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

2015.10 (Jan. 2019) Carn-07 MLT Report FOR ERP USE ONLY DO NOT DISTRIBUTE

b) Mobile Phase B (30 mM Ammonium Formate in water:ACN (50:50, v:v) with 0.2% Formic Acid): Weigh 1.89 ± 0.01g of Ammonium Formate into a 1-L mobile phase bottle containing 500mL of ultra-pure water. Add a stir bar and then mix on a stir plate until dissolved. Add 500 mL of acetonitrile to the mobile phase container, along with 2.00 mL of formic acid. Add a stir bar and mix on a stir plate until thoroughly mixed, typically for about 2 minutes. The solution is stable for 2 weeks when stored at room temperature. c) Injector wash: Mobile phase B is used as HPLC injector wash. ( b ) Standard solutions preparation. ( Notes : Stock, intermediate, and working standards are stable for 6 months when stored in a refrigerator set to maintain 2 to 8°C. Protect standard solutions from actinic light. Alternate weights or volumes may be used to scale these preparations.) (1) Preparation of choline/carnitine stock solutions .— a) Choline Stock Solution (~25 mg/mL): Weigh 0.5200 ± 0.01 g of choline bitartrate into a 20-mL polypropylene scintillation vial. Dissolve with 10.0 mL of water. b) Carnitine Stock Solution (~25 mg/mL): Due to the hygroscopic nature of L-Carnitine, a Karl Fischer Moisture analysis test must be performed to determine the water content of the material at the time of weighing. Weigh 0.2500 ± 0.01 g into a 20-mL polypropylene scintillation vial. Dissolve with 10.0 mL of UPW. c) L-Carnitine-d3 Stock Solution (~2.00 mg/mL) (IS1): Weigh 0.02500 ± 0.001 g of L-Carnitine-d3 HCl into a 20-mL polypropylene scintillation vial. Dissolve with 10.0 mL of UPW. d) Choline-1,1,2,2-d4 Stock Solution (~2.00 mg/mL) (IS2): Weigh 0.03100 ± 0.001 g of Choline-1, 1, 2, 2-d4 bromide into a 20-mL polypropylene scintillation vial. Dissolve with 10.0 mL of UPW. (2)Preparation of working standard solutions a) Prepare the working standard solutions according to Table 2015.10A b) L-Carnitine-d3/Choline-1,1,2,2-d4 [200 ug/mL] Internal Standard Working solution: Transfer 10 mL of IS1 solution and 10 mL of IS2 solution into a 100-mL digitube. Add 80 mL of UPW to bring to a final volume of 100 mL. ( c ) Sample preparation.— Sample Preparation for Samples Needing Reconstitution 1) Weigh 10.00 ± 0.500 g of sample in a suitable disposable cup or beaker. 2) Add UPW to bring the total weight (including the powder weight) to 100.00 ± 1.00 g. 3) Add a stir bar and stir as fast as possible without causing the sample to splatter. Stir for at least 10 minutes, but no longer than 30 minutes. Note: Weights may be adjusted as needed to accommodate different powder types and levels of vitamins. Free Analysis of Carnitine and Choline 1) Weigh 0.1-1.0 g of sample into a tared 50-mL Digitube depending on the concentration of each analyte (to fall within calibration curve).. 2) A reagent blank, a reagent blank + ISTD, and working standards must be included for each analysis and treated the same as samples through the analysis. a) Add 50 μL of each working solution to separate 50-mL Digitubes. The final nominal concentrations for these after they have gone through the sample preparation (diluted 1000X) are listed in the Standard Solutions Preparation section to be used for construction of the calibration curve. 3) Add 50 μL of Intermediate Mixed Internal Standard Solution to each sample, IWS, and reagent blank + ISTD.

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