Table I.
Method Validation Data for AP in Blood and Mammary Gland
| Validation parameter | Blood | Mammary |
|---|---|---|
| Matrix concentration range (ng/mL or ng/g)a | 5.00–500 | 100–5,000 |
| LODb (ng/mL or ng/g) | 0.499 | 17.7 |
| LOQc (ng/mL or ng/g) | 5.00 | 100 |
| Correlation coefficient (r) | ≥0.99 | ≥0.99 |
| Absolute recoveryd (%) | 67.1–79.0 | 1.26–3.70 |
| Relative recoverye (%) | 96.7–101 | 88.7–102 |
| Precision and accuracyf | ||
| Intraday precision (%RSD)g | ≤0.78 | ≤2.0 |
| Intraday accuracy (Mean %RE)h | ≤±13.4 | ≤±6.3 |
| Interday precision (%RSD) | ≤7.1 | ≤5.7 |
| Interday accuracy (Mean %RE) | ≤±9.6 | ≤±0.9 |
| Method extension (up to 1,500 ng/mL or 20,000 ng/g)i | ||
| Precision (%RSD) | 0.32 | 16.0 |
| Accuracy (mean %RE) | 18.7 | ≤±6.5 |
| Secondary matrix evaluation (HSD rat and B6C3F1 mouse blood and mammary)j | ||
| Male HSD precision (%RSD) | 2.4 | N/A |
| Male HSD accuracy (mean %RE) | 0.2 | N/A |
| Female HSD precision (%RSD) | 4.3 | ≤2.1 |
| Female HSD accuracy (mean %RE) | −1.2 | ≤±3.8 |
| Male B6C3F1 precision (%RSD) | 2.4 | N/A |
| Male B6C3F1 accuracy (mean %RE) | −3.9 | N/A |
| Female B6C3F1 precision (%RSD) | 1.7 | ≤2.0 |
| Female B6C3F1 accuracy (mean %RE) | −9.2 | ≤±3.2 |
Full validation was performed in SD rat blood and mammary gland, with matrix evaluation in HSD rat and B6C3F1 mouse blood and mammary.
Blood data are expressed as ng/mL; mammary data are expressed as ng/g.
LOD = limit of detection.
LOQ = lower limit of quantitation; lowest standard at which RE ≤ ±20% and RSD ≤ 20% for n = 6.
Absolute recovery expressed as percentage of AP peak area in matrix to AP peak area in solvent.
Relative recovery expressed as percentage of response ratio (AP:IS) in matrix to that in solvent.
Precision and accuracy determined for QCs at three levels in blood: 10, 100 and 250 ng/mL, and two levels in mammary gland: 250 and 2,500 ng/g. n = 3 for intraday; n = 9 for interday.
%RSD = percent relative deviation.
%RE = percent relative error.
Precision and accuracy determined for triplicate QCs at 1,500 ng/mL (blood) and 20,000 ng/g (mammary gland) analyzed using smaller injection volumes (150 µL for blood and 10–50 µL for mammary).
Precision and accuracy for secondary matrices determined for six replicate QCs at 10 ng/mL for blood and triplicate QCs at 250 and 2,500 ng/g for mammary gland.
N/A = not applicable (only female tissue assessed).