Table 3. Summary of bioanalytical method development results.
| 1 | Analyte name | Methotrexate |
| 2 | Internal standard | Aminoacetophenone |
| 3 | Analytical method type | LC-MS/MS |
| 4 | Extraction method | Protein precipitation |
| 5 | Sample volume | 10 μL |
| 6 | QC concentrations | 0.09, 0.5, 5, and 10 μM |
| 7 | Standard curve concentrations | 0.09, 0.198, 0.39, 0.78, 1.56, 3.12, 6.25, and 12.5 μM |
| 8 | Lower limit of quantitation | 0.09 |
| 9 | Upper limit of quantitation | 12.5 |
| 10 | Mean recovery of analyte (%) | 40 |
| 11 | Mean recovery of IS (%) | 40 |
| 12 | LLOQ QC intraday precision range (% CV) | 10.25–12.5 |
| 13 | LLOQ QC intraday accuracy range (% RE) | 11.1–18.9 |
| 14 | Analytical QC intraday precision range (% CV) | 1.0–2.18 |
| 15 | Analytical QC intraday accuracy range (% RE) | 0.01–15.2 |
| 16 | LLOQ QC interday precision range (% CV) | 10.25–19.4 |
| 17 | LLOQ QC interday accuracy range (% RE) | 11.1–23.3 |
| 18 | Analytical QC interday precision range (% CV) | 1.6–20 |
| 19 | Analytical QC interday accuracy range (% RE) | 79–103 |
| 20 | Dilution integrity | 1:5 in water |
| 21 | Selectivity at LLOQ | 86% for MTX and 99.7% for IS |
| 22 | Matrix effect | 4.56% for low QC and 2.1% for high QC |
Abbreviations: CV, coefficient of variation; IS, internal standard; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LLOQ, lower limit of quantification; QC, quality control; RE, relative error.