دانلود رایگان مقاله پروتکل عملکرد استاندارد برای تحلیل لیپید هیدروپروکسید در خونابه انسان

abstract

Standard operating procedures (SOPs) are of paramount importance in the analytical field to ensure the reproducibility of the results obtained among laboratories. SOPs gain special interest when the aim is the analysis of potentially unstable compounds. An SOP for analysis of lipid hydroperoxides (HpETEs) is here reported after optimization of the critical steps to be considered in their analysis in human serum from sampling to final analysis. The method is based on automated hyphenation between solid-phase extraction (SPE) and liquid chromatography–mass spectrometry (LC–MS). The developed research involves: (i) optimization of the SPE and LC–MS steps with a proper synchronization; (ii) validation of the method—viz. accuracy study (estimated as 86.4% as minimum value), evaluation of sensitivity and precision, which ranged from 2.5 to 7.0 ng/mL (0.25–0.70 ng on column) as quantification limit and precision below 13.2%), and robustness study (reusability of the cartridge for 5 times without affecting the accuracy and precision of the method); (iii) stability study, involving freeze–thaw stability, short-term and long-term stability and stock solution stability tests. The results thus obtained allow minimizing both random and systematic variation of the metabolic profiles of the target compounds by correct application of the established protocol.

4. Conclusions

A standard operation protocol for targeting analysis of hydroperoxy eicosatetraenoic and octadecadienoic acids in human serum has been developed by automated solid-phase extraction and liquid chromatography–mass spectrometry in selected reaction monitoring. A programmed validation study implemented robustness and stability tests for proper analysis of these metabolites of clinical relevance. After reception, the samples could be stored at −80 ◦C for 9 days with not statistically significant losses of the target lipid hydroperoxides.