Table 2. Method Validation: Linearity, Intra-, and Interday Precision and Accuracy.
| name | calibration curvea | R2 | linear range (nmol/L) | CV-interdayb (%) | accuracy-interday (%) | CV-day 1 (%) | accuracy-day 1 (%) | CV-day 2 (%) | accuracy-day 2 (%) | CV-day 3 (%) | accuracy-day 3 (%) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| PEA | y = 0.0009x – 0.015 | 0.9999 | 5–10 000 | 14.7 | 103.7 | 4.5 | 103.1 | 8.3 | 124.1 | 2.3 | 87.5 |
| 2-pyrrolidinone | y = 0.0014x + 0.02 | 0.9995 | 10–1000 | 8.0 | 105.4 | 12.1 | 97.7 | 12.9 | 116.4 | 13.7 | 107.6 |
| pyroglutamine | y = 0.0002x + 0.0187 | 0.9992 | 100–10 000 | 12.2 | 100.5 | 7.1 | 92.9 | 8.6 | 91.2 | 3.6 | 117.9 |
| 3-ohaa | y = 0.0038x – 1.0377 | 0.9988 | 100–10 000 | 3.7 | 101.3 | 4.7 | 99.3 | 0.6 | 107.0 | 0.9 | 99.1 |
| 4-methoxyindole | y = 0.0004x + 0.0019 | 0.9998 | 50–10 000 | 2.8 | 101.8 | 11.1 | 98.7 | 2.8 | 105.0 | 4.3 | 103.3 |
| N-methyl-5HT | y = 0.0005x + 0.0126 | 0.9923 | 20–1000 | 21.5 | 93.3 | 5.9 | 95.6 | 5.3 | 92.5 | 6.0 | 85.1 |
| 5-HIAA | y = 0.196x + 0.8928 | 0.9992 | 0.5–1000 | 5.4 | 93.6 | 4.6 | 90.3 | 4.4 | 89.0 | 14.8 | 95.1 |
| ACh | y = 0.0026x + 0.156 | 0.9982 | 0.5–10 000 | 18.1 | 116.0 | 2.6 | 106.1 | 10.3 | 146.7 | 11.4 | 111.0 |
| AMFK | y = 0.0042x + 0.3512 | 0.9966 | 5–10 000 | 17.4 | 116.1 | 10.6 | 145.2 | 8.6 | 114.2 | 4.9 | 105.1 |
| 2AA | y = 0.0009x – 0.0063 | 0.9999 | 2–10 000 | 11.8 | 114.7 | 11.6 | 124.4 | 7.8 | 106.5 | 5.4 | 128.0 |
| choline | y = 0.0004x + 0.0031 | 0.9984 | 2–10 000 | 10.1 | 108.6 | 11.2 | 123.9 | 8.6 | 101.4 | 4.1 | 109.2 |
| coumarin | y = 0.0024x – 0.0941 | 0.9999 | 20–10 000 | 8.7 | 113.4 | 19.7 | 123.6 | 19.7 | 113.9 | 9.9 | 116.3 |
| hippuric acid | y = 0.0003x – 0.0042 | 0.9996 | 5–10 000 | 7.5 | 107.8 | 10.5 | 102.8 | 13.8 | 110.9 | 4.8 | 117.7 |
| histamine | y = 0.097x – 1.7507 | 0.9986 | 10–500 | 18.7 | 82.3 | 7.7 | 90.2 | 7.1 | 67.6 | 3.2 | 71.4 |
| indole | y = 0.0004x – 0.0103 | 0.9985 | 100–5000 | 8.0 | 97.3 | 6.2 | 106.1 | 2.6 | 95.4 | 1.5 | 87.6 |
| IAA | y = 0.0008x + 0.0419 | 0.9995 | 10–10 000 | 4.8 | 95.0 | 6.7 | 88.0 | 16.0 | 96.5 | 4.6 | 100.6 |
| IAN | y = 0.00004 – 0.0025 | 0.9996 | 100–10 000 | 6.6 | 101.3 | 3.9 | 102.9 | 1.8 | 109.2 | 10.8 | 93.0 |
| IAcrA | y = 0.0005x – 0.0063 | 0.9997 | 5–10 000 | 14.0 | 103.9 | 7.6 | 86.7 | 0.6 | 121.4 | 4.9 | 107.7 |
| I3A | y = 0.0051x + 0.0802 | 0.9994 | 2–10 000 | 2.0 | 102.8 | 11.4 | 104.3 | 1.8 | 104.3 | 9.2 | 102.4 |
| indole-3-carboxylate | y = 0.0001x – 0.0106 | 0.9996 | 100–10 000 | 10.0 | 96.8 | 2.0 | 83.0 | 15.4 | 98.9 | 14.0 | 105.5 |
| IEt | y = 0.0042x + 0.065 | 0.9999 | 5–10 000 | 15.3 | 108.8 | 8.6 | 133.7 | 12.0 | 102.3 | 4.1 | 99.3 |
| indole-3-lactic acid | y = 0.0003x – 0.0174 | 0.9994 | 20–10 000 | 13.0 | 122.9 | 14.9 | 136.7 | 16.0 | 126.3 | 18.0 | 128.8 |
| IPA | y = 0.0011x + 0.0041 | 0.9997 | 20–10 000 | 5.2 | 106.7 | 3.6 | 104.6 | 16.1 | 109.6 | 2.0 | 112.6 |
| Kyna | y = 0.0025x + 0.0448 | 0.9994 | 50–10 000 | 12.4 | 120.2 | 7.1 | 119.3 | 9.4 | 135.2 | 11.2 | 126.1 |
