AOAC OMA# 2011.06 Final Action Review-OMB

2011.06 (September 2017)-Fol-22 MLT Report

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15. Prepare HPLC and Mass Spectrometer for analysis.

J. Analytical Procedure:

Calibration Curve: Calibration curve is drawn by analysis of the calibration standard solutions of different concentrations containing all folate compounds at Concentrations of: 1, 2, 5, 20, 50, 200 and 500 ng/mL in neutralized elution solvent (90% MeOH + 10% Formic acid + 1% ascorbic acid). Each level of calibration standard also contains three internal standards (labeled folic acid, methyl-THF & formyl-THF) at concentration of 40 ng/mL. The standard prepared as detailed in Table 4-C. LC-MS Analysis of Folates: Platform – Dionex Ultimate 3000 LC with Sciex 5500 (Q trap) LC-MS/MS system: HPLC Conditions: Dionex Ultimate 3000 LC System: LC Column: Waters ACQUITY UPLC HSS T3 Column (100Å, 1.8 µm, 2.1 mm X 50 mm). Mobile Phase: A: 1% Glacial acetic acid and B: MS grade methanol as mobile phase Mobile phase A and B are pumped in different ratios at different time of the analysis. The Pumping gradient of two solvents at different time is as the following.

Flow (mL/min)

Time (min)

%B

0

0

0.5

0.5

0

0.5

5.00

40

0.5

5.05

100

0.5

6.50

100

0.5

6.55

0

0.5

9.00 0.5 A = 1% Glacial Acetic Acid in Water; B = 100% Methanol 0

Collaborative Study Report: Method AOAC 2011.06 (Folate by LC-MS/MS) 38