.46

4.46

Wt of std x F
200
100
0.25(LOQ), 3(60%), 4(80), 5(100%), 6(120%) and 7(140%)

Reviewed by:
Date:

ATTACHMENT 4.0

le Benzyl Alcohol

Peak response
Linear (Peak response)
Benzyl Alcohol
Linear (Benzyl Alcohol)

0.040

0.050

0.060

0.070

/mL)

Reviewed by:
Date:

on

ATTACHMENT 4.0

Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Standard Diclofenac sodium

Finished Product

DicDiclofenac Sodium 75 mg /3 mL Injection

Standard and Preparation for Diclofenac sodium


Compound

Standard for Diclofenac sodium

Weight, mg

50.13
100

Purity, %
Stock concentration, mg/mL

Range (%)

0.5013

Conc (mg/ml)

Injection 1

Injection 2

Injection 3

Average

Std deviation

% RSD

Ycal

3 (LOQ)

0.000752

15619.7

15423.4

15806.1

15616.4

191

1.23 8846.780463087

60

0.015039

301574.8

305230.4

303112.1

303305.8

1835

0.61 306816.7348582

80

0.020052

403670.0

404613.4

404738.2

404340.5

584

100

0.025065

510601.2

514931.3

512070.7

512534.4

2202

0.14 411367.5960495
0.43 515918.4572407

120

0.030078

614968.9

617659.1

615343.8

615990.6

1457

140

0.035091

733035.5

737695.6

739223

736651.4

3223

0.24 620469.318432
0.44 725020.1796232

SUMMARY OUTPUT

Regression Statistics
Multiple R

0.9995763677

R Square

0.999152915

Adjusted R Square
Standard Error

0.9989411437
8281.44810666

y-intercept=

intercept

100

Concentration=

area average of 100%


y-intercept= -1.33

Where F:

Observations

ANOVA
df

SS

MS

Significance F

Regression

1 323576896916.746

323576896916.746 4718.0760418879 2.691584E-007

Residual

4 274329530.973196

68582382.7432989

Total

5 323851226447.719
Coefficients

Standard Error

t Stat

P-value

Lower 95%

0.397390538 -26883.416234

Upper 95%

Lower 95.0%

Upper 95.0%

Intercept

-6835.8487156

7220.5884715688

-0.9467162881

X Variable 1

20855946.7766

303631.93114692

68.6882525756 2.69158418E-007 20012929.3876 21698964.17 20012929.38762 21698964.16565

RESIDUAL OUTPUT
Observation

Prepared by:
Date:

Predicted Y

Residuals

Standard Residuals

1 8846.78046309

6769.6195369133

0.9139301045

2 306816.734858

-3510.9681915218

-0.4739970258

3 411367.596049

-7027.0627161189

-0.9486861303

4 515918.457241

-3384.0572407161

-0.4568634575

5 620469.318432

-4478.7184319799

-0.6046478066

6 725020.179623 11631.1870434231

1.5702643157

13211.7188 -26883.4162341 13211.71880293

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Standard Diclofenac sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Linearity and Peak Response of Standard Diclofenac sodium

800000.0

f(x) = 20855946.7766335x - 6835.8487156029


R = 0.999152915
Peak Area

300000.0

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.000

0.005

0.010

0.015

0.020

-200000.0

Concentration (mg/mL)

Prepared by:
Date:

0.025

0.030

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Sample Diclofenac sodium

Finished Product

Diclofenac sodium 75 mg/ 3mL

Standard and Preparation for Diclofenac sodium


Compound

Standard for Diclofenac sodium

Weight, mg

50.13

Purity, %
Stock concentration, mg/mL

100.0

Range (%)

0.5013

Conc (mg/ml)

Injection 1

Injection 2

Injection 3

Average

Std deviation

% RSD

Ycal

3 (LOQ)

0.00075

15598.2

15483.3

15622.4

15568.0

74

0.48 16613.54984165

60

0.01504

298008.7

293068.7

293739.2

294938.9

2680

0.91 293204.0676853

80

0.02005

380644.8

378744.8

383329.5

380906.4

2304

0.60 390253.3721918

100

0.02507

498416.2

498610.7

499066.4

498697.8

334

0.07 487302.6766983

120

0.03008

586963.6

589862.2

592651.8

589825.9

2844

0.48 584351.9812049

140

0.03509

670978.1

673274.8

675317.4

673190.1

2171

0.32 681401.2857114

SUMMARY OUTPUT

Regression Statistics
Multiple R

0.999428944

R Square

0.998858214

Adjusted R Square
Standard Error

0.9985727675
8926.14631561

y-intercept=

intercept

100

Concentration=

Wt of std x F
100
100

area average of 100%


y-intercept= 0.41

Where F: 0.15(LOQ), 3(60%), 4(8

Observations

ANOVA
df

SS

MS

Regression

278809220169.61

278809220169.61

Residual

4 318704352.190956

79676088.0477391

Total

5 279127924521.801
Coefficients

Standard Error

t Stat

7782.7003625397

0.2641954681

Intercept

2056.15416567

X Variable 1

19359526.1334 327269.217726562

Prepared by:
Date:

Predicted Y

Residuals

Significance F

3499.283498991 4.890644E-007

P-value

Lower 95%

Upper 95%

Lower 95.0%

Standard Residuals

1 16613.5498416

-1045.583174983

-0.1309632944

2 293204.067685

1734.7989813968

0.217290212

3 390253.372192

-9347.0055251367

-1.1707482155

4 487302.676698 11395.0899683299

1.4272786306

5 584351.981205

5473.8854617964

0.6856251041

6 681401.285711

-8211.1857114037

-1.0284824367

Upper 95.0%

0.8046829255 -19552.086161 23664.39449 -19552.0861611 23664.39449246

59.1547419823 4.89064398E-007 18450881.1157 20268171.15 18450881.11572

RESIDUAL OUTPUT
Observation

20268171.151

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Linearity of Sample Diclofenac sodium

Finished Product

Diclofenac sodium 75 mg/ 3mL

Linearity and Peak Response of Sample Diclofenac Sodium

1000000

Peak Response

f(x) = 19359526.1333599x + 2056.1541656699


R = 0.998858214

500000

0
0.000

0.005

0.010

0.015

Concentration (mg/mL)

Prepared by:
Date:

0.020

0.025

0.030

ATTACHMENT 4.0

Y-Ycal

Peak response

Ratio peak
response

6770

520547

1.013

-3511

505510

0.984

-7027

505426

0.983

-3384

512534

0.997

-4479

513326

0.999

11631

526180

1.024

513920

1.000

St Dev

8239

0.01603

% RSD

1.60

1.60

Mean

Wt of std x F
100
100
0.15(LOQ), 3(60%), 4(80), 5(100%), 6(120%) and 7(140%)

Reviewed by:
Date:

ATTACHMENT 4.0

Standard Diclofenac sodium

Diclofenac sodium
Linear (Diclofenac sodium)
Linear (Diclofenac sodium)
Linear (Diclofenac sodium)
Linear (Diclofenac sodium)
Linear (Diclofenac sodium)
Linear (Diclofenac sodium)

0.020

0.025

0.030

0.035

0.040

0.020

0.025

0.030

0.035

mL)

Reviewed by:
Date:

0.040

ATTACHMENT 4.0

Ratio peak
response

Y-Ycal

Peak response

-1046

518932

1.053

1735

491565

0.997

-9347

476133

0.966

11395

498698

1.012

5474

491522

0.997

-8211

480850

0.975

Mean

492950

1.000

St Dev

15108

0.03065

% RSD

3.06

3.06

Wt of std x F
100
100
0.15(LOQ), 3(60%), 4(80), 5(100%), 6(120%) and 7(140%)

Reviewed by:
Date:

ATTACHMENT 4.0

lofenac Sodium

Peak response
Linear (Peak response)
Diclofenac Sodium
Linear (Diclofenac Sodium)
Linear (Diclofenac Sodium)

.020

Linear (Diclofenac Sodium)

0.025

0.030

0.035

Reviewed by:
Date:

0.040

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Accuracy of Standard Benzyl Alcohol

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Preparation for Paracetamol


Compound

Benzyl Alcohol

Weight, mg

164.89
100.0

Purity, %
Intercept
X Variable 1

-204.670596745
1602022.78385236

Benzyl Alcohol

Area
Mean Area

Calculated
Conc, mg/mL

% Accuracy

40490.3

39740.3

0.02493

100.81

39144.0

39239.3

39191.7

0.02459

99.43

38566.6

40271.4

39419.0

0.02473

100.00

66480.7

66293.9

66387.3

0.04157

100.84

66900.8

66540.1

66720.5

0.04178

101.34

64761.6

65354.7

65058.2

0.04074

98.82

92487.5

92840.8

92664.2

0.05797

100.45

92809.9

91197.0

92003.5

0.05756

99.73

91789.9

91279.9

91534.9

0.05726

99.23

Sample

Level

Theoretical Conc,
mg/mL

60%

0.02473

Area
(Injection 1 )

(Injection 2)

38990.3

1
2

100%

140%

0.04122

0.05771

Average
Formula:
%Accuracy =

(Area - intercept) x 100%

Mean

100.08

100.33

99.80
100.07

SD

0.84

RSD

0.84

Slope

Significance level

Theoretical concentration

Sample size
95% Confidence
Interval (CI)

Prepared by:
Date:

AnalAnalyticytalicalMetMethodhodValValidatidiatonio:nLi:nLiearnearityity
(Least squares regression line, y = a + bx)

0.05
9
0.55

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Accuracy of Sample Benzyl Alcohol

Finished Product

Diclofenac Sodium 75 mg/ 3mL

Preparation for Benzyl Alcohol


Compound

Benzyl Alcohol

Weight, mg

164.89
100%

Purity, %
Intercept
X Variable 1

231.3809041591
1602672.35859031

Benzyl Alcohol

Area
Mean Area

Calculated
Conc, mg/mL

% Accuracy

40267.5

40586.0

0.02518

101.80

40177.2

40131.9

40154.6

0.02491

100.72

41418.3

41131.2

41274.8

0.02561

103.54

66259.0

66571.9

66415.5

0.04130

100.18

66477.3

66428.9

66453.1

0.04132

100.24

66046.2

66160.2

66103.2

0.04110

99.71

91584.2

88317.0

89950.6

0.05598

97.00

91725.4

91232.6

91479.0

0.05693

98.65

92133.8

92973.0

92553.4

0.05761

99.82

Sample

Level

Theoretical Conc,
mg/mL

1
2

60%

100%

0.02473

0.04122

3
1
2

140%

0.05771

Area
(Injection 1 )

(Injection 2)

40904.5

Average
Formula:

SD

Mean

102.02

100.04

98.49
100.18
1.84

%Accuracy =

(Area - intercept) x 100%


Slope
Theoretical concentration

RSD

1.83

Significance level

0.05

Sample size
95% Confidence
Interval (CI)

Prepared by:
Date:

AnAanlyatliyctaiclaMelMethothdoVdaVliadlidtiaotnio:nL:inLeinaeraitryity
(Least squares regres ionline,y= a + bx)

Analytical MethodValidation:Linearity

9
1.20

ATTACHMENT 5.0

SD

RSD

0.70

0.70

1.33

1.33

0.61

0.61

Confidence
Interval (Benzyl
Alcohol)

