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 1.5-

2.0 with dilute HCl in water/ methanol (60/40).