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

Abstract

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.

Rapid quantification of underivatized alloisoleucine and argininosuccinate using mixed-mode chromatography with tandem mass spectrometry

“…Chromatographic columns that combine two or more retention mechanisms (termed "mixed-mode" columns) have emerged as an alternative for LC-MS/MS analysis of amino acids [10,11]. Some mixedmode columns include MS compatible ion exchange binding properties, which may be useful in the chromatographic resolution of isomeric compounds such as alloisoleucine and isoleucine.…”

Determination of immunogenic proteins in biopharmaceuticals by UHPLC–MS amino acid analysis

“…The simplest way how to perform the AAA is direct analysis. In this case, the amino acids are mainly separated by ion-exchange chromatography (IEC) [10], ion-interaction chromatography (IIC) [11] or hydrophilic interaction chromatography (HILIC) [12, 13]. The main challenge in such type of analysis is lack of a moiety in the chemical structure of these molecules which allows UV, fluorescence and electrochemical detection.…”

The Performance Investigation of Bimodal Cation Exchange/Hydrophilic Interaction Liquid Chromatography–Electrospray Ionization Mass Spectrometry by Modifying Mobile Phase Composition in Amino Acid Separation

“…Thus, it is imperative to investigate and understand the role and effect of the mobile phase components to control chromatographic peaks of the compounds separated by CEX/HILIC. A number of studies have reported the retention behavior and applications in the IEX/RP modes, while studies using the IEX/HILIC MMC have been scarce. For the purpose of this study, amino acids are good model compounds because of properties, including zwitterion, as well as positive and negative charge properties.…”