Development of a Fast and Simple LC-MS/MS Method for Measuring the F(2)- Isoprostanes in newborns

BACKGROUND: Quantification of F(2)-IsoPs isomer has been regarded as the "gold standard" to assess oxidative stress status in various adult and neonatal human diseases. These methods require high amounts of plasma. OBJECTIVE: To develop a fast and simple LC-MS/MS method for measuring F(2)-isoprostanes in newborns. METHODS: A sample of heel blood (0.4 mL) was collected in a tube containing EDTA was collected from 20 term healthy newborns. For measurements, the tandem mass spectrometer has been run in multiple reaction monitoring (MRM) with the electrospray source operating in negative ion mode, and by exploiting the transitions m/z 353.3 > 193.2 for F2-IsoPs and 357.3 > 197.2 for the internal standard d4-8-iso PGF2α. RESULTS: The concentration of F(2)-IsoPs (in pg/mL) in the collected cord bloods was 60.50 ± 25.04 (mean ± S.D.). No statistical difference was found between male (57.09 ± 19.69) and female (64.67 ± 31.13) concentrations. The overall efficiency of the extraction has been over 80%, while the recovery on spiked samples has been around 94% for spikes of 100 pg/mL with a C.V. of 7.7%. CONCLUSIONS: We developed a suitable method for large-scale studies with a reduced sample requirement as it is mandatory in neonatal age. Small samples and quick answers are very useful in Neonatology allowing early diagnosis and preventive treatments' strategies of free radical related diseases of the newborn.