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

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

(k) Reference Internal Standard . – Choline-1,1,2,2-d4 Chloride (CDN Isotopes) or equivalent.

Note : All use of water in this method must be high purity MS grade water. It is recommended

that all preparation steps with nitric acid be performed within a fume hood, and the necessary

personal protective equipment used when handling.

C. Mobile Phase Preparation

Mobile phase A (5 mM ammonium formate in 50:50 (v/v) water:acetonitrile with 0.2%

formic acid) was prepared by weighing 0.63 g of ammonium formate into a 1 L graduated

cylinder. Water was added along with a stir bar and mixed to dissolve before diluting to volume

with water. The solution was transferred to a 2 L mobile phase container along with 1 L of

acetonitrile, 4 mL of formic acid, stir bar, and then thoroughly mixed. Mobile phase B (30 mM

ammonium formate in 50:50 (v/v) water:acetonitrile with 0.2% formic acid) was prepared by

weighing 3.78 g of ammonium formate into a 1 L graduated cylinder. Water was added along

with a stir bar and mixed to dissolve before diluting to volume with water. The solution was

transferred to a 2 L mobile phase container along with 1 L of acetonitrile, 4 mL of formic acid,

stir bar, and then thoroughly mixed. Mobile phase B was also used for the rinse solutions in the

autosampler.

D. Preparation of Standard Solutions

The carnitine stock standard was prepared at a concentration of 25 mg/mL by weighing

0.25 g of L-carnitine into a 20 mL polypropylene tube followed by 10 mL of water to dissolve.

The purity of L-carnitine from the Certificate of Analysis (CoA) and moisture determined by

Karl Fisher immediately at the time of weighing was used to calculate the final concentration of

carnitine. The choline stock standard was prepared at a concentration of 25 mg/mL choline by

weighing 0.62 g of choline bitartrate into a 20 mL polypropylene tube followed by 10 mL of