AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

Chol-08/Carn-07 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

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