LUMINARIN-3 AS A DERIVATIZATION REAGENT FOR THE LIQUID-CHROMATOGRAPHIC DETERMINATION OF CYTIDINE, ADENOSINE AND RELATED NUCLEOTIDES WITH FLUOROMETRIC DETECTION

Luminarin 3 is a quinolizinocoumarine labelling reagent for carbonyl c ompounds, reacting via its hydrazide function. It was found to be a hi ghly sensitive fluorescence reactive probe for nucleosides and nucleot ides in liquid chromatography. Luminarin 3 reacts with the dialdehyde induced by preliminary periodate oxidation of the ribose moiety of nuc leosides and nucleotides in 100 mM acetate buffer (at pH 3.0 or 4.0, d epending on the derivative), to give highly fluorescent morpholino der ivatives. The luminarin 3 derivatives of cytidine (Cyd), adenosine (Ad o) and corresponding nucleotides were separated by chromatography on a Spherisorb cyano 5-mu m column (300 x 4.6 mm i.d.), with a mixture of methanol and 100 mM phosphate buffer pH 6.0 (30/70, v/v), followed by fluorescence detection. In such a way the separation can easily be op timized for the eight nucleotides by variation of the methanol content of the mobile phase. Typical detection limits (in amount injected wit h signal/noise ratio of 3) were 40 fmol for Cyd; 52 fmol for CMP; 71 f mol for CDP; 36 fmol for CTP; 667 fmol for Ado, 70 fmol for AMP, 28 fm ol for ADP and 39 fmol for ATP.