2.7. Pharmacokinetics
Putrescine(1,4-diaminobutan) dihydrochloride and spermidine trihydrochloride were obtained from Acros Organics (Geel, Belgium), and spermine tetrahydrochloride was purchased from Alfa Aesar (Haverhill, MA, USA). Putrescine-d4(1,4-diaminobutan-2,2,3,3-d4) dihydrochloride, spermidine-(butyl-d8) trihydrochloride, The determination of the concentrations of spermidine, spermine, and putrescine in plasma and saliva was performed by protein precipitation following derivatization with Fmoc-OSu based on the method of Xiong and Zhai [Stock solutions, quality control spiking solutions, and internal standard solutions were prepared in methanol-water (20:80, A detailed overview of the sample preparation can be found in The LC system was equipped with a Phenomenex KinetexThe retention times of spermidine, spermine, and putrescine were 5.60, 6.90, and 3.65 min, respectively. To prevent contamination of the ionization unit, the eluent was initially directed to a waste bottle. For the analysis of plasma samples, after 3.4 min, the valve unit changed the direction of the flow to the mass spectrometer, and after 7.4 min, the valve switched back to its initial position. For the determination of putrescine in saliva, the valve switched the flow to the MS after 3.4 min and back after 4.6 min, while for the measurement of spermidine and spermine in saliva, the eluent was directed to the MS from 5.1 to 7.4 min.The detection of analytes was performed in the positive multiple reaction monitoring (MRM) mode (Data acquisition and analysis were performed using LabSolutions (Version 5.97 SP1). The calibration curves consisted of eight calibration standards and were constructed by plotting peak area ratios of the analytes and internal standards against the concentration of the analytes. A 1/cThe method was validated with respect to intra- and inter-day accuracy and precision at three concentration levels (QC-L, QC-M, and QC-H) in six replicates on the same day and 18 replicates over 2 days, respectively. Stability (freeze-thaw stability, long-term stability [6 weeks at −80 °C], and short-term stability [3 h at room temperature]) was evaluated at low (QC-L) and high (QC-H) quality control concentrations in four replicates. Furthermore, reinjection stability was investigated at three concentration levels (QC-L, QC-M, and QC-H) in six replicates after storage for 24 h in the autosampler. Plasma and saliva quality control standards were prepared by spiking QC working solutions (comprising 5% of the total volume) into the corresponding matrix. As no analyte-free plasma or saliva were available, the parameters of validation were evaluated by subtracting the concentration of nonspiked samples from spiked matrix samples. The matrix effect was evaluated by spiking the plasma and saliva of six individuals at low (QC-L) and high (QC-H) quality control concentrations and calculating the coefficient of variation (CV) of the measured concentration. As presented in Moreover, the stability investigation (data not presented) proved that the unprocessed plasma samples were stable for two freeze-thaw cycles, while unprocessed saliva samples were stable for three cycles. Unprocessed plasma and saliva samples were stable for a period of 6 weeks at −80 °C and for 3 h at room temperature. The reinjection of plasma and saliva samples after storage for 24 h in the autosampler revealed that the final extract was stable. The CV of the measured concentration of the six spiked individual plasma and saliva samples was below 15% at all investigated concentration levels (QC-L and QC-H).