Background: Reactive oxygen species (ROS) are produced in the body during normal metabolism by means of enzymes and non-enzymatic chemical reduction of molecular oxygen. In case of the prevalence of ROS formation over their elimination, highly reactive free radicals can be accumulated and can cause multiple damages to the biomolecules and cells. Determination of isoprostanes in biological matrices is most often used to register free radical damage and requires selective, sensitive and specific techniques.
Methods: This study presents the development and validation of the LC-MS/MS method for the determination of 8-iso-Prostaglandin F2α in human plasma utilising a modified liquid-liquid extraction procedure with phase separation.
Results: Modified sample preparation procedure assured higher extraction yield, clear separation of organic layer from the plasma water phase and protein precipitates, and better-purified product for instrumental analysis. Linearity was validated in the range 0.1-5.0 µg/L with R2 > 0.996; normalised matrix varied between 86.0% and 108.3%, accuracy ranged from 90.4 % to 113.9% and precision both within runs and between runs was less than 7%. With a run time of 10 min, a throughput of over 50 samples per working day could be performed.
Conclusions: The method meets all the current industrial validation criteria and allows the accurate and precise determination of 8-iso-PGF2α in human plasma at diagnostically significant concentration range.
Uvod: Reaktivne vrste kiseonika (ROS) nastaju u telu tokom normalnog metabolizma pomoću enzima i neenzimske hemijske redukcije molekularnog kiseonika. U slučaju bržeg formiranja ROS od njihovog eliminisanja, mogu se akumulirati visoko reaktivni slobodni radikali i mogu izazvati višestruka oštećenja biomolekula i ćelija. Određivanje izoprostana u biološkim matricama najčešće se koristi za registrovanje oštećenja prouzrokovana slobodnim radikalima i zahteva selektivne, osetljive i posebne metode rada.
Metode: Ovo istraživanje prikazuje razvoj i validaciju LC-MS/MS metode za određivanje 8-izo-prostaglandina F2α u ljudskoj plazmi pomoću modifikovanog postupka ekstrakcije tečno-tečno sa razdvajanjem faza.
Rezultati: Modifikovana procedura pripreme uzoraka osigurala je veći učinak ekstrakcije, jasno odvajanje organskog sloja od vodene faze u plazmi i taloga proteina, i bolje pročišćen proizvod za instrumentalnu analizu. Linearnost je potvrđena u opsegu 0,1-5,0 µg/L sa R2 > 0,996; normalizovana matrica varirala je između 86,0 i 108,3%, tačnost je bila u rasponu od 90,4% i 113,9%, a preciznost i u toku i između faza bila je manja od 7%. U toku radnog dana može se izvršiti propusnost od preko 50 uzoraka sa trajanjem od 10 minuta.
Zaključak: Ova metoda ispunjava sve trenutne kriterijume industrijske validacije i omogućava tačno i precizno određivanje 8-izo-PGF2α u ljudskoj plazmi u dijagnostički značajnom rasponu koncentracije.
Keywords: 8-iso-Prostaglandin F2a; LC-MS/MS; liquid extraction with phase separation.
2021 Desislav G. Tomov, Georgeta Bocheva, Vidka Divarova, Lilia Kasabova, Dobrin Svinarov, published by CEON/CEES.