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

F. Sample Preparation Powder infant formula and adult nutritionals were reconstituted by weighing 25 g and diluting with water to a final weight of 225 g. Viscous ready-to-feed (RTF) products that were being analyzed for total choline and carnitine were prediluted by weighing 1.0 g and diluting with water to a final weight of 5.0 g. (a)  Free choline and carnitine .—Samples were prepared by weighing 1.0 g of reconstituted product into a 50 mL polypropylene tube. Six additional tubes were designated for the working standards along with two tubes for the reagent blank and reagent blank + internal standard to monitor any interference or carryover. The working standards, reagent blank, and reagent blank + internal standard were included with each free analysis and treated the same as samples through the sample preparation. The working standard tubes received 50 μL of the appropriate intermediate working standard level. All tubes except the reagent blank received 50 μL of the intermediate internal standard solution. The tubes were diluted to 25 mL with water and thoroughly mixed on a horizontal shaker. The reagent blank + internal standard solution was used as the diluent if dilutions were needed. A 0.5 mL aliquot the sample solution was mixed with 0.5 mL of acetonitrile in a microcentrifuge tube, and then filtered through a 0.45 μm GHP syringe filter into a silanized injection vial. Aliquots of 0.5 mL of the working standard and reagent blank solutions were mixed with 0.5 mL acetonitrile directly in the silanized injection vials. (b)  Total choline and carnitine .—Samples were prepared by weighing 1.0 g of reconstituted or diluted product into a 55 mL MARSXpress liner. Six additional liners were designated for the working standards along with two liners for the reagent blank and reagent blank + internal standard to monitor any interference or carryover. The working standards, reagent blank, and reagent blank + internal standard were included with each total analysis and treated the same as samples through the sample preparation. Liners designated for the working standards received 50 μLof the appropriate intermediate working standard level. All liners except the reagent blank received 50 μL of the intermediate internal standard solution. A 5 mL volume of water followed by 2.5 mL of 70% (w/w) nitric acid delivered with a bottle top dispenser were then added to each liner, capped, and vortexed to mix. The microwave program used was a ramp to temperature of 120°C over 10 min, followed by a 40 min

Figure 2015.10A. Extracted ion chromatogram (XIC) of choline.

prepared by weighing 3.78 g 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 Lmobile phase container along with 1 L acetonitrile, 4 mL formic acid, a stir bar, and then thoroughly mixed. Mobile phase B was also used for the rinse solutions in the autosampler. E. Preparation of Standard Solutions The carnitine stock standard was prepared at a concentration of 25 mg/mL by weighing 0.25 g l-carnitine into a 20 mL polypropylene tube followed by 10 mL water to dissolve. The purity of l-carnitine from the Certificate of Analysis (CoA) and moisture determined by Karl Fischer titration 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 choline bitartrate into a 20 mL polypropylene tube followed by 10 mL water to dissolve. The purity of choline bitartrate from the CoA along with a molecular weight conversion from choline bitartrate to choline of 0.41133, was used to calculate the final concentration of choline. Intermediate working standards were prepared at concentrations of 10, 20, 500, 2000, 4000, and 5000 μg/mL for each analyte using both the stock and higher concentration intermediate working standard solutions using appropriate volumes into 20 mL polypropylene tubes with water as the diluent. All stock and intermediate standard solutions were stable for 2 months when stored at 5 ± 3°C and protected from light. Aliquots of the intermediate working standards were treated through the sample analysis, so the concentrations used for the calibration curves for both free and total analyses were the same numerical values as the intermediate working standards but in ng/mL. Internal stock standards were prepared at a concentration of 2 mg/mL by weighing 25 mg l-carnitine-d 3 and 35 mg choline-1,1,2,2-d 4 into separate 20 mL polypropylene tubes. A volume of 10 mL water was added to each to dissolve, and then both solutions quantitatively transferred to a 100 mL polypropylene tube and diluted to volume with water to prepare an intermediate solution at 200 μg/mL. The purity from the CoA was used to calculate the final concentration of each internal standard. Stability of these solutions was monitored while being stored at 5 ± 3°C and protected from light.

Figure 2015.10B. Extracted ion chromatogram (XIC) of carnitine.

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