AOAC SPIFAN ERP Meeting Book-March 16, 2016

2011.06 (Fol-22) w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

Ion source gas1(zero air): Ion source gas2(zero air):

60 psi; 50 psi;

Peak Area Ratio

The peak area ratio of the two MRM transitions of each analyte was determined by the average value of those at difference concentration levels ( Table ). All of the following data, including peak area and calculated concentration, are based on the quantification (more intense MRM transitions). Table 11: Peak Area Ratios of the six analytes in solvent .

Conc (ng/mL)

FA

5-CH3-THF 5-CHO-THF

THF

10-CH3-FA 10-CHO-FA

1 2 5

2.7853 3.0791 1.9507 2.0907 2.1094 2.1657 2.2014 2.3403

3.0872 3.4956 3.4167 3.7024 3.9070 4.0045 3.9217 3.6479

4.1603 5.3346 4.4296 4.4652 4.0784 4.1413 4.4295 4.4341

1.7700 1.4988 2.5811 2.3251 2.4488 2.2653 2.1387 2.1468

1.8942 1.8534 1.9350 2.1522 1.8941 2.0275 2.0199 1.9680

1.9322 2.0930 1.7459 1.6303 1.7760 1.7895 1.7018 1.8098

20 50

200 500

Average

Optimization of HPLC Conditions:

Selection of the Column provides best performance: Attempts were made to get the best resolution possible of the analytes in scope. A series of HPLC column were selected for evaluation purposes based on the AOAC method and the available literature. We used HPLC (Shimadzu LC 20) for this study. The Medallion used Waters UPLC in developing the AOAC method. The column used by the AOAC method is Waters ACQUITY UPLC HSS T3 Column, 100Å, 1.8 µm, 2.1 mm X 150 mm. The following columns were evaluated by us for the best possible resolution of the analytes in scope.

Poly LC PolyHYDROXYETHYLA ™ 200x 2.1 mm, 5 um Agilent Zorbax Eclipse xdb-c8, 4.6x150 mm, 3.5 um

Phenomenex Kinetex C18, 2.1x50 mm, 1.7 um Phenomenex Kinetex HILIC, 2.1x50 mm, 1.7 um The Nest Group Zic HILIC 2.1x100 mm, 3.5 um Waters ACQUITY UPLC BEH C18, 2.1 mm X 50 mm, 1.7 µm Waters ACQUITY UPLC HSS T3 Column, 100Å, 1.8 µm, 2.1 mm X 50 mm Thermo Hypersil GOLD aQ, 2.1 mm X 50 mm, 1.9 µm

2. Mobile Phase Tested: Evaluation of the best Mobile Phase Buffer: Different buffers were evaluated for best analytical response of different analytes in the scope in the

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