Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry

Abstract

Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15 min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20 ng/ml for a range of analytes and a dynamic range from 2.5-20 to 500-4000 ng/ml. This LC-MS/MS method was validated for biomarker discovery in models of human neurological disorders.

Fig 1

A representative chromatogram of the endogenous levels of AAs in mouse brain before spiking with analyte standards (A), and after spiking with analyte standard solutions (concentration) and IS (concentration) (B)

Fig 2

Degradation profile of some AAs from brains homogenized (A) in (1:3 w/v) water and (B) in (1:3 w/v) methanol. The % relative peak area is calculated by taking the ratio of peak area of analytes at various time points to peak area of analytes at time “0 hr”.