= Mean assay Confidence Interval


= 100.07% 0.55
= 99.52% - 100.62%

Reviewed by:
Date:

ATTACHMENT 5.0

SD

RSD

1.43

1.40

0.29

0.29

1.41

1.44

= Mean assay Confidence Interval


Confidence
Interval
= 100.18% 1.20
(Benzyl Alcohol)
= 98.98% - 101.38%

Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Accuracy of Standard Diclofenac sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Preparation for Pamabrom


Compound

Diclofenac sodium

Weight, mg

50.13
100.0

Purity, %
Intercept

-6835.8487156029

X Variable 1

20855946.7766335

Diclofenac sodium

Area
Mean Area

Calculated
Conc, mg/mL

% Accuracy

305230.4

303402.6

0.01488

98.91

304207.3

303234.4

303720.9

0.01489

99.01

301866.0

301818.7

301842.4

0.01480

98.41

510601.2

514931.3

512766.3

0.02491

99.40

510105.2

510714.8

510410.0

0.02480

98.95

510339.1

507274.6

508806.9

0.02472

98.64

733035.5

737695.6

735365.6

0.03559

101.41

715214.8

720710.1

717962.5

0.03475

99.04

706207.1

706704.6

706455.9

0.03420

97.46

Sample

Level

Theoretical Conc,
mg/mL

60%

0.01504

Area
(Injection 1 )

(Injection 2)

301574.8

1
2

100%

140%

0.02507

0.03509

3
Formula:
%Accuracy =

(Area - intercept) x 100%

Mean

98.78

98.99

99.30

Average

99.03

SD

1.05

RSD

1.06

Slope

Significance level

Theoretical concentration

Sample size
95% Confidence
Interval (CI)

Prepared by:
Date:

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi:nLiearnearityity
(Least squares regression line, y = a + bx)

0.05
9
0.69

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Accuracy of Sample Diclofenac Sodium

Finished Product

Diclofenac Sodium 75 mg/ 3mL

Preparation for Diclofenac Sodium


Compound

Diclofenac sodium

Weight, mg

50.13
100%

Purity, %
Intercept
X Variable 1

2056.1541656696
19359526.1333599

Diclofenac Sodium

Area
Mean Area

Calculated
Conc, mg/mL

% Accuracy

293068.7

295538.7

0.01516

100.80

295018.5

302259.2

298638.9

0.01532

101.87

303019.0

301305.6

302162.3

0.01550

103.08

498416.2

498610.7

498513.5

0.02564

102.31

485537.0

486306.4

485921.7

0.02499

99.72

491446.9

492207.5

491827.2

0.02530

100.93

671371.5

669438.1

670404.8

0.03452

98.38

670978.1

673274.8

672126.5

0.03461

98.63

688970.4

687174.1

688072.3

0.03544

100.98

Sample

Level

Theoretical Conc,
mg/mL

Area
(Injection 1 )

(Injection 2)

298008.7

1
2

60%

0.01504

3
1
2

100%

140%

0.02507

0.03509

Average
Formula:
%Accuracy =

(Area - intercept) x 100%


Slope

Mean

101.92

100.99

99.33
100.74

SD

1.59

RSD

1.6

Significance level

0.05

Theoretical concentration

Sample size
95% Confidence
Interval (CI)

Prepared by:
Date:

AnAanlyatliyctaiclaMel MethothdoVdaVliadlaidtiaotnio:nL:inLeinaeraitryity
(Least squares regres ionline,y= a + bx)

AnalyticalMethod Validation :Linearity

9
1.04

ATTACHMENT 5.0

SD

RSD

0.32

0.3

0.38

0.4

1.99

2.0

= Mean assay Confidence Interval


Confidence Interval
= 99.03% 0.69
(Diclofenac Sodium)
= 98.34% - 99.72%

Reviewed by:
Date:

ATTACHMENT 5.0

SD

RSD

1.14

1.12

1.30

1.29

1.43

1.44

= Mean assay Confidence Interval


Confidence Interval
= 100.74% 1.04
(Diclofenac sodium)
= 99.70% - 101.78%

Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics

AMVP-06/15
Precision -Repetability

Finished Product

Diclofenac sodium 75 mg/ 3mL

Standard Preparations
Compound

Benzyl Alcohol

Diclofenac Sodium

Sample

Weight, mg

167.64

100.09

4.07

Purity

100%

100%
NA

NA

Expiry Date

NA

Precision for Benzyl Alcohol Standard Preparation

Number of Injection

Retention Time, Min

Area

Injection-1
Injection-2
Injection-3
Injection-4
Injection-5
Injection-6
Mean
SD
RSD

3.966
3.966
3.960
3.964
3.961
3.962
3.963
0.00
0.1

66085.0
65634.0
66106.0
66515.0
66491.0
66151.0
66163.67
322.74
0.5

NA

Precision for Diclofenac Sodium Standard Preparation

Number of Injection

Retention Time, Min

Area

Injection-1
Injection-2
Injection-3
Injection-4
Injection-5
Injection-6
Mean
SD
RSD

13.880
13.883
13.878
13.882
13.878
13.878
13.880
0.00
0.0

525656.0
523081.0
526085.0
527332.0
525523.0
523476.0
525192.17
1618.82
0.3

Prepared by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Precision for Benzyl Alcohol - Sample Preparation
Sample preparation
Retention Time, Min
3.962
Sample-1, 60%
3.963
3.967
Sample-2, 60%
3.963
3.961
Sample-3, 60%
3.964
3.968
Sample-1, 100%
3.967
3.965
Sample-2, 100%
3.965
3.967
Sample-3, 100%
3.971
3.964
Sample-4, 100%
3.962
3.964
Sample-5, 100%
3.965
3.962
Sample-6, 100%
3.961
3.964
Sample-1, 140%
3.961
3.958
Sample-2, 140%
3.958

Area
40175.1
40182.9
40651.4
40277.6
39947.8
39820.5
67500.2
67968.1
64247.4
67563.1
69998.7
66977.4
67539.5
67173.2
67088.2
67687.2
67834.4
67962.5
92625.2
92683.9
92203.8
92108.1

% Assay
106.294
106.315
107.554
106.565
105.693
105.356
107.154
107.897
101.990
107.254
111.120
106.324
107.217
106.635
106.500
107.451
107.685
107.888
105.028
105.095
104.550
104.442

3.962
3.962
3.96

Sample-3, 140%
Mean
SD
RSD

92776.6
91831.5
66867.7

105.200
104.128
106.3
1.76
1.7

Confidence Interval
at 95%

0.70

Calculation: Aspl

x Wstd x
Astd
200

5 x
100

100 x 200 x Wt/mL x P


Wspl
3
LC

(Benzyl alcohol 60%)

Aspl x Wstd x
Astd
200

5 x
100

100 x 200 x Wt/mL x P


Wspl
5
LC

(Benzyl alcohol 100%)

Aspl x Wstd x
Astd
200

5 x
100

100 x 200 x Wt/mL x P


Wspl
7
LC

(Benzyl alcohol 140%)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL
Prepared by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Precision for Diclofenac Sodium - Sample Preparation
Sample preparation
Retention Time, Min
13.928
Sample-1, 60%
13.951
13.948
Sample-2, 60%
13.942
13.885
Sample-3, 60%
13.900
13.908
Sample-1, 100%
13.909
13.907
Sample-2, 100%
13.908
13.908
Sample-3, 100%
13.911
13.912
Sample-4, 100%
13.911
13.915
Sample-5, 100%
13.919
13.918
Sample-6, 100%
13.916
13.921
Sample-1, 140%
13.915
13.916
Sample-2, 140%
13.913

Area
313396.9
312056.1
311007.0
313381.4
313844.4
311517.2
517414.70
520946.90
516043.60
515896.90
516788.10
517413.70
517210.80
516075.10
517070.90
517837.60
517088.10
516452.60
718239.40
720390.00
721367.60
721620.80

% Assay
99.789
99.362
99.028
99.784
99.931
99.190
98.850
99.525
98.588
98.560
98.730
98.850
98.811
98.594
98.784
98.931
98.788
98.666
98.012
98.306
98.439
98.473

Sample-3, 140%
Mean
SD
RSD

13.922
13.919
13.92

727917.50
722765.90
517239.30

Confidence Interval
at 95%

0.20

Aspl x Wstd x 5
x
60%)
Calculation:
Astd
50
100
Aspl x Wstd x 5
x
100%)
Astd
50
100
Aspl x Wstd x 5
x
140%)
Astd
50
100
Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL
Prepared by:
Date:

99.333
98.630
98.9
0.49
0.5

100
Wspl
100
Wspl
100
Wspl

x 200 x Wt/mL x P
3

LC

x 200 x Wt/mL x P
5

(Diclofenac sodium

LC

x 200 x Wt/mL x P
7

(Diclofenac sodium

LC

(Diclofenac sodium

ATTACHMENT 6.0

Sample
4.07
NA
NA

Reviewed by:
Date:

ATTACHMENT 6.0

% Assay
106.294
106.315
107.554
106.565
105.693
105.356
107.154
107.897
101.990
107.254
111.120
106.324
107.217
106.635
106.500
107.451
107.685
107.888
105.028
105.095
104.550
104.442

105.200
104.128
106.3
1.76
1.7

= Mean assay Confidence Interval


Confidence Interval at 95%
(Benzyl Alcohol)

= 106.3% 0.70
= 105.6% - 107.0%

0.70

Reviewed by:
Date:

ATTACHMENT 6.0

% Assay
99.789
99.362
99.028
99.784
99.931
99.190
98.850
99.525
98.588
98.560
98.730
98.850
98.811
98.594
98.784
98.931
98.788
98.666
98.012
98.306
98.439
98.473

99.333
98.630
98.9
0.49
0.5
0.20

= Mean assay Confidence Interval


Confidence Interval at 95%
(Diclofenac Sodium)

= 98.9% 0.20
= 98.70% - 99.10%

Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Benzyl Alcohol

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Standard and Preparation for Benzyl Alcohol


Standard for
Benzyl Alcohol

Sample

Weight, mg

167.64

4.06

Purity, %

100%

NA

Compound

Condition

Analyst

Date

Instrument ID

Yaakob

31-Oct-2015

Alliance III

Repeatability for Benzyl Alcohol Standard Preparation


Std

Std-2

Number of Injection

Area

Average area

SD

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

% RSD
Benzyl Alcohol

64112.5

153.26

0.24

Injection-1

63993.8

Injection-2

63878.6

Injection-3

64088.8

Injection-4

64215.2

Injection-5

64241.0

Injection-6

64257.5

Mean
SD
RSD

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi:nLiearineartyity

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi:nLiearineartyity
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Benzyl Alcohol

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Repeatability for Benzyl Alcohol Sample Preparation


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Assay Calculation:

Area

% Assay

% Mean assay

SD

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

% RSD
Benzyl Alcohol

Injection-1

64374.4

105.72

Injection-2

64660.3

106.19

105.96

0.33

0.31

Injection-1

65910.5

108.24

Injection-2

64939.8

106.65

107.45

1.13

1.05

Injection-1

64869.1

106.53

Injection-2

65388.4

107.39

106.96

0.60

0.56

Injection-1

65651.2

107.82

Injection-2

65430.3

107.46

107.64

0.26

0.24

Injection-1

65213.3

107.10

Injection-2

65581.8

107.70

107.40

0.43

0.40

Injection-1

65875.5

108.19

Injection-2

64315.8

105.63

106.91

1.81

1.69

Number of Injection

Mean

107.05

SD

0.608

RSD

0.6

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

(Benzyl alcohol)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

Analytical Method Validation : Linearity


Prepared by:

