AOAC Final Action Methods in 2019

Table 2015.10A. Preparation of working standard solutions a Standard solution Source solution ID Source concn, mg/mL

Source volume, mL

MS grade water, mL

Final volume, mL

Concn (µg/mL) extracted (ng/mL)

STD6 STD5 STD4 STD3 STD2 STD1

Stock solutions Stock solutions Stock solutions

25.0 25.0 25.0 5.00

2.00 each 1.60 each 0.800 each

6.00 6.80 8.40 9.00 9.60 9.80

10.0 10.0 10.0 10.0 10.0 10.0

5000 4000 2000

STD6 STD3 STD3

1.00

500

0.500 0.500

0.400 0.200

20 10

a  Alternate weights or volumes may be used to scale these preparations

( b )  Standard solutions preparation .—( 1 )  Preparation of choline/carnitine stock solutions .—( a )  Choline stock solution (~25 mg/mL) .—Weigh 0.5200 ± 0.01 g choline bitartrate into a 20 mL polypropylene scintillation vial. Dissolve with 10.0 mL water. ( b )  Carnitine stock solution (~25 mg/mL) .—Due to the hygroscopic nature of L-carnitine, perform a Karl Fischer moisture analysis test to determine the water content of the material at the time of weighing.Weigh 0.2500 ± 0.01 g into a 20mLpolypropylene scintillation vial. Dissolve with 10.0 mL UPW. ( c )  L-Carnitine-d3 stock solution (~2.00 mg/mL; internal standard I; IS1) .—Weigh 0.02500 ± 0.001 g L-carnitine-d3 HCl into a 20 mL polypropylene scintillation vial. Dissolve with 10.0 mL UPW. ( d )  Choline-1,1,2,2-d4 stock solution (~2.00 mg/mL; IS2) .— Weigh 0.03100 ± 0.001 g choline-1,1,2,2-d4 bromide into a 20 mL polypropylene scintillation vial. Dissolve with 10.0 mL 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 μ g/mL) IS working solution .—Transfer 10 mL IS1 solution and 10 mL IS2 solution into a 100 mL Digitube. Add 80 mL UPW to bring to a final volume of 100 mL. ( Note : 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.) ( c )  Sample preparation .—( 1 )  Sample preparation for samples needing reconstitution.— ( a ) Weigh 10.00 ± 0.500 g sample in a suitable disposable cup or beaker. ( b ) Add UPW to bring the total weight (including the powder weight) to 100.00 ± 1.00 g. ( c ) Add a stir bar and stir as fast as possible without causing the sample to splatter. Stir for at least 10 min, but no longer than 30 min. Note : Adjust weights as needed to accommodate different powder types and levels of vitamins. ( 2 )  Free analysis of carnitine and choline.— ( a ) Weigh 0.1–1.0 g sample into a tared 50 mL Digitube depending on the concentration of each analyte (to fall within calibration curve). ( b ) A reagent blank, a reagent blank + IS, and working standards must be included for each analysis and treated the same as samples through the analysis. Add 50 μL of each working solution to separate 50 mL Digitubes. 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. ( c ) Add 50 μL intermediate mixed IS solution to each sample, internal working solution (IWS), and reagent blank + IS. ( d ) Dilute to 25 mL with MS grade water, cap, and thoroughly mix sample on a horizontal shaker. Sample extract is stable for 3 days when stored refrigerated and protected from light. ( e ) If dilutions are required, dilute samples to appropriate concentrations using the reagent blank + IS using microcentrifuge tubes to do the dilutions. ( f ) Aliquot 0.5 mL extract into a microcentrifuge tube, along with 0.5 mL acetonitrile. Mix well. ( g ) Filter samples through a 0.45 μm GHP syringe filter into a silanized injection vial. Standards do not need to be filtered through the syringe filter. Sample extract is stable for 3 days when stored refrigerated and protected from light. ( 3 )  Total analysis of carnitine and choline.— ( a ) Weigh 0.1– 1.0 g sample into a tared 55 mL MarsExpress vessel depending on the concentration of each analyte (to fall within calibration curve). For viscous (high-fat, high-protein) ready-to-feed samples, perform a predilution by weighing 1.0 g sample and adding MS grade water

Table 2015.10B. HPLC parameters Column Zorbax 300-SCX, 3.0 × 50 mm, 5 µm (Agilent, Santa Clara, CA, USA) Mobile phase A 5 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid Mobile phase B 30 mM ammonium formate in water:ACN (50 + 50, v/v) with 0.2% formic acid Injection volume 5–10 μL Run time 4.2 min

Flow rate, mL/min

Phase B, %

Gradient program Time, min

0

1.00 1.00 1.00 1.00 1.00

0 0

1.0 1.5 2.5 3.0 4.2

100 100

0 0

1.00

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