Table 1.
Results of basic validation.
| Intraday | Interday | Operator | Autosampler Stability | Recovery | Carryover | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| QC-L QC-M QC-H |
5 Injections of One Sample Set | Independent Preparations on 6 Days | 10 Preparations on One Day | 24 h in Between Injections | Ratio Peak Area Pre-/Post Extraction (%) |
Ratio Peak Area Blank/ LLOQ (%) |
|||||||
| Exp. Conc (µM) | Mean (µM) | Accuracy (%) | Precision (%CV) | Mean (µM) | Accuracy (%) | Precision (%CV) | Mean (µM) | Accuracy (%) | Precision (%CV) | Ratio Conc. d1/d0 (%) | |||
| Itaconate | 0.3 | 0.3 | 102.7 | 2.6 | 0.3 | 101.1 | 3.0 | 0.3 | 106.4 | 4.6 | 96.4 | 100.1 | |
| 3 | 3.1 | 102.6 | 1.0 | 3.1 | 104.2 | 4.7 | 3.1 | 103.4 | 2.3 | 99.7 | 101.4 | ||
| 80 | 87.1 | 108.8 | 1.4 | 86.2 | 107.7 | 3.8 | 84.3 | 105.4 | 1.9 | 100.2 | 105.2 | 3.8 ± 5.1 | |
| Citraconate | 0.15 | 0.2 | 100.7 | 4.0 | 0.2 | 100.7 | 6.7 | 0.2 | 114.5 | 4.6 | 90.5 | 103.8 | |
| 3 | 3.1 | 102.9 | 2.4 | 3.1 | 103.7 | 5.2 | 3.0 | 101.0 | 2.8 | 102.3 | 101.0 | ||
| 40 | 44.0 | 110.0 | 1.2 | 43.4 | 108.4 | 3.8 | 42.9 | 107.2 | 2.1 | 98.2 | 106.3 | 5.1 ± 6.5 | |
| Mesaconate | 0.3 | 0.3 | 107.7 | 1.8 | 0.3 | 104.1 | 4.9 | 0.3 | 105.4 | 7.0 | 95.2 | 99.2 | |
| 3 | 3.1 | 104.9 | 2.6 | 3.2 | 105.4 | 4.8 | 3.1 | 102.9 | 1.5 | 97.9 | 101.7 | ||
| 80 | 86.9 | 108.7 | 1.4 | 86.1 | 107.6 | 3.4 | 85.2 | 106.5 | 2.1 | 99.0 | 106.1 | 4.5 ± 6.7 | |
| 4-Octyl-Itaconate | 0.03 | 0.03 | 110.9 | 2.2 | 0.03 | 107.9 | 12.2 | 0.04 | 119.3 | 6.8 | 96.6 | 88.8 | |
| 0.3 | 0.3 | 90.5 | 2.3 | 0.3 | 95.4 | 10.6 | 0.3 | 99.0 | 2.3 | 96.1 | 114.9 | ||
| 2 | 2.3 | 112.7 | 1.9 | 2.3 | 113.8 | 5.2 | 2.1 | 107.1 | 3.0 | 97.3 | 170.4 | 21.8 ± 5.2 | |
| cis-Aconitate | 0.15 | 0.2 | 100.5 | 1.3 | 0.1 | 99.9 | 4.8 | 0.1 | 98.1 | 4.1 | 102.6 | 99.3 | |
| 3 | 3.1 | 102.5 | 0.9 | 3.1 | 102.8 | 4.0 | 3.0 | 101.5 | 2.0 | 98.8 | 101.3 | ||
| 40 | 43.7 | 109.2 | 1.6 | 43.5 | 108.8 | 2.9 | 42.5 | 106.3 | 1.4 | 99.3 | 109.2 | 3.3 ± 5 | |
| Citrate | 1.2 | 1.3 | 105.3 | 0.8 | 1.3 | 105.4 | 1.1 | 1.3 | 104.8 | 3.8 | 101.3 | 94.9 | |
| 6 | 6.4 | 106.2 | 1.3 | 6.4 | 106.0 | 2.0 | 6.2 | 103.5 | 1.0 | 101.0 | 97.2 | ||
| 20 | 22.0 | 109.9 | 2.1 | 21.8 | 108.9 | 3.8 | 20.9 | 104.7 | 1.4 | 99.8 | 104.4 | 0.2 ± 0.2 | |
| Fumarate | 5 | 5.5 | 109.3 | 2.4 | 5.4 | 107.3 | 2.7 | 5.3 | 107.0 | 2.0 | 100.0 | 97.6 | |
| 12 | 13.0 | 108.0 | 2.4 | 13.1 | 108.9 | 2.4 | 12.6 | 105.1 | 1.3 | 102.9 | 100.8 | ||
| 40 | 45.1 | 112.8 | 1.9 | 45.8 | 114.4 | 3.3 | 43.2 | 107.9 | 1.4 | 98.8 | 108.4 | 0.5 ± 1 | |
| Isocitrate | 0.6 | 0.6 | 99.9 | 2.2 | 0.6 | 101.9 | 3.3 | 0.6 | 102.5 | 3.5 | 99.1 | 98.4 | |
| 6 | 6.2 | 103.8 | 1.7 | 6.4 | 106.6 | 2.8 | 6.5 | 109.0 | 1.6 | 98.3 | 101.4 | ||
| 40 | 40.9 | 102.3 | 2.2 | 42.1 | 105.2 | 4.8 | 43.2 | 107.9 | 2.5 | 97.3 | 112.1 | 15.6 ± 11.3 | |
| Lactate | 50 | 54.5 | 109.0 | 2.6 | 55.6 | 111.2 | 6.2 | 55.0 | 109.9 | 5.9 | 97.3 | 103.0 | |
| 150 | 160.6 | 107.1 | 1.5 | 159.8 | 106.6 | 4.9 | 161.6 | 107.7 | 3.4 | 99.4 | 100.5 | ||
| 400 | 449.2 | 112.3 | 2.7 | 436.8 | 109.2 | 5.5 | 427.5 | 106.9 | 2.0 | 100.8 | 103.9 | 2.1 ± 1.3 | |
| Malate | 0.6 | 0.7 | 109.8 | 3.8 | 0.7 | 110.4 | 4.1 | 0.7 | 116.6 | 3.9 | 97.9 | 99.8 | |
| 6 | 6.4 | 107.5 | 2.1 | 6.6 | 109.3 | 3.2 | 6.7 | 111.5 | 2.0 | 98.3 | 102.5 | ||
| 40 | 41.4 | 103.4 | 2.0 | 42.0 | 104.9 | 4.9 | 43.0 | 107.5 | 2.3 | 99.7 | 110.7 | 18.8 ± 12.8 | |
| Succinate | 5 | 4.6 | 92.5 | 1.4 | 4.4 | 87.6 | 7.0 | 3.9 | 78.5 | 2.7 | 98.6 | 99.1 | |
| 12 | 11.3 | 94.2 | 2.0 | 10.8 | 90.0 | 6.1 | 9.3 | 77.6 | 1.7 | 102.2 | 101.5 | ||
| 40 | 41.2 | 103.0 | 1.7 | 39.7 | 99.2 | 6.6 | 35.3 | 88.3 | 1.9 | 97.8 | 106.5 | 13 ± 4.7 | |
Deviations > 10% are underlined once; deviations > 15%, which do not meet FDA recommendations, are underlined twice.