Development of a fast and reliable methodology for the determination of polyamines in urine by using a guard column as a low-resolution fractioning step prior to mass spectrometry. Comparison with flow injection-mass spectrometry analysis

Abstract

In this paper, we propose a fast and reliable method for the determination of polyamines and related compounds in urine samples using flow injection analysis directly coupled to a triple quadrupole mass spectrometer, with the inclusion of a guard column (XBridge® C18 guard column; 2.1 × 10 mm, 3.5 µm) as a low-resolution fractioning step prior to MS analysis. The use of the guard column resulted in a sensitivity enhancement, a critical issue because of the low concentration of the target compounds found in urine samples. Moreover, it represents an interesting alternative to the use of other sample preparation techniques. Using this instrumental setup, each sample can be analyzed in 2.7 min, which makes this method appropriate for screening analysis. A method including a chromatographic separation step is also proposed, if confirmation of the results is required (samples with abnormal concentrations of the compounds). In this case, the total analysis time was 33.3 min. All the steps involved in sample preparation and instrumental conditions were optimized. In both methods, an ion-pairing reagent was added to the mobile phase and to the diluted sample. For the screening method, the limits of detection were found to be between 0.6 and 21 µg/L and the limits of quantification between 2.0 and 70 µg/L. Influence of the matrix was confirmed; thus quantification was performed using the one-point standard addition method and normalization to IS. Comparable results were obtained with the screening and the confirmatory methods when applied to urine samples of apparently healthy volunteers, showing that the proposed screening methodology is suitable for rapid and reliable analysis of polyamines and related compounds in these samples.