Cosmosil 5c18 ar 2 column

Organic acids in the TBE-8 culture were analyzed using Chinese National Standard methods (CNS12635-N6224) and methods reported by Han et al.^{51 (link)} with some modifications. TBE-8 cultures were collected from different incubation time points (i.e., 24, 48, and 72 h) and centrifuged at 5000 rpm for 10 min. The resulting supernatants were filtered through a 0.22-μm-pore-size membrane filter for further chromatographic analysis. A total of 20 μL of the filtered sample was loaded into the Cosmosil 5 C18-AR-II column (4.6 × 250 mm, 5 µm, Cosmosil, Japan) with 0.01 M KH₂PO₄ aqueous solution (pH 2.5; including 0.06 mmol tetra-butylammonium phosphate) as the mobile phase and a flow rate of 1.0 mL/min. The UV detection wavelength was set at 210 nm (Waters TaperSlit, USA). The specific fingerprint chromatograms of eight organic acids, namely oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid, and succinic acid, were identified through comparison with the reference standards. The standard curves of the eight organic acids were prepared using six concentrations (1%, 0.5%, 0.25%, 0.125%, 0.0625%, and 0.03125%) to calculate the organic acid content of the culture. Each experiment was performed in three replicates.

A Waters liquid chromatography system (Waters Associates Inc., Bedford, MA, USA), equipped with a double pump and a photodiode array detector was used. Separation was carried out on a Cosmosil 5C₁₈-AR-II column (4.6 mm × 250 mm, 5 μm) with a column temperature set to 25°C. The mobile phases consisted of 0.4% aqueous phosphoric acid (A) and acetonitrile (B). For gradient elution the conditions were: 15% B (v/v) at 0–16 min, 15–25% B at 16–30 min, 25% B at 30–32 min, 25–30% B at 32–35 min, 30% B at 35–37 min, 30–33% B at 37–40 min, 33% B at 40–45 min, 33–48% B at 45–55 min, 48–55% B at 55–75 min, 55–80% B at 75–82 min, 80% B at 82–88 min, and the re-equilibration time of the gradient elution was 15 min. Flow rate was 0.8 mL/min, and injection volume was 10 μL. The detection wavelength for the QCT analyte was set to 360 nm. The absorption spectrum of the compound was recorded at 200–500 nm. The compound was identified by comparing the retention time and UV spectrum with those of standard markers.

To investigate the main constituents of the ethanol extracts of EF qualitatively, we undertook high-performance liquid chromatography (HPLC). We undertook HPLC of the chemical constituents in the ethanol extract of the male flower of EU using a 1260 system from Agilent Technologies (Santa Clara, CA, USA). Mobile phase acetonitrile (phase A) was 0.1% phosphoric acid aqueous solution (phase B). The order was: 0 min, 4% A; 5 min, 10% A; 35 min, 23% A; 45 min, 60% A; 50 min, 90% A; 60 min, 90% A; 60.01–80 min, 4% A. We used a Cosmosil 5C18 AR-II column (Cosmosil, Japan). The absorbance wavelengths were 238 nm and 194 nm. The elution speed was 1.0 mL/min and the temperature was 25 °C. As stated above, the concentration of the extract was 1 g/mL. The extract was transferred to a 100-mL volumetric flask, and made up to 100 mL. The concentration of the reference standards was formulated as follows: pinoresinol diglucoside (0.0816 mg/mL), geniposidic acid (0.0368 mg/mL), chlorogenic acid (0.038 mg/mL), geniposide (0.0396 mg/mL), genipin (0.0408 mg/mL), and quercetin (0.0204 mg/mL). The content of these components could be calculated using the external standard two point method according to the standard concentration.

We analyzed the chemical composition of HE-CE using HPLC (Thermo Scientific Vanquish Horizon UHPLC System), while E-A and E-S were separated using a COSMOSIL 5C18-AR-II column (250 × 4.6 mm; particle size 5 μm, Nacalai USA, Inc.) [22 (link)–25 (link)].

A small amount of 7 was dissolved in 5 μL of acetonitrile in the reaction vial. A measure of 10 μL of 1% acetic acid in acetonitrile, 3 μL of [¹²⁵I]NaI (3.7 MBq) solution, and 15 μL of N-chlorosuccinimide (NCS) in acetonitrile (1 mg/mL) were added to the vial. After heating at 80 °C for 15 min, the reaction mixture was quenched with 15 μL of NaHSO₃ solution (1 mg/mL). After evaporating the solvent using N₂ gas, the residue was treated in 100 μL of a mixture of 95% TFA, 2.5% water, and 2.5% TIS for 90 min at room temperature, and then purified by RP-HPLC with a Cosmosil 5C₁₈-AR-II column (4.6 × 150 mm) at a flow rate of 1 mL/min with a gradient mobile phase of 35% methanol in water with 0.1% TFA to 55% methanol in water with 0.1% TFA for 20 min. The radiochemical yield and the radiochemical purities of [¹²⁵I]6 were 38% and >96%, respectively.

[¹²⁵I]IMT, which we synthesized, uptake assay was performed as previously described (25 (link)). High-performance liquid chromatography (HPLC) was performed with a COSMOSIL 5C18-AR-II column (4.6 inner diameter [ID] mm × 150 mm; Nacalai Tesque) at a flow rate of 1 mL/min with a gradient mobile phase of 20% methanol in water with 0.1% trifluoroacetic acid (TFA) to 40% methanol in water with 0.1% TFA for 20 minutes. The column temperature was maintained at 40°C. Microdissected ARC/VMH-rich region of hypothalamus was washed twice with HBSS and subsequently incubated in HBSS at 37°C for 10 minutes in a 5% CO₂ incubator. Samples were then incubated with [¹²⁵I]IMT at 4 or 37°C for 30 minutes. Samples were treated with JPH203 (MilliporeSigma SML1892) at 30 μM. The reaction was terminated by the aspiration of buffer, followed by superficial rinsing with ice-cold HBSS containing 1 mM unlabeled l-α-methyltyrosine (MilliporeSigma 286680) at 4°C 3 times to remove extracellular [¹²⁵I]IMT. Samples were lysed using 0.1 M NaOH. The radioactivity was measured by a γ-counter (Hitachi Ltd., ARC-7010). Protein concentration was determined with a Protein Assay Bicinchoninate Kit (Nacalai Tesque).