HIGH-RESOLUTION REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHYANALYSIS OF POLYAMINES AND THEIR MONOACETYL CONJUGATES BY FLUORESCENCE DETECTION AFTER DERIVATIZATION WITH N-HYDROXYSUCCINIMIDYL 6-QUINOLINYL CARBAMATE

A highly sensitive, accurate, and reproducible HPLC method for the det ermination of all natural polyamines and their monoacetyl conjugates i s described. The presented method is based on precolumn derivatization with N-hydroxysuccinimidyl 6-quinolinyl carbamate (HSQC) followed by C-18-HPLC separation using a ternary gradient and fluorescence detecti on (lambda(Ex) = 248 nm, lambda(Em) = 398 nm). The derivatives of the four main polyamines (putrescine, cadaverine, spermidine, and spermine ) and the internal standard were synthesized on a preparative scale an d characterized, especially with respect to their molar absorptivities and fluorescence quantum yields. The limits of detection of the highl y stable derivatives ranged from 30 to 130 fmol (injection volume 10 m u l) for putrescine and N-acetylcadaverine, respectively (signal to no ise ratio = 10), with excellent Linearity within the range hom 1 to 10 0 mu M. High reproducibility for both retention times and peak areas, with coefficients of variation ranging from 0.14 to 0.88% and from 1.8 3 to 3.80%, respectively, were achieved. Provided that deproteinizatio n of the samples was carried out immediately, recoveries of the analyt es from homogenates of pancreatic cancer xenografts were high and repr oducible. The optimized method was applied to the determination of the polyamine con tent of cultured pancreatic cancer cells and of tissue from colorectal adenocarcinoma, proving precise and reproducible quant ification in these complex biological matrices. (C) 1997 Academic Pres s.