Table 4.
Method validation data.
| Test | Details | Acceptance Criteria | Results | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Imp-E | Imp-A | Imp-H | Imp-D | MPM | Imp-F | Imp-B | Imp-C | Imp-G | |||
| Specificity |
Retention time n = 1, each impurity at 1.0 µg mL−1 |
Each peak should elute at a different retention time | 4.953 | 5.418 | 6.137 | 6.509 | 6.871 | 7.146 | 7.289 | 7.658 | 9.820 |
| There are no peak interferes with placebo peaks. All the individual impurity peaks eluted at different retention times | |||||||||||
| Linearity | Range (µg mL−1) | Range (µg mL−1) | 0.0371–0.7545 | 0.0321–0.7321 | 0.0233–0.7508 | 0.0467–0.7509 | 0.0333–0.7497 | 0.0293–0.7678 | 0.0363–0.7584 | 0.0347–4.9634 | 0.0436–0.7636 |
| Minimum 6 concentration levels in the range LOQ–150% of specification | Correlation coefficient should be > 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | 0.999 | |
| Slope | 44,357.242 | 60,717.374 | 138,890.406 | 66,685.223 | 71,979.649 | 86,647.962 | 90,671.303 | 101,629.307 | 83,134.619 | ||
| Intercept | 1759.467 | 58.533 | − 237.267 | − 501.467 | 1041.667 | − 82.800 | − 121.667 | − 770.800 | − 475.467 | ||
| STEYX SD | 520.919 | 121.679 | 264.279 | 515.648 | 71.593 | 229.146 | 390.461 | 719.195 | 283.291 | ||
| Limit of detection | 0.01 µg mL−1 (lowest detectable concentration) | S/N ratio > 3 | 0.011 | 0.010 | 0.007 | 0.014 | 0.010 | 0.009 | 0.011 | 0.010 | 0.013 |
| Limit of quantitation | 0.03 µg mL−1 (lowest quantitatable concentration) | S/N ratio > 10 | 0.037 | 0.032 | 0.023 | 0.046 | 0.033 | 0.029 | 0.035 | 0.034 | 0.043 |
| Accuracy | n = 9 (3 determinations each at LOQ, 50%, 100% and 150% specification level | Recovery at each level should be 90–110% |
99.8 ± 1.1 100.2 ± 3.0 98.0 ± 2.1 99.7 ± 3.0 |
100.6 ± 2.6 101.0 ± 3.2 98.2 ± 0.8 102.4 ± 2.1 |
102.1 ± 2.9 100.5 ± 3.8 99.7 ± 2.0 100.7 ± 2.4 |
98.5 ± 2.5 98.2 ± 3.1 98.0 ± 2.2 98.1 ± 1.2 |
102.7 ± 2.0 100.1 ± 2.9 99.9 ± 0.5 100.5 ± 3.3 |
96.2 ± 1.9 96.7 ± 1.0 99.3 ± 0.5 98.2 ± 1.9 |
99.8 ± 2.3 100.3 ± 2.9 98.3 ± 2.3 101.5 ± 2.6 |
98.9 ± 3.8 100.6 ± 2.1 97.8 ± 1.2 100.0 ± 2.9 |
98.4 ± 1.3 101.4 ± 1.7 101.3 ± 2.5 99.5 ± 1.7 |
| Precision (Analyst 1) | n = 6 (6 determinations each at 1.0 µg mL−1) | RSD should be < 5% | 1.6% | 2.5% | 2.8% | 2.8% | 2.2% | 1.6% | 3.0% | 1.5% | 2.1% |
| Intermediate precision (Analyst 2) | n = 6 (6 determinations at µg mL−1) | RSD should be < 5% | 4.5% | 0.8% | 1.6% | 2.1% | 3.3% | 1.9% | 1.2% | 1.8% | 2.2% |