G4212a diode array detector

The sample preparation for total toxin concentration was as follows: 100 μL of plasma was initially diluted with 260 μL of UPLC grade water (Thermo Scientific, Geel, Belgium). For heat deproteinization, the samples were placed at 95 °C for 30 min, cooled in an ice bath for 10 min, and centrifuged at 18,000× g for 10 min. The supernatant was centrifuged through a 30 kDa cutoff centrifugal filter (Amicon Ultra 0.5, Merck KGaA, Darmstadt, Germany) for 20 min at 4500× g. For the free concentration, 260 μL of untreated plasma was initially centrifuged through a 30 kDa cutoff centrifugal filter at 4500× g for 20 min, and 100 μL of the ultrafiltrate was diluted with 260 μL of UPLC-grade water followed by the same heat treatment as described above. Finally, 180 μL of the ultrafiltrate was transferred into a vial, and internal standard (fluorescein; 50 ppm) was added. UPLC (Agilent 1290 Infinity device; Agilent, Santa Clara, CA, USA) was used to separate the uremic toxins. HA and CMPF were detected with an Agilent G4212A diode array detector at 245 nm and 254 nm, respectively. Indoxyl sulfate (λex: 280 nm, λem: 376 nm), p-cresyl sulfate and p-cresyl glucuronide (λex: 264 nm, λem: 290 nm), indole-3-acetic acid (λex: 280 nm, λem: 350 nm), and fluorescein (λex: 443 nm, λem: 512 nm) were detected by an Agilent G1316C fluorescence detector.

Relative abundances of unmethylated (dC) and methylated (m4C) were determined using liquid chromatography and mass spectrometry (LC-MS). In this case DNA samples were isolated from a dcm- E.coli host strain to eliminate m5C contamination which would otherwise confound the analyses. DNA samples were hydrolyzed to nucleosides using the Nucleoside Digestion Mix (NEB). LC-MS/MS analysis was performed in duplicate by injecting digested polynucleotide samples on an Agilent 1290 UHPLC equipped with a G4212A diode array detector and a 6490A Triple Quadrupole Mass Detector operating in the positive electrospray ionization mode. UHPLC was carried out using a Waters XSelect HSS T3 XP column (2.1 × 100 mm, 2.5 μm) with the gradient mobile phase consisting of methanol and 10 mM aqueous ammonium formate (pH 4.4). Data acquisition was performed in the dynamic multiple reaction monitoring (DMRM) mode. Each nucleoside was identified in the extracted chromatogram associated with its specific MS/MS transition: dC [M+H]⁺ at m/z 228 → 112 and m4C [M+H]⁺ at m/z 242 → 126. External calibration curves with known amounts of the nucleosides were used to calculate their ratios within the samples analyzed.