Agilent 1290 infinity device

For the quantification of uremic toxins (UTs), venous blood was collected at all time points (except week 4) in K-EDTA tubes (9 mL). Blood was immediately centrifuged at 2100× g for 10 min at 4 °C. Plasma was aliquoted on ice at 500 µL in sterile tubes, then stored at −80 °C before it was sent on dry ice to the Nephrology laboratory of the Ghent University Hospital in Belgium for batch analysis. Total and free concentrations of pCS, IxS, pCG and IAA were determined by liquid chromatography and fluorescence detection as previously described [31 (link)]. In brief, for quantification of the total toxin concentrations, plasma samples were deproteinized by heat, centrifuged and filtered through an Amicon^® Ultra 0.5 µL (Merck Millipore Ltd. Carrigtwohill, Ireland) (molecular weight cut-off 30 kDa filter). For the free fraction, the untreated plasma was first filtered through the Amicon filter. The ultrafiltrate was transferred into an autosampler vial, and fluorescein was added as an internal standard. Analysis was performed by ultra-performance liquid chromatography with an Agilent 1290 Infinity device (Agilent, Santa Clara, United States of America). IxS (λex: 280 nm, λem: 376 nm), pCS and pCG (λex: 264 nm λem: 290 nm), IAA (λex: 280 nm, λem: 350 nm), and fluorescein (λex: 443 nm, λem: 512 nm) was detected by an Agilent G1316C fluorescence 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.