TABLE 3.
Validation of the UHPLC-MS/MS-sMRM method to quantify free endocannabinoids and ERCs in human plasma
| Recovery (%) n = 6 | Intraday CV (%) n = 6 | Interday CV (%) 3x n = 4 | Literature | |||||||||||||
| Amount spiked | Q | q | Q | q | ||||||||||||
| Low | Med | High | Unspiked Plasma | Low | Med | High | Unspiked NAPEs | Unspiked Plasma | Low | Med | High | LOQ | LOD | LOQ | LOD | |
| 2-AG | 91 | 92 | 92 | 3 | 4 | 4 | 5 | < LOD | 8 | 9 | 7 | 4 | 0.27 | 0.1 | 0.5-3.2a,d,e,f | 0.3a |
| 1-AG | 107 | 101 | 96 | 8 | 7 | 1 | 5 | < LOD | 15 | 21 | 5 | 7 | 0.27 | 0.1 | ||
| 2-LG | 107 | 93 | 95 | 9 | 9 | 6 | 4 | < LOD | 8 | 10 | 4 | 4 | 0.62 | 0.24 | 2.5-29.3d,e | — |
| 1-LG | 98 | 96 | 99 | 7 | 7 | 5 | 4 | < LOD | 10 | 10 | 6 | 5 | 0.62 | 0.24 | ||
| 2-OG | 82 | 86 | 88 | 9 | 11 | 9 | 6 | < LOD | 11 | 12 | 7 | 10 | 1.82 | 0.36 | 2.5e | — |
| NE | 93 | 95 | 98 | < LOD | 7 | 5 | 3 | < LOD | < LOD | 13 | 8 | 4 | 0.07 | 0.07 | 1.8-3.2a,d | 3.2a |
| PEA | 96 | 98 | 100 | 5 | 3 | 2 | 2 | 6 | 10 | 9 | 5 | 2 | 0.21 | 0.06 | 0.17-3.2a,c,d,e | 0.5a |
| SEA | 101 | 107 | 103 | 9 | 4 | 3 | 1 | 6 | 22 | 12 | 14 | 21 | 0.66 | 0.66 | 0.27-8a,c,d,e | 0.5a |
| POEA | 112 | 97 | 102 | 5 | 4 | 1 | 4 | 4 | 4 | 6 | 3 | 3 | 0.08 | 0.08 | 0.02-1.6d,e | — |
| OEA | 91 | 100 | 101 | 7 | 5 | 1 | 2 | 5 | 6 | 6 | 2 | 2 | 0.15 | 0.15 | 0.12-0.5a,c,d,e | 0.2a |
| VEA | 128 | 102 | 112 | 7 | 5 | 3 | 4 | 9 | 6 | 5 | 6 | 5 | 0.15 | 0.14 | — | — |
| tVEA | 113 | 103 | 108 | 14 | 6 | 5 | 7 | 5 | 16 | 10 | 13 | 10 | 0.04 | 0.04 | — | — |
| EEA | 93 | 97 | 103 | < LOD | 9 | 4 | 4 | < LOD | < LOD | 13 | 15 | 11 | 0.04 | 0.04 | — | — |
| tPeEA | 87 | 85 | 90 | < LOD | 13 | 3 | 6 | < LOD | < LOD | 12 | 5 | 6 | 0.02 | 0.01 | — | — |
| ALEA | 98 | 97 | 99 | 9 | 7 | 4 | 4 | 10 | 9 | 9 | 5 | 4 | 0.04 | 0.04 | 0.11c | — |
| GLEA | 96 | 96 | 96 | < LOD | 5 | 5 | 3 | 11 | < LOD | 7 | 4 | 3 | 0.02 | 0.02 | — | — |
| DALEA | 99 | 92 | 98 | < LOD | 10 | 4 | 7 | < LOD | < LOD | 9 | 6 | 6 | 0.12 | 0.12 | — | — |
| DGLEA | 109 | 97 | 98 | 7 | 6 | 3 | 4 | 7 | 10 | 8 | 4 | 4 | 0.02 | 0.02 | 0.02e | — |
| LEA | 108 | 100 | 102 | 3 | 3 | 1 | 2 | 3 | 5 | 5 | 3 | 2 | 0.08 | 0.06 | 0.1-0.24a,c,d,e | 0.6a |
| AEA | 98 | 95 | 98 | 3 | 5 | 2 | 2 | 5 | 6 | 5 | 4 | 3 | 0.06 | 0.02 | 0.02-0.36a,c,d,e,f | 0.1a |
| ETEA | 95 | 93 | 97 | < LOQ | 6 | 3 | 3 | 9 | < LOQ | 5 | 3 | 3 | 0.01 | 0.01 | — | — |
| EPEA | 95 | 99 | 96 | 6 | 6 | 6 | 4 | 6 | 14 | 6 | 4 | 4 | 0.01 | 0.01 | 0.02-0.36c,e | — |
| DHEA | 105 | 94 | 94 | 6 | 5 | 1 | 3 | 6 | 7 | 6 | 3 | 3 | 0.03 | 0.01 | 0.02-2.6a,d | 2.6a |
| NAGly | 108 | 99 | 102 | 5 | 8 | 5 | 3 | < LOD | 9 | 8 | 5 | 4 | 0.12 | 0.12 | 0.32-0.73b,d | 0.63b |
Accuracy (recovery, %) was determined by spiking three calibrator solutions at the lower and limits of quantification (LOQ), in an intermediate and in a high concentration range as indicated in supplemental Table S3. Interday and intraday precision (CV %) was assessed with unspiked plasma and with three different concentrations of spiked analytes. Precision of NAPE hydrolysis was determined with unspiked plasma. LOQ of the quantifier transition Q and LOD of the qualifier transition q are given in ng/ml. References: a (24), b (29), c (27), d (26), e (22), and f (28).