Table 2.
Summary of Analytical Method Validation and Stability Data of BPB and 3-OH BPB.
| Plasma/ Amniotic Fluidh |
Pup/Fetal Homogenatei |
|||
|---|---|---|---|---|
| BPB | 3-OH-BPB | BPB | 3-OH-BPB | |
| Free BPB | ||||
| LOD | ||||
| ng/mLa | 0.17 | 0.0697 | 0.477 | 0.121 |
| ng/gb | N/A | N/A | 1.91 | 0.484 |
| ELOQs | ||||
| ng/mLa | 1.01 | 0.5 | 2.02 | 1.01 |
| ng/gb | N/A | N/A | 8.08 | 4.04 |
| Concentration Range | ||||
| ng/mLa | 1 to 1000 | 0.5 to 500 | 2 to 1000 | 1 to 500 |
| ng/ga | N/A | N/A | 8 to 4000 | 4 to 2000 |
| Total BPB | ||||
| LOD | ||||
| ng/mLa | 0.292 | 0.133 | 0.573 | 0.219 |
| ng/gb | N/A | N/A | 2.29 | 0.876 |
| ELOQ | ||||
| ng/mLa | 1.01 | 0.5 | 2.01 | 0.988 |
| ng/gb | N/A | N/A | 8.04 | 3.95 |
| Concentration Range | ||||
| ng/mLa | 1 to 1000 | 0.5 to 500 | 2 to 1000 | 1 to 500 |
| ng/gb | N/A | N/A | 8 to 4000 | 4 to 2000 |
| Mean Accuracy Rangec, d, e | -8.2 to 15% | -8.9 to 11.3% | -6.8 to 6.0% | -7.6 to 11% |
| Intra-Day Precisiond, e | 1.6 to 9.3% | 1.5 to 11% | 2.2 to 6.2% | 3.4 to 6.7% |
| Inter-Day Precisiond, e | 1.6 to 25%j | 1.1 to 14% | 1.4 to 3% | 1.6 to 10% |
| Mean Extraction Recoverye | ||||
| Free | 88.2 to 120% | 91 to 115% | 85.3 to 120% | 77.4 to 95.4% |
| Total | 82.9 to 132% | 82.3 to 124% | 90.6 to 135% | 64.9 to 86.9% |
| Stability | ||||
| Extract Stability (4 days)e, f | 80 to 99.8% | 89.4 to 115% | 91.5 to 108% | 99.2 to 122% |
| Matrix Freeze/Thaw Stability at −70°C(4 or 9 days)g | 87.7 to 97.4% | 88 to 99.3% | 93.7 to 118% | 99.6 to 119% |
| Matrix Stability at −70 °C | ||||
| (60 days) | 104 to 106% | 108 to 110% | 100 to 105% | 101 to 110% |
| Matrix Stability at −70°C | ||||
| (206 days) | 90.6 to 116% | 86.8 to 108% | N/A | N/A |
The limit of detection (LOD) and experimental limit of quantitation (ELOQ) values are shown for free (unconjugated) BPB, total (unconjugated+ conjugated) BPB, free 3-OH BPB and total 3-OH BPB. ELOQ is the lowest standard and LOD was estimated as three times the standard deviation of the ELOQ.
Values indicate either maternal plasma or pup homogenate (1 g pup in 3 mL water, total volume 4 mL).
Values indicate mass concentration per gram of pup.
Calculated as the percent relative error.
Accuracy and Precision were calculated using calibration and QC samples.
Values indicate the range across 8 concentrations.
Values indicate mean percent recovered after refrigerator, autosampler or ambient air storage for 4 days.
Spiked matrix stored at −70 °C and subjected to 3 freeze-thaw cycles over 4 days (plasma) or 9 days (pup homogenate).
Amniotic fluid and pup plasma were cross-validated using maternal plasma calibration curves.
Fetal homogenate was cross-validated using pup homogenate calibration curves.
The 1 ng/mL sample had an inter-day precision of 25%, all other concentrations were within 11%.