Use of microextraction by packed sorbents and gas chromatography-mass spectrometry for the determination of polyamines and related compounds in urine

摘要

A novel methodology for the determination of ornithine, putrescine, cadaverine, spermidine and gamma amino butyric acid in urine samples has been developed. The method uses in situ aqueous derivatization followed by automated microextraction by packed sorbent coupled to a gas chromatography mass spectrometry system equipped with a programmed temperature vaporizer. This instrumental configuration minimizes sample manipulation due to from the mixing of the reagents, the process is completely automated. The analytes were derivatized using ethyl chloroformate as derivatization reagent. The reaction occurred in aqueous medium and was carried out in 1 min in the vial of an autosampler used to perform microextraction by packed sorbent. The parameters affecting derivatization, extraction and separation were optimized in order to obtain maximum sensitivity. Calibration curves were obtained for five calibration levels in three different matrices. All the calibration models displayed good linearity, with R-2 values higher than 0.95. The validity of the models was checked using ANOVA, and it was observed that they did not exhibit any lack of fit. Repeatability and reproducibility was evaluated, with values below 15% in both cases. LOD and LOQ values were found to be in the low mu g/L level. Influence of the matrix was confirmed, thus quantification was performed using the standard additions method and normalization to IS. The method developed was applied to the analysis of these compounds in urine samples from healthy individuals and cancer diagnosed patients (Internal Medicine Unit of the Virgen de la Vega Hospital, Salamanca, Spain). Significant differences (Mann-Whitney U test) were observed for putrescine and ornithine concentrations. (C) 2016 Elsevier B.V. All rights reserved.