Table 4.
Inter-day (n = 3 days) recovery, accuracy and precision for validation of the analytical method developed.
| Recovery | Accuracy | Precision | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Analyte (Conc.) | Spike level | day1 | day2 | day3 | inter-day | %Deviation | %RSD | ||||||
| day1 | day2 | day3 | inter-day | day1 | day2 | day3 | inter-day | ||||||
| AA (nM) | 100 | 102 | 108 | 104 | 104 ± 3 | 3.4 | 6.4 | 5.9 | 5 ± 1 | 5.9 | 10 | 11 | 9 ± 2 |
| 501 | 93.8 | 94.5 | 89 | 92 ± 2 | 5.3 | 8.7 | 9.4 | 8 ± 2 | 4.4 | 10 | 4.2 | 6 ± 3 | |
| 1002 | 104 | 104 | 105 | 104 ± 4 | 3.9 | 4 | 5.4 | 4.4 ± 0.7 | 3.1 | 3.8 | 5 | 4.0 ± 0.8 | |
| PA (nM) | 96 | 96.6 | 105 | 84.7 | 95 ± 8 | 4.9 | 6.6 | 18 | 10 ± 6 | 5.1 | 6.2 | 16 | 9 ± 5 |
| 561 | 103 | 100 | 101 | 101 ± 1 | 3.3 | 1.5 | 2.2 | 2.3 ± 0.7 | 0.2 | 1.7 | 2 | 1.3 ± 0.8 | |
| 962 | 102 | 106 | 104 | 104 ± 2 | 2.1 | 6.5 | 3.8 | 4 ± 2 | 0.9 | 4.2 | 3.4 | 3 ± 1 | |
| KA (nM) | 50 | 114 | 117 | 61 | 97 ± 26 | 13.3 | 11 | 20 | 15 ± 4 | 24 | 18 | 12 | 18 ± 5 |
| 100 | 90.3 | 107 | 90.4 | 96 ± 8 | 6.4 | 6.6 | 6.5 | 6.5 ± 0.1 | 5.1 | 7.4 | 10 | 8 ± 2 | |
| 1003 | 101 | 94.3 | 98.5 | 98 ± 3 | 2.8 | 5.5 | 2.9 | 4 ± 1 | 2.6 | 4.5 | 1.6 | 3 ± 1 | |
| QA (nM) | 197 | 110 | 89.6 | 116 | 105 ± 11 | 4.7 | 6.1 | 6.9 | 5.9 ± 0.9 | 9.2 | 14 | 15 | 13 ± 3 |
| 1077 | 106 | 107 | 121 | 111 ± 7 | 8.4 | 5.8 | 16 | 10 ± 4 | 11 | 7.3 | 0.6 | 6 ± 4 | |
| 5385 | 98 | 103 | 98 | 100 ± 2 | 5 | 3.7 | 2.3 | 4 ± 1 | 5.9 | 3.5 | 3 | 4 ± 1 | |
| TRP (μM) | 71 | 92.4 | 89.4 | 87.4 | 90 ± 2 | 4.8 | 6.2 | 7.3 | 6 ± 1 | 6.8 | 0.9 | 1.8 | 3 ± 3 |
| 142 | 103 | 101 | 104 | 103 ± 1 | 2.5 | 1 | 3.1 | 2.2 ± 0.9 | 2.1 | 1.9 | 1.3 | 1.8 ± 0.3 | |
| 282 | 104 | 103 | 104 | 103.7 ± 0.5 | 3.8 | 2.2 | 3.2 | 3.1 ± 0.7 | 3.2 | 0.8 | 1.8 | 2 ± 1 | |
| KYN (μM) | 4 | 114 | 118 | 123 | 118 ± 4 | 11 | 14 | 18 | 14 ± 3 | 5 | 0.6 | 0.8 | 2 ± 2 |
| 8 | 90 | 89.6 | 90.6 | 90.1 ± 0.4 | 8.7 | 9.2 | 8.1 | 8.7 ± 0.5 | 0.9 | 2.2 | 3 | 2.0 ± 0.9 | |
| 16 | 98.3 | 99.4 | 98.8 | 98.8 ± 0.5 | 1.5 | 3.1 | 2.3 | 2.3 ± 0.7 | 0.5 | 3.3 | 2.3 | 2 ± 1 | |
The assay recovery for the validation was calculated as follows: spiked recovery = 100 × (measured concentration for spiked sample - the measured concentration for nonspiked sample)/spiked value.
The measured concentration of spiked sample = Mean basal concentration (X) + spiked conc.
the measured concentration for nonspiked sample = Mean basal concentration (X).
Mean basal concentration is determined as an unknown concentration value obtained from the calibration curve shown in Fig. S2 and text box A.
Spiked value = added standard concentration.
The assay accuracy (% deviation) = 100 × І(measured concentration for spiked sample - the theoretical concentration for spiked sample)І/ the theoretical concentration for the spiked sample. Theoretical concentration for spiked sample = Mean Basal concentration + added standard concentration.