Liquid Chromatographic Determination of Ornithine and Lysine

with Krebs–Henseleit cycle and polyamine synthesis.1–3 It was reported that their high concentration levels in plasma and urine were indicative in diagnoses of some congenital metabolic disorders like cystinuria or hyperlysinemia.2–4 Thus, the selective determination of ornithine and lysine might be more valuable for the diagnoses of these disorders. An amino acids analyzer based on derivatization has been used for the determination of the two amino acids. However, the analyzer method allows the quantification of many amino acids including these two amino acids. Accordingly, the method is timeconsuming for the liquid chromatographic (LC) separation. Therefore, a simple and selective determination method for ornithine and lysine is required in biomedical and clinical fields. A large number of fluorescence derivatization reagents for amino acids in LC have been reported.5–14 Some fluorogenic Edman-type derivatization reagents for the sequencing of peptides have also been developed.15–19 All of these reagents react with almost all amino acids to form the corresponding fluorescence derivatives. They are thus useful for the simultaneous determination of many amino compounds including amino acids analysis. However, when they were used for the determination of specific amino compound(s), troublesome clean-up procedure(s) like liquid-liquid or solidphase extraction and/or elegant LC separation conditions like gradient elution or column-switching technique are needed to avoid the interferences from impurities having amino moiety and/or the excess reagents. Only a few fluorogenic derivatization methods for specific amino acid(s) have been introduced: i.e., ninhydrin,20 9,10phenanthraquinone,21 and benzoin-type reagents22,23 for arginine, o-phthalaldehyde24 in the absence of a thiol for cysteine, 1,2diamino-4,5-dimethoxybenzene25 and borate-hydroxylamineCo(II) reagent26 for tyrosine related compounds, and glyoxaltype reagents27,28 for tryptophan. These methods are so selective that they do not require complicated separations of analytes from other amino concomitants and reagent components. However, selective derivatization methods for ornithine and lysine have not been reported. In our previous research, we developed a highly selective and sensitive determination method for polyamines in LC based on intramolecular excimer-forming fluorescence derivatization.29 In this method, all the primary and secondary amino moieties in polyamines were labeled with a pyrene reagent, 4-(1pyrene)butyric acid N-hydroxysuccinimide ester (PSE), and the excited polypyrene-labeled derivative afforded an intramolecular excimer to fluoresce in the wavelength region (450 – 520 nm) longer than that for usual pyrene derivatives (360 – 420 nm). By using these characteristics, polyamines were determined highly selectively, even though the sample was contaminated with monoamino compounds. In this paper, we have reported the determination method for ornithine and lysine by using the above-mentioned intramolecular excimer-forming fluorescence derivatization. Ornithine and lysine, which have two amino moieties in each molecule, were converted to the corresponding dipyrene-labeled derivatives by PSE, and the derivatives generated excimer fluorescence from the intramolecular dipyrene sites (Fig. 1). The present method allowed highly sensitive and selective determination of ornithine and lysine. The structures of 107 ANALYTICAL SCIENCES JANUARY 2001, VOL. 17 2001 © The Japan Society for Analytical Chemistry