Rp c18 column

Five catechin compounds, EGC, C, EGCG, EC, and ECG, in each extract were analyzed using HPLC by following the protocol of Bronner and Beecher.28 (link) All the compounds were separated on an RP-C18 column (25 cm × 4 mm i.d. and 5 μm particle diameter; Phenomenex, CA, USA) with a mobile phase of water/acetonitrile/methanol/ethyl acetate/glacial acetic acid (89:6:1:3:1 v/v/v/v/v) at a flow rate of 0.7 mL/min. Catechin compounds were detected by measuring ultraviolet (UV) absorbance at 280 nm, and those in each VC Less extract were calculated by comparing the absorbance of each standard compound from 3.12 to 100 μg/mL.

Nitrite and nitrate were analyzed using HPLC by following the protocol of Lulla et al.30 (link) Compounds were separated on an RP-C18 column (25 cm × 4 mm i.d. and 5 μm particle diameter; Phenomenex) with a mixed solution of (0.2%) sodium acetate and (2.5%) glacial acetic acid at a flow rate of 1.0 mL/min at 313 nm. The standard references were sodium and potassium nitrate.

Caffeine was evaluated using HPLC by following the protocol of Harris.31 It was separated on an RP-C18 column (25 cm × 4 mm i.d. and 5 μm particle diameter; Phenomenex) with a mixed solution of 0.5% phosphoric acid and 40% methanol at a flow rate of 1.0 mL/min. Caffeine was identified by measuring UV absorbance at 280 nm, and the concentration was calculated by comparing with standard caffeine.

Flavonoids such as quercetin, kaempferol, and myricetin were analyzed in each extract using HPLC by following the protocol of Tokusoglu et al.29 Each extract of 5 g was acidified with 10 mL of acidic methanol solution (1% HCl) and refluxed at 70°C for 2 h before measuring by HPLC. Each compound was separated on an RP-C18 column (25 cm × 4 mm i.d. and 5 μm particle diameter; Phenomenex) with a mixed solution of 25:75 (v/v) acetonitrile:pH 2.4 phosphate buffer (25% acetonitrile in 0.025 M NaH₂PO₄) as a mobile phase at a flow rate of 1.2 mL/min. Quercetin, kaempferol, and myricetin were detected in each extract by measuring UV absorbance at 266 nm and calculated by comparing the absorbance of each standard (100–3 μg/mL).

Levels of Hg in serum were determined using a Perkin Elmer A-Analyst 800 (Shelton, USA) atomic absorption spectrometer and expressed as μg/L.
TEGDMA and Bis-GMA analyses were performed using the method described by Pelka et al,²² with some modifications. High-pressure liquid chromatography (HPLC) method was used to detect Bis-GMA and TEGDMA. HPLC was performed using Agilent 1100 modular systems (including a gradient pump, auto sampler, column oven, and UV detector). The UV detector was set at 205 nm. Mobile phase A was a mixture of 90% water, 9.8% acetonitrile, and 2% tetrahydrofuran with 25mM/l KH₂PO₄ adjusted pH = 3. Mobile phase B was a water–acetonitrile mixture at a ratio of 10:90% (vol/vol). All reagents were obtained as HPLC grade. RP-C18 column (250 mm × 4.6 mm × 5.0 μm particle size, Phenomenex, CA, USA) was used for chromatographic separation. The flow rate was constant at 1 mL/minute. HPLC equipment gave us the concentrations of the samples according to the peak areas in parallel with the retention time of the monomers in the column, and the amount of monomers (Bis-GMA and TEGDMA) in serum was calculated directly from the standard calibration curve. Levels of TEGDMA and Bis-GMA were expressed as μM.