514 / 696
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