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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
15
13
C
5
-FA
447.2
295.2
20
4.5
39
31
6
13
C
5
-5-CH3-
THF
465.2
313.2
35
5
23.9
29
6
DP: Declustering potential;
EP Entrance potential;
CEP collision cell entrance potential;
CE Collision cell;
CXP collision cell exit potential.
The General MS parameters for API 5500 (Qtrap):
Curtain gas (nitrogen):
25 psi;
Collision Assisted Dissociation: Medium
Ion Spray voltage:
5500 V;
Temperature:
700 °C;
Ion source gas1 (zero air):
60 psi;
Ion source gas2 (zero air):
50 psi;
Calculations :
Calculations and reporting
Ratio of the folate vitamer with respective internal standard is determined by
HPLC-MS analysis. 5-CH3-THF, 5-CHO-THF and THF use labeled 5-CH3-THF as
internal std.
Folic acid and 10-CH3-Folic acid 10-CHO-Folic acid use labeled Folic acid as internal
std. Calibration curve is drawn based on concentration of calibration standard of
specific folate compound plotted against ratio of peak area (for the analyte) to the
relevant internal standard. The calibrations curve are prepared for all the targeted
folate compounds. The ratio peak areas of the folate compounds and respective
internal standard is calculated in the standard sample which was processed like
sample. Concentration of each of the folate compound as ng/mL in the standard
processed like sample is calculated based on calibration curve. This is usually
determined by the MS software (multquant). The ratio of peak areas of the
detected folate compounds and respective internal standard is calculated in the
blank which was processed like sample in the batch. Concentration of each of the
detected folate compound as ng/mL is calculated in blank based on calibration
curve. . Concentration of each of the folate compound as ng/mL in the standard
processed like sample is adjusted by substracting the amount of respective folate
compound detected in blank. Recovery factors (Rfs) for all the targeted folate
compounds are determined based on adjusted concentration of respective analyte
determined in standard processed like sample using the following relationship.
Rf = Adjusted concentration (ng/mL) of folate compound estimated in std.
/amount of the specific folate compound expected to be present in the std.
The ratio peak areas of the folate compounds and respective internal standard is
calculated in a sample. Concentration of each of the folate compound as ng/mL
2011.06 (Fol-22) w/SLV
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