Table 2 Extraction methods used in 4 participating laboratories
From: A round robin approach to the analysis of bisphenol a (BPA) in human blood samples
| Â | Laboratory #1 | Laboratory #2 | Laboratory #3 | Laboratory #4 |
|---|---|---|---|---|
Volume used in analysis | 200 μL | 500 μL | 250 μL | 1000 μL for spiked samples, 550 - 1000 μL for environmental samples |
Extraction protocol | Protein precipitation used Honeywell Burdick & Jackson LC-MS grade acetonitirile containing 10 ng/mL [d6]-BPA. | Solid phase extraction: A Strata® NH2 cartridge (#8B-S009-FBJ; 200 mg/3 cc; Phenomenex, Torrance, CA) mounted on an Oasis® MCX cartridge (#186000254; 60 mg/3 cc; Waters, Milford, MA) was used. | Solid phase extraction: Waters Oasis HLB cartridge, 1 cc, 10 mg REF 18600383. | Solid phase extraction: Thermo Hypersep C18 (Thermo Fisher Scientific, cat # 60108–518), pre-washed with 15 ml methanol. |
Vortex mixed, centrifuged and removed 900 μL of supernatant; evaporated supernatant to dryness under stream of nitrogen. | Formic acid (98.2%; #F-4636) Ammonium acetate (98%; #0596-01), acetic acid (99.9%; #V194-04), hydrochloric acid (HCl, 37%; #H611), ammonium hydroxide (NH4OH, 29.5% assayed as NH3; #1177-04), and methanol (HPLC grade; #9093-03) | Honeywell B&J Methanol REF BJ230-4 | HPLC-grade methanol (Fisher A452-4), water (Fisher W5-4) and ammonium acetate (≥97%, Fisher A639-500). Cartridges were washed with 25 mM ammonium acetate and water. Analytes were eluted with MeOH and dried under N2. | |
Aqua Solutions Ultra Pure Water, HPLC grade, BPA free REF W1089-10 L | ||||
Reconstituted in 50 μL acetonitrile/water (50:50; v/v) (Honeywell Burdick & Jackson) and immediately analyzed using LC-MS-MS. | SPE column was washed with 5 column volumes methanol | |||
Column activated with 1000 μL BPA free water | ||||
Load sample | Â | |||
Wash with 1000 μL 5% (v/v) methanol | ||||
Elute with 1000 μL pure methanol | ||||
Extraction pH | not monitored | 5.0 | 7 – 7.5 | 5.0 |
Containers for extraction | Fisher polypropylene micro-centrifuge tubes | 16 × 100 mm Borosilicate Glass Disposable Culture Tube (#73500-16100, Kimble Chase). | VWR 16X100 mm Test Tubes REF 60825–425 Kimble and Chase 13x110 Conical Tubes REF 73785–5 | Borosilicate glass tubes (Fisher Scientific cat#14-961-26). |
Drying agent for extraction | Nitrogen | Nitrogen | Nitrogen | Nitrogen |
Water type & source | Honeywell Burdick & Jackson LC-MS grade | Milli-Q water was purified by an ultrapure water system (Barnstead International, Dubuque, IA) and verified to be BPA free | BPA-free water (verified to be BPA free) | HPLC-grade water from Fisher Scientific. It has always tested BPA-free. |
Temperature for extraction | RT | RT for extraction, 30°C for concentration with nitrogen | Ambient RT (20-25°C) | RT for extraction, 37°C for concentration with nitrogen |