High-Throughput and Sensitive Analysis of Free and Total 8-Isoprostane in Urine with Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Oxidative stress (OS) plays a major role in the pathogenesis of various diseases in humans. OS is a result of an imbalance between reactive oxygen species (ROS) and the biologically available antioxidants that prevent or repair damage that ROS inflict on the host cells. ROS are naturally generated during normal mitochondrial respiration and by oxidative burst during the immune response. Many factors may influence OS, including genetics, diet, exercise, and exposure to environmental toxicants (e.g., tobacco smoke). A nonenzymatic peroxidation product of arachidonic acid (AA), 8-iso-PGF2α (8-isoprostane), is a validated biomarker of OS that is present in urine as both glucuronide conjugate and free acid. Previous studies report that the conjugated forms of 8-isoprostane can vary between 30 and 80% of the total 8-isoprostane levels. By hydrolyzing the conjugated forms, it is possible to obtain a total (free + conjugated) measurement of 8-isoprostane in urine samples. Here, we describe a robust, automated, and high-throughput method for measuring total urinary 8-isoprostane using a polymeric weak anion-exchange solid-phase extraction (SPE) and isotope-dilution ultrahigh performance liquid chromatography electrospray ionization–tandem mass spectrometry (UHPLC–MS/MS). This method, using a 96-well plate platform, showed good sensitivity (8.8 pg/mL LOD) and used only 400 μL of the sample volume with a cycle time of 11 min. The inter- and intraday precision, calculated from 20 repeated measurements of two quality control pools, varied from 4 to 10%. Accuracy, calculated from the recovery percentage at three spiking levels, ranged from 92.7 to 106.7%. We modified this method to allow for the exclusive measurement of free 8-isoprostane by removing the hydrolysis step. We measured both free and total 8-isoprostane in urine collected from 30 cigarette smokers (free: 460 ± 78.8 pg/mL; total: 704 ± 108 pg/mL) and 30 nonusers of tobacco products (free: 110 ± 24.2 pg/mL; total: 161 ± 38.7 pg/mL). This method is robust, accurate, and easily adaptable for large population studies.

Novel Fast Chromatography-Tandem Mass Spectrometric Quantitative Approach for the Determination of Plant-Extracted Phytosterols and Tocopherols

“…Consequently, the use of natural products as food additives is favored. Regardless of the source for the bioactives, a fast, simple, and sensitive analytical method is required for their identification and quantification [ 11 , 12 ]. Traditionally, gas chromatography (GC) with flame ionization detector (FID) [ 13 , 14 , 15 , 16 , 17 ] or mass spectrometry (MS) [ 18 , 19 , 20 , 21 ] has been utilized for the determination phytosterols and tocopherols.…”

Isoprostanes in wastewater as biomarkers of oxidative stress during COVID-19 pandemic

“… Ryu et al (2016) also reported a 20-fold increase in 8-iso-PGF2α level in wastewater after enzyme hydrolysis. The glucuronide conjugated forms of 8-isoprostane in human urine samples can vary between 30 to 80% of the total iso-prostane ( Holder et al, 2020 ; Yan et al, 2010 ). However, 2,3-dinor-iPF2α-III was not detected even after enzyme hydrolysis.…”

A Novel UHPLC-MS/MS Method for Measuring 8-iso-Prostaglandin F2α in Bronchoalveolar Lavage Fluid

“…Our previous assay of 8-isoprostane in urine resulted in recoveries ranging from 92.7 to 106.7% with a mean recovery of 99.7%, indicating that the results from our BAL fluid assay are consistent from analysis of a different physiological matrix. ( Holder et al, 2020 ) We compared the ISTD responses between unextracted calibration samples and extracted samples to evaluate sample recovery and calculated an average recovery of 55% for all extracted samples.…”

“…The LC-MS/MS instrument parameters were kept the same as our previously published CLIA urinary assay. ( Holder et al, 2020 ) In brief, chromatographic separation was achieved using a Waters ACQUITY reversed-phase column (150 mm × 2.1 mm, particle size 1.8 μm, C18) and a Waters ACQUITY reversed-phase pre-column (5 mm × 1 mm, particle size 1.7 μm, C18) (Milford, MA, United States) on an ultra high performance liquid chromatographic system from Shimadzu Corp. (Columbia, MD, United States) A gradient program was performed with a combination of 0.15% formic acid in water (mobile phase A) and acetonitrile in 0.15% formic acid in water (v/v 1:1) (mobile phase B). The combined chromatographic flow rate was 0.65 ml/min, and acetonitrile was infused, post-column, at 0.15 ml/min.…”