Gemini nx

The method used to measure the concentrations of 8-OHdG, 8-isoPGF_2α and HNE-MA in urine by liquid chromatography–tandem mass spectrometry (LC–MS/MS), with some modifications, has been described in detail previously (28 (link)). The procedure and QC standard were similar to that used in a previous TCS assay. First, the sample was centrifuged at 10,000 rpm for 10 min and the supernatant (100 μl) was diluted with deionized water (1,500 μl). Second, the internal standard (50 μl) was added to the diluted sample for solid-phase extraction, the eluate was evaporated to dryness, and then redissolved in 5% methanol/water (200 μl). The target compounds were separated by high-performance liquid chromatography (HPLC) on a C18 column (3 μm, 100 × 2 mm Phenomenex Gemini-NX) and identified using an Agilent 6,460 triple quadrupole mass spectrometer. To assess the reliability of the analytical method, two quality controls and one blank sample from each batch were simultaneously measured. The results showed that the recoveries of the target compounds were in the range of 87.4–102.0%, and the intra-batch and inter-batch precision are 6.91% and 8.36%. The limits of qualification (LOQs) for 8-OHdG, 8-isoPGF_2α and HNE-MA were 0.08, 0.06 and 0.03 ng/ml, respectively.

NMR spectra were analyzed on a Bruker 600 MHz Avance III HD spectrometer (Bruker, Fällande, Switzerland) equipped with a triple resonance probe at 298 K, using TMS (trimethylsilane) as an internal standard. ESI-MS data were taken on an Agilent 6210 LC-MS spectrometer (Agilent Technologies, Santa Clara, CA, USA). Purification of all compounds was performed on Waters D600 apparatus (Waters, San Diego, CA, USA), Agilent 1200 apparatus (Agilent, USA), equipped with a preparative column (Phenomenex Gemini-NX, 50 × 21.2 mm), a semi-preparative column (Agilent Eclipse XDB-C18, 9.4 × 250 mm, 5 µm, USA), and an analytical column (Synergi Hydro-RP, 250 mm × 4.6 mm, 4 µm, Phenomenex, Torrance, CA, USA). The acetonitrile (Merck, Darmstadt, Germany), methanol (Sigma-Aldrich Co., St. Louis, MO, USA) and H₂O that were used in HPLC system were of chromatographic grade, and all other chemicals were analytical.