An Efficient Sample Preparation Method for High-Throughput Analysis of 15(S)-8-iso-PGF2 in Plasma and Urine by Enzyme Immunoassay

Although several methods have been reported on the analysis of the oxidative stress marker 15(S)-8-iso-prostaglandin-F2alpha (8-iso-PGF2) in biological fluids, they either involve extensive sample preparation and costly technology or require high sample volume. This study presents a sample preparation method that utilizes low sample volume for 8-iso-PGF2 analysis in plasma and urine by an enzyme immunoassay (EIA). In brief, 8-iso-PGF2 in deproteinized plasma or native urine sample is complexed with an antibody and then captured by molecular weight cut-off filtration. This method was compared with two other sample preparation methods that are typically used in the analysis of 8-iso-PGF2 by EIA: Caymans affinity column purification method and solid-phase extraction on C-18. The immunoaffinity purification method described here was superior to the other two sample preparation methods and yielded recovery values of 99.8 and 54.1 for 8-iso-PGF2 in plasma and urine, respectively. Analytical precision (relative standard deviation) was 5 for plasma and 15 for urine. The analysis of healthy human plasma and urine resulted in basal 8-iso-PGF2 levels of 31.8 5.5 pg/mL and 2.9 2.0 ng/mg creatinine, respectively. The robustness and analytical performance of this method makes it a promising tool for high-throughput screening of biological samples for 8-iso-PGF2.