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Jing et al.: J ournal of AOAC I nternational V ol. 98, N o . 5, 2015  1397

( f )  Working standard solution (WS). –Prepare fresh on day of analysis. Use 90% acetonitrile in final working solution. ( 1 ) Pipet 200 µL of each IWS (IWS 1–3) into three 10 mL volumetric flasks. ( 2 ) Add 800 µL laboratory water to each volumetric flask. ( 3 ) Bring to volume with acetonitrile and mix well. ( 4 ) Transfer to autosampler vials for analysis. ( 5 ) Table 2014.04B summarizes the concentrations of choline and carnitine and their associated ISs in the three IWS and in the three WS. F. Procedure ( a )  Liquids .—For liquids and ready-to-feed products, select sample weights between 1 and 5 g based on expected concentration of the two nutrients in each sample. ( b )  Powders. —Powder products are reconstituted with water prior to analysis, typically 11.1% (w/w; 25 g powder added to 200 g water). Typically a 5 g sample aliquot is taken from the reconstituted material for analysis, but adjust as needed. ( c )  Sample analysis.— ( 1 ) Weigh sample into a tarred microwave reaction vessel. ( 2 ) Add 125 µL MIX-IS. ( 3 ) Add water to sample to achieve a total sample volume of approximately 8 mL. ( 4 ) Add 2.2 mL of approximately 70% nitric acid and seal vessels. The final volume should be close to 10 mL (sample + acid + water + MIX-IS = 10 mL) prior to microwave digestion which makes acid concentration approximately 3.5 M. ( 5 ) Vortex vessels for 30 s, and then place vessels into turntable and insert into microwave system. ( 6 ) Complete microwave digestion using the conditions defined in Table 2014.04C . Operate with microwave venting to a fume hood. ( 7 ) After cool down (by air), uncap each vessel, and add 3.5 mL concentrated ammonium hydroxide to each vessel. Operate in a fume hood. ( 8 ) Vortex each sample for 30 s. ( 9 ) Allow samples to stand in a hood for 30 min to react any acylcarnitines under basic conditions. ( 10 ) Vortex samples for 30 s. ( 11 ) Transfer 1 mL digested sample into a disposable centrifuge tube containing 25 mL laboratory water. ( 12 ) Vortex for 30 s. ( 13 ) Filter about 1 mL diluted sample using a 0.45 µm nylon syringe filter. ( 14 ) Transfer 0.1 mL filtered solution into autosampler vial containing 0.9 mL acetonitrile. Mix well. Sample is ready for analysis. ( 15 ) Samples diluted with acetonitrile are stable for 24 h

in a vacuum oven at 65°C (±3°C) for 4 h. ( b ) Weigh 170 (±17) mg into an appropriate weighing boat. ( c ) Quantitatively transfer into a 50 mL volumetric flask. ( d ) Dissolve and bring to volume with laboratory water. ( Note : choline chloride is the form weighed, but many laboratories prefer working in units of choline hydroxide, which is how the method is presented here.) ( 2 ) L-Carnitine (approximately 750 µg/mL).— ( a ) Weigh 150.0 ± 8 mg into an appropriate weighing boat as quickly as possible. ( b ) Transfer into a 200 mL volumetric flask. ( c ) Dissolve and bring to volume with laboratory water. ( d ) Correct weight for moisture content. ( e ) Aliquot the stock solution into 2 mL plastic vials, with each vial receiving at least 1 mL and store ≤–15°C. Expiration: 1 year. ( c )  Internal standard (IS) stock solutions.— ( 1 ) Choline chloride-d 9 (approximately 20000 µg/mL).— ( a ) Transfer 1 g choline chloride-d 9 into a 50 mL volumetric flask. ( b ) Dissolve and bring to volume with laboratory water. ( c ) Aliquot the stock solution into 2 mL plastic vials with each vial receiving about 1 mL and store at ≤–15°C. Expiration: 1 year. ( 2 )  L-Carnitine chloride-d 3 HCl (approximately 10000 µg/mL).— ( a ) Transfer 0.5 g L-carnitine chloride-d 3 to a 50 mL flask. ( b ) Dissolve and bring to volume with laboratory water. ( c ) Aliquot the stock solution into 2 mL plastic vials with each vial receiving about 1 mL and store at ≤–15°C. Expiration: 1 year. ( d )  Mixed intermediate internal standard solution (MIX-IS) .—Prepare fresh on day of analysis. Must use same MIX-IS solution for both calibration working standards and samples. ( 1 ) Transfer 400 µL of each choline chloride-d 9 and 400 µL carnitine chloride-d 3 HCl stock solution by a calibrated pipet into a 10 mL volumetric flask. ( 2 ) Bring to volume with laboratory water and mix well. ( e )  Intermediate working standards (IWS). —Prepare fresh on day of analysis. ( 1 ) Pipet 100 µL MIX-IS into three individual 50 mL volumetric flasks. ( 2 ) Using Table 2014.04A , add the required volumes of the stock choline and carnitine solution by proper sized calibrated pipets. ( 3 ) Bring to volume with laboratory water and mix well. Table 2014.04C. Microwave digestion parameters Power 100% (1600 W) Ramp to temperature, min 10 Hold time, min 40 Temperature, ° C 120

Table 2014.04D. Mass analysis parameters (Xevo TQ-S)

Molecular ion (precursor), amu Product ion, amu Dwell, s

Standard

Retention time typical, min

Cone voltage, V Collision energy, V

Choline

2.5 2.5 4.4 4.4

104.2 113.4

60.3 69.3 103 103

0.025 0.025 0.025 0.025

40 40 30 30

32 30 28 26

Choline chloride-d 9

Carnitine

162 165

Carnitine chloride-d 3