Cto 10 as vp column

Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured using the commercially available kits (BT-LABS, Shanghai, China) according to the manufacturer’s procedure. Sensitivities were 3.04 ng/ml, 052 ng/ml, and 2.21 U/ml for SOD, CAT, and GPx, respectively, and intra- and inter-assays were CV <8% and CV <10% for all. For MDA analyses, an HPLC apparatus of Shimadzu UV–vis SPD-10 AVP detector, a CTO-10 AS VP column, and 30 mM KH₂PO₄ and methanol (82.5: 17.5, v/v, pH 3.6) at a flow rate of 1.2 ml/min were used (Shimadzu, Japan). Column waste was monitored at 250 nm. For antioxidant enzymes and MDA analyses, tissue samples were rinsed in PBS and minced and homogenized in PBS with a glass homogenizer on ice, then thawed at 2–8°C and centrifuged at 2000–3000 rpm for 20 min.

The levels of serum glucose, creatinine, blood urea nitrogen (BUN), total protein (TP), albumin (ALB), globulin (GLOB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL) were analyzed using a portable automated chemistry analyzer (Samsung LABGEO PT10V, Samsung Electronics Co., Suwon, Korea). Serum TNF-α, IL-1β, IL-6, IL-10, cartilage oligomeric matrix protein (COMP), and C-reactive protein (CRP) levels were analyzed using commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer instructions (Cayman Chemical, Ann Arbor, MI, USA).
The levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) were determined using the commercially available kits according to the manufacturer instructions (Cayman Chemical, Ann Arbor, MI, USA). For malondialdehyde (MDA) analysis, an HPLC apparatus of Shimadzu UV–vis SPD-10 AVP detector, a CTO-10 AS VP column, and 30mM KH₂PO₄ and methanol (82.5: 17.5, v/v, pH 3.6) at a flow rate of 1.2 ml/min were used (Shimadzu, Japan). Column waste was monitored at 250 nm.

Samples were analysed using a liquid chromatographic system (Shimadzu, Kyoto, Japan) equipped with a CBM-20A system controller connected to an LC-20AD solvent delivery unit, DGU-20A3 degasser system, CTO-10ASVP column oven, SIL-20AHT autosampler and SPD-20A ultraviolet-visible detector. The isocratic mobile phase used was methanol and 0.1% of trifluoroacetic acid in distilled water at a volume ratio of 85:15 and a flow rate of 1 mL/min. The mobile phase was filtered through a 0.45 µm nylon membrane filter under a vacuum condition and sonicated before use. A Phenomenex^® Luna C18 column (250 × 4.6 mm ID, 5 μm; Torrance, CA, USA) fitted with a Thermo Scientific Uniguard™ guard column (Waltham, Boston, MA, USA) with an ODS C18 cartridge (10 × 4 mm ID, 5 μm) was used as stationary phase. UV detection was set at 305 nm and a sample injection volume of 40 μL was used with a column temperature of 40 °C. An internal standard of MS was used during method development and validation. The retention time of MS was ~4.6 min. The limits of detection and quantification for MS were 0.08 and 0.25 µg/mL for a low drug concentration range of 0.25–7 µg/mL, respectively, as well as 4.20 and 12.72 µg/mL for a high drug concentration range of 5–150 µg/mL, respectively.