Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method |
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| Authors: | Takehiko Yokoyama Masaharu Tokuda Masafumi Amano Koji Mikami |
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| Institution: | 1. School of Marine Biosciences, Kitasato University, Sagamihara, Japan;2. National Research Institute of Aquaculture, Fisheries Research and Education Agency, Minami-ise, Japan;3. Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan |
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| Abstract: | This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids. |
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| Keywords: | d-amino acid" target="_blank">d-amino acid d-proline" target="_blank">d-proline o-phthalaldehyde (OPA) l-cysteine (NAC)" target="_blank">N-acetyl- l-cysteine (NAC) 1-(9-fluorenyl)ethyl chloroformate (FLEC) |
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