421 / 670
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
Chol-08/Carn-07 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE