A Sensitive LC-MS/MS Method for the Quantification of Serum Epoxyeicosatrienoic and Dihydroxyeicosatrienoic Acids in the Identification of Diabetic Kidney Disease

Epoxyeicosatrienoic acids (EETs) are vasoactive eicosanoids with vasodilatory, anti-inflammatory, and nephroprotective properties, and metabolized to low-bioactive dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. Evidence from animal studies suggests that EETs may be potential biomarkers for diabetic kidney disease (DKD). The aim of this study was to establish a simple, sensitive and accurate LC-MS/MS method for quantifying EETs and DHETs in human serum. Samples were prepared by solid phase extraction and quantified using a "one-to-one" isotope internal standard approach. The assay required a 9-min run time per sample. EETs and DHETs demonstrated good linearity over the investigated concentration range (r > 0.99). The limits of quantification were 0.01 ng/mL, with accuracies ranging from 85.62 to 110.95%, intra- and inter-day variations of less than ± 15%, and matrix effects of less than ± 10%. However, 5,6-EET showed poor performance due to chemical instability and low response peaks. We successfully applied this method to rapidly analyze serum samples from 54 patients with diabetes and DKD. Levels of EETs and DHETs were downregulated in DKD patients compared to diabetic patients, and these eicosanoids showed significant negative correlations with proteinuria. This study presented a sensitive and robust LC-MS/MS method for monitoring low levels of EETs and DHETs in human serum and showed the potential for its application in the diagnosis and staging of DKD.