A simple analytical workflow is described for gas chromatographic-mass spectrometric (GC-MS)-based chiral profiling of secondary amino acids (AAs) in biological matrices. The sample preparation is carried out directly in aqueous biological sample extracts and involves in situ heptafluorobutyl chloroformate (HFBCF) derivatization-liquid-liquid microextraction of nonpolar products into hexane phase followed by subsequent formation of the corresponding methylamides from the HFB esters by direct treatment with methylamine reagent solution. The (O, N) HFB-butoxycarbonyl-methylamide AA products (HFBOC-MA) are separated on a Chirasil-L-Val capillary column and quantitatively measured by GC-MS operated in selected ion monitoring (SIM) mode. The protocol includes 12 simple pipetting steps and covers the quantitative analysis of 8 L, D pairs of secondary amino acids, including proline, isomeric 3-, 4-hydroxyprolines, pipecolic acid, nipecotic acid, azetidine-2-carboxylic acid, and cis- and trans-5-hydroxy-L-pipecolic acid using 13C5 -L-proline as an internal standard. The individual analytical steps are commented on and explained, with emphasis on the chiral GC-MS analysis of secondary amino acids in human urine, serum, and peptide hydrolysate samples.

• Keywords
• Alkyl chloroformate derivatization, Amino acids, Chiral analysis, GC-MS, Peptide hydrolysates, Quantitative analysis, Secondary amino acids, Serum, Urine,
• MeSH
• Amino Acids * chemistry   urine   analysis   MeSH
• Formates chemistry   MeSH
• Humans MeSH
• Liquid Phase Microextraction methods   MeSH
• Gas Chromatography-Mass Spectrometry * methods   MeSH
• Stereoisomerism MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Amino Acids * MeSH
• Formates MeSH