AOAC SPIFAN Nutrients ERP-Final Review (Oct. 2018) Pubs

2011.06 (September 2017)-Fol-22 MLT Report

FOR ERP USE ONLY DO NOT DISTRIBUTE

Prepare method blank, (no sample but with all

Reconstituted /RTF Sample + ext. buffer & int std +

Add 1 mL protease and incubate at 37 ° C for 3 hours under Nitrogen

Deactivate protease in boiling water bath for 5 min.

Load extract on SPE adsorbent, wash 3x w/ water (1 ml ea.)

Add 1 ml alpha-amylase + 300 µL plasma conjugase. Incubate @ 37 ° C for 16 hrs

Elute with 1 ml elution solvent & add 50 µl Ammonium hydroxide

Keep in Boiling water bath for 5 min.

Centrifuge & filter, 45 micron PVDF filter

Analyze by LSMS/MS

Condition SPE with methanol and water

D. APPARATUS

1. Precision balance (reading 0.1 mg) 2. Agilent 1290 Infinity Binary LC System or Dionex 3000 Ulimate LC or (used in validation) or equivalent. 3. Sciex 6500 or 5500 Triple QUAD LC-MS/MS System (used in validation) or equivalent. 4. pH Meter 5. 24-well SPE Vacuum manifold

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 24