Table 1.
Validation results for DTNB and DLM methods.
| Validation test | Acceptance criteria | Method result | |
|---|---|---|---|
| DTNB | DyLight 488 | ||
| Precision (repeatability) | <20% | RSD: 2.52% (denaturing-reducing conditions). RSD: 17.3% (native conditions). |
RSD: NT% (denaturing-reducing conditions). RSD: 13.3% (native conditions). |
|
| |||
| Accuracy | Total recovery between 95 and 105% in respect to theoretical value. | 99.5% recovery using Somatropin (Cys/Protein ratio 3.98) | 101.0% recovery using HSA (Cys/Protein ratio 1.01) |
|
| |||
| Intermediate precision | RSD < 10% | RSD: 3.8% | RSD: 0.66% |
|
| |||
| Linearity |
r
2 ≥ 0.980 Ordinate = 0 |
r
2 = 0.999 native and denaturing-reducing conditions. Slope = 0.01285 μM−1 r 2 = 0.983 denaturing conditions. Slope = 0.0093 μM−1 Forced through zero. |
r
2 = 0.990 native and denaturing conditions. Slope = 465,834 μM−1 Forced through zero. |
|
| |||
| Quantification limit | Report value | 5 μM | 0.1 μM |
|
| |||
| Selectivity | Cysteine-free protein has the same signal of placebo. | GA: 0.0010 Placebo: 0.0005 |
PM: 0.0005 Placebo: 0.0005 |
NT%: not tested.