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Silliker

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

10

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 1.0 to 500 ng/mL 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 obtained using Agilent 1290 Infinity LC system and Sciex 6500 LC/MS detector are listed

in Table 4.

Table 4: Evaluation of Response Linearity against concentration of seven target folate compounds

Compound

Slope

Intercept

r2

FA

0.02763

7.27E-04

0.999

THF

0.00684

-1.92E-03

0.999

5,10-methenyl-

THF**

0.02377

4.14E-04

0.999

10-CH3-FA

0.04616

-3.28E-04

0.9999

5-CH3-THF*

0.02118

-3.30E-03

0.999

10-CHO-FA

0.06558

9.01E-03

0.999

5-CHO-THF*

0.03902

7.98E-03

0.9999

*weighting: 1/x

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

FA

10-CH3-FA

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

Max. 1.5e4

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

Intensity,

6.15

THF

5-CH3-THF

10-CHO-FA

5-CHO-THF

FA

10-CH3-FA

2011.06/Fol-22 (February 2016)

FOR ERP USE ONLY

DO NOT DISTRIBUTE