Analysis of guanidino compounds in plasma and urine by RP-HPLC with fluorescence detection

Several guanidino compounds occur in human physiological fluids and tissues. Serum and urinary guanidinosuccinic acid (GSA) levels decrease significantly in cirrhotic patients, while a extremely high plasmatic and urinary guanidinoacetic acid (GAA) concentration is the most specific abnormality in the guanidine acetate methyltransferase deficiency. Guanidine analysis is quite difficult due to their poor detectability owing to the absence of a strong chromophore and fluorophore. Pre- or postcolumn chemical derivatization in combination with fluorescence detection constitutes an effective approach to overcome the problem. Recently, we have proposed anisoin, the 4,4’-dimethoxy analogue of benzoin, as reagent of guanidines (1). On the basis of the encouraging results, to develop additional useful fluorogenic reagents, our attention was directed to furoin (I). This communication describes the application of the compound (I) as pre-column fluorogenic labelling reagent in the HPLC analysis of guanidino compounds in human plasma and urine. Furoin was found to react rapidly (5 min at 100°C) and selectively with the guanidino function to give stable and highly fluorescent adducts (II). The quantitation limit was in the range of 5-25 fmol. The RP-HPLC separations were performed at 33±2°C on a Phenomenex Synergi MAX-RP (250 x 3.0 mm i.d.) with gradient elution. The proposed method could be useful for diagnostic purpose to investigate the biological levels of guanidino compounds implicated as toxins in patients with uremia, chronic renal failure and other diseases. (1) R. Gatti, M.G. Gioia, J. Pharm. Biomed. Anal. 42 (2006) 11-16