*5.2. Biochemical and Anthropometic Analyses*

After an HD session, the body weight and height were measured to the nearest half kilogram and half centimeter, respectively, with the patients wearing light clothing. Body mass index was calculated as the weight (kg)/height (m)2.

Before HD, approximately 5 mL of blood was collected from each patient. This blood sample was centrifuged at 3000× *g* for 10 min, stored at 4 ◦C, and used for biochemical analyses within 1 h after collection. The serum levels of blood urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total calcium, and phosphorus were examined by an autoanalyzer (Siemens Advia 1800, Siemens Healthcare GmbH, Henkestr, Germany). The adequacy of HD was calculated as the fractional clearance index for urea (Kt/V) and the urea reduction ratio, using the single compartment dialysis urea kinetic model. The levels of intact parathyroid hormone were measured by a commercially available enzyme-linked immunosorbent assay (Diagnostic Systems Laboratories, Webster, TX, USA).

### *5.3. Determination of Serum P-Cresyl Sulfate Levels*

A Waters e2695 HPLC system that comprised a mass spectrometer (ACQUITY QDa, Waters Corporation, Milford, MA, USA) was used in this study [26]. The analytical column was a Phenomenex Luna ® C18 (2) (5 μ, 250 × 4.60 mm, 100 Å) with the following settings: column temperature 40 ◦C, flow 0.8 mL/min, and 30-μL injection. A binary gradient was applied on the mobile phase: the initial composition (95% (A) water with 0.1% formic acid/5% (B) methanol with 0.1% formic acid) was kept constant for 1 min; solvent B was then increased linearly up to 70% over 12 min and was kept constant for 2 min. For column equilibration, solvent B was reduced to 50% over 1 min and was kept constant for 2 min.

The liquid chromatography–mass spectrometry (LC–MS) gradient condition was modified as the pretreated samples were synchronously assessed in positive- or negative-ion (i.e., PCs) mode electrospray ionization. The instrument settings were as follows: desolvation temperature 600 ◦C, capillary voltage 0.8 kV, and sample cone 15.0 V. The mass spectrometer was operated in full scan at 50 to 450 *m*/*z* for positive-ion mode and 100 to 350 *m*/*z* for negative-ion mode. The single ion recording mode was used to monitor the individual masses of each compound (PCs: 187.0 *m*/*z*). Empower ® 3.0 software (Waters Corporation, Milford, MA, USA) was used for data acquisition and processing. The

retention time for PCs was approximately 16.56 min. Endogenous compounds were quantified by measuring and comparing the peak areas with the calibration curve obtained from standard solutions. All the determination coe fficients of linearity (r2) were more than 0.995. LC–MS single ion recording mode was used for single ion analysis.
