AOAC SPIFAN Review Team Meeting Book-July 22, 2015

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.

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.

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

Max.1.4e4cps.

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

2000.0

4000.0

6000.0

8000.0

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

6.15

6.66

7.80

7.08 6.95 7.14

6.86

7.90

7.44

6.05

7.607.69

5.80

4.70

4.02

THF

5-CH3-THF

10-CHO-FA

5-CHO-THF

10-CH3-FA

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

Spray)

Max. 1.5e4

cps.

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

111

130

148

166

184

202

220

239

257

275

293

311

329

348