Reviewed by:

Date:

Date:

ATTACHMENT 7.0

Benzyl Alcohol

tion

ATTACHMENT 7.0

Benzyl Alcohol

tion

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Diclofenac sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Standard and Preparation for Diclofenac sodium


Standard for Diclofenac
Sodium

Compound

Sample

Weight, mg

100.09

Purity, %

100.0

4.060
NA

Condition

Analyst

Date

Instrument ID

Yaakob

31-Oct-2015

Alliance III

Area

Average area

SD

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

% RSD
Diclofenac sodium

521912

2705.33

0.52

Repeatability for Diclofenac sodium Standard Preparation


Std

Std-2

Number of Injection
Injection-1

525911.1

Injection-2

520713.1

Injection-3

523102.1

Injection-4

523450.3

Injection-5

519368.9

Injection-6

518926.4

Mean
SD
RSD

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi:nLiearinearityty
east squares regression line, y = a + bx)
Reviewed (Lby:

Date:

Date:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearnearityity
(Least squares regression line, y = a + bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Diclofenac Sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Repeatability for Diclofenac Sodium Sample Preparation


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Assay Caculation:

Area

% Assay

% Mean assay

SD

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

497731.6

95.92

Injection-2

494980.3

95.39

95.66

0.37

Injection-1

510026.4

98.29

Injection-2

514605.3

99.17

98.73

0.62

Injection-1

520881.2

100.38

Injection-2

512866.1

98.84

99.61

1.09

Injection-1

516520.6

99.54

Injection-2

515755.5

99.40

99.47

0.10

Injection-1

499107.4

96.19

Injection-2

499520.3

96.27

Injection-1
Injection-2
Mean
SD
RSD

494874.1
498580.6

95.37
96.09

Number of Injection

Aspl x Wstd x 5 x 100 x 200 x Wt/mL x P


Astd
200
100
Wspl
5
LC

96.23
95.73
97.57
1.896
1.9

(Diclofenac sodium)

AnalAnalytiyctalicalMetMethodhodValValid0.06
atidationio:nLi: nLiearnearityity
(Least squares regression line, y = a + bx)
0.51

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

Analytical Method Validation :Linearity


Prepared by:
Date:

Reviewed by:
Date:

ATTACHMENT 7.0

dium

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearnearityity
(Least squares regression line, y = a + bx)

ATTACHMENT 7.0

dium

% RSD
Diclofenac sodium
0.39
0.63
1.10
0.10

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearnearityity
(Least squares regression line, y = a + bx)

0.06
0.53

Analytical Method Validation : Linearity

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Precision (Intermediate Precision) of Benzyl Alcohol
Diclofenac Sodium 75 mg /3 mL Injection

Standard and Preparation for Benzyl Alcohol done Analyst 2 (Aina), Day 2
Standard for Benzyl
Alcohol

Compound

Sample

Weight, mg

169.82

Purity, %

100%

4.067
NA

Condition

Analyst

Date

Instrument ID

Aina

8-Nov-15

Alliance VI

Intermediate precision for Benzyl Alcohol Standard Preparation done by Analyst 2 (Aina), Day 2 using Instrument 2
Area
Average area
SD
Std
Number of Injection
Benzyl Alcohol
Benzyl Alcohol
Benzyl Alcohol

Std-2

Injection-1

63718

Injection-2

64242

Injection-3

64325

Injection-4

64173

Injection-5

64335

Injection-6

64070

64144

230.93

% RSD
Benzyl Alcohol

0.36

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearineartyity
(Least squares regression line, y = a + bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Precision (Intermediate Precision) of Benzyl Alcohol
Diclofenac Sodium 75 mg /3 mL Injection

Intermediate precision for Benzyl Alcohol Sample Preparation done by Analyst 2 (Aina), Day 2 using Instrument 2
Area
% Assay
% Mean assay
Sample
Number of Injection
Benzyl Alcohol
Benzyl Alcohol
Benzyl Alcohol
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Assay Calculation:

Injection-1

64479.6

107.03

Injection-2

63619.0

105.61

Injection-1

63506.2

105.42

Injection-2

64045.3

106.31

Injection-1

64272.4

106.69

Injection-2

64128.0

106.45

Injection-1

65793.1

109.21

Injection-2

66921.4

111.09

Injection-1

64807.1

107.58

Injection-2

65716.4

109.09

Injection-1

64822.9

107.60

Injection-2

64817.1

107.59

1.01

105.87

0.63

106.57

0.17

110.15

1.32

108.33

1.07

107.60

0.01

107.47

SD

1.591

RSD

1.5

100 x 200 x Wt/mL x P


Wspl
5
LC

(Benzyl alcohol)

Benzyl Alcohol

106.32

Mean

Aspl x Wstd x 5 x
Astd
200
100

SD

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearineartyity
(Least squares regression line, y = a + bx)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi:nLiearnearityity
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Precision (Intermediate Precision) of Benzyl Alcohol
Diclofenac Sodium 75 mg /3 mL Injection

Assay (%) Comparison of Benzyl Alcohol between Day 1 and Day 2


Day 1 (Yaakob)_Set 1
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Day 2 (Aina)_Set 2

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

105.72

Injection-2

106.19

Injection-1

108.24

Injection-2

106.65

Injection-1

106.53

Injection-2

107.39

Injection-1

107.82

Injection-2

107.46

Injection-1

107.10

Injection-2

107.70

Injection-1

108.19

Injection-2

105.63

105.96
107.45
106.96
107.64
107.40
106.91

107.0341264017
105.6055572235
105.4183127391
106.3132019373
106.6901808594
106.4504813598
109.2144954646
111.0874382995
107.5777661947
109.087175855
107.6039937022
107.594365883

106.32
105.87
106.57
110.15
108.33
107.60

Mean

107.1

107.5

SD

0.608

1.590

%RSD

0.6

1.5

AnAanlyatliyctaiclaMel MethothdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity

AnAanlyatliyctaiclaMel MethothdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity
(Least squares regres ion line, y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Precision (Intermediate Precision) of Benzyl Alcohol
Diclofenac Sodium 75 mg /3 mL Injection

Summary for Intermediate Precision (Benzyl Alcohol)


Parameter

Mean Assay (%)

Day

Analyst

Instrument

Location

Analyst 1, Day 1, Instrument 1

Yaakob

HPLC III

Analytical Laboratory
Bukit Raja

107.1

Analyst 2, Day 2, Instrument 2

Aina

HPLC VI

Analytical Laboratory
Bukit Raja

107.5

Difference

-0.4

Benzyl Alcohol

Assay (%) Benzyl Alcohol Comparison between Analyst 1, Day 1 and Analyst 2, Day 2
Benzyl Alcohol
Day 1 (Yaakob)_set 1
105.72

Day 2 (Aina)_set 2

106.19

105.61

108.24

105.42

Average (Assay)

106.65

106.31

Significance Level

106.53

106.69

Stdev

107.39

106.45

% RSD

1.2

107.82

109.21

Sample size

24

107.46

111.09

95% Confidence Interval (CI)

107.10

107.58

107.70

109.09

108.19

107.60

105.63

107.59

107.03
Benzyl Alcohol

Confidence Interval (Benzyl


Alcohol)

107.26
0.05
1.3137

0.53
= Mean assay Confidence Interval
= 107.26% 0.53
= 106.73% - 107.79%

AnAanlyatliyctaiclaMleMtheothdoVdaVliadlidtaotnio:nL:inLeinaeraitryit

AnAanlyatliyctaiclaMleMtheothdoVdaVliadlidtaotnio:nL:inLeinaeraitryit
(Leastsquares regres ionline,y=a +bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Characteristics
Finished Product

AMVP-06/15
Solution stability

AnalyticalMethodValidation:Linearity

AnalyticalMethodValidation:Linearity
(Leastsquaresregres ionline,y=a+bx)
Prepared by:

Reviewed by:

Date:

Date:

ATTACHMENT 7.0

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearineartyity
(Least squares regression line, y = a + bx)

ATTACHMENT 7.0

% RSD
Benzyl Alcohol
0.95
0.60
0.16
1.20

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearineartyity 0.99


(Least squares regression line, y = a + bx)
0.01

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi:nLiearnearityity
(Least squares regression line, y = a + bx)

ATTACHMENT 7.0

nzyl Alcohol
n

AnAanlyatliyctaiclaMel MethothdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity

AnAanlyatliyctaiclaMel MethothdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity
(Least squares regres ion line, y= a + bx)

ATTACHMENT 7.0

% RSD
Benzyl Alcohol
0.6
1.5

AnAanlyatliyctaiclaMleMtheothdoVdaVliadlidtaotnio:nL:inLeinaeraitryit

AnAanlyatliyctaiclaMleMtheothdoVdaVliadlidtaotnio:nL:inLeinaeraitryit
(Leastsquares regres ionline,y=a +bx)

ATTACHMENT 11.0

AnalyticalMethodValidation:Linearity

AnalyticalMethodValidation:Linearity
(Leastsquaresregres ionline,y=a+bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 7.0

Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Diclofenac Sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Standard and Preparation for Diclofenac Sodium done Analyst 2 (Aina), Day 2
Standard for
Diclofenac Sodium

Sample

Weight, mg

100.14

4.0671

Purity, %

100.0

NA

Compound

Condition

Analyst

Date

Instrument ID

Aina

4-Nov-15

Alliance VI

Intermediate precision for Diclofenac Sodium Standard Preparation done by Analyst 2 (Aina), Day 2 using Instrument 2
Std

Std-2

Number of Injection

Area

Average area

SD

% RSD

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

515209

5066.86

0.98

Injection-1

518113

Injection-2

509254

Injection-3

517982

Injection-4

522158

Injection-5

510107

Injection-6

513640

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 7.0

Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Diclofenac Sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Intermediate precision for Diclofenac Sodium Sample Preparation done by Analyst 2 (Aina), Day 2 using Instrument 2
Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Assay Caculation:

Area

% Assay

% Mean assay

SD

% RSD

Diclofena sodium

Diclofenac sodium

Diclofena sodium

Diclofena sodium

Diclofena sodium

Injection-1

506353

98.73

Injection-2

500465

97.58

98.16

0.81

0.83

Injection-1

504377

98.35

Injection-2

510797

99.60

98.97

0.89

0.89

Injection-1

503361

98.15

Injection-2

500029

97.50

97.82

0.46

0.47

Injection-1

497710

97.05

Injection-2

494054

96.33

96.69

0.50

0.52

Injection-1

499262

97.35

Injection-2

493716

96.27

96.81

0.76

0.79

Injection-1

495295

96.57

Injection-2

495561

96.63

96.60

0.04

0.04

Number of Injection

Mean

97.51

SD

0.964

RSD

1.0

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

(Diclofenac sodium)

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi:nLiearnearityity
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

AnalAnalytiyctalicalMetMethodhodValValidiatdatioinon: Li: Linearinearityty


(Least squares regression line, y = a + bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 7.0

Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Diclofenac Sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Assay (%) Comparison of Diclofenac Sodium between Day 1 and Day 2


