Tailored 96-well μElution solid-phase extraction combined with UFLC-MS/MS: a significantly improved approach for determination of free 3-nitrotyrosine in human urine.

We developed and validated a simple and fast UFLC-MS/MS method for the accurate determination of 3-nitrotyrosine (3-NT) in human urine as a noninvasive biomarker for oxidative stress. The method, involving tailored 96-well μElution solid-phase extraction (SPE) combined with UFLC-MS/MS, allows 3-NT to be determined in biological samples without the need for hydrolysis, derivatization, evaporation, and two-dimensional LC for the first time. Using ammonium acetate (pH 9, 25 mM) as an elution buffer was found to improve SPE selectivity. Fast chromatographic elution of 3-NT with a total run time of 7 min was achieved on a PFPP column (150 mm × 2.1 mm, 3 μm). This fine-tuned integrated method delivered significantly improved throughput, specificity, and sensitivity while reducing the matrix effect, solvent usage, and waste disposal. Using this simple and rapid method, two plates of urine samples (n = 192) can be processed within 24 h. The lower limit of quantification for 3-NT is 10 pg/mL, which represents a notable sensitivity enhancement over reported methods. Less than 6.0 % variations for intraday and interday assay precisions and 97.7-106.3% for accuracies in terms of recovery were obtained. The applicability and reliability of the method were demonstrated by determining the reference range in human urine for 82 healthy people. Considering the noninvasive and inexpensive nature of urine sampling, this novel method could be used to re-evaluate the role of 3-NT as an oxidative stress biomarker in pre-clinical and clinical studies.