Review Team (February 2016).pdf

down (3). 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 of acetonitrile in a microcentrifuge tube, and then filtered through a 0.45 μm

PTFE syringe filter into a silanized injection vial. Aliquots of 0.5 mL of the working standard

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

injection vials.

F. LC/MS/MS Parameters

A Shimadzu Prominence liquid chromatography system equipped with an Agilent Zorbax

300-SCX column (3.0 x 50 mm, 5 μm) was utilized. A flow rate of 1.0 mL/min was maintained

over the 4.2 minute total run time. The mobile phase conditions were 100% mobile phase A

until 1.0 minute, ramped to 100% mobile phase B by 1.5 minutes, and ramped back to 100% A

by 3.0 minutes. 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 ion

electrospray (ESI) ionization was used in multiple reaction monitoring (MRM) mode. The

MS/MS overall settings used are described in Table 1. The MS/MS settings may need to be

modified except for ionization, mode, and gas types to obtain optimum chromatography and

sensitivity. Figures 1 and 2 show typical extracted ion chromatograms (XIC) of NIST SRM

1849a for choline and carnitine.

G. 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 2) versus the corresponding

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

Chol-08/Carn-07 (February 2016)

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