Table I: Critical analytical attributes

Target
CAA’s
Apixaban Dabigatran

Retention time 2-3 5-6

Theoretical plates High High

Table II: Risk estimation matrix (REM) for Bioanalytical RP-HPLC method of apixaban
and dabigatran
Run Factor 1 Factor 2 Response 1 Response 2 Response 3 Response 4
A- pH retention
B-Amount time of Retention Theoretica Theoretical
of apixaban time of l plates of plates of
acetonitrle dabigatran apixaban dabigatran

1 6 80 2 3.2 3958 2650

2 4 70 2.2 8.5 5750 4300

3 5 80 2.6 7.5 2587 2117

4 4 80 2.1 7 6523 2560

5 5 60 3.1 5.4 4527 3498

6 4 60 2 6 3020 4992

7 6 70 2.3 3.5 3436 5234

8 5 70 2.8 4 6005 4826

9 6 60 2.4 4.2 4772 3334

Table III: ANOVA for response surface quadratic model

 Model terms F value p value Ramark

Model 11.87 0.0343 Significant

A-pH 1.45 0.3153 Insignificant

B- Amt of ACN 5.79 0.005 Significant

AB 3.39 0.00162 Significant

A2 48.24 0.0061 Significant

B2 0.48 0.5373 Insignificant

Table IV: ANOVA for response surface quadratic model

Model terms F value p value Remark

Model 6.30 0.0336 Significant

A- pH 12.12 0.0131 Significant

B- amt of ACN 0.48 0.51 Insignificant

Table V: ANOVA for response surface quadratic model

Model terms F value p value Ramark

Model 11.20 0.0443 Significant

A-pH 9.2 0.03153 Significant

B-Model
amt ofterms
ACN F0.053
value 0.8
p value Insignificant
Ramark

AB
Model 2.63
14.88 0.16
0.01143 Insignificant
Significant

A-pH
Table VII: 17 surface quadratic0.09
ANOVA for response model Significant

B- amt of ACN 8.61 0.051 Significant

AB 1.95 0.25 Insignificant

A2 0.68 0.47 Insignificant

B2 13 0.036 Significant
 Table VIII: Confirmation report of the model
Factor Selected level Predicted response Predicted response
of dabigatran
of apixaban

pH 5.4 2.9 5.4

Amount of ACN 60 4591 3739

Table IX: Linearity of Apixaban

Sr. No. Standard concentration (µg/mL) Mean area

1 10 59832

2 20 87566

3 30 126352

4 40 153902

5 50 183894

6 60 219915

Table X A: Accuracy result of Apixaban (10 µg/mL)

Sr. Conc. Area % recovery Recovered conc.
(µg/mL)
No. (µg/mL)
Sr. Conc. Area % recovery Recovered conc.
1 10 61666 98.14% 9.8
No. (µg/mL)
(µg/mL)
2 10 59872 98.81% 9.8
1 30 135503 99.7% 29.9
3 10 59664 96.74% 9.6
2 30 138680 101.9% 30.5
4 10 59769 97.96% 9.7
3 30 138752 102 % 30.5
5 10 59780 96.58% 9.6
4 30 135691 99.7% 29.7
Mean 9.7
5 30 136215 99.4% 29.8
Standard deviation 0.1
Mean 30.08
% Coefficient of variation 1.03
Standard deviation 0.38
% Accuracy 97%
% Coefficient of variation 1.26
Table X B: Accuracy of Apixaban (30µg/mL)
% Accuracy 100.2%

Table X C: Accuracy result of Apixaban (60µg/mL)

Sr. Conc. (µg/mL) Area % recovery Recovered conc.
No. (µg/mL)

1 60 247652 100.4% 60.2

2 60 247730 99.14% 59.4

3 60 248568 99.44% 59.6

4 60 242442 97.23% 58.3

5 60 245512 98.68% 59.2

Mean 59.35

Standard deviation 0.70

 % Coefficient of variation 1.1

% Accuracy 98.91%

Acceptance Criteria: The % mean accuracy for all samples should be within 85.00-
115.00%

Table XI A: Interday precision result of Apixaban

Day 1 Day 2 Day 3

A
Concentration 10µg/mL (LQC)

1 59873 58769 59883

2 59716 58835 59956

3 59881 59782 59862

4 58835 5980 59984

5 59933 59961 59989

Mean 59647.6 59429.4 59924.8

SD 561.44 577.43 72.4

% CV 0.77 0.97 0.12

B Concentration 30µg/ mL (MQC)

1 135996 129886 130025

2 136697 129783 131128

 3 136418 129654 131856

4 137658 130889 130087

5 136552 129987 130217

Mean 136664.2 130039.8 130662.6

SD 614.0865 490.5 802.68

% CV 0.44 0.37 0.61

C Concentration 60 µg/ mL (HQC)

1 222998 221532 221587

2 221957 221965 221669

3 222698 221984 221182

4 222658 222947 221198

5 222984 221899 221365

Mean 222659 222065.4 221400.2

SD 422.755 525.86 221.85

% CV 0.18 0.23 0.1

Table XI B: Intraday precision results of Apixaban

1st session 2nd session 3rd session
A
Concentration 10µg/mL (LQC)

