Table 3.
Validation and Regression parameters of the proposed HPLC–DAD and LC–MS/MS methods
| Method Parameter | HPLC–DAD method | LC–MS/MS method | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| HCT | CT | DSA | VAL | AML | ATN | AMI | CAN | CT | SAL | |
| Linearity rangea | 0.10–100.00 | 0.05–8.00 | 0.10–100.0 | 0.05–100.00 | 0.10–100.00 | 1.0–200.0 | 5.0–200.0 | |||
| Regression equation parameters | ||||||||||
| Slope | 0.0530 | 0.4789 | 0.4957 | 0.0449 | 0.0493 | 0.0492 | 0.4940 | 0.0502 | 92.062 | 1.4412 |
| Intercept | 0.0051 | 0.0343 | 0.0134 | 0.0889 | 0.0169 | 0.0084 | 0.0155 | 0.0020 | 29.337 | 0.8209 |
| Correlation Coefficient (r) | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 1.0000 | 0.9999 | 0.9999 |
| Accuracy b (Mean ± SD) | 99.51 ± 0.68 | 100.44 ± 0.59 | 100.52 ± 0.69 | 100.18 ± 0.86 | 100.57 ± 1.05 | 99.25 ± 0.77 | 99.97 ± 0.69 | 99.03 ± 0.75 | 99.92 ± 0.60 | 99.64 ± 0.77 |
| Precision (± %RSD) | ||||||||||
| Repeatability c | 0.85 | 0.38 | 0.69 | 0.75 | 0.48 | 0.37 | 0.25 | 0.55 | 0.88 | 0.61 |
| Intermediate precision d | 0.91 | 0.80 | 0.86 | 0.87 | 0.53 | 0.48 | 0.48 | 0.76 | 1.00 | 0.78 |
| LOD e | 0.01 | 0.01 | 0.01 | 0.02 | 0.01 | 0.01 | 0.01 | 0.03 | 0.19 | 0.14 |
| LOQ e | 0.05 | 0.02 | 0.02 | 0.06 | 0.02 | 0.03 | 0.03 | 0.08 | 0.59 | 0.41 |
| Robustness f | 0.96 | 0.79 | 0.88 | 0.79 | 0.88 | 1.15 | 0.85 | 0.98 | 0.65 | 0.52 |
a For HPLC–DAD method: in μg/mL and for LC–MS/MS method: in ng/mL
b Accuracy was checked using concentrations for HPLC–DAD method: 0.2, 40.0 and 80.0 µg/mL for HCT, VAL, AML, ATN, AMI and CAN and 0.3, 3.0 and 7.0 µg/mL for CT and SAL while for LC–MS/MS method: 40.0, 80.0 and 120.0 ng/mL for CT and SAL
c and d are repeatability and intermediate precision, respectively (n = 9) percentage relative standard deviation of three different concentrations in triplicate for HPLC–DAD method: 0.5, 20.0 and 60.0 µg/mL for HCT, VAL, AML, ATN, AMI and CAN and 0.1, 2.0 and 5.0 µg/mL for CT and SAL while for LC–MS/MS method: 5.0, 50.0 and 150.0 ng/mLfor CT and 10.0, 50.0 and 150.0 ng/mL for SAL
e LOD and LOQ were calculated as per ICH, 3.3 × SD of the intercept/slope and 10 × SD of the intercept/slope, respectively
f For HPLC–DAD: average %RSD of the change in detection wavelength (± 1 nm) and flow rate (± 0.1 mL/min), while for LC–MS/MS: average %RSD of the change in the mobile phase composition (± 2%)