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Indirect Enantioseparation of Amino Acids by CE Using Automated In-Capillary Derivatization with ortho-Phthalaldehyde and N-Acetyl-l-Cysteine

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Abstract

A simple CE method for the assessment of enantiomeric purity of individual amino acids (AAs) is presented. The AAs were derivatized with ortho-phthalaldehyde and a thiol in the capillary by means of successive hydrodynamic injections (5 s, 0.5 psi) of derivatization solution, sample and derivatization solution. A voltage of 1 kV was applied to blend the solutions and to start the reaction. Subsequently, the derivatives were separated using a background electrolyte (BGE) composed of 50 mM borate buffer (pH = 9.25) and 7.5 mM of a cyclodextrin (CD). To find the optimal derivatization reagent and CD, the impact of the thiols 3-mercaptopropionic acid (MPA), N-acetyl-l-cysteine (NAC) and N-isobutyryl-l-cysteine (NIBLC), and various neutral and negatively charged CDs on the resolution were assessed. The use of NAC and γ-CD enabled the best indirect enantioseparation of 16 AAs. Serine could be separated with β-CD, and proline and cysteine are not suitable for automated OPA-derivatization. The resolutions were ranging from 2.3 to 16.6. A method was validated representative for the AAs methionine, aspartic acid, tryptophan and phenylalanine. The recovery (n = 3) of the minor enantiomers was found to be within 95.1 and 107.5 %. The relative standard deviation (RSD) was ranging from 1.2 to 6.4 %. The method is accurate, precise, linear, and robust. For methionine it could be demonstrated that the ratio of l- and d-AA ranging from 1 to 99 % can be evaluated by direct comparison of the corrected peak areas with satisfactory accuracy ranging between 92.1 and 115.9 %.

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Abbreviations

AA:

Amino acid

BGE:

Background electrolyte

CD:

Cyclodextrin

CDA:

Chiral derivatization agent

CMBCD:

Carboxymethyl-β-cyclodextrin

DMCD:

2,6-Di-O-methyl-β-cyclodextrin

HPBCD:

(2-Hydroxypropyl)-β-cyclodextrin

HPGCD:

(2-Hydroxypropyl)-γ-cyclodextrin

IS:

Internal standard

MPA:

3-Mercaptopropionic acid

NAC:

N-Acetyl-l-cysteine

NIBLC:

N-Isobutyryl-l-cysteine

OPA:

ortho-Phthalaldehyde

SBCD:

Sulfated β-cyclodextrin sodium salt

TMCD:

2,3,6-Tri-O-methyl-β-cyclodextrin

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Acknowledgments

Thanks are due to the Federal Institute of Drugs and Medical Devices (BfArM, Bonn, Germany) for financial support.

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  1. Institute for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074, Würzburg, Germany

    Raphael Kühnreich & Ulrike Holzgrabe

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  1. Raphael Kühnreich
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Correspondence to Ulrike Holzgrabe.

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Kühnreich, R., Holzgrabe, U. Indirect Enantioseparation of Amino Acids by CE Using Automated In-Capillary Derivatization with ortho-Phthalaldehyde and N-Acetyl-l-Cysteine. Chromatographia 79, 1013–1022 (2016). https://doi.org/10.1007/s10337-016-3122-0

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  • DOI: https://doi.org/10.1007/s10337-016-3122-0

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