In line filter

The assay used d₉-betaine chloride, d₉-choline chloride, and d₆-dimethylglycine HCl as internal standards. EDTA plasma samples (100 µL) were diluted using 300 µL internal standard mix dissolved in acetonitrile. After precipitation of the proteins, the samples were centrifuged for 5 min at 10,000× g at room temperature and the supernatant was transferred to glass vials. Sealed vials were immediately measured using an Acquity Ultra Performance LC system coupled to a MicroMass Quattro Premier XE tandem quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA). The analytes were separated on an Acquity UPLC BEH HILIC column (100 mm × 2.1 mm (i.d.); 1.7 µm particle size) with an Acquity HILIC VanGuard pre-column (5 mm × 2.1 mm (i.d.); 1.7 µm particle size) and a 0.2 µm in-line filter (Waters Corporation). The column temperature was 30 °C and the flow rate was 0.6 mL/min. The separation was performed using ammonium formate (solvent A) and acetonitrile (solvent B) as described in detail [29 (link)]. The interassay CVs were ≤7% for betaine and choline, and 9.0% for dimethylglycine. The measurements of choline, betaine and dimethylglycine were performed at the Central Laboratory of the University Hospital of the Saarland, Germany.

The frozen urine samples were thawed, mixed, and heated to 37°C for 5 min and then centrifuged at 10,000 g for 5 min. The content of the oxidized nucleosides 8-oxodG and 8-oxoGuo was quantified using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) assay. On an Acquity UPLC system (Waters, Milford, MA), chromatographic separation was performed with an Acquity UPLC BEH Shield RP18 column (1.7 μm, 2.1 × 100 mm²; Waters Corp.), which was protected with an in-line filter (4 × 2 mm², 0.2 μm; Waters Corp.). Column temperature was 4°C. The MS detection of the nucleosides 8-oxodG and 8-oxoGuo was performed on an API 3,000 triple quadrupole mass spectrometer (Sciex, Toronto, Canada) equipped with an ESI ion source (Turbospray) operated in positive mode. For a detailed description, see elsewhere [31 (link)]. The creatinine concentrations in the urine were measured to divide the 8-oxodG and 8-oxoGuo levels by creatinine levels according to Jaffe’s reaction [32 (link)]. All laboratory technicians were blinded to participant diagnoses. The analysis was done at the Laboratory of Clinical Pharmacology, Rigshospitalet, Copenhagen, Denmark.