Hplc apparatus

At the end of the study, the rats were sacrificed, and blood samples were collected. The blood samples were centrifuged, and the collected sera were kept at − 80 °C. Serum biochemical parameters, namely glucose, blood urea nitrogen (BUN), and creatine levels, as well as ALT and AST activities, were assessed biochemistry analyzer (Samsung Electronics Co., Suwon, Korea). Enzyme-linked immunosorbent assay (ELISA) kits (Cayman Chemical, Ann Arbor, MI, USA) were used in analyzing serum inflammation parameters of IL‐1β, IL‐6, TNF-α, cartilage oligomeric matrix protein (COMP), and C-reactive protein (CRP) according to the manufacturer instructions. Serum malondialdehyde (MDA) was analyzed using an HPLC apparatus of Shimadzu (Shimadzu, Japan) equipped with 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^{26 (link)}. Column waste was monitored at 250 nm. Antioxidant levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were measured using the relevant commercial kits (Cayman Chemical, Ann Arbor, MI, USA) according to the ELISA method.

The concentrations of drugs in the receptor medium or rabbit plasma were determined using a validated HPLC method. The HPLC system was equipped with a Shimadzu HPLC apparatus, including an LC-20AT pump, a SIL20A syringe, an SPD-20A detector, and a CTO-20A column oven. The propylparaben was used as an internal standard, and the flow rate of chromatographic analysis was set 1.0 mL/min. For in vitro analysis of SR-FP, thermo (USA) C18 column (150 mm × 4.6 mm, 5 μm particle) maintained at 40 °C was used to elute SR-FP, the mobile phase was used methanol and 1% aqueous glacial acetic acid (60:40, v/v) and the wavelength was set at 247 nm. The HPLC conditions of S-FP and R-FP were as follows: The column was CHIRALPAK®IA (4.6 mm × 250 mml, 5 μm particle diameter) of DAOCEL (Shanghai, China). The mobile phase consisted of acetonitrile and trifluoroacetic acid (0.05%, 50:50, v/v), the wavelength was set at 254 nm. For in vivo analysis of S-FP and R-FP, the mobile phase consisted of acetonitrile and trifluoroacetic acid (0.05%, 35:65, v/v), and other conditions were the same to in vitro analysis.

Liquid chromatography (LC) was performed with a Shimadzu HPLC apparatus (Kyoto, Japan) consisting of a gradient pump (model LC-30AD), an automatic injector (model SIL-30AC) and an on-line degasser (model DGU-20A_5R). Analytes were detected with an AB Sciex® 6500⁺ triple quadrupole tandem mass spectrometer (Sciex, Framingham, MA) equipped with a turbo ion spray interface (API 6500 + MS/MS). Data acquisition and integration were carried out with Analyst software (Analyst 1.7, Sciex) linked directly to the LC–MS/MS.

The determination of whole-blood spermine concentration was performed as previously described [20 (link)]. Briefly, blood samples were vortexed and sonicated twice for 5 min before centrifugation at 18,000 g for 10 min. Then, the supernatants transferred to a new microtube, and an equal volume of 20% trichloroacetic acid, containing 20 µM N-(3-aminopropyl) cadaverine as an internal standard, was added. After centrifugation at 18,000 g for 10 min, the supernatant was separated and injected into a high performance liquid chromatography (HPLC) apparatus (Shimadzu, Kyoto, Japan) for analysis. Post-column derivatisation of eluted polyamines with o-phthalaldehyde yielded fluorescent products detectable at a wavelength of 450 nm after excitation at 345 nm.