Liquid chromatography-mass spectrometry methods for multiplexed detection and quantitation of free amino acids

A method of analyzing a sample suspected of containing free amino acids using liquid chromatography-mass spectrometry, comprising:

• preparing a sample to obtain free amino acids;
• separating the free amino acids by liquid chromatography using a series of at least two chromatographic columns including (a) a first chromatographic column including a graphitic carbon packing material to separate negatively-charged interferents from the free amino acids, followed by (b) a second, reversed phase, solid core chromatographic column to separate the free amino acids from one another, wherein separating the free amino acids includes eluting the free amino acids off the first chromatographic column in a flow-reverse direction into the second chromatographic column;
• ionizing the sample to generate ions;
• fragmenting the ions to produce parent and daughter ions;
• detecting the parent and daughter ions by selected reaction monitoring (SRM);
• and
• detecting the presence of the free amino acids in accordance to the detected parent and daughter ions.