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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
6
16x125mm
2.) 50mL centrifuge tubes
Fisher
Cat. No. 14-375-150
3.) PVDF syringe filters
4.) Microcentrifuge tubes; 2mL
Fisher cat. No. 02-681-344
5.) SPE cartridge: Phenomenex Part# 8B-S038UBJ.
A mix mode weak anion exchange and reverse phase solid-phase extraction cartridge
Reagents:
1.)
Folate Stock Solution Solvent (15.6 mM ammonium acetate buffer with 25% ascorbic acid
and 1% mercaptoethanol, pH = 5.5):
a.)
weigh accurately 1.27g of ammonium acetate and transfer into a 1000mL beaker.
b.)
In a fume hood, add approximately 700ml of DI water and 4.8 mL of glacial acetic acid
slowly.
c.)
Add 250g of ascorbic acid and 10mL of 2-mercaptoethanol. Stir to completely dissolve
using a stir bar. Add concentrated ammonium hydroxide drop wise as necessary to dissolve
completely.
d.)
Check the pH of the solution and adjust to pH 5.5, if necessary, using concentrated
ammonium hydroxide or glacial acetic acid.
e.)
Transfer the solution into a 1L volumetric flask and bring to volume with DI water.
2.)
Folate Intermediate Standard Solution Solvent (1.6 mMAmmoniumAcetate buffer, with 1%
ascorbic acid, pH 5.5):
a.)
Weigh accurately 0.12 g ammonium acetate and transfer into a 1000mL beaker.
b.)
In a fume hood, add approximately 700ml of DI water and 0.5081g (484 uL)of glacial
acetic acid slowly. Add 10g of ascorbic acid and stir until completely dissolved.
c.)
Adjust pH to 5.5 if necessary with concentrated ammonium hydroxide or glacial acetic acid.
d.)
Transfer the solution to a 1L volumetric flask and bring to volume with DI water.
3.)
Solvent for SPE Elution and Folates Standard (Methanol with 10% formic acid and 1%
ascorbic acid):
a.)
Into a 100mL volumetric flask, transfer 10mL of formic acid and 1g of ascorbic acid. Add
approximately 50ml methanol.
b.)
Sonicate in a water bath set at approximately 25
o
C until complete dissolution.
c.)
Bring to volume with methanol. Use solution to elute folates from SPE sorbent after clean
up, to make analytical standard of folates and as a blank in UPLC-MS-MS analysis.
4.)
Folate Stock Solution (500ug/mL)
a.)
Accurately weigh out about 5mg of each standard in a separate 10mL low-actinic
volumetric flask.
b.)
Add approximately 7.5mL of the Folates Stock Solution Solvent to each flask and sonicate
in a bath set at 40
o
C for about 20min or until complete dissolution. Add several drops
Ammonium hydroxide to aid in dissolution if necessary.
c.)
Bring to volume with Folates Stock Solution Solvent and transfer to freezer safe containers.
d.)
The Folic acid,
13
C-Folic Acid (IS), (6S)-5-Formyl-5,6,7,8-Tetrahydrofolate, 10-methyl-
folic acid and 10-formyl-folic acid solutions are stable for up to 180 days stored in the
freezer at -70
o
C.
e.)
The (6S,R)-5-Methyl-5,6,7,8-Tetrahydrofolate, and 5,6,7,8-Tetrahydrofolate solutions are
2011.06 (Fol-22) w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE