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

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

water to dissolve. The purity of choline bitartrate from the CoA along with a molecular weight

conversion from choline bitartrate to choline of 0.41133 was used to calculate the final

concentration of choline. Intermediate working standards were prepared at concentrations of 10,

20, 500, 2000, 4000, and 5000 µg/mL for each analyte using both the stock and higher

concentration intermediate working standard solutions using appropriate volumes into 20 mL

polypropylene tubes with water as the diluent. All stock and intermediate standard solutions

were stable for 2 months when stored at 5 ± 3°C and protected from light. Aliquots of the

intermediate working standards were treated through the sample analysis, so the concentrations

used for the calibration curves for both free and total analyses were the same numerical values as

the intermediate working standards but in ng/mL. Internal stock standards were prepared at a

concentration of 2 mg/mL by weighing 25 mg of L-carnitine-d3 and 35 mg choline-1,1,2,2-d4

into separate 20 mL polypropylene tubes. A volume of 10 mL of water was added to each to

dissolve, and then both solutions quantitatively transferred to a 100 mL polypropylene tube and

diluted to volume with water to prepare an intermediate solution at 200 µg/mL. The purity from

the CoA was used to calculate the final concentration of each internal standard. Stability of these

solutions was monitored while being stored at 5 ± 3°C and protected from light.

E. Sample Preparation

Powder infant formula (IF) and adult nutritionals (AN) were reconstituted by weighing

25 g and diluting with water to a final weight of 225 g. Viscous ready to feed (RTF) products

that were being analyzed for total choline and carnitine were pre-diluted by weighing 1.0 g and

diluting with water to a final weight of 5.0 g.

(a) Free choline and carnitine . – Samples were prepared by weighing 1.0 g of reconstituted

product into a 50 mL polypropylene tube. Six additional tubes were designated for the working