Table 3.
Detection and analytical methods utilized in the four participating laboratories
| Laboratory #1 | Laboratory #2 | Laboratory #3 | Laboratory #4 | |
|---|---|---|---|---|
|
Detection method |
UHPLC-MS/MS using a Shimadzu (Kyoto, Japan) Nexera UHPLC system and Shimadzu LCMS-8080 triple quadrupole tandem mass spectrometer |
Agilent 1100 series HPLC (Agilent Technologies Inc.,) interfaced with an Applied Biosystems API 5500 electrospray triple-quadrupole mass spectrometer (ESI-MS/MS; Applied Biosystems). |
LC-MS/MS (Agilent LC 1260- AB Sciex 5500 Triple Quadrupole) |
LC-MS/MS using a Thermo Surveyor TSQ plus connected to an integrated Thermo-Accela LC system. |
|
Blanks |
Charcoal/dextran stripped human serum |
Milli-Q water. Trace levels of free BPA were found in procedural blanks in some batches (0.40-0.46 for Phase 2, 0.19-0.28 for Phase 3). |
Double-charcoal stripped human serum |
HPLC-grade water (Fisher Scientific; cat# W5-4). |
|
Blanks: parallel? |
Yes |
Yes |
Yes |
Yes |
|
Blank values subtracted from environmental samples? |
Yes |
Yes |
Not required – no contamination found in blanks |
Yes |
|
LLOQ |
0.10 ng/ml (uBPA) |
0.01 ng/ml (uBPA) |
0.1 ng/ml (uBPA and BPA-G) |
0.13 ng/ml (uBPA) |
| 0.01 ng/ml (BPA-G) |
0.05 ng/ml (BPA-G) |
|
0.06 ng/ml (BPA-G) |
|
|
ULOQ |
50 ng/mL |
|
40 ng/ml (uBPA and BPA-G) |
|
|
Calculations for determining LLOQ, ULOQ |
FDA Guidance for industry biomedical method validation |
The LOD and LOQ were calculated as 3 times (3S) and 10 times (10S) of the standard deviations (S) of five replicate analyses, using the lowest calibration standard (0.01 ng/mL) |
a signal that has a S/N of at least 10 and is the lowest calibrant that allows a linear regression coefficient of at least 0.95 |
The LOD and LOQ were calculated as 3 times (3S) and 10 times (10S) of the standard deviations (S) of three replicate analyses, using the lowest calibration standard. The means of three assays are given. |
|
http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf | ||||
| Standard procedure. C1 pg. 6 of Guidance | ||||
| LOD | 0.02 ng/ml (uBPA) 0.002 ng/ml (BPA-G) | 0.003 ng/ml (uBPA) 0.02 ng/ml (BPA-G) | 0.05 ng/ml (uBPA and BPA-G) | 0.04 ng/ml (uBPA) 0.02 ng/ml (BPA-G) |