Methods to be Reviewed by OMB in 2016

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 5mL volume of water followed by 2.5mL of 70% (w/w) nitric acid delivered with a

bottle top dispenserwere then added to each liner, capped, and vortexed to mix. The microwave

program used was a ramp to temperature of 120°C over 10minutes, followed by a 40minute hold at a

power of 1000W, ending in a cool down (6). The contents of the vessels were transferred into 50mL

polypropylene tubes withwater and diluted to a volume of 25mL with water. A 0.5mL aliquot of the

sample solution was mixed with 0.5mL of acetonitrile in a microcentrifuge tube, and then filtered

through a 0.45 µm PTFE syringe filter into a silanized injection vial. Aliquots of 0.5mL of the working

standard and reagent blank solutions were mixed with 0.5mL of acetonitrile directly in the silanized

injection vials.

LC/MS/MS Parameters

A Shimadzu Prominence liquid chromatography system equipped with an Agilent Zorbax 300-SCX

column (3.0 x 50mm, 5 µm) was utilized. A flow rate of 1.0mL/min was maintained over the 4.2minute

total run time. The mobile phase conditions were 100% mobile phase A until 1.0minute, ramped to

100% mobile phase B by 1.5minutes, and ramped back to 100% A by 3.0minutes. A column

temperature of 40°C, and an autosampler temperature of 5°C was maintained. A 1 µl injectionwas

used. Autosampler rinse settings were adjusted to eliminate carryover as much as possible. An ABSciex

API 4000mass spectrometer with positive ion electrospray (ESI) ionization 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 (XIC) of NIST SRM1849a for choline and carnitine.

Quantification and Confirmation

The quantification of choline and carnitine was accomplished by generation of calibration curves using

the peak area ratio of the chosen transition (Table

2015.10B

) versus the corresponding deuterated

internal standards. Least square regression analysis using a linear model with 1/x

weighting was used

for both analytes. Confirmation was achieved through 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

Candidates for 2016 Method of the Year

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