AOAC OMA# 2011.06 Final Action Review-OMB

2011.06 (September 2017)-Fol-22 MLT Report

FOR ERP USE ONLY DO NOT DISTRIBUTE

that charcoal mixes with plasma uniformly. Stand for 5 minutes at 4-8 °C. Filter through PDVF syringe filter. Usually you can recover about 4.3-4.4 mL from 5 mL after charcoal treatment. Prepare fresh on day of use. Store at 4-8 °C if required but not over 6 hours. Each sample requires 0.3mL of the charcoal treated conjugase. 15.) TCEP Solution (approx. 1M): 1g of TCEP is dissolved in 3.5mL H 2 O in a test tube or a vial. 16.) Extraction Buffer (10mM phosphate buffer, 1% ascorbic acid, pH 6.0) : a.) Accurately weigh 0.355g of sodium phosphate, dibasic, anhydrous Na 2 HPO 4 in a 250mL beaker. Add 2.5g of ascorbic acid and approximately 200mL of HPLC water. Stir on a stir plate until completely dissolved. b.) Adjust pH with phosphoric acid and/or 10M sodium hydroxide (10M NaOH) to pH 6.0 c.) Transfer into a 250 mL volumetric flask and dilute to volume with HPLC water. Mix. Prepare fresh on day of use. d.) Add 0.05mL of the 20 ppm Internal Intermediate Standard Solution to 250 mL of the buffer. Mix thoroughly (Internal std concentration in Extraction 17.) HPLC Mobile Phase : a.) Mix 500mL Water (Optima LC/MS) and 5 mL glacial acetic acid thoroughly b.) Methanol (Optima LC/MS). H - Checking Efficiency of Rat Plasma Conjugase : 1. To check the efficiency of the rat plasma conjugase, use 150uL of Rat Plasma Test Solution (20ug/mL Pteroyltri-y-glutamate) in a 50mL centrifuge tube. Add 10mL of Extraction buffer (10mM phosphate buffer, 1% ascorbic acid, pH 6.0, Internal std 4 ng/mL of each of the targeted folate compound). Add 50uL TCEP Solution. Vortex to mix well. 2. Add 300uL of rat plasma conjugase (freshly treated with Charcoal). Fill the head space with nitrogen, cover and incubate for 16hrs in a shaking water bath at 37 °C. Shaking speed low, about 60 rpm. 3. Go to the step 7 of the extraction section as described in the following section and follow all the steps (7 to 15) of the method leading to analysis of folates including folic acid. 4. The viability of the rat plasma conjugase, expressed as the percent conversion of 3 µg pteroyltri-γ-L-glutamate (FA-glu3) to folic acid. Buffer = 4 ng/mL of each targeted folate compound). e.) Each sample requires 10 mL of the extraction buffer.

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 35