Table 3. Validation parameters of the analytical method by UPLC-MS/MS, to quantify ifosfamide in DBS at 30% HTC.
| LLQ (100 ng/mL) | |||
|---|---|---|---|
| Intra-day variability | Day 1 | Day 2 | Day 3 |
| Mean± SD (n = 5) | 109.2±3.4 | 96.7± 5.3 | 102.7± 5.0 |
| % CV | 3.1 | 5.5 | 4.8 |
| % Deviation | -9.2 | 3.2 | -2.7 |
| Inter-day variability | |||
| Mean± SD (n = 15) | 102.9 ± 6.8 | N/R | N/R |
| % CV | 6.6 | N/R | N/R |
| % Deviation | -2.9 | N/R | N/R |
| Inter-day variability | QC1 (ng/mL) | QC2 (ng/mL) | QC3 (ng/mL) |
| Mean± SD (n = 15) | 306.0 ± 26.9 | 3930.9 ± 228.0 | 7519.8 ± 293.9 |
| % CV | 8.7 | 5.8 | 3.9 |
| % Deviation | -2.0 | 1.7 | 6.0 |
| Intra-day variability | |||
| Mean± SD (n = 5) a | 269.3±8.2 | 3819.0± 167.4 | 7681.7 ± 263.6 |
| % CV | 3.0 | 4.3 | 3.4 |
| % Deviation | 10.2 | 4.5 | 3.9 |
| Mean± SD (n = 5) b | 327.2±8.1 | 4135.3 ± 239.0 | 7479.6 ± 306.5 |
| % CV | 3.4 | 5.8 | 4.1 |
| % Deviation | -9.08 | -3.3 | 6.5 |
| Mean± SD (n = 5) c | 316.35 ± 7.7 | 3830.8 ± 58.7 | 7293.8 ± 294.09 |
| % CV | 2.4 | 1.5 | 4.0 |
| % Deviation | -5.4 | 4.2 | 8.8 |
| Stability assays | QC1 | QC2 | QC3 |
| Room temperature (25 o C)/ 3 days | |||
| Mean± SD (n = 5) | 301.9±16.9 | N/R | 7789.8 ± 351.7 |
| % CV | 5.6 | N/R | 4.5 |
| % Deviation | -0.6 | N/R | 2.6 |
| Short term stability (4h at 25 o C) | |||
| Mean± SD (n = 5) | 304.6 ± 8.7 | N/R | 7922.6 ± 357.1 |
| % CV | 2.8 | N/R | 4.5 |
| % Deviation | -1.5 | N/R | 0.97 |
| Autosampler (8h) | |||
| Mean± SD (n = 5) | 310.1 ± 16.5 | N/R | 7182.8 ± 161.0 |
| % CV | 5.3 | N/R | 2.2 |
| % Deviation | -3.3 | N/R | 10.2 |
| Freeze-thaw (3 cycles) | |||
| Mean± SD (n = 5) | 280.4 ± 7.9 | N/R | 7120.1 ± 124.5 |
| % CV | 2.8 | N/R | 1.7 |
| % Deviation | 6.5 | N/R | 11.8 |
| Processed sample (20 h) | |||
| Mean± SD (n = 5) | 311.9 ± 16.3 | N/R | 7328.3 ± 483.5 |
| % CV | 5.2 | N/R | 6.6 |
| % Deviation | 3.9 | N/R | 8.4 |
| Long term stability (52 d at -80 ° C) | |||
| Mean± SD (n = 5) | 293.7 ± 14.5 | N/R | 7764.4 ± 293.6 |
| % CV | 4.9 | N/R | 3.7 |
| % Deviation | 2.0 | N/R | 2.9 |
SD: Standard Deviation. CV: Coefficient of Variation. N/R: Not required according to the guidelines. Theoretical amounts for QC1, QC2 and QC3 were 300, 4000 and 8000 ng/mL, respectively.