Acetonitrile adduct analysis of underivatised amino acids offers improved sensitivity for hydrophilic interaction liquid chromatography tandem mass-spectrometry

LC-MS/MS method development for native amino acid detection can be problematic due to low ionisation efficiencies, in source fragmentation, potential for cluster ion formation and incorrect application of chromatography techniques. This has led to the majority of the scientific community derivatising amino acids for more sensitive analysis. Derivatisation has several benefits including reduced signal-to-noise ratios, more efficient ionisation, and a change in polarity, allowing the use of reverse phase chromatography. However, derivatisation of amino acids can be expensive, requires additional sample preparation steps, is more time consuming and increases sample instability, due to the most derivatised amino acids only be stable for finite amount of time. While showing initial promise, development of reliable hydrophilic interaction liquid chromatography (HILIC) separation methods has presented difficulties for the analyst including irreproducible separation and poor sensitivity. This study aimed to find a means to improve the detection sensitivity of the 20 protein amino acids by HILIC-MS/MS. We describe the use of previously undescribed amino acid-acetonitrile (ACN) adducts to improve detection of 16 out of the 20 amino acids. While all amino acids examined did form an ACN adduct, 4 had low intensity adduct formation compared to their protonated state, 3 of which are classified as basic amino acids. For 15 of the 20 amino acids tested, we used the ACN adduct for both quantification and qualification ions and demonstrated a significant enhancement in signal-to-noise ratio, ranging from 23 to 1762% improvement. Lower LODs, LOQs and lower ranges of linearity were also achieved for these amino acids. The optimised method was applied to a human neuroblastoma cell line (SH-SY5Y) with the potential to be applied to other complex sample types. The improved sensitivity this method offers simplifies sample preparation and reduces the costs of amino acid analysis compared to those methods that rely on derivatisation for sensitivity.