Day 1 (Yaakob)_Set 1
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Day 2 (Aina)_Set 2

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Diclofenac Sodium

Diclofenac Sodium

Diclofenac Sodium

Diclofenac Sodium

Injection-1

95.92

Injection-2

95.39

Injection-1

98.29

Injection-2

99.17

Injection-1

100.38

Injection-2

98.84

Injection-1

99.54

Injection-2

99.40

Injection-1

96.19

Injection-2

96.27

Injection-1

95.37

Injection-2

96.09

98.73

95.66

97.58
98.35

98.73

99.60
98.15

99.61

97.50
97.05

99.47

96.33
97.35

96.23

96.27
96.57

95.73

96.63

98.16
98.97
97.82
96.69
96.81
96.60

Mean

97.57

97.51

SD

1.896

0.964

%RSD

1.944

1.0

AnAanlyatliyctaiclaMel MethothdoVdaVliadlaidtiaotnio:nL:inLeinaeraitryity
(Least squares regres ion line, y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 7.0

Reference Protocol No

AMVP-06/15

Characteristics

Precision (Intermediate Precision) of Diclofenac Sodium

Finished Product

Diclofenac Sodium 75 mg /3 mL Injection

Summary for Intermediate Precision (Diclofenac Sodium)


Parameter

Mean Assay (%)

% RSD

Diclofenac Sodium

Diclofenac Sodium

Analytical Laboratory Bukit


Raja

97.57

1.9

Analytical Laboratory Bukit


Raja

97.51

1.0

Difference

0.064

Day

Analyst

Instrument

Location

Analyst 1, Day 1, Instrument 1

Yaakob

HPLC III

Analyst 2, Day 2, Instrument 2

Aina

HPLC VI

Assay (%) Diclofenac sodium Comparison between Analyst 1, Day 1 and Analyst 2, Day 2
Diclofenac Sodium
Day 1 (Yaakob)_set 1

Day 2 (Aina)_set 2

95.92

98.73

95.39

97.58

98.29

98.35

Average (Assay)

99.17

99.60

Significance Level

100.38

98.15

Stdev

1.4709

98.84

97.50

%RSD

1.51

99.54

97.05

Sample size

99.40

96.33

95% Confidence Interval (CI)

96.19

97.35

96.27

96.27

95.37

96.57

96.09

96.63

Diclofenac Sodium

Confidence Interval (Diclofenac


Sodium)

97.54
0.05

24
0.59
= Mean assay Confidence Interval
= 97.54% 0.59
= 96.95% - 98.13%

AnAanlyatliyctaiclaMelMtheothdoVdaVliadlidtaotnio:nL:inLeinaeraitryity
(Least squares regres ionline,y=a +bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 9.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium


Compound

Standard for Benzyl Alcohol

Standard for
Diclofenac Sodium

Sample

Weight, mg

165.21

100.12

4.06

Purity, %

100.0

100.0

NA

Benzyl Alcohol Standard Injection (pH 2.4)

Std (Reduced)

Std-2

Assay Calculation:

Benzyl Alcohol Standard Injection (pH 2.6)


Area

Number of Injection

Retention time

Injection-1

4.028

Injection-2

4.026

Injection-3

4.026

64453.1

Injection-4

4.028

65763.5

Injection-5

4.033

Injection-6

4.035

Area

Number of Injection

Retention time

66312.7

Injection-1

4.014

65697.8

66035.7

Injection-2

4.008

67760.6

Injection-3

4.002

65845.6

Injection-4

4.004

67247.5

65102.2

Injection-5

4.007

68045.4

66957.6

Injection-6

4.007

65472.1

Mean

65770.8

Mean

66678.2

SD

889.46

SD

1137.89

RSD

1.35

RSD

1.71

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

Std (Increased)

Benzyl Alcohol

Std-2

(Benzyl alcohol)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

Benzyl alcohol

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 9.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Robustness for Benzyl Alcohol Sample Preparation (pH 2.4)

Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

68206.7

107.61

Injection-2

67493.3

106.48

Injection-1

67914.6

107.15

Injection-2

66479.5

104.88

Injection-1

67789.0

106.95

Injection-2

66917.2

105.57

Injection-1

67660.6

106.75

Injection-2

66842.1

105.46

Injection-1

67284.1

106.15

Injection-2

67242.9

106.09

Injection-1

66462.8

104.86

Injection-2

66213.2

104.46

Number of Injection

107.05
106.02
106.26
106.10
106.12

AnalAnalytiyctalicalMetMethodhodValValidatidatioinon: Li: Linearinearityty


(Least squares regression line, y = a + bx)

104.66

Mean

106.03

SD

0.771

RSD

0.727

Robustness for Benzyl Alcohol Sample Preparation (pH 2.6)

Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

67613.1

105.22

Injection-2

68814.5

107.09

Injection-1

67168.3

104.53

Injection-2

67645.0

105.27

Injection-1

68423.9

106.48

Injection-2

68087.2

105.96

Injection-1

67478.1

105.01

Injection-2

67413.8

104.91

Injection-1

67969.6

105.77

Injection-2

67750.6

105.43

Injection-1

67023.9

104.30

Injection-2

67309.0

104.75

Number of Injection

106.15
104.90
106.22
104.96
105.60
104.52

Mean

105.39

SD

0.706

Prepared by:

An0.670
AnaalytliyctaiclaMetl MehtohdoVad Vallidaidtaiotnio:nL:inLieaneraitryity
(Least squares regression line, y= a + bx)
Reviewed by:

Date:

Date:

RSD

AnalAnalytiyctalicalMetMethodhodValValidatidatioinon: Li: Linearnearityity


(Least squares regression line, y = a + bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 9.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Benzyl Alcohol


Reduced(pH 2.4)
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Actual(pH 2.5)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol
105.72

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

107.61

Injection-2

106.48

Injection-1

107.15

Injection-2

104.88

Injection-1

106.95

Injection-2

105.57

Injection-1

106.75

Injection-2

105.46

Injection-1

106.15

Injection-2

106.09

Injection-1

104.86

Injection-2

104.46

107.05

106.19
108.24

106.02

106.65
106.53

106.26

107.39
107.82

106.10

107.46
107.10

106.12

107.70
108.19

104.66

105.63

105.96
107.45
106.96
107.64
107.40
106.91

105.22
107.09
104.53
105.27
106.48
105.96
105.01
104.91
105.77
105.43
104.30
104.75

106.15
104.90
106.22
104.96
105.60
104.52

Mean

106.03

107.05

105.39

SD

0.771

0.608

0.706

%RSD

0.727

0.568

0.670

Summary for Robustness (pH)-Benzyl Alcohol


Mean Assay (%)

% RSD

Difference in Sample (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Actual (pH 2.5)

107.05

0.6

Reduced (pH 2.4)

106.03

0.7

1.0

Increased (pH 2.6)

105.39

0.7

-1.5

Parameter

Increased(pH 2.6)

Assay (%)

AnAanlyatliyctaiclaMelMethothdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity
(Least squares regres ionline, y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 9.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Benzyl Alcohol

Finished Product

Diclofenac sodium 75mg/3mL Injection

Assay (%) Comparison of Benzyl Alcohol


Benzyl Alcohol
Actual(pH 2.5)
105.72

Reduced(pH 2.4)
107.61

Increased(pH 2.6)
105.22

106.19

106.48

107.09

108.24

107.15

104.53

Average (Assay)

106.65

104.88

105.27

Significance Level

106.53

106.95

106.48

Stdev

1.1220

107.39

105.57

105.96

%RSD

1.06

107.82

106.75

105.01

Sample size

107.46

105.46

104.91

95% Confidence Interval (CI)

107.10

106.15

105.77

Confidence Interval

= Mean assay Confidence Interval

107.70

106.09

105.43

(Benzyl Alcohol)

= 106.16% 0.37

108.19

104.86

104.30

105.63

104.46

104.75

Benzyl Alcohol
106.16
0.05

36
0.37

= 105.79% - 106.53%

AnalyticalMethodValidation:Linearity
Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium


Compound

Standard for Benzyl Alcohol

Standard for
Diclofenac Sodium

sample

Weight, mg

165.21

100.12

4.06

Purity, %

100.0

100.0

NA

Diclofenac sodium Standard Injection (pH 2.4)


Std (Reduced)

Std-2

Assay Caculation:

Diclofenac sodium Standard Injection (pH 2.6)

Number of Injection

Retention time

Injection-1

14.956

Injection-2

14.959

Injection-3

Area

Area

Number of Injection

Retention time

495226.3

Injection-1

14.214

498176.4

495325.3

Injection-2

14.176

496043.3

14.965

497768.6

Injection-3

14.156

495719.8

Injection-4

14.971

497902.0

Injection-4

14.157

496452.0

Injection-5

14.977

497920.1

Injection-5

14.163

494473.7

Injection-6

14.982

498876.9

Injection-6

14.161

493980.2

Mean

497169.9

Mean

495807.6

SD

1519.95

SD

1497.22

RSD

0.31

RSD

0.30

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

Std (Increased)

Diclofenac sodium

Std-2

(Diclofenac sodium)

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

Diclofenac sodium

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Robustness for Diclofenac sodium Sample Preparation (pH 2.4)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

472244.4

95.57

Injection-2

476647.1

96.46

Injection-1

473907.2

95.91

Injection-2

474708.9

96.07

Injection-1

471607.0

95.44

Injection-2

469800.2

95.07

Injection-1

473823.1

95.89

Injection-2

476065.8

96.34

Injection-1

471112.5

95.34

Injection-2

470954.6

95.31

Injection-1

470316.3

95.18

Injection-2

478320.2

96.80

Number of Injection

96.01
95.99
95.26
96.12
95.32

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

95.99

Mean

95.78

SD

0.383

RSD

0.400

Robustness for Diclofenac sodium Sample Preparation (pH 2.6)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

471494.5

95.68

Injection-2

476443.0

96.68

Injection-1

471620.2

95.70

Injection-2

471846.1

95.75

Injection-1

479403.7

97.28

Injection-2

475638.5

96.52

Injection-1

478940.5

97.19

Injection-2

480665.4

97.54

Injection-1

472406.7

95.86

Injection-2

470988.4

95.58

Injection-1

475076.7

96.41

Injection-2

474839.0

96.36

Number of Injection

96.18
95.73
96.90
97.37
95.72
96.38

Mean

96.38

SD

0.655

RSD

0.679

Prepared by:

AnAnaalytliyctaiclaMel MetthohdodVaVallidaidtationio:nL:inLiearneaitryity
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearnearityity
(Least squares regression line, y = a + bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Diclofenac sodium


Reduced(pH 2.4)
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Actual(pH 2.5)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium
95.92

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

95.57

Injection-2

96.46

Injection-1

95.91

Injection-2

96.07

Injection-1

95.44

Injection-2

95.07

Injection-1

95.89

Injection-2

96.34

Injection-1

95.34

Injection-2

95.31

Injection-1

95.18

Injection-2

96.80

96.01

95.39
98.29

95.99

99.17
100.38

95.26

98.84
99.54

96.12

99.40
96.19

95.32

96.27
95.37

95.99

96.09

95.66
98.73
99.61
99.47
96.23
95.73

95.68
96.68
95.70
95.75
97.28
96.52
97.19
97.54
95.86
95.58
96.41
96.36

96.18
95.73
96.90
97.37
95.72
96.38

Mean

95.78

97.57

96.38

SD

0.383

1.896

0.655

%RSD

0.400

1.944

0.679

Summary for Robustness (pH)-Diclofenac sodium


Mean Assay (%)

