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standards along with two tubes for the reagent blank and reagent blank + internal standard to
monitor any interference or carry over. The working standards, reagent blank, and reagent blank
+ internal standard were included with each free analysis and treated the same as samples
through the sample preparation. The working standard tubes received 50 μl of the appropriate
intermediate working standard level. All tubes except the reagent blank received 50 μl of the
intermediate internal standard solution. The tubes were diluted to 25 mL with water and
thoroughly mixed on a horizontal shaker. The reagent blank + internal standard solution was
used as the diluent if dilutions were needed. A 0.5 mL aliquot of the sample solution was mixed
with 0.5 mL of acetonitrile in a microcentrifuge tube, and then filtered through a 0.45 μm GHP
syringe filter into a silanized injection vial. Aliquots of 0.5 mL of the working standard and
reagent blank solutions were mixed with 0.5 mL of acetonitrile directly in the silanized injection
vials.
(b)
Total choline and carnitine
.
–
Samples were prepared by weighing 1.0 g of reconstituted
or diluted product into a 55 mL MarsExpress liner. Six additional liners were designated for the
working standards along with two liners for the reagent blank and reagent blank + internal
standard to monitor any interference or carryover. The working standards, reagent blank, and
reagent blank + internal standard were included with each total analysis and treated the same as
samples through the sample preparation. Liners designated for the working standards received
50 μl of the appropriate intermediate working standard level. All liners except the reagent blank
received 50 μl of the intermediate internal standard solution. A 5 mL volume of water followed
by 2.5 mL of 70% (w/w) nitric acid delivered with a bottle top dispenser were then added to each
liner, capped, and vortexed to mix. The microwave program used was a ramp to temperature of
120°C over 10 minutes, followed by a 40 minute hold at a power of 1000 W, ending in a cool
Chol-08/Carn-07 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE