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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
10
Table 4: Preparation of Calibration Standard Solutions:
11.) Folate Rat Plasma Test Solution (20ug/mL Pteroyltri-y-glutamic acid (FA-glu
3
)):
a.)
Pipette and appropriate amount of FA-glu
3
stock solution into a 10mL low-actinic
volumetric flask.
b.)
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.
c.)
Bring to volume with Folate Intermediate Standard Solvent and store approximately -70
o
C.
Stable for up to 180days.
12.) Protease Solution (2 mg/mL):
Dissolve 0.1g protease in 50 mL of water. Filter through
glass wool if necessary and store at 4-8 °C. until use. Prepare fresh on day of use. Each sample requires
1mL.
K STD
Intermed
iate STD
Soln.
Internal
Std Soln.
(2
µ
g/mL)
(µL)
Working
Internal
Std Soln.
(40ng/mL)
(µL)
Neutr
Elution
Solv.
ml
Final
Vol
(mL)
Conc.
#8
(ng/
mL)
Conc.
#3
(ng/mL
)
Conc.
#1
(ng/mL
)
Conc.
#7
(ng/mL
)
Conc.
#4
(ng/mL
)
Conc.
#2
(ng/m
L)
Conc. #5
(ng/mL)
A
25 uL
Intermed
. Std.
soln. # I,
II, III
20
0
905
1.0
mL
499.5
541.66
540.23
499.99
5
499.90
5
499.9
875
540.04
B
10 uL
Intermed
. Std.
soln. # I,
II, III
20
0
950
1.0
mL
199.8
216.66
4
216.09
2
199.99
8
199.96
2
199.9
95
216.016
C
0.1 mL
Work std
Soln A
0
900
0
1.0
mL
49.95
54.166
54.023
49.999
5
49.990
5
49.99
875
54.004
D
0.1 mL
Work std
Soln B
0
900
0
1.0
mL
19.98
21.666
4
21.609
2
19.999
8
19.996
2
19.99
95
21.6016
E
0.1 mL
Work std
Soln C
0
900
0
1.0
mL
4.995
5.4166
5.4023
4.9999
5
4.9990
5
4.999
875
5.4004
F
0.1 mL
Work std
Soln D
0
900
0
1.0
mL
1.998
2.1666
4
2.1609
2
1.9999
8
1.9996
2
1.999
95
2.16016
G
0.05 mL
Work std
Soln D
0
950
0
1.0
mL
0.999
1.0833
2
1.0804
6
0.9999
9
0.9998
1
0.999
975
1.08008
2011.06 (Fol-22) w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE