Water-Soluble Vitamins from Serum using

Strata X 30mg/3mL
(Part # 8B-S100-TBJ)
Specimen Pretreatment:
Mix sample 250L in 2.5-mL of water and 2.5-mL of dichloromethane/hexane (2:1 v/v),
vortex for 3 minutes and filter. Separate the (organic) lipid- and hydro-soluble layers in a
separatory funnel. Add 50mg NaCl to the aqueous layer and mix.
Take the hydro-layer for sample loading. Do no discard the organic layer until you have
completed the method.

Condition and Equilibration:

Pass through the cartridge to waste:
300 L of Methanol
Apply vacuum and leave on (est. 2-4 in Hg)
300 L D.I. Water
NOTE: It is OK to leave the vacuum on after the application of the methanol above. Strata X
cartridges are not damaged by drying. Safe for up to 10 minutes.

Load:
Apply 5-10 mL of sample at a flow rate of 1-2 mL/ min. Slow is
Safe.
Wash:
Pass through the cartridge to waste,
2x 300 L D.I. water
Dry column 30 60 seconds at full vacuum to remove all traces of wash solvents.

Elution:
Turn off vacuum, remove manifold lid, and place collection tubes in rack.
Place lid back on manifold.
With the vacuum OFF apply,
2x 600 L Methanol
Allow to sit on the cartridge for 30 seconds then slowly aspirate through the
cartridge under very low vacuum, at a rate not to exceed 2 mL/ min. Slow is
safe.

Analyze: Evaporate and analyze per standard protocol.

*For Thiamine, higher recoveries can be obtained using Strata X-C (500mg/6ml):
Condition: 5-mL methanol + 6-mL 20mM sodium acetate (pH 4.6). Buffer sample
with 20mM sodium acetate (pH 4.6) and load at 3-mL/min. Wash: 6-mL 20mM
sodium acetate (pH 4.6) and dry. Elute with 6mL of 2M KCl adjusted to pH 1.52.0 with dilute HCl in water/ methanol (60/40).