% RSD

Difference in Sample (%)

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Actual (pH 2.5)

97.57

1.9

Reduced (pH 2.4)

95.78

0.4

1.8

Increased (pH 2.6)

96.38

0.7

-1.2

Parameter

Increased(pH 2.6)

Assay (%)

AnAanlyatliyctaiclaMelMethtohdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity
(Least squares regres ion line,y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (pH)-Diclofenac Sodium

Finished Product

Diclofenac sodium 75mg/3mL Injection

Assay (%) Comparison of Diclofenac Sodium


Diclofenac Sodium
Actual(pH 2.5)

Reduced(pH 2.4)

95.92

95.57

Increased(pH 2.6)
95.68

95.39

96.46

96.68

98.29

95.91

95.70

Average (Assay)

99.17

96.07

95.75

Significance Level

100.38

95.44

97.28

Stdev

1.3779

98.84

95.07

96.52

%RSD

1.43

99.54

95.89

97.19

Sample size

99.40

96.34

97.54

95% Confidence Interval (CI)

96.19

95.34

95.86

Confidence Interval

= Mean assay Confidence Interval

96.27

95.31

95.58

(Diclofenac sodium)

= 96.58% 0.45

95.37

95.18

96.41

96.09

96.80

96.36

Diclofenac sodium
96.58
0.05

36
0.45

= 96.13% - 97.03%

AnalyticalMethodValidation:Linearity
Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Flow rate)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium


Compound

Standard for Benzyl Alcohol

Standard for
Diclofenac Sodium

sample

Weight, mg

162.4

100.6

4.05

Purity, %

100.0

100.0

NA

Benzyl Alcohol Standard Injection (0.8 mL/min)


Std (Reduced)

Std-2

Assay Calculation:

Benzyl Alcohol Standard Injection (1.2 mL/min)

Number of Injection

Retention time

Injection-1

4.997

Injection-2

4.998

Injection-3

Area

Area

Number of Injection

Retention time

77774.6

Injection-1

3.357

50375.7

77971.9

Injection-2

3.358

50281.1

4.997

77798.0

Injection-3

3.359

50657.4

Injection-4

5.000

77580.8

Injection-4

3.357

50854.4

Injection-5

5.000

77102.8

Injection-5

3.356

50954.6

Injection-6

5.004

78686.4

Injection-6

3.356

50370.7

Mean

77819.1

Mean

50582.3

SD

519.20

SD

281.59

RSD

0.67

RSD

0.56

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

Std (Increased)

Benzyl Alcohol

Std-2

(Benzyl alcohol)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

Prepared by:

Reviewed by:

Date:

Date:

Benzyl Alcohol

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Flow rate)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Robustness for Benzyl Alcohol Sample Preparation (0.8 mL/min)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Benzyl alcohol

Benzyl alcohol

Benzyl alcohol

Injection-1

80780.3

106.14

Injection-2

80834.1

106.21

Injection-1

80811.0

106.18

Injection-2

80381.1

105.62

Injection-1

81514.0

107.11

Injection-2

81704.7

107.36

Injection-1

81210.1

106.71

Injection-2

80853.6

106.24

Injection-1

80896.7

106.30

Injection-2

80620.3

105.93

Injection-1

81732.1

107.39

Injection-2

80952.6

106.37

Number of Injection

106.18
105.90
107.23
106.47
106.11

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearneariityty
(Least squares regression line, y = a + bx)

106.88

Mean

106.46

SD

0.507

RSD

0.476

Robustness for Benzyl Alcohol Sample Preparation (1.2 mL/min)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

52431.1

105.99

Injection-2

52279.7

105.68

Injection-1

52353.7

105.83

Injection-2

53256.2

107.66

Injection-1

53133.0

107.41

Injection-2

52546.1

106.22

Injection-1

52058.4

105.24

Injection-2

51974.1

105.07

Injection-1

52049.0

105.22

Injection-2

51585.1

104.28

Injection-1

52766.9

106.67

Injection-2

51940.6

105.00

Number of Injection

105.84
106.74
106.81
105.15
104.75
105.83

Mean

105.85

SD

0.829

RSD

0.783

Prepared by:

AnalAnalytiyctaiclalMetMethodhoVald Validatidationio:nLi:nLienareaitryity
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi:nLiearnearityity
(Least squares regression line, y = a + bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Flow rate)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Benzyl Alcohol


Reduced(0.8 mL/min)
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Actual(1.0 mL/min)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol
105.721275485

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

106.14

Injection-2

106.21

Injection-1

106.18

Injection-2

105.62

Injection-1

107.11

Injection-2

107.36

Injection-1

106.71

Injection-2

106.24

Injection-1

106.30

Injection-2

105.93

Injection-1

107.39

Injection-2

106.37

106.18

106.1908054948
108.2439933864

105.90

106.6498248643
106.5337151347

107.23

107.386555058
107.8181482254

106.47

107.4553669062
107.0989905085

106.11

107.7041734696
108.1865133223

106.88

105.6250374348

105.96
107.45
106.96
107.64
107.40
106.91

105.99
105.68
105.83
107.66
107.41
106.22
105.24
105.07
105.22
104.28
106.67
105.00

105.84
106.74
106.81
105.15
104.75
105.83

Mean

106.46

107.05

105.85

SD

0.507

0.608

0.829

%RSD

0.476

0.568

0.783

Summary for Robustness (Flow rate)-Benzyl Alcohol


Mean Assay (%)

% RSD

Difference in Sample (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Actual

107.05

0.6

Reduced

106.46

0.5

0.5

Increased

105.85

0.8

-1.1

Parameter

Increased(1.2 mL/min)

Assay (%)

AnAanlyatliyctaiclaMelMethothdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity
(Leastsquares regres ionline, y=a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Flow rate)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Assay (%) Comparison of Benzyl Alcohol


Benzyl Alcohol
Actual(1.0 mL/min)
105.72

Reduced(0.8 mL/min)
106.14

Increased(1.2 mL/min)
105.99

106.19

106.21

105.68

108.24

106.18

105.83

Average (Assay)

106.65

105.62

107.66

Significance Level

106.53

107.11

107.41

Stdev

0.9587

107.39

107.36

106.22

%RSD

0.90

107.82

106.71

105.24

Sample size

107.46

106.24

105.07

95% Confidence Interval (CI)

107.10

106.30

105.22

Confidence Interval

= Mean assay Confidence Interval

107.70

105.93

104.28

(Benzyl Alcohol)

= 106.46% 0.31

108.19

107.39

106.67

105.63

106.37

105.00

Benzyl Alcohol
106.46
0.05

36
0.31

= 106.15% - 106.77%

AnalyticalMethodValidation:Linearity
Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Flow rate)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium


Compound

Standard for Benzyl Alcohol

Standard for
Diclofenac Sodium

sample

Weight, mg

162.4

100.6

4.05

Purity, %

100.0

100.0

NA

Diclofenac sodium Standard Injection (0.8 mL/min)

Std (Reduced)

Std-2

Assay Caculation:

Diclofenac sodium Standard Injection (1.2 mL/min)


Area

Number of Injection

Retention time

Injection-1

17.903

Injection-2

17.906

Injection-3

17.904

676461.7

Injection-4

17.905

679593.5

Injection-5

17.906

Injection-6

17.918

Area

Number of Injection

Retention time

679352.9

Injection-1

12.042

437417.6

675288.9

Injection-2

12.040

440646.6

Injection-3

12.044

440992.8

Injection-4

12.040

440803.5

674127.0

Injection-5

12.043

437173.8

676749.5

Injection-6

12.039

435876.1

Mean

676928.9

Mean

438818.4

SD

2180.21

SD

2251.00

RSD

0.32

RSD

0.51

Aspl x Wstd x 5 x 100 x 200 x Wt/mL x P


Astd
200
100
Wspl
5
LC

Std (Increased)

Diclofenac sodium

Std-2

(Diclofenac sodium)

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidiatonio:nLi: nLiearinearityty
Reviewed by:(Least squares regression line, y = a + bx)

Date:

Date:

Diclofenac sodium

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Flow rate)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Robustness for Diclofenac sodium Sample Preparation (0.8 mL/min)

Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

645002.8

96.57

Injection-2

643630.5

96.36

Injection-1

643146.2

96.29

Injection-2

642555.6

96.20

Injection-1

654093.6

97.93

Injection-2

649405.4

97.22

Injection-1

642353.5

96.17

Injection-2

640857.9

95.94

Injection-1

650696.2

97.42

Injection-2

645208.3

96.60

Injection-1

654128.8

97.93

Injection-2

643044.9

96.27

Number of Injection

96.46
96.24
97.58
96.06
97.01

AnalAnalytiyctalicalMetMethodhodValValidatidatioinon: Li: Linearinearityty


(Least squares regression line, y = a + bx)

97.10

Mean

96.74

SD

0.582

RSD

0.601

Robustness for Diclofenac sodium Sample Preparation (1.2 mL/min)

Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

420957.6

97.22

Injection-2

420151.0

97.03

Injection-1

419115.6

96.79

Injection-2

417453.4

96.41

Injection-1

422675.1

97.62

Injection-2

420789.9

97.18

Injection-1

417981.8

96.53

Injection-2

415555.8

95.97

Injection-1

421546.7

97.36

Injection-2

420339.9

97.08

Injection-1

420549.8

97.13

Injection-2

419384.8

96.86

Number of Injection

97.13
96.60
97.40
96.25
97.22
96.99

Mean

96.93

SD

0.427

Prepared by:

Ana0.440
AnalytliyctalicaMel MethothdoVald Vaidlaidtaiotnio:nL:inLienaeraitryity
(Least squares regres ion line, y = a + bx)
Reviewed by:

Date:

Date:

RSD

AnalAnalytiyctalicalMetMethodhodValValidatidatioinon: Li: Linearnearityity


(Least squares regression line, y = a + bx)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-16/15

Characteristics

Robustness (Flow rate)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Diclofenac sodium


Reduced(0.8 mL/min)
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Actual(1.0 mL/min)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium
95.92

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

96.57

Injection-2

96.36

Injection-1

96.29

Injection-2

96.20

Injection-1

97.93

Injection-2

97.22

Injection-1

96.17

Injection-2

95.94

Injection-1

97.42

Injection-2

96.60

Injection-1

97.93

Injection-2

96.27

96.46

95.39
98.29

96.24

99.17
100.38

97.58

98.84
99.54

96.06

99.40
96.19

97.01

96.27
95.37

97.10

96.09

95.66
98.73
99.61
99.47
96.23
95.73

97.22
97.03
96.79
96.41
97.62
97.18
96.53
95.97
97.36
97.08
97.13
96.86

97.13
96.60
97.40
96.25
97.22
96.99

Mean

96.74

97.57

96.93

SD

0.582

1.896

0.427

%RSD

0.601

1.944

0.440

Summary for Robustness (Flow rate)-Diclofenac sodium


Mean Assay (%)

% RSD

Difference in Sample (%)

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Actual (1.0 mL/min)

97.57

1.9

Reduced (0.8 mL/min)

