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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
8
a.)
Pipette and appropriate amount of each folate stock solution (EXCPET THE INTERNAL
STANDARD STOCK SOLUTION AND PTEROLYTRI-y-GLUTAMIC ACID) into a
10mL low-actinic volumetric flask.
b.)
Three separate solutions (one mixture and two individual solutions) are prepared as detailed
in the following table.
c.)
Add approximately 5mL of Folate Intermediate Standard Solution Solvent and sonicate in
water bath set at 40
o
C for about 10min to thoroughly mix.
d.)
Bring to volume with Folate Intermediate Standard Solvent and store approximately -20
o
C.
Stable for up to 30days.
Table 3: Preparation intermediate standard solutions
Intermed
std Soln
#
Folate
compd
#
Stock Standard
Mixture
Intermed
or
Individual
std. Soln
Stock
std.
Concn
(mcg/mL)
Stock
std.
Volume
(uL)
Final
Volume*
(mL)
Concen
(mcg/mL)
I
1
Shircks (6S)-5-Formyl-5,6,7,8-
tetrahydrofolic acid
Mixture
Intermed
Std. Soln
607
356
10
21.6092
2
10-Formylfolic acid
597
335
19.9995
4
10-methylfolic acid
529
378
19.9962
5
Shircks (6R,S)-5,10-methenyl-
5,6,7,8-tetrahydrofolic acid
587
368
21.6016
7
Shircks (6S)-5,6,7,8-
tetrahydrofolic acid
542
369
19.9998
II
3
Shircks (6R,S)-5-methyl-5,6,7,8-
tetrahydrofolic acid
Indiv.
Intermed.
Std soln
584
371
10
21.6664
III
8
Shircks Folic acid
Indiv.
Intermed.
Std soln
555
360
10
19.98
Conjugase
check
soln
6
pteroyltri-y-L-glutamic acid
Conjugase
check
soln
911
222
10
20.2242
*Make up final volume to 10 mL with Folate Intermediate std. solvent.
7.) Internal Standard Intermediate Standard Solution (20ug/mL):
2011.06 (Fol-22) w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE