| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1212070 | Journal of Chromatography B | 2015 | 9 Pages |
•Putrescine, cadaverine, spermidine, spermine determined by HPLC–ESI–ITMS/MS.•4-fluoro-3-nitrobenzenotrifluoride used for pre-column derivatization.•DLs/QLs consistent with those reported elsewhere for more powerful instrumentation.•Versatility demonstrated by analyzing plant extracts and human 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 0.62–2.14 fmol (0.039–0.215 ng/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.