96.74

0.6

0.9

Increased (1.2 mL/min)

96.93

0.4

-0.7

Parameter

Increased(1.2 mL/min)

Assay (%)

AnAanlyatliyctaiclaMelMethothdoVdaVliadlaidtiaotnio:nL:inLeinaeraitryity
(Least squares regres ion line,y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 10.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Flow rate)-Diclofenac Sodium

Finished Product

Diclofenac sodium 75mg/3mL Injection

Assay (%) Comparison of Diclofenac Sodium


Diclofenac Sodium
Actual(1.0 mL/min)
95.92

Reduced(0.8 mL/min)
96.57

Increased(1.2 mL/min)
97.22

95.39

96.36

97.03

98.29

96.29

96.79

Average (Assay)

99.17

96.20

96.41

Significance Level

100.38

97.93

97.62

Stdev

1.1968

98.84

97.22

97.18

%RSD

1.23

99.54

96.17

96.53

Sample size

99.40

95.94

95.97

95% Confidence Interval (CI)

96.19

97.42

97.36

Confidence Interval

= Mean assay Confidence Interval

96.27

96.60

97.08

(Diclofenac Sodium)

= 97.08% 0.39

95.37

97.93

97.13

96.09

96.27

96.86

Diclofenac Sodium
97.08
0.05

36
0.39

= 96.69% - 97.47%

AnalyticalMethodValidation:Linearity
Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Temperature)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium


Compound

Standard for Benzyl Alcohol

Standard for Diclofenac


Sodium

sample

Standard for Benzyl Standard for Diclofenac


Alcohol
Sodium

Weight, mg

162.4

100.6

4.08

163.0

25.12

4.06

Purity, %

100.0

100.0

NA

100.0

100.0

NA

Benzyl Alcohol Standard Injection (25C)


Std (Reduced)

Std-2

Assay Calculation:

sample

Benzyl Alcohol Standard Injection (35C)

Number of Injection

Retention time

Injection-1

3.952

Injection-2

3.952

Injection-3

Area

Area

Number of Injection

Retention time

62027.0

Injection-1

3.969

63193.7

62004.0

Injection-2

3.969

63322.2

3.951

63577.3

Injection-3

3.969

63305.1

Injection-4

3.950

61858.0

Injection-4

3.968

66324.6

Injection-5

3.955

60582.7

Injection-5

3.965

64412.7

Injection-6

3.948

62052.2

Injection-6

3.968

64244.3

Mean

62017

Mean

64134

SD

950.64

SD

1193.22

RSD

1.53

RSD

1.86

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

Std (Increased)

Benzyl Alcohol

Std-2

(Benzyl alcohol)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

Prepared by:

Reviewed by:

Date:

Date:

Benzyl Alcohol

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Temperature)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Robustness for Benzyl Alcohol Sample Preparation (25C)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Benzyl alcohol

Benzyl alcohol

Benzyl alcohol

Injection-1

66817.7

109.36

Injection-2

66304.2

108.52

Injection-1

66785.2

109.30

Injection-2

67178.4

109.95

Injection-1

67038.5

109.72

Injection-2

66810.8

109.35

Injection-1

66121.2

108.22

Injection-2

66974.2

109.61

Injection-1

66656.9

109.09

Injection-2

66611.5

109.02

Injection-1

66951.7

109.58

Injection-2

66542.6

108.91

Number of Injection

108.94
109.63

Assay Calculation:

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

109.53
108.92
109.06

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

109.24

Mean

109.22

SD

0.304

RSD

0.278

Robustness for Benzyl Alcohol Sample Preparation (35C)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

68056.3

108.64

Injection-2

67275.3

107.39

Injection-1

67355.7

107.52

Injection-2

66974.1

106.91

Injection-1

67196.5

107.27

Injection-2

69597.2

111.10

Injection-1

67309.3

107.45

Injection-2

67494.0

107.74

Injection-1

68192.3

108.86

Injection-2

67655.7

108.00

Injection-1

66406.9

106.01

Injection-2

68786.8

109.80

Number of Injection

108.02
107.22
109.18
107.59
108.43
107.90

Mean

108.06

SD

0.686

RSD

0.635

Prepared by:

AnaAnalytliyctaiclaMetl MehothdoVald Vaidlaidtaiotnio:nLi:nLeinaeraitryity


(Least squares regres ion line, y = a + bx)
Reviewed by:

Date:

Date:

Aspl x Wstd x 5 x
Astd
200
100

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearnearityity
(Least squares regression line, y = a + bx)

100 x 200 x Wt/mL x P


Wspl
5
LC

(Benzyl alcohol)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Temperature)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Benzyl Alcohol


Reduced(25C)
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Actual(30C)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol
105.72

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

109.36

Injection-2

108.52

Injection-1

109.30

Injection-2

109.95

Injection-1

109.72

Injection-2

109.35

Injection-1

108.22

Injection-2

109.61

Injection-1

109.09

Injection-2

109.02

Injection-1

109.58

Injection-2

108.91

108.94

106.19
108.24

109.63

106.65
106.53

109.53

107.39
107.82

108.92

107.46
107.10

109.06

107.70
108.19

109.24

105.63

105.96
107.45
106.96
107.64
107.40
106.91

108.64
107.39
107.52
106.91
107.27
111.10
107.45
107.74
108.86
108.00
106.01
109.80

108.02
107.22
109.18
107.59
108.43
107.90

Mean

109.22

107.05

108.06

SD

0.304

0.608

0.686

%RSD

0.278

0.568

0.635

Summary for Robustness (temperature)-Benzyl Alcohol


Mean Assay (%)

% RSD

Difference in Sample (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Actual

107.05

0.6

Reduced

109.22

0.3

2.0

Increased

108.06

0.6

0.9

Parameter

Increased(35C)

Assay (%)

AnAanlyatliyctaiclaMelMethtohdoVdaVliadlaidtaiotnio:nL:inLienaeraitryity
(Least squares regres ion line,y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 8.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Temperature)-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Assay (%) Comparison of Benzyl Alcohol


Benzyl Alcohol
Actual(30C)
105.72

Reduced(25C)
109.36

Increased(35C)
108.64

106.19

108.52

107.39

108.24

109.30

107.52

Average (Assay)

106.65

109.95

106.91

Significance Level

106.53

109.72

107.27

Stdev

1.3157

107.39

109.35

111.10

%RSD

1.22

107.82

108.22

107.45

Sample size

107.46

109.61

107.74

95% Confidence Interval (CI)

107.10

109.09

108.86

Confidence Interval

= Mean assay Confidence Interval

107.70

109.02

108.00

(Benzyl Alcohol)

= 108.11% 0.43

108.19

109.58

106.01

105.63

108.91

109.80

Benzyl Alcohol
108.11
0.05

36
0.43

= 107.68% - 108.54%

AnalyticalMethodValidation:Linearity
Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 13.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Temperature)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium


Compound

Standard for Benzyl Alcohol

Standard for Diclofenac


Sodium

sample

Standard for Benzyl


Alcohol

Standard for Diclofenac


Sodium

sample

Weight, mg

162.4

100.6

4.08

163.0

25.12

4.06

Purity, %

100.0

100.0

NA

100.0

100.0

NA

Diclofenac sodium Standard Injection (35 C)

Diclofenac sodium Standard Injection (25C)


Std (Reduced)

Std-2

Assay Caculation:

Number of Injection

Retention time

Injection-1

15.034

Injection-2

14.965

Injection-3

Area

Area

Number of Injection

Retention time

516517.1

Injection-1

13.296

541961.3

516422.7

Injection-2

13.292

545330.5

14.904

519214.7

Injection-3

13.284

542997.8

Injection-4

14.855

519905.3

Injection-4

13.277

542881.3

Injection-5

14.830

519051.9

Injection-5

13.270

542998.4

Injection-6

14.816

519559.7

Injection-6

13.264

542089.4

Mean

518445

Mean

543043

SD

1558.35

SD

1211.80

RSD

0.30

RSD

0.22

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

Std (Increased)

Diclofenac sodium

Std-2

(Diclofenac sodium)

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidatioino:nLi: Linearinearityty
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

Diclofenac sodium

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 13.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Temperature)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Robustness for Diclofenac sodium Sample Preparation (25C)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

506278.6

98.24

Injection-2

500065.0

97.03

Injection-1

497051.9

96.45

Injection-2

496295.5

96.30

Injection-1

490237.8

95.13

Injection-2

488056.6

94.70

Injection-1

485344.3

94.18

Injection-2

485618.5

94.23

Injection-1

499950.1

97.01

Injection-2

500032.4

97.03

Injection-1

489475.3

94.98

Injection-2

483875.3

93.89

Number of Injection

Assay Caculation:

97.64
96.38

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

94.91
94.20
97.02

AnalAnalytiyctalicalMetMethodhodValValidatidationion: Li: Linearinearityty


(Least squares regression line, y = a + bx)

94.44

Mean

95.76

SD

1.441

RSD

1.504

Robustness for Diclofenac sodium Sample Preparation (35C)


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

525285.3

97.67

Injection-2

524765.2

97.58

Injection-1

515041.1

95.77

Injection-2

517937.0

96.31

Injection-1

515142.9

95.79

Injection-2

515521.7

95.86

Injection-1

511553.9

95.12

Injection-2

511282.0

95.07

Injection-1

521132.7

96.90

Injection-2

519802.0

96.65

Injection-1

513385.8

95.46

Injection-2

512983.8

95.39

Number of Injection

97.62
96.04
95.82
95.09
96.78
95.42

Mean

96.13

SD

0.930

RSD

Prepared by:

AnAnaalytliyctaiclaMel MethtohdoVald Vaidlaidtaiotino:nL:iLinenaeraitryity


(Least squares regres ion line, y = a + bx)
Reviewed by:

Date:

Date:

0.968

Aspl x Wstd x 5 x
Astd
200
100

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearnearityity
(Least squares regression line, y = a + bx)

100 x 200 x Wt/mL x P


Wspl
5
LC

(Diclofenac sodium)

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 13.0

Reference Protocol No

AMVP-16/15

Characteristics

Robustness (Temperature)-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Diclofenac sodium


Reduced(25C)
Sample

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Actual(30C)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium
95.92

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Injection-1

98.24

Injection-2

97.03

Injection-1

96.45

Injection-2

96.30

Injection-1

95.13

Injection-2

94.70

Injection-1

94.18

Injection-2

94.23

Injection-1

97.01

Injection-2

97.03

Injection-1

94.98

Injection-2

93.89

97.64

95.39
98.29

96.38

99.17
100.38

94.91

98.84
99.54

94.20

99.40
96.19

97.02

96.27
95.37

94.44

96.09

95.66
98.73
99.61
99.47
96.23
95.73

97.67
97.58
95.77
96.31
95.79
95.86
95.12
95.07
96.90
96.65
95.46
95.39

97.62
96.04
95.82
95.09
96.78
95.42

Mean

95.76

97.57

96.13

SD

1.441

1.896

0.930

%RSD

1.504

1.944

0.968

Summary for Robustness (Temperature)-Diclofenac sodium


Mean Assay (%)

% RSD

Difference in Sample (%)

Diclofenac sodium

Diclofenac sodium

Diclofenac sodium

Actual (1.0 mL/min)

97.57

1.9

Reduced (0.8 mL/min)