1 58926 59897 59874

2 59362 59948 59869

 3 59366 59732 58982

4 59402 59639 59689

5 59366 59926 59861

Mean 59284.4 59828.4 59655

SD 201.0095 135.7398 384.1478

% CV 0.33 0.22 1.2

B Concentration 30µg/ mL (MQC)

1 137847 136482 136987

2 137665 136488 136785

3 137781 136625 136654

4 136978 135961 137021

5 136997 136565 130217

Mean 137453.6 136424.2 136890.4

SD 430.5006 265.5702 161.1833

% CV 0.31 0.19 0.11

C Concentration 60 µg/ mL (HQC)

1 223281 222967 222541

2 223535 223965 223154

3 223434 223785 223965

4 223621 222997 223682

5 222998 223961 223994

Mean 223373.8 223535 223467.2

SD 245.2319 510.133 617.94

% CV 0.1 0.22 0.27

Table XII A: Recovery results of apixaban (10µg/mL) (LQC)

Replicate Peak area of standard drug Peak area of drug spiked in

plasma

1 77985 59984

2 78736 59763

3 77884 59937

Mean 78198 59894

SD 469.27 116.42

% CV 0.6 0.19

% mean 76.59%
recovery

Table XII B: Recovery results of apixaban (30µg/mL) (MQC)

 Replicate Peak area of standard drug Peak area of drug spiked in
plasma

1 179288 130968

2 182354 131784

3 181787 131098

Mean 181143 131283.3

SD 1613.3 438.43

% CV 0.9 0.33

% mean recovery 72.47%

Table XII C: Recovery results of apixaban (60µg/mL) (HQC)

Replicate Peak area of standard drug Peak area of drug spiked in
plasma

1 315198 235984

2 316215 223978

3 315185 235998

Mean 315532.7 235320

SD 590.95 1162.22

% CV 0.18 0.49

% mean recovery 74.57%

Table XIII A: Robustness results of Apixaban (10µg/mL)

 Replicate Parameter Peak area of sample

Flow rate mL/min

1 0.7 58552

2 0.8 59179

3 0.9 61325

Mean 59352
SD
899
% CV
1.5

Table XIII B: Robustness results of Apixaban (10µg/mL)

Replicate Parameter Peak area of sample

Wavelength (nm)

1 278 59547

2 280 59739

3 282 59792

Mean 59326
SD
678.95
% CV
1.1

Table XIII C: Robustness results of Apixaban (10µg/mL)

Replicate Parameter Peak area of sample

pH of mobile phase

1 5.3 61882
 2 5.5 62351

3 5.7 60887

Mean 61706.67
SD
747.58
% CV
1.2

Table XIV A: Freeze and thaw stability result of Apixaban (10µg/mL) (LQC)
Replicate Peak area of standard Peak area of drug spiked in
drug plasma

1 59638 56971

2 59912 56628

3 59352 55715

Mean 59364 56438

SD 280.02 649.19

% CV 0.46 1.1

% mean stability 95%

Table XIV B: Freeze and thaw stability result of Apixaban (60µg/mL) (HQC)
Replicate Peak area of standard Peak area of drug spiked in
drug plasma

1 249172 223691
 2 245582 225894

3 244935 227958

Mean 246563 225847.7

SD 2282.5 2133.87

% CV 0.92 0.94

% mean stability 91.59%

a. Short –term temperature stability:

Table XV A: Short-term stability result of Apixaban (30µg/mL) (LQC)

Replicate Peak area of standard Peak area of drug spiked in

drug plasma

1 59786 57649

2 59862 57590

3 58857 58577

Mean 59501.67 57705.33

SD 559.58 844.90

% CV 0.93 1.4

% mean stability 96.98%

Table XV B: Short-term stability result of Apixaban (60µg/mL) (HQC)

Replicate Peak area of standard Peak area of drug spiked in plasma
drug

1 238586 232650
 2 238861 228967

3 236894 227684

Mean 236894 229767

SD 1065.174 2577

% CV 0.44 1.1

% mean stability 95.65%

c.Long-term stability:
Table XVI A: Long-term stability result of Apixaban (30µg/mL) (LQC)

Replicate Peak area of standard drug Peak area of drug spiked in

plasma

1 59806 53981

2 60758 53562

3 59872 52123

Mean 60145.33 53222

SD 531.61 974.54

% CV 1 1.8

% mean stability 88%

Table XVI B: Long-term stability result of Apixaban (60µg/mL) (HQC)

Replicate Peak area of standard Peak area of drug spiked in
 drug plasma

1 249839 214356

2 249662 211638

3 248796 216542

Mean 249432.3 214178.7

SD 558.1419 2456.805

% CV 0.22 1.1

% mean stability 85.8%

d. Stock-solution stability:
Table XVII A: Stock-solution stability result of Apixaban (30µg/mL) (LQC)

Replicate Peak area of standard drug Peak area of drug spiked in

plasma

1 231259 219689

2 232254 219333

3 229872 214358

Mean 231128.3 217793.3

SD 1196.364 2980.406

% CV 0.5 1.3

% mean stability 94.23%

Table XVII B: Stock-solution stability result of Apixaban (60µg/mL) (HQC)

Replicate Peak area of standard drug Peak area of drug spiked in
 plasma

1 58823 56917

2 58706 57069

3 57989 58706

Mean 58506 57564

SD 541.54 991.91

% CV 0.77 1.7

% mean stability 98.38