Table 6 Robustness of the validated analytical method

Optimized parameters: 4.0 pH, 0.4 mL/min flow rate, 85:15 mobile phase ratio, and column temperature 30 °C

BCA

0.5

–

1.10 (± 0.026)

14,162 (± 89.8)

4

–

1.13 (± 0.03)

14,642.6 (± 24.3)

32

–

1.065 (± 0.005)

17,226 (± 23.6)

LTB

0.5

6.53 (± 0.021)

1.16 (± 0.01)

11,863 (± 7.54)

4

6.60 (± 0.005)

1.16 (± 0.005)

12,042 (± 38.09)

32

6.70 (± 0.006)

1.17 (± 0.021)

12,424 (± 22.91)

Change in pH 3.5

BCA

0.5

–

1.16 (± 0.038)

15,173 (± 97)

4

–

1.12 (± 0.01)

15,095 (± 100)

32

–

1.124 (± 0.016)

17,199.3 (± 10.61)

LTB

0.5

5.91 (± 0.17)

1.07 (± 0.03)

12,822.6 (± 33)

4

6.34 (± 0.17)

1.15 (± 0.017)

12,757.3 (± 130.7)

32

6.76 (± 0.03)

1.15 (± 0.015)

12,819 (± 12.5)

Change in pH 4.5

BCA

0.5

–

1.087(± 0.08)

13,819 (± 111)

4

–

1.153 (± 0.03)

14,049 (± 68)

32

–

1.117 (± 0.021)

16,522 (± 139)

LTB

0.5

6.53 (± 0.05)

1.34 (± 0.03)

12,297 (± 78.42)

4

6.6 (± 0.02)

1.16 (± 0.03)

12,331.3 (± 57.3)

32

6.76 (± 0.034)

1.17 (± 0.03)

12,612 (± 99.14)

Change in flow rate of mobile phase: 0.38 mL/min

BCA

0.5

–

1.104 (± 0.043)

1444 (± 13.11)

4

–

1.141 (± 0.017)

14,757 (± 30.805)

32

–

1.078 (± 0.051)

17,210.93 (± 6.957)

LTB

0.5

6.687 (± 0.001)

1.153 (± 0.038)

12,098.3 (± 18.823)

4

6.673 (± 0.005)

1.1736 (± 0.040)

12,294.6 (± 32.02)

32

6.864 (± 0.018)

1.1673 (± 0.011)

12,653 (± 60.32)

Change in flow rate of mobile phase: 0.42 mL/min

BCA

0.5

–

1.128 (± 0.007)

14,030 (± 18.248)

4

–

1.224 (± 0.035)

14,030 (± 18.24)

32

–

1.109 (± 0.046)

16,689 (± 103.79)

LTB

0.5

6.522 (± 0.008)

1.140 (± 0.027)

12,239.67 (± 37.44)

4

6.592 (± 0.038)

1.1894 (± 0.060)

12,253.3 (± 74.14)

32

6.787 (± 0.025)

1.1738 (± 0.0182)

12,572 (± 62.628)

Change in mobile phase ratio: 83: 17% (ACN: Acidified water)

BCA

0.5

–

0.910 (± 0.009)

16,352 (± 19.38)

4

–

1.115 (± 0.022)

14,738.6 (± 36.82)

32

–

1.124 (± 0.016)

17,199.3 (± 10.61)

LTB

0.5

7.727 (± 0.071)

1.108 (± 0.048)

12,332.6 (± 12.61)

4

7.537 (± 0.020)

1.139 (± 0.018)

12,210.6 (± 43.775)

32

7.777 (± 0.023)

1.1392 (± 0.027)

12,496.3 (± 28.676)

Change in mobile phase ratio: 87: 13% (ACN: Acidified water)

BCA

0.5

–

1.029 (± 0.067)

15,617 (± 17.16)

4

–

1.115 (± 0.0264)

15,453.66 (± 19.25)

32

–

1.092 (± 0.020)

16,853.6 (± 14.08)

LTB

0.5

5.957 (± 0.037)

1.139 (± 0.019)

12,790.3 (± 15.42)

4

5.902 (± 0.014)

1.1623(± 0.0297)

12,712.66 (± 57.23)

32

5.999 (± 0.02)

1.160 (± 0.0297)

12,917 (± 11.53)

Change in column temperature 28 °C

BCA

0.5

–

0.90 (± 0.02)

16,361 (± 79.03)

4

–

1.11 (± 0.01)

15,178.8 (± 59.3)

32

–

 ± 0.01)

17,245 (± 86.5)

LTB

0.5

6.90 (± 0.05)

1.15 (± 0.04)

12,101 (± 26.3)

4

6.86 (± 0.01)

1.15 (± 0.03)

12,479.6 (± 73.07)

32

7.023 (± 0.0321)

1.157 (± 0.024)

12,748 (± 7.63)

Change in column temperature 32 °C

BCA

0.5

–

0.92 (± 0.01)

16,405 (± 76.3)

4

–

1.109 (± 0.026)

15,100.8 (± 57.9)

32

–

1.08 (± 0.032)

16,927 (± 55.03)

LTB

0.5

6.77 (± 0.082)

1.14 (± 0.02)

12,396.7 (± 41)

4

6.59 (± 0.01)

1.149 (± 0.013)

12,268.9 (± 46.5)

32

6.701 (± 0.002)

1.14(± 0.015)

12,545 (± 96.26)