ABSTRACT
Objective: To establish an ultra performance liquid chromatography(UPLC)method for determination of prot- amine peptide analogul(R15),and investigate the metabolism in biological samples of R15 in vitro by the method. Methods: Chro- matographic separation was performed by pumping the mobile phase into a BioBasic C 18 (2.1 mm×100 mm,5 μm). The mobile phase was composed of 0.05% trifluoroacetic acid-water and 0.05% trifluoroacetic acid acetonitrile(98:2,V/V),using 10% trifluoroacetic ac- id-acetonitrile-water as precipitant. The stability of R15 in rat whole blood,liver and kidney homogenate was studied. Results: The in- tra-and inter-batch relative standard deviations (RSD)were less than 15%,the relative errors (RE)were in the range of ±15%. The range of calibration curve was 25-1000 μg/ml with a good correlation coefficient. The extraction recoveries of R15 were 99.43-100.43%. R15 was stable under various experimental storage conditions. Half-lives of R15 in rat liver,rat kidneys,rat blood,beagle dog blood and human blood were 27.4 min,9.1 min,15.6 min,11.9 min and 21.0 min,respectively. Compared with the control group without inhibi- tor,the inhibitory rates of EDTA,pepstatin and benzamidine HCl protease inhibitor in the experimental group was 92.7%,41.3% and 33.0%,respectively. Protamine analogue peptide was mainly metabolized by metal carboxyl peptidases. Compared with the control group,AEBSF,leupeptin and trypsin protease inhibitors had significant difference,but the inhibitory rate was low. Conclusion: The method is specific,sensitive,accurate and reliable. The metabolism of R15 in rat kidney is the fastest. The degradation of R15 in whole blood is possibly related to metal carboxypeptidase.
ABSTRACT
The effects of storage temperatures, repeated freeze-thaw cycles, or delays in separating plasma or serum from blood samples are largely unknown for heat shock protein 27 (HSP27). We evaluated (1) the imprecision of the HSP27 assay used in this study; (2) the in vitro stability of HSP27 in blood samples stored at 4℃ for up to 6 hr with immediate and delayed serum/plasma separation from cells; and (3) the in vitro stability of HSP27 in blood samples stored at -80℃ after repeated freeze-thaw cycles. The ELISA to detect HSP27 in this study showed a within-run CV of <9% and a total CV of <15%. After 4-6 hr of storage at 4℃, HSP27 concentrations remained stable when using serum tubes irrespective of sample handling, but HSP27 concentrations decreased by 25-45% when using EDTA plasma tubes. Compared with baseline HSP27, one freeze-thaw cycle had no effect on serum concentrations. However, plasma concentrations increased by 3.1-fold after one freeze-thaw cycle and by 7.3-fold after five freeze-thaw cycles. In conclusion, serum is an appropriate biological sample type for use in epidemiological and large-scale clinical studies.