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

Table 13: Response (peak area) of targeted folate compounds at 50 ppb levels as impacted by change in Organic Phase of the HPLC mobile phase during the analysis

5-CH3- THF

5-CHO- THF

10-CH3- FA

10-CHO- FA 9.43E+04 9.69E+04 9.81E+04 9.64E+04 8.16E+04 8.24E+04 8.34E+04 8.25E+04 2.0%

Organic Phase

FA

THF

2.26E+04 1.19E+05 2.34E+04 1.13E+05 2.34E+04 1.18E+05 2.31E+04 1.17E+05 2.73E+04 9.73E+04 2.77E+04 9.44E+04 2.75E+04 8.24E+04 2.75E+04 9.14E+04 2.0% 2.8%

7.31E+04 4.69E+04 5.01E+04 7.02E+04 4.88E+04 5.07E+04 7.25E+04 4.93E+04 5.08E+04 7.19E+04 4.83E+04 5.05E+04 5.82E+04 4.63E+04 3.88E+04 5.25E+04 5.08E+04 3.19E+04 5.17E+04 4.80E+04 3.19E+04 5.41E+04 4.84E+04 3.42E+04 2.1% 2.6% 0.7%

Methanol

Average

%RSD

Acetonitrile

Average

%RSD

0.7%

8.6%

6.5%

4.7%

11.6% 67.7%

1.1%

ACN/MeOH

118.9%

78.3%

75.3%

100.1%

85.5%

HPLC Conditions: Flow: 0.5 mL/min; Column Temperature: 40 ° C All the HPLC columns listed above (pages 15-16) were evaluated using different and relevant mobile phase combinations. Waters ACQUITY UPLC HSS T3 Column (100Å, 1.8 µm, 2.1 mm X 50 mm) with 1% acetic acid and methanol as mobile phase with a varied flow rate provided the best sensitivity and separation. The gradient is shown below: The Pumping gradient of different solvents and flow at different time

Flow (mL/min)

Time (min)

%B

0

0 0

0.5 0.5 0.5 0.5 0.5 0.5 0.5

0.5

5.00 5.05 6.50 6.55 9.00

40

100 100

0 0

Investigation of different Injection Solvents: Various solvent mixture were evaluated for their effect on chromatography and resolution of the analytes. The most important solvent mixture tried are mixtures A and G and are listed in the following section. The solvent A is used for elution of the analytes during solid-phase extraction. We tried a step of the evaporation of the extract concentrated and purified by SPE to improve sensitivity of the method. The extract residue following the evaporation is reconstituted in the following solvent mixture G which is some what similar to the HPLC mobile phase used in the analysis. A: 90% MeOH + 10% Formic acid + 1% ascorbic acid

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