6-aminoquinolyl-n-hydroxysuccinimidyl-carbamate and Colorectal-Neoplasms

The relationship between amine submetabolome and cancer has been increasingly investigated. However, no study was performed to evaluate the current methods of amine submetabolomics comprehensively, or to use such quantification results to provide an applicable approach for cancer screening. In this study, a highly sensitive and practical workflow for quantifying amine submetabolome, which was based on 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC)-labeled-HPLC-MS/MS analysis combined with multiple statistical data processing approach, was established and optimized. Comparison and optimization of two analytical approaches, HILIC separation and precolumn derivatization, and three types of surrogate matrices of plasma were performed systematically. The detection sensitivities of AQC-labeled amines were increased by 50-1000-fold compared with the underivatization-HILIC method. Surrogate matrix was also used to verify the method after a large dilution factor was employed. In data analysis, the specific amino-index for each cancer sample was identified and validated by univariate receiver operating characteristic (ROC) curve analysis, partial least-squares discrimination analysis (PLS-DA), and multivariate ROC curve analysis. These amino indexes were innovatively quantified by multiplying the raised markers and dividing the reduced markers. As a result, the numerical intervals of amino indexes for healthy volunteers and cancer patients were provided, and their clinical value was also improved. Finally, the integrated workflow successfully differentiated the value of the amino index for plasma of lung, breast, colorectal, and gastric cancer samples from controls and among different types of cancer. Furthermore, it was also used to evaluate therapeutic effects. Taken together, the developed methodology, which was characterized by high sensitivity, high throughput, and high practicality, is suitable for amine submetabolomics in studying cancer biomarkers and could also be applied in many other clinical and epidemiological research.

Topics: Amines; Aminoquinolines; Biomarkers, Tumor; Breast Neoplasms; Carbamates; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Humans; Lung Neoplasms; Molecular Structure; Tandem Mass Spectrometry

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.

Topics: Acetylation; Aminoquinolines; Animals; Carbamates; Chromatography, High Pressure Liquid; Colorectal Neoplasms; Drug Stability; Evaluation Studies as Topic; Fluorescein; Fluoresceins; Humans; Indicators and Reagents; Magnetic Resonance Spectroscopy; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Polyamines; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence; Transplantation, Heterologous; Tumor Cells, Cultured