Journal article
Direct analysis of free amino acids by mixed-mode chromatography with tandem mass spectrometry

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Publication Details
Author list: Vilches, Ana Paola;Norström, Sara H;Bylund, Dan;
Publication year: 2017
Journal: Journal of Separation Science
Start page: 1482
End page: 1492
Number of pages: 11
ISSN: 1615-9306
eISSN: 1615-9314
View additional information: View in Web of Science™


We developed a straightforward, robust, and relatively fast method for the analysis of amino acids by mixed-mode high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The method does not involve derivatization and allows the detection of 21 amino acids, representing a wide range of isoelectric points, in less than 40 min. Chromatographic separation was governed by a silica-based mixed-mode column providing simultaneous hydrophobic and ion exchange separation mechanisms. The use of tandem mass spectrometry increased selectivity, reducing potential problems associated with poor selectivity in the chromatographic system. For an injection volume of 1 µL, we obtained detection limits <3 µM for the majority of analytes. For all analytes, a linearity of r > 0.99 was obtained, recovery in matrix was >86%, and the retention times were highly reproducible. The method was successfully applied to soil solution and fungal culture samples, demonstrating the advantages in successfully avoiding issues associated with high amounts of substances that may interfere with derivatization-based methods. This method represents an alternative to derivatization-based methods and can be applied in areas where sample matrices are highly complex.

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