Analysis of urinary 8-isoprostane as an oxidative stress biomarker by stable isotope dilution using automated online in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry

•Online in-tube solid-phase microextraction (SPME) of 8-isoprostane (8-IP) was optimized.•8-IP was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS).•In-tube SPME coupled with LC–MS/MS method is automated, simple, rapid, selective and sensitive.•8-IP could be directly and quantitatively analyzed from human urine sample without any other pre-treatment.•This method is a useful tool for assessing oxidative stress and the effects of antioxidant intake.

We have developed a simple and sensitive method for the determination of the oxidative stress biomarker 8-isoprostane (8-IP) in human urine by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a Zorbax Eclipse XDB-8 column and 0.1% formic acid/methanol (25/75, v/v) as a mobile phase. Electrospray MS/MS for 8-IP was performed on an API 4000 triple quadruple mass spectrometer in negative ion mode. The optimum in-tube SPME conditions were 20 draw/eject cycles with a sample size of 40 μL using a Carboxen 1006 PLOT capillary column for the extraction. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Total analysis time of this method including online extraction and analysis was about 30 min for each sample. The in-tube SPME LC–MS/MS method showed good linearity in the concentration range of 20–1000 pg/mL with a correlation coefficient r = 0.9999 for 8-IP using a stable isotope-labeled internal standard, 8-IP-d4. The detection limit of 8-IP was 3.3 pg/mL and the proposed method showed 42-fold higher sensitivity than the direct injection method. The intra-day and inter-day precisions (relative standard deviations) were below 5.0% and 8.5% (n = 5), respectively. This method was applied successfully to the analysis of urine samples without pretreatment or interference peaks. The recovery rates of 8-IP spiked into urine samples were above 92%. This method is useful for assessing the effects of oxidative stress and antioxidant intake.

Graphical abstractOxidative stress biomarker 8-isoprostane in human urine can be analyzed by automated online in-tube SPME LC–MS/MS without any other pre-treatment.Figure optionsDownload full-size imageDownload as PowerPoint slide