Journal of Pharmaceutical and Biomedical Analysis 69 (2012) 28–41 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j ourna l ho me p a ge: www.elsevier.com/locate/jpba Recent advances in the direct and indirect liquid chromatographic enantioseparation of amino acids and related compounds: A review István Ilisz, Anita Aranyi, Zoltán Pataj, Antal Péter ∗ Department of Inorganic and Analytical Chemistry, University of Szeged, H-6720 Szeged, Dóm tér 7, Hungary a r t i c l e i n f o Article history: Received 15 December 2011 Received in revised form 18 January 2012 Accepted 19 January 2012 Available online 2 February 2012 Keywords: Amino acids Indirect methods Chiral derivatizing agent (CDA) Direct methods Chiral stationary phases (CSPs) a b s t r a c t Amino acids are essential for life, and have many functions in metabolism. One particularly impor- tant function is to serve as the building blocks of peptides and proteins, giving rise to complex three dimensional structures through disulfide bonds or crosslinked amino acids. Peptides are fre- quently cyclic and contain protein as well as non-protein aminoacids in many instances. Since most of the proteinogenic -amino acids contain a chiral carbon atom (with the exception of glycine), the stereoisomers of all these amino acids and the peptides in which they are to be found may pos- sess differences in biological activity in living systems. The impetus for advances in chiral separation has been highest in the past decade and this still continues to be an area of high focus. The impor- tant analytical task of the separation of isomers is achieved mainly by chromatographic methods. This review surveys indirect and direct HPLC separations of biologically and pharmaceutically impor- tant enantiomers of amino acids and related compounds, with emphasis on the literature published from 2005. © 2012 Elsevier B.V. All rights reserved. Abbreviations: Ac, acetyl; AITC, 2,3,4-tri-O-acetyl--d-arabinopyranosyl isothiocyanate; ANPAD, (1S,2S)-(+)-2-amino-1-(4-nitrophenyl)-1,3- propanediol; APPI, 1-acetoxy-1-phenyl-2-propyl isothiocyanate; Bis–Tris, bis(2-hydroxyethyl)imino–tris(hydroxymethyl)methane; BGIT, 2,3,4,6-tetra-O- benzoyl--d-glycopyranosyl isothiocyanate; Boc, tert-butyloxycarbonyl; Bzl, benzyl; Cbz, carbobenzyloxy; Chirobiotic T (T), teicoplanin-based CSP; Chi- robiotic TAG (TAG), teicoplanin aglycone-based CSP; Me-TAG, methylated teicoplanin aglycone-based CSP; Chirobiotic R (R), ristocetin A-based CSP; Chirobiotic V (V), vancomycin-based CSP; CDA, chiral derivatizing agent; CSP, chiral stationary phase; DANI, 1,3-diacetoxy-1-(4-nitrophenyl)-2-propyl isothiocyanate; DBD-PyNCS, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N- dimethylaminosulfonyl)-2,1,3-benzoxadiazole; DNFB, 2,4-dinitrofluorobenzene; DNP, dinitrophenyl; DNB, dinitrobenzoyl; ESI-MS, electrospray ionization mass spectrometry; FITC, fluorescein isothiocyanate; FDAA, 1-fluoro-2,4-dinitrophenyl- 5-l-alanine amide; FDLA, 1-fluoro-2,4-dinitrophenyl-5-l-leucine amide; FDMA, 1-fluoro-2,4-dinitrophenyl-l-methionine amide; FDPA, 1-fluoro-2,4-dinitrophenyl- 5-l-phenylalanine amide; FDVA, 1-fluoro-2,4-dinitrophenyl-5-l-valine amide; Fmoc, 9-(fluorenylmethyl)oxycarbonyl; GITC, 2,3,4,6-tetra-O-acetyl- -d-glucopyranosyl isothiocyanate; IBDC, N-isobutyryl-d-cysteine; IBLC, N-isobutyryl-l-cysteine; IPA, 2-propanol; MeCN, acetonitrile; R-NMC, R- mandelyl-l-cysteine; NAC, N-acetyl-l-cysteine; NAP, N-acetyl-d-penicillamine; NBD-PyNCS, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole; NIFE, N-(4-nitrophenoxycarbonyl)phenylalanine methoxyethyl ester; OPA, ortho- phthalaldehyde; NPM, normal phase mode; PIM, polar ionic mode; POM, polar organic mode; RPM, reversed-phase mode; Reagent 1, (5R,6S,9R)-4-(2-fluoro- 3,5-dinitrobenzoyl)-6-isopropyl-9-methyl-1.4-diazaspiro[4.5]decan-2-one; SFP 1. Introduction The existence of enantiomers has been known for many years and the importance of chirality with respect to biological activity has been recognized by Pasteur more than a century ago [1]. The physiological environment within a living organism is chiral, and the biological activities of enantiomeric forms of molecules can dif- fer dramatically. With the exception of glycine, all encoded protein amino acids have at least one chiral center (enantiomers) or even two (epimers). Some 20 genetically encoded amino acids comprise the building blocks of proteins, which besides nucleotides, polysaccharides or lipids are the most important constituents of all living sys- tems. Proteins of multicellular organisms are usually based on l-amino acids but the d-forms of amino acids in peptides can differ significantly in biological systems [2–5]. The preparation of enantiomerically pure analogs of amino acids is a challenging task that requires accurate analytical methods with which to determine enantiomeric excesses during asymmetric syntheses. Improved Reagent, succinimidyl ferrocenyl propionate; TATG, 1-thio--d-glucose tetraacetate; Z, benzyloxycarbonyl. ∗ Corresponding author. Tel.: +36 62 544000/3656; fax: +36 62 420505. E-mail address: apeter@chem.u-szeged.hu (A. Péter). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2012.01.020