95.76

1.5

1.9

Increased (1.2 mL/min)

96.13

1.0

-1.5

Parameter

Increased(35C)

Assay (%)

AnAanlyatliyctaiclaMelMethtohdoVdaVliadlaidtaiotino:nL:iLnienaeraitryity
(Least squares regres ionline,y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 13.0

Reference Protocol No

AMVP-06/15

Characteristics

Robustness (Temperature)-Diclofenac Sodium

Finished Product

Diclofenac sodium 75mg/3mL Injection

Assay (%) Comparison of Diclofenac Sodium


Diclofenac sodium
Actual(30C)
95.92

Reduced(25C)
98.24

Increased(35C)
97.67

95.39

97.03

97.58

98.29

96.45

95.77

Average (Assay)

99.17

96.30

96.31

Significance Level

100.38

95.13

95.79

Stdev

1.6115

98.84

94.70

95.86

%RSD

1.67

99.54

94.18

95.12

Sample size

99.40

94.23

95.07

95% Confidence Interval (CI

96.19

97.01

96.90

Confidence Interval

= Mean assay Confidence Interval

96.27

97.03

96.65

(Diclofenac Sodium)

= 96.49% 0.53

95.37

94.98

95.46

96.09

93.89

95.39

Diclofenac sodium\
96.49
0.05

36
0.53

= 95.96% - 97.02%

AnalyticalMethodValidation:Linearity
Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution stability (Standard)

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium


Compound

Standard for Benzyl Alcohol

Standard for
Diclofenac Sodium

sample

Standard for Benzyl


Alcohol

Standard for Diclofenac


Sodium

sample

Weight, mg

162.4

100.6

4.08

163

25.12

4.06

Purity, %

100.0

100.0

NA

100.0

100.0

NA

Number of Injection

Retention time

Benzyl Alcohol Standard Injection (Intial)


Std (Reduced)

Std-2

Diclofenc Sodium Standard Injection (Intial)


Area

Std (Increased)

Area

Number of Injection

Retention time

Injection-1

3.878

62494.8

Injection-1

13.047

501915.5

Injection-2

3.882

61968.5

Injection-2

13.047

502067.7

Injection-3

3.886

61173.9

Injection-3

13.054

505743.1

Injection-4

3.873

63491.1

Injection-4

13.037

501622.8

Injection-5

3.885

62407.9

Injection-5

13.044

502614.6

Injection-6

3.871

62867.4

Injection-6

13.023

502763.3

Mean

62400.6

Mean

502787.8

SD

788.36

SD

1510.04

RSD

1.26

RSD

0.30

Benzyl Alcohol

Std-2

AnalAnalytiyctalicalMethod
MethodValValidatiidatonio:nLi: nLiearinearityty
Prepared by:

(Least squares
Reviewed
by:regression line, y = a + bx)

Date:

Date:

Diclofenac sodium

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution Stability (Standard)

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Benzyl Alcohol Standard Injection (24 hours-Room temperature)


Std (Reduced)

Diclofenc Sodium Standard Injection (24 Hours-Room temperature)


Area

Number of Injection

Retention time

Injection-1

3.890

Injection-2

3.889

Injection-3

3.902

63569.7

Injection-4

3.904

62327.0

Injection-5

3.888

Injection-6

3.891

Std-2

Std (Increased)

Retention time

63128.6

Injection-1

13.200

503752.0

64388.1

Injection-2

13.209

503687.2

Injection-3

13.224

503344.2

Injection-4

13.218

504260.7

63245.4

Injection-5

13.195

503055.7

63050.8

Injection-6

13.182

504637.4

Mean

63284.9

Mean

503789.5

SD

677.72

SD

581.31

RSD

1.07

RSD

0.12

Std-2

Benzyl Alcohol Standard Injection (24 hours-4C)


Std (Reduced)

Std-2

Area

Number of Injection

Benzyl Alcohol

Diclofenac sodium

Diclofenc Sodium Standard Injection (24 Hours-4C)

Number of Injection

Retention time

Injection-1

3.884

Injection-2

3.889

Injection-3

Area

Std (Increased)

Area

Number of Injection

Retention time

61743.0

Injection-1

13.126

512181.8

62962.7

Injection-2

13.128

512500.0

3.880

62803.8

Injection-3

13.114

512433.7

Injection-4

3.885

62873.6

Injection-4

13.166

513149.1

Injection-5

3.891

62621.6

Injection-5

13.198

513596.4

Injection-6

3.890

62206.6

Injection-6

13.217

513234.7

Mean

62535.2

Mean

512849.3

SD

471.69

SD

554.41

RSD

0.75

RSD

0.11

Benzyl Alcohol

Std-2

Summary of Standard Solution Stability

Diclofenac sodium

Summary of Standard Solution Stability

Condition

Area
Benzyl Alcohol

Initial

62400.6

Room Temperature

63284.9

24 Hours
in Refrigerator 4C 3C

62535.2

Difference, %

-1.4

Conclusion

Complies

-0.2

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi:nLiearnearityity
(Least squares regression line, y = a + bx)
Reviewed by:

Date:

Date:

Condition

Area
Diclofenac Sodium

Initial

502787.8

Room Temperature

503789.5

24 Hours
in Refrigerator 4C 3C

512849.3

Difference, %

Conclusion

-0.2
-2.0

Complies

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution Stability-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium-Initial

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium-Fresh standard

Compound

Standard for Benzyl Alcohol

Standard for Diclofenac


Sodium

sample

Standard for Benzyl


Alcohol

Standard for
Diclofenac Sodium

sample

Weight, mg

163.0

25.12

4.06

164.06

25.13

4.06

Purity, %

100.0

100.0

NA

100.0

100.0

NA

Number of Injection

Retention time

Injection-1

3.878

Injection-2

3.882

Injection-3

3.886

61173.9

Injection-4

3.873

63491.1

Injection-5

3.885

62407.9

Injection-6

3.871

Benzyl Alcohol Standard Injection-Initial


Std

Std-2

Assay Calculation:

Benzyl Alcohol Standard Injection-Fresh standard


Area

Area

Number of Injection

Retention time

62494.8

Injection-1

3.891

61968.5

Injection-2

3.898

61134.8

Injection-3

3.888

62888.4

Injection-4

3.903

63279.5

Injection-5

3.89

62508.1

62867.4

Injection-6

3.898

61573.1

Mean

62400.6

Mean

62415.2

SD

788.36

SD

872.60

RSD

1.26

RSD

1.40

Aspl x Wstd x 5 x
Astd
200
100

100 x 200 x Wt/mL x P


Wspl
5
LC

Std

Benzyl Alcohol

Std-2

(Benzyl alcohol)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

AnalAnalytiyctalicalMetMethodhodValValidatidatioinon: Li: Linearinearityty


Prepared by:

(Least squares
Reviewed
by: regression line, y = a + bx)

Date:

Date:

Benzyl Alcohol
63107.3

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution Stability-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Solution Stability for Benzyl Alcohol Sample Preparation-Initial


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Area

% Assay

% Mean assay

Benzyl alcohol

Benzyl alcohol

Benzyl alcohol

Injection-1

63424.4

104.06

Injection-2

62381.9

102.35

Injection-1

63678.5

104.47

Injection-2

63650.9

104.43

Injection-1

63196.6

103.68

Injection-2

63257.8

103.78

Injection-1

62766.2

102.98

Injection-2

62995.6

103.35

Injection-1

63518.8

104.21

Injection-2

63825.1

104.71

Injection-1

63833.1

104.73

Injection-2

63544.5

104.25

Number of Injection

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

103.20
104.45

104.46
104.49
103.92
0.636

RSD

0.612

% Assay

% Mean assay

Benzyl Alcohol

Benzyl Alcohol

63145.3

104.25

Injection-2

63980.9

105.63

Injection-1

62801.0

103.68

Injection-2

63673.9

105.12

Injection-1

64280.6

106.12

Injection-2

64954.0

107.23

Injection-1

64102.4

105.83

Injection-2

63421.6

104.70

Injection-1

64240.8

106.06

Injection-2

64127.8

105.87

Injection-1

63595.4

104.99

Injection-2

63729.1

105.21

100 x 200 x Wt/mL x P


Wspl
5
LC

(Benzyl alcohol)

Remarks: Wt/mL
= 1.02 g/mL
LC Benzyl Alcohol = 40 mg/mL

103.16

SD

Injection-1

Aspl x Wstd x 5 x
Astd
200
100

103.73

Mean

Solution stability for Benzyl Alcohol Sample Preparation-24 hours (Room Temperature)
Area
Sample
Number of Injection
Benzyl Alcohol

Assay Calculation:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

Solution stability for Benzyl Alcohol Sample Preparation-24 hours (4C)


Area
% Assay
Sample
Number of Injection
Benzyl Alcohol
Benzyl Alcohol

104.94

Sample-1

104.40

Sample-2

106.68

Sample-3

105.27

Sample-4

105.96

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi:nLiearnearitySample-5
ity
(Least squares regression line, y = a + bx)

105.10

Sample-6

Injection-1

62866.8

103.79

Injection-2

63429.8

104.72

Injection-1

63711.3

105.18

Injection-2

63554.9

104.92

Injection-1

63001.8

104.01

Injection-2

62733.3

103.57

Injection-1

63924.5

105.53

Injection-2

63331.9

104.56

Injection-1

63748.8

105.24

Injection-2

62840.3

103.74

Injection-1

64581.8

106.62

Injection-2

64479.4

106.45

% Mean assay
Benzyl Alcohol
104.25
105.05
103.79
105.05
104.49
106.53

Mean

105.39

Mean

104.86

SD

0.809

SD

0.951

RSD

0.767

RSD

0.907

Prepared by:

AnAnalalytiyctaiclaMel MetthohdoVad Vallidaidtaiotnio:nL:inLeinaeraitryity


(Least squares regression line, y= a + bx)
Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution Stability-Benzyl Alcohol

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Benzyl Alcohol


Initial
Sample

Number of Injection

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

24 Hours-Room temperature

24 Hours-Refrigerator 4C

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol
104.25

Benzyl Alcohol

Benzyl Alcohol

Benzyl Alcohol

Injection-1

104.06

Injection-2

102.35

Injection-1

104.47

Injection-2

104.43

Injection-1

103.68

Injection-2

103.78

Injection-1

102.98

Injection-2

103.35

Injection-1

104.21

Injection-2

104.71

Injection-1

104.73

Injection-2

104.25

103.20

105.63
103.68

104.45

105.12
106.12

103.73

107.23
105.83

103.16

104.70
106.06

104.46

105.87
104.99

104.49

105.21

104.94
104.40
106.68
105.27
105.96
105.10

103.79
104.72
105.18
104.92
104.01
103.57
105.53
104.56
105.24
103.74
106.62
106.45

104.25
105.05
103.79
105.05
104.49
106.53

Mean

103.92

105.39

104.86

SD

0.636

0.809

0.951

%RSD

0.612

0.767

0.907

Summary of Standard Solution Stability


Condition

Assay,%
Benzyl Alcohol

Initial

103.9

Room Temperature

105.4

24 Hours
in Refrigerator 4C 3C

104.9

Difference, %

-1.4

Conclusion

Complies

-0.9

AnAanlyatliyctaiclaMelMethtohdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity
(Least squares regres ion line, y= a +bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution Stability-Diclofenac Sodium

