Eclipse plus c18 rrhd

Optical rotations were determined with a Unipol L1000 Schmidt + Haensch polarimeter at the sodium D line (589.3 nm), at 20 °C, with a 10 cm cell. UV spectra were acquired in spectroscopic grade CHCl₃ or MeOH on a Varian, Cary 100 UV-Vis spectrophotometer. IR spectra were recorded on a Perkin Elmer 400 or a Perkin Elmer Spectrum One ATR FT-IR spectrometer. NMR spectra were acquired using a Jeol 400 MHz, a Varian 500 MHz or an Agilent 600 MHz spectrometer. Chemical shifts are given on the δ (ppm) scale, referenced to the residual solvent signal (CDCl₃: δ_H 7.24, δ_C 77.0 or C₆D₆: δ_H 7.16, δ_C 128.0), and J values in Hz. High-resolution mass spectrometric data were measured on an Agilent QTOF 6540 MS system, with electrospray ionisation (ESI) in the positive ion mode, coupled to an Agilent 1290 Infinity UPLC system, operating on the elution gradient: 50% B for 8 min, increasing to 100% B in 3 min, maintaining 100% B for 5 min (solvent A: H₂O + 0.1% formic acid, solvent B: MeCN + 0.1% formic acid), on a Zorbax Eclipse Plus C18 RRHD (50 × 2.1 mm, 1.8 μm) column, at 0.5 mL/min, with UV detection at 200−600 nm. Thin layer chromatography (TLC) was performed with Kieselgel 60 F254 aluminium support plates (Merck) and spots were detected after spraying with 6% vanillin and 15% H₂SO₄ in MeOH reagent. All solvents were of HPLC or LCMS grade and were purchased from Sigma-Aldrich.

Betulin was isolated from the nanoemulsion by employing methylene chloride for extraction, followed by centrifugation and subsequent evaporation of the organic layer to yield a desiccated residue that was reconstituted in HPLC-grade methanol. The betulin content was determined using an Agilent 1290 Infinity chromatographic system (Agilent, Carpinteria, CA, USA) on a reversed stationary phase column (ZORBAX Eclipse Plus C18 RRHD with dimensions of 2.1 × 50 mm and 1.8 µm) with an operating pressure of up to 1200 bar. The mobile phase consisted of a mixture of water and acetonitrile (20:80 (v/v)) with a flow rate of 0.35 mL/min, an injection volume of 2 µL at 35 °C, a detection wavelength of 210 nm, and a slit width of 4 nm. Identification and quantification of betulin were performed using a calibration curve of 24–56 µg/mL, and 2 µL of betulin was dispensed into the chromatographic system in triplicate. To determine the concentration of betulin in the emulsion, samples packed in vials were enclosed in a constant climate chamber “Binder KBF 240” (Binder, Tuttlingen, Germany) at a temperature of 55 °C. Every 180 h, the vial was removed, cooled, and visually assessed for the degree of emulsion separation, as well as the mass concentration of betulin using HPLC.

Global DNA methylation analyses were performed using reverse-HPLC (Quinlivan and Gregory, 2008 (link)) by measuring total 5-methylcytosine (5mC) content in 136 patients. Briefly, 2500 ng genomic DNA were RNase-treated in NEB2.1 buffer and incubated for 1 h at 37 C, purified with ethanol and lyophilised. The sample was re-suspended in 50 μl RNase-free water and hydrolysed overnight in a solution containing 45 mM NaCl, 9 mM MgCl₂, 9 mM Tris pH 7.9, ⩾250 U/ ml⁻¹ Benzonase (Sigma), 50 mU/ ml⁻¹ Phosphodiesterase I, ⩾20 U/ ml⁻¹ Alkaline phosphatase, 46.8 ng/ ml⁻¹ EHNA hydrochloride, 8.64 μM deferoxamine. Protein components were removed with Amicon centrifugal filter units (3 kDa cutoff, Millipore, Watford, UK) and resolved with an Agilent UHPLC 1290 (Stockport, UK) instrument fitted with Eclipse Plus C18 RRHD 1.8 μm, 2.1 × 150 mm column. Buffer A was 100 mM ammonium acetate, pH 6.5; buffer B was 40% acetonitrile, and the flow rate 0.4 ml min⁻¹. The gradient was between 1.8 and 100 of 40% acetonitrile. Methylation was also assayed in blood and bowel DNA separately.