High performance liquid chromatography (hplc)

One piece of the agar plate (2 cm × 2 cm) containing fungal mycelia was inoculated into 50 mL ISP2 medium (Difco, Basel, Switzerland), fermented on shaker incubator at 180 rpm for 10 days at 30 °C. At the end of fermentation process, about 50 g L⁻¹ diaion HP20 resin was added to the fermentation flask, left shaking for 6 h, then centrifuged at 10,000 rpm for 5 min. The precipitate was then extracted with methanol twice and the combined methanolic extract was evaporated under vacuum to a residue. For GC-MS analysis, approximately 10 mg of the fungal methanolic extract was re-dissolved in 10 mL methanol, then fractioned with 2 × 10 mL n-hexane in separating funnel successively. The hexane extract was evaporated and 1 mg of the residue was dissolved in 10 mL of hexane. About 1 mL of this solution was filtered through 0.2 µm PTFE filter (Milian, Geneva, Switzerland) into HPLC (Thermo Fisher Scientific, Foster City, CA, USA) vial where it is submitted to GC-MS analysis. For LC-MS analysis, 1 mg of the methanolic extract was accurately weighted and dissolved in 10 mL methanol and about 1 mL of this solution was filtered through 0.2 µm PTFE filter into an HPLC vial where it was submitted to LC-MS analysis.

For HPLC analysis, the procedure was as described by Kildegaard et al. (2017 (link)). A total of 100 μL of ethyl acetate extract was evaporated on SpeedVac, and the dry extracts were redissolved in 1 mL 99% ethanol + 0.01% BHT. Then, the extracts were analyzed by HPLC (Thermo Fisher Scientific) equipped with a Discovery HS F5 150 mm x 2.1 mm column (particle size 3 mm). The column oven temperature was set to 30°C. All organic solvents used were HPLC grade (Sigma Aldrich, St. Louis, MO). The flow rate was set to 0.7 mL/min with an initial solvent composition of 10 mM ammonium formate (pH = 3, adjusted with formic acid; solvent A) and acetonitrile (solvent B; 3:1) until minute 2.0. Solvent composition was then changed at minute 4.0 following a linear gradient until % A = 10.0 and % B = 90.0. The solvent composition was kept until 10.5 min when the solvent was returned to initial conditions, and the column was re-equilibrated until 13.5 min. The injection volume was 10 µL. The peaks obtained from the sample analysis were identified by comparison to prepared standards and integration of the peak areas was used to quantify carotenoids from obtained standard curves. β-carotene was detected at a retention time of 7.6 min by measuring absorbance at 450 nm. The β-carotene standard (C4582–5 mg) was purchased from Sigma-Aldrich.

Reagents including CRANAD-2 were purchased from Merck (Darmstadt, Germany) and 4-(methylamino)benzaldehyde was from Manchester Organics (Cheshire, UK). β-Amyloid(1–42) peptide was purchased from Bachem (Torrance, CA, USA). ¹H NMR spectra were obtained using a Bruker Avance III 500 (500 MHz) spectrometer (Rheinstetten, Germany), and chemical shifts (δ) were reported as the ppm downfield of the internal tetramethylsilane. ¹⁹F NMR spectra were obtained using a Bruker Avance III HD (300 MHz) spectrometer. The electron impact (EI) and fast atom bombardment (FAB) mass spectra were obtained using a JMS-700 Mstation (JEOL Ltd, Tokyo, Japan). Purification of the non-radioactive ligands was performed using HPLC (Thermo Scientific, Waltham, MA, USA) that was equipped with a semi-preparative column (YMC-Pack C18, 10 × 250 mm, 5 µm). Purity of the ligands was determined using HPLC equipped with an analytical column (YMC-Pack C18, 4.6 × 250 mm, 5 µm) (Supplementary Figs S9–S12). The eluent was monitored using a UV (254 nm) detector.

IBU and its derivatives analysis were performed by HPLC (Knauer, Berlin, Germany) using Hypersil ODS (C18) 125 mm × 4 mm column (Thermo Scientific™ Waltham, MA, USA) and a mobile phase composed of 0.02 mol·dm⁻³ potassium dihydrogen phosphate-acetonitrile-methanol (45/45/10, v/v/v) at 1 mL/min. The column temperature was set at 25 °C. UV detection at 264 nm was employed.

The study screened feed additives targeting the ACE‐2 receptor that could be an alternative antibiotic strategy for treating pneumonia and diarrhea in weanling piglets. We proposed a natural product "Guizhi Li‐Zhong Tang Extract Granules (GLZ)" as an alternative antibiotic feed additive for weanling piglets. GLZ is a herbal formula, which was obtained from Sun‐Ten Pharmaceutical Company (New Taipei City, Taiwan). The bioactive marker substances of GLZ were analyzed using 3D chromatographic fingerprint analysis through high‐performance liquid chromatography (HPLC, Thermo Fisher Scientific Inc.); the substances were graded with acetonitrile and methanol (Burdick & Jackson Korea, Seoul, Korea) and were qualitatively determined within 70 min under the selected HPLC condition. The ingredients of GLZ were dissolved in purified water provided by the Milli‐Q water purification system (Millipore).

The cDNAs encoding wild-type (WT) or codon-optimized (OP) structural protein C-E3-E2-6K-E1 genes from CHIKV strain LR2006 OPY1 (23 (link)) were cloned into plasmid ABOP-028 (GENEWIZ), which contained 5′ and 3′ untranslated regions (UTRs) and a poly A tail. The mRNA was synthesized in vitro using T7 polymerase-mediated transcription from the linearized plasmid DNA template.
Formulation was performed as described previously for the COVID-19 vaccine (24 (link)). Briefly, lipids were dissolved in ethanol containing an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and PEG-lipid (molar ratios of 50:10:38.5:1.5). The lipid mixture was run through a T-mixer with mRNA dissolved in 20 mM citrate buffer (pH = 4.0) at a ratio of 1:2. The generated mRNA-LNPs were diafiltrated against PBS (pH = 7.4) in a dialysis cassette with 20 kD MWCO overnight, passed through a 0.22-μm filter, and stored at 2°C–8°C until use. The product was characterized for particle size and distribution using a particle size analyzer (Malvern Panalytical), RNA concentration using HPLC (Thermo Fisher Scientific), and encapsulation using RiboGreen reagent (Thermo Fisher Scientific).