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the time of weighing was used to calculate the final concentration of carnitine. The choline stock
standard was prepared at a concentration of 25mg/mL choline by weighing 0.62 g of choline bitartrate
into a 20mL polypropylene tube followed by 10mL of 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 20mL
polypropylene tubes withwater as the diluent. All stock and intermediate standard solutions were
stable for 2months 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 2mg/mL
by weighing 25mg of L-carnitine-d3 and 35mg choline-1,1,2,2-d4 into separate 20mL polypropylene
tubes. A volume of 10mL of waterwas added to each to dissolve, and then both solutions
quantitatively transferred to a 100 mL polypropylene tube and diluted to volume withwater 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 monitoredwhile 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 50mL polypropylene tube. Six additional tubes were designated for the working 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 25mL with water and thoroughly mixed on a horizontal shaker. The reagent blank + internal
standard solutionwas used as the diluent if dilutions were needed. A 0.5mL aliquot of the sample
solution was mixedwith 0.5mL of acetonitrile in a microcentrifuge tube, and then filtered through a
0.45 µm GHP syringe filter into a silanized injection vial. Aliquots of 0.5mL of the working standard and
reagent blank solutions were mixed with 0.5mL 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 55mL 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
Candidates for 2016 Method of the Year
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