LC-MS/MS-Based Separation and Quantification of Marfey’s Reagent Derivatized Proteinogenic Amino Acid dl-Stereoisomers

  • Navid J. Ayon
  • Amar Deep Sharma
  • William G. GutheilEmail author
Research Article


d-Amino acids are important biological molecules. Improved analytical methods for their resolution and quantification remain of keen interest. In this study, we investigated the use of Marfey’s reagent (chiral) derivatization coupled with LC-MS/MS-based separation and detection of the resulting diastereomers for quantification of the 19 common l- and d-amino acids and glycine. Standard formic acid (pH 2)-based separations on reverse phase media were unable to separate all 19 amino acid dl pairs. In contrast, a water/acetonitrile/ammonium acetate (pH 6.5) solvent system allowed all 19 amino acid dl pairs to be chromatographically resolved on a 30 min gradient, with negative mode detection at pH 6.5 giving good sensitivity. Derivatization reaction rates between amino acids varied substantially, with overnight derivatization required for some amino acids. Chromatography at pH 6.5 combined with MS/MS quantification in negative mode demonstrated good linearity over a wide concentration range for all 20 amino acids. Matrix effects, assessed with an MRSA extract, were negligible. Marfey’s derivatized analytes were stable for 24 h at room temperature. This method was demonstrated by determining the levels of these analytes in mid-log phase MRSA extracts. This approach provides for the chromatographic resolution and MS/MS-based quantification of all 20 common l- and d-amino acids in complex matrices.

Graphical Abstract


Amino acid Marfey’s reagent Chiral separation LC-MS/MS 



The authors acknowledge support by grants from National Institute of Health (R21-AI121903 and R15-GM126502) to WGG.

Supplementary material

13361_2018_2093_MOESM1_ESM.docx (6.7 mb)
ESM 1 (DOCX 6.71 mb)


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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  1. 1.Division of Pharmacology and Pharmaceutical Sciences, School of PharmacyUniversity of Missouri-Kansas CityKansas CityUSA

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