AOAC OMA# 2011.06 Final Action Review-OMB

2011.06 (September 2017)-Fol-22 MLT Report

FOR ERP USE ONLY DO NOT DISTRIBUTE

V - Preparation of substrate to test activity of plasma conjugase:

11.) Folate Rat Plasma Test Solution (20ug/mL Pteroyltri-y-glutamic acid (FA-glu 3 )): a.) Pipette 0.4 mL, (500 mcg/mL) of FA-glu 3 stock solution into a 10mL low- actinic volumetric flask. b.) Add approximately 5 mL of Folate Intermediate Solvent and sonicate in water bath set at room temperature for about 20 seconds to thoroughly mix. c.) Bring to 10 mL volume with Folate Intermediate Solvent. Mix thoroughly by a brief 1 min sonication and 5x inversion of the flask. d.) Store at -20 o C. VI - Other reagents for folate analysis: 12.) Protease Solution (2 mg/mL) (Proteinase from Bacillus licheniformis , Subtilisin, Sig ma-Aldrich, cat. No. P3910 or equivalent): Prepare fresh before use. Dissolve 0.1g protease in 50 mL of water in a 100 mL beaker or Erlenmeyer flask. Store at 4°C if necessary but not over 6 hours. Each sample requires 1mL. 13.) α-Amylase Solution (20 mg/mL; 40 units/mL) (α-Amylase from Aspergillus oryzae , > 150 units/mg protein, Sigma-Aldrich, Cat. No. A9857 or equivalent) : Prepare fresh before use. Dissolve 0.5 g in about 20 mL of water in a 25 mL volumetric flask or a centrifuge tube. Mix by gentle swirling . Make up volume to 25 mL. Mix gently by inversion of the flask/tube till complete mixing, 5x inversions or so. The contents in the centrifuge tube can be mixed by gentle vortex for about 30 seconds. Make sure no foams develop. The amylase solution, as prepared above is transferred into a 50 mL centrifuge tube if already not in that. Treat amylase solution with charcoal (20 mg charcoal per mL of solution) (0.5 g in 25 mL). Vortex gently for about 45 seconds. Filter through 0.45µ PDVF syringe filter. Prepare fresh before use. Store at 4°C if necessary but not over 6 hours. Each sample requires 1mL. 14.) Male Rat Plasma Conjugase: The plasma usually supplied by manufactures in a 5 mL aliquot in a tube. It can be kept frozen till use at -20C. Don’t store plasma over 120 days. Thaw the plasma as required. Treat the plasma with 20mg charcoal per mL (100mg for 5 mL) of rat plasma. Take 5 mL of plasma in a tube. Add 100mg of charcoal. Vortex gently for about 30 seconds so

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 34