AOAC OMA# 2011.06 Final Action Review-OMB

2011.06 (September 2017)-Fol-22 MLT Report

FOR ERP USE ONLY DO NOT DISTRIBUTE

5. Inactivate the protease enzyme by placing the tube in a boiling water bath for 5 minutes with intermittent shaking (3 times in 5 minutes). Cool the tubes to room temperature. 6. Add 1mL α-amylase (freshly charcoal treated) solution and 0.3 mL rat plasma (conjugase; freshly charcoal treated), fill the head space with nitrogen, cap the tube. Incubate for 16hours in a shaking water bath at 37 °C (shaking speed is low). 7. After the 16hr incubation, place the tubes in a boiling water bath (100°C) for 5 minutes with mixing every 1.5 minute (3 times during 5 minute period). Cool to room temperature. 8. Centrifuge tubes at 4-5°Cfor 10 min at 1400 x g (rotor diameter =10.4 cm) 9. Filter supernatant through 0.45um PVDF filter into disposable glass culture tubes (16x125mm) 10. Set up 24-well manifold 11. Condition the SPE as follows (gravitational, no vacuum used, may require a push with a syringe plunger or piette bulb to initiate flow): a.) Condition the SPE with 2mL methanol (1mL x 2). b.) Before SPE sorbent is dry repeat with 2 mL water (1mL x 2). 12. Load Samples onto conditioned SPE sorbent (gravitational; no vacuum, may require a push with a syringe plunger or piette bulb to initiate flow): a.) Add all of filtered supernatant (approximately 12 mL) to SPE sorbent. b.) Let the loaded solution go through the SPE by gravity. c.) Wash each SPE with 1 mL water. Repeat wash twice with 1mL of water. (Total 3 water washes). d.) Use a syringe plunger or pipette bulb to push out last remnants of water 13. Eluting the analyte from the sorbent: a.) Empty waste reservoir and add disposable microfuge tubes to collect eluent. b.) Add 1 mL of Solvent for SPE Elution (methanol containing 10% formic and 1% ascorbic acid) to each of the analyte loaded SPE cartridge. Elute the folates into the freshly set tubes. c.) Allow gravity to elute the eluent into the tubes. Use a syringe plunger or pipette bulb to push out residual solvent d.) Add 50 µ Ammonium hydroxide (28-30%) for neutralization of the eluent. e.) Immediately Vortex for 20 seconds for mixing. f.) centrifuge in ultrafuge (Micofuge) at about 9500 x g for 5min. 14. Transfer supernatant from Microfuge tubes into separate respective HPLC vials for LC-MS/MS analysis.

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 37