| Kyn | y = 0.0041x – 0.005 | 0.9997 | 10–10 000 | 2.6 | 96.5 | 13.7 | 96.6 | 5.8 | 95.0 | 6.0 | 94.3 |
| Arg | y = 0.003x – 0.1939 | 0.9989 | 5–10 000 | 14.6 | 100.7 | 8.4 | 87.7 | 6.2 | 121.4 | 13.1 | 93.7 |
| Glu | y = 0.0014x + 0.1077 | 0.9985 | 5–10 000 | 8.1 | 93.2 | 3.5 | 85.7 | 11.5 | 99.4 | 12.1 | 87.8 |
| Met | y = 0.00006x – 0.0034 | 0.9984 | 100–10 000 | 14.9 | 128.0 | 5.6 | 135.2 | 8.6 | 134.5 | 5.5 | 142.4 |
| Phe | y = 0.002x – 0.0058 | 0.9999 | 5–10 000 | 4.8 | 94.0 | 2.0 | 91.7 | 6.3 | 93.4 | 3.8 | 97.1 |
| Pro | y = 0.0064x + 0.2074 | 0.9994 | 5–5000 | 3.5 | 99.7 | 16.9 | 94.9 | 4.6 | 100.3 | 3.1 | 103.4 |
| PCA | y = 0.0005x – 0.0163 | 0.9994 | 50–10 000 | 7.1 | 96.6 | 7.7 | 100.5 | 13.2 | 86.4 | 17.6 | 99.7 |
| Trp | y = 0.0012x + 0.0038 | 0.9998 | 2–10 000 | 3.0 | 102.5 | 7.3 | 100.2 | 9.6 | 106.6 | 19.8 | 103.0 |
| Tyr | y = 0.0001x + 0.0164 | 0.9977 | 500–10 000 | 5.3 | 92.7 | 8.9 | 90.2 | 18.8 | 90.8 | 2.6 | 89.6 |
| melatonin | y = 0.0184x + 0.0187 | 0.9995 | 1–1000 | 7.1 | 96.2 | 3.6 | 100.3 | 7.8 | 98.4 | 6.5 | 86.0 |
| meIAA | y = 0.003x – 0.0259 | 0.9999 | 1–10 000 | 8.3 | 112.9 | 15.3 | 118.4 | 8.7 | 111.9 | 4.7 | 121.2 |
| PAA | y = 0.0016x – 0.0188 | 0.9999 | 5–10 000 | 6.9 | 107.8 | 9.1 | 114.7 | 14.9 | 113.6 | 11.0 | 103.0 |
| AFMK | y = 0.0006x – 0.0009 | 1 | 2–500 | 8.7 | 92.4 | 0.8 | 90.7 | 7.6 | 97.1 | 7.2 | 81.8 |
| NAS | y = 0.023x – 0.6326 | 0.9994 | 2–5000 | 8.7 | 100.7 | 2.4 | 111.8 | 2.7 | 90.3 | 3.9 | 100.6 |
| NMPEA | y = 0.0015x + 0.041 | 0.9991 | 5–10 000 | 15.5 | 113.5 | 7.7 | 139.1 | 4.8 | 109.9 | 10.7 | 105.1 |
| nicotinic acid | y = 0.00006x + 0.0003 | 0.9984 | 50–10 000 | 16.1 | 88.8 | 9.6 | 98.1 | 10.6 | 68.8 | 11.9 | 88.4 |
| niacin | y = 0.0021x – 0.0145 | 0.9987 | 10–1000 | 4.8 | 99.9 | 2.0 | 100.6 | 7.0 | 105.5 | 6.3 | 93.7 |
| Nα-acetyl-l-glutamine | y = 0.0007x – 0.0281 | 0.9998 | 20–10 000 | 4.8 | 104.9 | 11.4 | 108.0 | 3.6 | 101.2 | 1.6 | 110.2 |
| GIcNAc | y = 0.0001x – 0.0217 | 0.9961 | 100–10 000 | 18.7 | 106.7 | 4.2 | 81.4 | 5.4 | 122.9 | 6.1 | 122.5 |
| phenylacetyl l-glutamine | y = 0.0001x – 0.0217 | 0.9961 | 100–10 000 | 2.2 | 101.2 | 4.2 | 101.4 | 5.4 | 104.2 | 6.1 | 99.1 |
| PAG | y = 0.0022x + 0.1629 | 0.999 | 20–10 000 | 7.9 | 110.9 | 15.1 | 119.9 | 3.9 | 107.9 | 16.0 | 115.7 |
| 5-HT | y = 0.0002x – 0.00003 | 0.999 | 1–500 | 5.6 | 103.6 | 8.0 | 100.0 | 5.9 | 102.3 | 6.1 | 112.1 |
| TMAO | y = 0.001x – 0.0053 | 0.9997 | 5–500 | 13.2 | 97.7 | 2.8 | 108.9 | 4.2 | 102.7 | 8.5 | 79.2 |
| tyramine | y = 0.0049x + 0.6964 | 0.9959 | 5–10 000 | 8.8 | 91.3 | 10.1 | 82.1 | 15.0 | 95.7 | 12.8 | 87.6 |
| XA | y = 0.0012x – 0.011 | 0.9998 | 5–10 000 | 4.8 | 94.6 | 6.3 | 92.7 | 9.0 | 89.3 | 6.2 | 96.2 |
| GABA | y = 0.0002x + 0.0037 | 0.9998 | 20–10 000 | 6.2 | 91.6 | 1.4 | 89.6 | 7.3 | 87.8 | 8.7 | 88.8 |
a
The calibration curves listed here are those obtained by plotting ASD-to-ISD peak area ratio (y-axis) over the absolute concentration of targeted analytes (x-axis).
b
Intra- and interday precision and accuracy at the LLOQ level.