Quantitative determination of 8-isoprostaglandin F2α in human urine using microfluidic chip-based nano-liquid chromatography with on-chip sample enrichment and tandem mass spectrometry

Urinary 8-isoprostaglandin F2α (8-isoPGF2α) has been reported as an important biomarker to indicate the oxidative stress status in vivo. In order to quantitatively determine the low contents of 8-isoPGF2α (in sub- to low ng mL−1 range) in physiological fluids, a sensitive detection method has become an important issue. In this study, we employed a microfluidic chip-based nano liquid chromatography (chip-nanoLC) with on-chip sample enrichment coupled to triple quadrupole mass spectrometer (QqQ-MS) for the quantitative determination of 8-isoPGF2α in human urine. This chip-nanoLC unit integrates a microfluidic switch, a chip column design having a pre-column (enrichment column) for sample enrichment prior to an analytical column for separation, as well as a nanospray emitter on a single polyimide chip. The introduction of enrichment column offers the advantages of online sample pre-concentration and reducing matrix influence on MS detection to improve sensitivity. In this study, the chip-nanoLC consisting of Zorbax 300A SB-C18 columns and Agilent QqQ Mass spectrometer were used for determining 8-isoPGF2α in human urine. Gradient elution was employed for effective LC separation and multiple reaction monitoring (MRM) was utilized for the quantitative determination of 8-isoPGF2α (m/z 353 → 193). We employed liquid–liquid extraction (LLE)/solid-phase extraction (SPE) for extracting analyte and reducing matrix effect from urine sample prior to chip-nanoLC/QqQ-MS analysis for determining urinary 8-isoPGF2α. Good recoveries were found to be in the range of 83.0–85.3%. The linear range was 0.01–2 ng mL−1 for urinary 8-isoPGF2α. In addition, the proposed method showed good precision and accuracy for 8-isoPGF2α spiked synthetic urine samples. Intra-day and inter-day precisions were 1.8–5.0% and 4.3–5.8%, respectively. The method accuracy for intra-day and inter-day assays ranged from 99.3 to 99.9% and 99.4 to 99.7%, respectively. Due to its rapidity, enhanced sensitivity, and high recovery, this chip-nanoLC/QqQ-MS system was successfully utilized to determine the physiological biomarkers such as 8-isoPGF2α in human urine for clinical diagnosis.