Given the biological importance of L- and D-amino acids, improved analytical methods for their resolution and quantification remain of interest. In this study, we investigated the use of Marfey’s reagent derivatization coupled with reversed phase separation and MS/MS-based detection for the resolution and quantification of the 20 common amino acids in their L- and D- forms. Using standard formic acid based separations (water/acetonitrile/formic acid) on a C8 column with MS/MS detection in positive mode gave good separation of 15 of the 20 amino acid d/l pairs (except Ser, Asn, Gln, Lys and Cys), and with good sensitivity for all derivatized amino acids. Chromatography at pH 6.5 in water/acetonitrile/ammonium acetate allowed all 20 d/l amino acid pairs to be readily resolved. However, early eluting Marfey’s-derivatized amino acids gave very low sensitivity with positive mode MS/MS detection. MS/MS negative mode detection using the pH 6.5 gradient gave good sensitivity and resolution of all 20 d/l amino acid pairs. The resulting approach was then demonstrated for the determination of d/l amino acids in several biological samples of interest, including rat serum, normal and cancer patient serum, extracts of Staphylococcus aureus and Vancomycin resistant enterococcus.

LC-MS/MS-based analytical method for Marfey’s reagent derivatized amino acid stereoisomers and it’s application to biological samples

255th National Meeting (2018)

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