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

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

(v) Desiccator . – glass (VWR) or equivalent. Note : Non-specific binding can occur with these analytes when using glassware, so plasticware should be used at all times for standard/sample preparation. All laboratory plasticware should be single-use whenever possible. Positive displacement pipets are also mandatory for pipeting to avoid contamination and for accuracy with organic solvents. C. Chemicals and Reagents (a) Water/Ultra-pure water (UPW) . – Optima MS grade (Thermo Fisher Scientific, Waltham, MA) or equivalent. (b) Acetonitrile . – Optima MS grade (Thermo Fisher Scientific) or equivalent. (c) Ammonium formate . – Optima MS grade (Thermo Fisher Scientific) or equivalent. (d) Formic acid . – Optima MS grade (Thermo Fisher Scientific) or equivalent. (e) Nitric acid . – 70% w/w, ACS grade (Avantor, Center Valley, PA) or equivalent. (f) Isopropanol . – Optima MS grade (Thermo Fisher Scientific) or equivalent. (g) Desiccant . – (VWR) 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. D. Reference Standards (a) Reference Standard . – L-Carnitine (USP, Rockville, MD) or equivalent. (b) Reference Standard . – Choline Bitartrate (TCI, Tokyo, Japan) or equivalent. (c) Reference Internal Standard . – L-Carnitine-d3 HCl (CDN Isotopes, Pointe Claire, Québec, Canada) or equivalent. (d) Reference Internal Standard . – Choline-1,1,2,2-d4 Chloride (CDN Isotopes) or equivalent.

E. Procedure ( a ) Reagent preparation. (1) Preparation of mobile phase

Noted: All mobile phase bottles should be rinsed thoroughly prior to use with ultra-pure water and isopropanol. An additional rinse with concentrated acid may be used if high background signal is present. a) Mobile Phase A (5 mM Ammonium Formate in water:ACN (50:50, v:v) with 0.2% Formic Acid): Weigh 0.32 ± 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.

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