Table 1.
Method validation parameters in melatonin-free matrix and real nail matrix. Calculated melatonin-free matrix parameters were linearity (in terms of weighting and range), LLOQ, LQC, MQC, HQC and LOD (in terms of accuracy and CV). Calculated real nail matrix parameters were within-run and total accuracy and CV; matrix effect (in terms of recovery and CV); the endogenous range of selected samples’ melatonin; and specificity of the method by comparing standard and samples. Units of range, level, LOD and concentration are fg/mg.
| Melatonin-Free Matrix (CDER Regulations) | ||||||
|---|---|---|---|---|---|---|
| Linearity | Weight. | Range (fg/mg) | ||||
| 1/x | 6.5–830 | |||||
| LLOQ | LQC | MQC | HQC | LOD | ||
| Level | 6.5 | 13 | 0.104 | 830 | 3.5 | |
| Acc. (%) | 113 | 115 | 104 | 103 | - | |
| CV (%) | 8 | 6 | 4 | 4 | - | |
| Real nail matrix (Standard additions) | ||||||
| Within-run (n = 6) | Total (n = 12) | M.E. (n = 6) | Endog Range | |||
| Acc. (%) | 100 | 104 | Rec. (%) | 105 | Conc. | 30–110 |
| CV (%) | 10 | 13 | CV (%) | 7 | (fg/mg) | |
| Specificity | ||||||
| Standard | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | |
| Q/q ratio | 0.22 | 0.23 | 0.21 | 0.24 | 0.23 | 0.22 |
Abbreviations: CDER: Center for Drug Evaluation and Research; Weight: weighting; LLOQ: lowest limit of quantification; LQC: lowest QC; MQC: medium QC; HQC: highest QC; LOD: limit of detection; Acc.: accuracy; CV: coefficient of variation; M.E.: matrix effect; Endog Range: endogenous range; Rec.: recovery; Conc: concentration; Q/q: ion ratio between quantitative transition (233 > 174) and qualitative transition (233 > 159).