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

G: 20% methanol + 80% of 1% acetic acid + 1% ascorbic acid A standard mixture was taken in either solvent mixture A or the solvent mixture G and analyzed by the chromatographic and MS method described above. A representative chromatogram of the analytes in two different injection solvents is provided in the following Figure 1. The analytes could be satisfactorily resolved by the chromatography and the injection solvent did not have any considerable effect.

XICof+MRM(12pairs):442.200/295.200DaID:FA-295fromSample50(100ppbG)ofHSST3_120821_01.wiff (TurboSpray)

Max.1.4e4cps.

10-CHO-FA

1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4 4.6e4 4.8e4 Intensity, cps

5-CH3-THF

5-CHO-THF

10-CH3-FA

THF

FA

6.15

2000.0 4000.0 6000.0 8000.0

6.66

7.08 6.95 7.14

7.80

6.86

7.44

7.90

4.02

4.70

5.80

6.05

7.607.69

4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 2 20 39 57 75 93 111 130 148 166 184 202 220 239 257 275 293 311 329 348 Time,min 0.0

XICof+MRM (12 pairs): 442.200/295.200Da ID:FA-295 fromSample 43 (100ppbA)ofHSST3_120821_01.wiff (Turbo Spray)

Max. 1.5e4 cps.

10-CHO-FA

1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 3.2e4 3.4e4 3.6e4 3.8e4 4.0e4 4.2e4 4.4e4

5-CH3-THF

5-CHO-THF

10-CH3-FA

Intensity, cps

FA

THF

6.15

2000.0 4000.0 6000.0 8000.0

0.0

4.0

4.2 20

4.4 39

4.6 57

4.8 75

5.0 93

5.2

5.4 130

5.6 148

5.8 166

6.0 184

6.2 202

6.4 220

6.6 239

6.8 257

7.0 275

7.2 293

7.4 311

7.6 329

7.8 348

2

111

Time,min

Figure 1 . A representative chromatograms of folate standards at 100 ppb level in G (Top) and A (Bottom). There were two Q1/Q3 transitions for each compound. Evaluation of the linearity of analytical response against concentration of the respective analyte The linearity of the analytical response of the analytes against their concentration was evaluated in various injection solvent mixtures including the solvent mixtures A and G as listed above. All six compounds show good linearity in the range of 2.5 to 500 ng/mL irrespective of the injection solvent.. The intermediate standard (IMS, 20 µg/mL in 10 mM ammonium acetate, 1% ascorbic acid & 1% mercaptoethanol pH=5.5) was series diluted to 2.5, 5, 100 and 500 ng/mL with 1% acetic acid /methanol/1% ascorbic acid (v/v/w 80:20:1)) and 2 µL were injected into the LC-MS/MS system. The stronger Q1/Q3 transitions were used for the quantification and the peak area gave good linearity vs. the corresponding concentrations with 1/x weighing factor. A representative slope, intercept and correlation coefficient for each target compound are listed in Table . The back-calculated concentrations and the accuracies using the calibration curve were also listed.

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