Development of a method for the determination of 8-iso-PGF2α in sheep and goat plasma using solid-phase microextraction and ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE

Isoprostane 8-iso-PGF2α is a biomarker of lipid peroxidation in cell membranes. The method developed to measure plasma total levels (esterified + free) of 8-iso-PGF2α must be reproducible and be able to reduce the use of solvents in solid phase extraction. It should be useful to evaluate oxidative stress due to the excess of free radicals that are generated by some disorder or disease.

METHODS

The method was developed using solid-phase microextraction with Oasis®MAX μElution plates and ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). Electrospray ionization was performed in the negative mode (ESI-); the multiple reaction monitoring mode (MRM) was used. The development of the method included the optimization of the chromatographic conditions to achieve the separation of PGF2α and 8-iso-PGF2α as well as the optimization of the microextraction conditions of the analyte of interest in ovine and goat plasma.

RESULTS

The developed method was validated with a calibration curve of plasma samples fortified with standards at five concentration levels in the range 49-639 pg/mL. The average recovery was 89% with a standard deviation of 10.73%. The inter-day precision was evaluated, obtaining a coefficient of variance (CV) less than 15%. The limit of quantification was 20 pg/mL and the limit of detection was 10 pg/mL. 8-iso-PGF2a was determined in the plasma of 14 sheep and 20 goats of 5 months of age and 6 goats of 24 months of age. The concentrations found were 50-300 pg/mL.

CONCLUSIONS

The method developed is precise, accurate and reliable with low reagent consumption compared with conventional solid-phase extraction. The analysis time was decreased because, with the use of the microextraction plate, the step of the evaporation and reconstitution of the analyte was avoided. The method is applicable to quantify the plasma total levels (esterified + free) of 8-iso-PGF2α.