Improved high-performance liquid chromatographic determination of guanidino compounds by pre-column dervatization with ninhydrin and fluorescence detection

• Published in: Journal of separation science Vol. 27; no. 15-16; pp. 1309 - 1312
• Main Authors: Buchberger, Wolfgang, Ferdig, Matthias
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.11.2004; WILEY‐VCH Verlag; Wiley
• Subjects: Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Derivatization; Exact sciences and technology; Guanidino compounds; HPLC; Ninhydrin; Other chromatographic methods; Human; Biological fluid; Fluorescence detector; Chemical analysis; Ninhydrin; Precolumn; HPLC chromatography; Guanidines; Guanidino compounds; Derivatization; Blood; α-Aminoacid; Reversed phase chromatography; Arginine; Fluorescent labelling; Clinical biology; HPLC; Quantitative analysis
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.200401866

Ninhydrin has been investigated as a pre‐column derivatization reagent for guanidino compounds. The reaction takes place under strongly alkaline conditions, followed by a second step at low pH and elevated temperature. This procedure yields derivatives with favourable fluorescence properties (excitation at 390 nm, emission at 470 nm). Amino acids do not react with ninhydrin under these conditions so that the method can easily be used for biological samples. Reversed‐phase HPLC separations of the derivatives of several representative guanidino compounds in human blood have been achieved with gradients consisting of aqueous formic acid and methanol. Fluorescence detection yields quantification limits of about 20 μg L–1. Hyphenation with electrospray mass spectrometry has been used to confirm the identity of the derivatives.