6. AOACSPIFANMethods-2018Awards

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206 E llingson et al .: J ournal of AOAC I nternational V ol . 99, N o . 1, 2016

described in Table 2015.10A . The MS/MS settings may need to be modified except for ionization, mode, and gas types to obtain optimum chromatography and sensitivity. Figures 2015.10A and 2015.10B show typical extracted ion chromatograms (XICs) from National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1849a for choline and carnitine.

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 μL of 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 hold at a power of 1000 W, ending in a cool down (6). The contents of the vessels were transferred into 50 mL polypropylene tubes with water and diluted to a volume of 25 mL with water. A 0.5 mL aliquot of the sample solution was mixed with 0.5 mL acetonitrile in a microcentrifuge tube, and then filtered through a 0.45 μm PTFE 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 of acetonitrile directly in the silanized injection vials. A Shimadzu Prominence LC system equipped with an Agilent Zorbax 300-SCX column (3.0 × 50 mm, 5 μm) was used. A flow rate of 1.0 mL/min was maintained over the 4.2 min total run time. The mobile phase conditions were 100% mobile phase A until 1.0 min, ramped to 100% mobile phase B by 1.5 min, and ramped back to 100% phase Aby 3.0 min. A column temperature of 40°C, and an autosampler temperature of 5°C was maintained. A 1 μL injection was used. Autosampler rinse settings were adjusted to eliminate carryover as much as possible. An ABSciex API 4000 mass spectrometer with positive ESI was used in multiple reaction monitoring (MRM) mode. The MS/MS overall settings used are G. LC/MS/MS Parameters

H. Quantification and Confirmation

The quantification of choline and carnitine was accomplished by the generation of calibration curves using the peak area ratio of the chosen transition (Table 2015.10B ) versus the corresponding deuterated internal standards. Least-squares regression analysis using a linear model with 1/ x 2 weighting was used for both analytes. Confirmation was achieved through the analysis of ion ratios between samples and reference standards for at least one additional transition listed in the table. The concentration of each analyte in a sample was calculated by the following equation:

100 10 ng mg 6

R

C V D S = × × ×

Table 2015.10A. MS/MS settings Ionization

Positive ion electrospray (ESI+)

Mode

MRM

Ion spray voltage

1000 V 550°C

Turbo ion spray temp. Declustering potential

120 V

Dwell time

100 msec

Entrance potential

10 V 25 V

Collision cell exit potential

Collision gas Curtain gas

Nitrogen, 5 psig Nitrogen, 20 psig Nitrogen, 60 psig Nitrogen, 60 psig

Nebulizing gas (gas 1) Auxiliary gas (gas 2)

Needle position

Y = 5 mm, X = 5 mm

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