A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC 
LC-MS/MS) method was developed and validated to simultaneously quantify six 
aqueous choline-related compounds and eight major phospholipids classes in a 
single run. HILIC chromatography was coupled to positive ion electrospray mass 
spectrometry. A combination of multiple scan modes including precursor ion scan, 
neutral loss scan and multiple reaction monitoring was optimized for the 
determination of each compound or class in a single LC/MS run. This work 
developed a simplified extraction scheme in which both free choline and related 
compounds along with phospholipids were extracted into a homogenized phase using 
chloroform/methanol/water (1:2:0.8) and diluted into methanol for the analysis 
of target compounds in a variety of sample matrices. The analyte recoveries were 
evaluated by spiking tissues and food samples with two isotope-labeled internal 
standards, PC-d(3) and Cho-d(3). Recoveries of between 90% and 115% were 
obtained by spiking a range of sample matrices with authentic standards 
containing all 14 of the target analytes. The precision of the analysis ranged 
from 1.6% to 13%. Accuracy and precision was comparable to that obtained by 
quantification of selected phospholipid classes using (31)P NMR. A variety of 
sample matrices including egg yolks, human diets and animal tissues were 
analyzed using the validated method. The measurements of total choline in 
selected foods were found to be in good agreement with values obtained from the 
USDA choline database.