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Determination of putrescine, cadaverine, spermidine and spermine in different chemical matrices by high performance liquid chromatography–electrospray ionization–ion trap tandem mass spectrometry (HPLC–ESI–ITMS/MS)

Ibarra, Alan Alexander González, Wrobel, Katarzyna, Escobosa, Alma Rosa Corrales, Elguera, Julio Cesar Torres, Garay-Sevilla, Ma Eugenia, Wrobel, Kazimierz
Journal of Chromatography B 2015 v.1002 pp. 176-184
1,6-hexanediamine, Lepidium sativum, biomarkers, cadaverine, cadmium, chromatography, derivatization, detection limit, diabetes, formic acid, humans, ions, methanol, methylene chloride, monitoring, patients, plant extracts, putrescine, seedlings, spermidine, spermine, t-test, tandem mass spectrometry, urine
The goal of this work was to establish a simple HPLC–ESI–ITMS/MS procedure, suitable for the determination of four common aliphatic polyamines in two different types of biological matrices. To this end, 1,6-diaminohexane was used as the internal standard (IS) and 4-fluoro-3-nitrobenzenotrifluoride (FNBT) as the derivatizing agent. Formation of fully derivatized compounds was confirmed by high resolution ESI–QTOFMS and MS/MS analysis. Reversed phase chromatographic separation was carried out by gradient elution with 0.1% (v/v) formic acid and methanol. In a positive ESI mode, the following pairs of precursor/quantifier ions were used for multiple reaction monitoring: 467.4/261.0 for PUT, 481.2/461.1 for CAD, 713.7/261.0 for SPD, 959.8/507.2 for SPM and 495.3/475.2 for IS. On-column instrumental detection limits of four polyamines were in the range 0.62–2.14fmol (0.039–0.215ng/ml). Versatility was demonstrated by analyzing plant extracts and human urine; prior to derivatization, all samples were cleaned-up by dichloromethane extraction. The evaluated signal suppression/enhancement was in the range 82.3–115.4% and the percentage recoveries obtained in the method of standard addition were in the range 83.7–114.4%. Statistically significant differences in polyamines concentrations were found in garden cress exposed to Cd(II) versus control seedlings (t-test, p<0.05); results obtained for urine from healthy volunteers and diabetic patients at different clinical conditions suggested possible utility of free polyamines as biomarkers of progressive diabetes.