High-resolution reversed-phase high-performance liquid chromatography analysis 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 determination of all natural polyamines and their monoacetyl conjugates is described. The presented method is based on precolumn derivatization with N-hydroxysuccinimidyl 6-quinolinyl carbamate (HSQC) followed by C18-HPLC separation using a ternary gradient and fluorescence detection (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 and characterized, especially with respect to their molar absorptivities and fluorescence quantum yields. The limits of detection of the highly stable derivatives ranged from 30 to 130 fmol (injection volume 10 microliters) for putrescine and N-acetylcadaverine, respectively (signal to noise ratio = 10), with excellent linearity within the range from 1 to 100 microM. High reproducibility for both retention times and peak areas, with coefficients of variation ranging from 0.14 to 0.88% and from 1.83 to 3.80%, respectively, were achieved. Provided that deproteinization of the samples was carried out immediately, recoveries of the analytes from homogenates of pancreatic cancer xenografts were high and reproducible. The optimized method was applied to the determination of the polyamine content of cultured pancreatic cancer cells and of tissue from colorectal adenocarcinoma, proving precise and reproducible quantification in these complex biological matrices.
Weiss
T
T
Department of Surgery, University of Regensburg, Germany. Thomas.Weiss@klinik.uni-regensburg.de
Bernhardt
G
G
Buschauer
A
A
Jauch
K W
KW
Zirngibl
H
H
D000107:Acetylation; D000634:Aminoquinolines; D000818:Animals; D002219:Carbamates; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000706:statistics & numerical data; D015179:Colorectal Neoplasms / Q000737:chemistry; D004355:Drug Stability; D005069:Evaluation Studies as Topic; D019793:Fluorescein; D005452:Fluoresceins; D006801:Humans; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D010190:Pancreatic Neoplasms / Q000737:chemistry; D011073:Polyamines / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
C078178:6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; D000634:Aminoquinolines; D002219:Carbamates; D005452:Fluoresceins; D007202:Indicators and Reagents; D011073:Polyamines; D019793:Fluorescein