Methods to be Reviewed by OMB in 2016

206

llingson

et al

ournal of

AOAC I

nternational

99, N

1, 2016

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.

G. LC/MS/MS Parameters

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% phaseAby 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

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.

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/

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:

C V D

100

10 ng mg

= × × ×

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

Table 2015.10A. MS/MS settings

Ionization

Positive ion electrospray (ESI+)

Mode

MRM

Ion spray voltage

1000 V

Turbo ion spray temp.

550°C

Declustering potential

120 V

Dwell time

100 msec

Entrance potential

10 V

Collision cell exit potential

25 V

Collision gas

Nitrogen, 5 psig

Curtain gas

Nitrogen, 20 psig

Nebulizing gas (gas 1)

Nitrogen, 60 psig

Auxiliary gas (gas 2)

Nitrogen, 60 psig

Needle position

= 5 mm,

= 5 mm

Candidates for 2016 Method of the Year

126