Spd 20av uv detector

Butyrate, succinate, lactate, and acetate concentrations in culture supernatants were quantified as described previously (Clark et al., 2021 (link)). Supernatant samples were thawed in a room temperature water bath before addition of 2µL of ${\text{H}}_2{\text{SO}}_4$ to precipitate any components that might be incompatible with the running buffer. The samples were then centrifuged at 2400xg for 10 min and then 150µL of each sample was filtered through a 0.2µm filter using a vacuum manifold before transferring 70µL of each sample to an HPLC vial. HPLC analysis was performed using a Shimadzu HPLC system equipped with a SPD-20AV UV detector (210 nm). Compounds were separated on a 250×4.6 mm Rezex OA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.2 ml min_-1 and at a column temperature of -50°C. The samples were held at 4°C prior to injection. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N ${\text{H}}_{2}S{O}_4$ ( $415\mu {\text{LL}}^{-1}$ ). At least two standard sets were run along with each sample set. Standards were 100, 20, and 4 mM concentrations of butyrate, succinate, lactate, and acetate, respectively. The injection volume for both sample and standard was 25µL. The resultant data was analyzed using the Shimadzu LabSolutions software package.

Mucin disks were prepared using the method of Tsuchiya et al. [25 ]. Four-hundred microliters of 10% mucin solution was spread on a 25-mm diameter filter paper. The filter paper was dried at room temperature for 24 h. An artificial saliva solution (pH 6.8) consisting of 0.8% NaCl, 0.019% KH₂PO₄, and 0.238% Na₂HPO₄ was prepared. The mucin disk was moistened with artificial saliva, and 3 mg of IM bulk powder or the IM microparticles were mounted on the mucin disk, and the mucin disk was dried at room temperature for 5 min. Each mucin disk with sample was fixed to a slide glass with a clip and immersed in 150 mL of artificial saliva at 37 °C and incubated with shaking at 50 rpm. Mucin disks were taken out at 5, 10, and 15 min and the amount of IM remaining on the disk was measured using high-performance liquid chromatography (HPLC). The HPLC system consisted of an LC-6AD pump (Shimadzu Corporation, Kyoto, Japan) and a Chromato-PRO (Run Time Corporation, Tokyo, Japan) equipped with a Capcell Pak C18 MG II column (4.6 × 250 mm, OSAKA SODA CO., LTD., Osaka, Japan) and an SPD-20AV UV detector (Shimadzu Corporation). Chromatography was carried out at 40 °C. The mobile phase comprised 60% (v/v) acetonitrile in 0.02 M sodium acetate buffer adjusted to pH 3.6 using orthophosphoric acid. The flow rate was 1 mL/min. The detection wavelength was 320 nm.

The solution was then transferred to HPLC vials for injection onto an HPLC system. The HPLC system consisted of an LC-20AD solvent delivery unit (Shimadzu Corporation, Kyoto, Japan) equipped with an SIL-20AC autosampler (Shimadzu Corporation) and an SPD-20AV UV detector (Shimadzu Corporation) set at a wavelength of 206 nm. Samples were injected onto a SHISEIDO CAPCELL PAK C18 (particle size 5 µm, 250 mm × 4.6 mm; SHISEIDO, Tokyo, Japan) and eluted at 1 mL/min with a mobile phase comprising buffer:acetonitrile at a ratio of 98:2. The buffer was prepared from 10 mM KH₂PO₄ and H₃PO₄ at pH 2.5.