Finished Product

Diclofenac Sodium 75mg/ 3mL Injection

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium-Initial


Compound

Standard for Diclofenac


Sodium

sample

Standard for Benzyl


Alcohol

Standard for
Diclofenac Sodium

sample

163

25.12

4.06

164.06

25.13

4.06

100.0

100.0

NA

100.0

100.0

NA

Weight, mg
Purity, %

Standard and Preparation for Benzyl Alcohol and Diclofenac Sodium-Fresh standard

Standard for Benzyl Alcohol

Diclofenac Sodium Standard Injection-Intial


Std

Std-2

Assay Caculation:

Diclofenac Sodium Standard Injection-Fresh standard


Area

Number of Injection

Retention time

Injection-1

13.047

Injection-2

13.047

Injection-3

13.054

505743.1

Injection-4

13.037

501622.8

Injection-5

13.044

Injection-6

13.023

Std

Area

Number of Injection

Retention time

501915.5

Injection-1

13.241

508541.7

502067.7

Injection-2

13.246

506831.6

Injection-3

13.239

505236.6

Injection-4

13.255

505911.8

502614.6

Injection-5

13.244

505072.3

13.248

507223.7

Diclofenac Sodium

Std-2

Diclofenac Sodium

502763.3

Injection-6

Mean

502787.8

Mean

506470

SD

1510.04

SD

1324.93

RSD

0.30

RSD

0.26

Aspl x Wstd x 5 x
Astd
50
100

100 x 200 x Wt/mL x P


Wspl
5
LC

(Diclofenac sodium)

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: nLiearinearityty
(Least squares regression line, y = a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution Stability-Diclofenac sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Solution Stability for Diclofenac Sodium Sample Preparation-Initial


Sample
Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

Number of Injection

Area

% Assay

% Mean assay

Diclofenac Sodium

Diclofenac Sodium

Diclofenac Sodium

Injection-1

491150.2

98.64

Injection-2

504852.5

101.39

Injection-1

490758.5

98.56

Injection-2

494174.0

99.25

Injection-1

496714.7

99.76

Injection-2

492025.0

98.81

Injection-1

491343.8

98.68

Injection-2

490785.5

98.56

Injection-1

487275.7

97.86

Injection-2

484595.4

97.32

Injection-1

487224.3

97.85

Injection-2

489207.9

98.25

100.01
98.90

97.59

98.74

SD

0.867

RSD

0.878

Diclofenac sodium

Diclofenac sodium

Injection-1

483881.9

96.51

Injection-2

495808.5

98.89

Injection-1

485090.3

96.75

Injection-2

486948.5

97.12

Injection-1

501527.5

100.03

Injection-2

503488.7

100.42

Injection-1

484778.0

96.69

Injection-2

480678.6

95.87

Injection-1

492495.6

98.23

Injection-2

483739.8

96.48

Injection-1

486593.3

97.05

Injection-2

485640.0

96.86

Sample-4
Sample-5
Sample-6

x 200 x Wt/mL x P
5

(Diclofenac

LC

Remarks: Wt/mL
= 1.02 g/mL
LC Diclofenac Sodium = 25 mg/mL

AnalAnalytiyctalicalMetMethodhodValValidiatdationion: Li: Linearinearityty


(Least squares regression line, y = a + bx)

98.05

Mean

% Mean assay

Sample-3

100
Wspl

98.62

% Assay

Sample-2

Aspl x Wstd x 5 x
sodium)
Astd
50
100

99.28

Solution stability for Diclofenac Sodium Sample Preparation-24 hours (Room Temperature)
Area
Sample
Number of Injection
Diclofenac sodium
Sample-1

Assay Caculation:

97.70
96.94

Solution stability for Diclofenac Sodium Sample Preparation-24 hours (4C)


Area
% Assay
Sample
Number of Injection
Diclofenac Sodium
Diclofenac Sodium
Sample-1
Sample-2

100.23

Sample-3

96.28

Sample-4

97.35

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi: Linearnearityity Sample-5


(Least squares regression line, y = a + bx)

96.96

Sample-6

Injection-1

483120.4

96.36

Injection-2

482278.2

96.19

Injection-1

481659.4

96.07

Injection-2

490574.4

97.84

Injection-1

481033.6

95.94

Injection-2

490810.0

97.89

Injection-1

482883.2

96.31

Injection-2

480958.4

95.93

Injection-1

481274.4

95.99

Injection-2

482111.0

96.16

Injection-1

492969.5

98.32

Injection-2

494126.5

98.55

% Mean assay
Diclofenac Sodium
96.27
96.96
96.92
96.12
96.07
98.44

Mean

97.58

Mean

96.80

SD

1.382

SD

0.893

RSD

1.417

RSD

0.923

Prepared by:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi:nLiearnearityity
(Least squares regression line, y= a + bx)
Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 11.0

Reference Protocol No

AMVP-06/15

Characteristics

Solution Stability-Diclofenac Sodium

Finished Product

Diclofenac Sodium 75mg/3mL Injection

Assay (%) Comparison of Diclofenac Sodium


Initial
Sample

Number of Injection

Sample-1
Sample-2
Sample-3
Sample-4
Sample-5
Sample-6

24 Hours-Room temperature

24 Hours-Refrigerator 4C

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Assay (%)

Mean Assay (%)

Diclofenac Sodium

Diclofenac Sodium

Diclofenac Sodium
96.51

Diclofenac Sodium

Diclofenac Sodium

Diclofenac Sodium

Injection-1

98.64

Injection-2

101.39

Injection-1

98.56

Injection-2

99.25

Injection-1

99.76

Injection-2

98.81

Injection-1

98.68

Injection-2

98.56

Injection-1

97.86

Injection-2

97.32

Injection-1

97.85

Injection-2

98.25

100.01

98.89
96.75

98.90

97.12
100.03

99.28

100.42
96.69

98.62

95.87
98.23

97.59

96.48
97.05

98.05

96.86

97.70
96.94
100.23
96.28
97.35
96.96

96.36
96.19
96.07
97.84
95.94
97.89
96.31
95.93
95.99
96.16
98.32
98.55

96.27
96.96
96.92
96.12
96.07
98.44

Mean

98.74

97.58

96.80

SD

0.867

1.382

0.893

%RSD

0.878

1.417

0.923

Summary of Sample Solution Stability


Condition

Assay,%
Diclofenac Sodium

Initial

98.7

Room Temperature

97.6

24 Hours
in Refrigerator 4C 3C

96.8

Difference, %

1.18

Conclusion

Complies

1.97

AnAanlyatliyctaiclaMelMethothdoVdaVliadlaidtaiotnio:nL:inLeinaeraitryity
(Least squares regres ionline,y= a + bx)

Prepared by:

Reviewed by:

Date:

Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 12.0

Reference Protocol No

AMVP 06/15

Parameter

Limit of Detection

Finished Product

Diclofenac Sodium 75 mg/ 3mL Injection

LOD Benzyl Alcohol (Concentration: 0.0008 mg/mL)


Injection
Retention time
Area
Injection 1
4.009
1432.0

Height
216.4

Injection 2
Injection 3
Injection 4
Injection 5
Injection 6
Injection 7
Injection 8
Injection 9

3.998
4.000
3.999
4.005
4.005
4.001
4.006
4.001

1325.6
1374.6
1392.5
1343.4
1327.6
1274.6
1398.9
1387.2

209.0
217.6
199.3
211.8
185.5
196.6
218.8
207.2

Injection 10
Mean
SD
%RSD

3.996
4.002
0.00
0.10

1387.4
1364.4
46.03
3.37

198.5
206.1
10.85
5.27

LOD Diclofenc Sodium (Concentration: 0.00025 mg/mL)


Injection
Retention time
Area
Injection 1
15.471
6765
Injection 2
15.443
6425
Injection 3
15.393
6731
Injection 4
15.404
6922
Injection 5
15.424
6710
Injection 6
15.382
6614
Injection 7
15.411
6720
Injection 8
Injection 9
Injection 10
Mean
SD
%RSD

15.447
15.343

6739
6852

15.478
15.420
0.04
0.27

6422
6690.0
163.00
2.44

Height
319.1
292.0
330.2
323.6
337.5
309.4
325.5

Analytical Method Validation : Linearity

314.4
331.6
297.3
318.1
14.89
4.68

Summary of Limit of Detection


Peak
Benzyl Alcohol
Diclofenac Sodium

Prepared by:
Date:

S/N ratio
3.7:1
3.8:1

Analytical Method Validation :Linearity


Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION

ATTACHMENT 13.0

Reference Protocol No

AMVP 06/15

Parameter

Limit of Quantitation

Finished Product

Diclofenac Sodium 75 mg/ 3mL Injection

LOQ Benzyl Alcohol (Concentration: 0.002 mg/mL)


Injection
Retention time
Area
Injection 1
4.000
3030.3

Height
507.0

Injection 2
Injection 3
Injection 4
Injection 5
Injection 6
Injection 7
Injection 8
Injection 9

4.002
3.998
3.998
4.000
4.001
4.001
4.001
3.998

3085.8
2968.1
3074.1
3049.2
3080.5
3081.7
2988.0
3131.2

500.6
504.4
507.8
508.2
529.7
533.3
485.2
492.9

Injection 10
Mean
SD
%RSD

4.000
4.000
0.00
0.04

2951.6
3044.1
58.37
1.92

492.4
506.2
15.41
3.04

LOQ Diclofenc Sodium (Concentration: 0.00075 mg/mL)


Injection
Retention time
Area
Injection 1
15.576
17197.9
Injection 2
15.556
17353.4
Injection 3
15.515
17356.4
Injection 4
15.492
17253.4
Injection 5
15.495
17414.4
Injection 6
15.492
17563.0
Injection 7
15.470
17443.1
Injection 8
Injection 9
Injection 10
Mean
SD
%RSD

15.483
15.511

17350.6
17512.7

15.493
15.508
0.03
0.21

15323.7
17176.9
660.24
3.84

Height
867.0
868.3
873.9
878.5
885.9
881.6
902.6

Analytical Method Validation : Linearity

847.5
857.1
844.2
870.7
17.90
2.06

Summary of Limit of Quantitation


Peak
Benzyl Alcohol
Diclofenac Sodium

Prepared by:
Date:

S/N ratio
10.7:1
11.7:1

Analytical Method Validation :Linearity


Reviewed by:
Date:

PHARMANIAGA RESEARCH CENTRE SDN BHD


PRODUCT DEVELOPMENT DEPARTMENT
ANALYTICAL METHOD VALIDATION
Reference Protocol No
Test
Finished Product

Sample
RD1503012

AMVP-06/15
Weight per mL
Diclofenac Sodium 75mg/ 3mL Injection

Weight of empty
pycnometer in g-W1
20.1479

Weight per ml = W2 - W1 x F
W3 - W1
note : in this case, F=0.99718

Weight of sample and


pycnometer in g -W2
46.0648

Weight of water -W3

Weight per ML

45.5252

1.02

Prepared by:
Date:

AnalAnalytiyctalicalMetMethodhodValValidatidationio:nLi:nLiearineartyity
(Least squares regression line, y =Reviewed
a + bx)
by:
Date:

ATTACHMENT 6.0

earineartyity
=Reviewed
a + bx)
by: