Lichrospher 100 rp 18

The amount of drug loaded into PCL and PHBV microparticles was determined by means of a previously developed method by high performance liquid chromatography with UV detection (HPLC/UV). An amount of microparticles, equivalent to 50 mg of MAN, was weighted and magnetic stirred with 100 mL of methanol for 12 h in order to completely extract the drug from microparticles. Samples were suitable diluted in methanol and filtered through a poly(vinylidene fluoride) membrane filter (Durapore membrane, 0.45 μm pore size, Millipore, Bedford, MA, USA). The concentration of MAN was obtained chromatographically through a LiChroCart (Merck, Darmstadt, Germany) analytical column (4 × 250 mm) filled with LiChrospher 100 RP-18 (5 μm) with UV detection at 265 nm in triplicate. The mobile phase was composed of phosphate buffer pH 5.0 and acetonitrile (9 : 1 v/v) with a flow rate of 0.5 mL·min⁻¹.
The amount of manidipine was calculated and reported as loading efficiency, following (1)
$\begin{array}{c}\text{Consider}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{loading}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{efficiency}\%\\ \hspace{1em}=\frac{\text{mass}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{of}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{MAN}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{in}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{microparticles}}{\text{theoretical}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{mass}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{of}\mathrm{\hspace{0.17em}\hspace{0.17em}}\text{MAN}}\times \mathrm{100}.\end{array}$

In the microsomal stability assay, an analytical high-performance liquid chromatography (HPLC) system was used with the following condition: column, Merck Lichrospher 100 RP18 125 mm 4.6 mm (5 µm); mobile phase A = 0.1% trifluoroacetic acid in water, B = 0.1% trifluoroacetic acid in acetonitrile, isocratic; flow rate, 1 mL/min; detection, UV at 400 nm injection, 20 µL; column temperature, ambient. The metabolic stability assays were carried out using the cosolvent method, appropriate for assessing the metabolic stability of compounds poorly soluble in aqueous medium [40 (link)]. For the cosolvent method, a 0.5 mM DMSO stock solution of Oxa12 was prepared. Then, a diluted solution of the compound was prepared by adding 50 µL of the previous 0.05 mM solution with 200 µL of acetonitrile, to make a 0.1 mM solution of Oxa12 in 20% DMSO/80% acetonitrile. Cosolvent assay conditions were: substrate concentration, 1 µM; microsomal protein, 0.5 mg/mL; organic solvents, 0.2% DMSO, 0.8% acetonitrile; incubation time, 30 min; number of assays, duplicates for T0 and T30 min. Time 0 and time 30 batches, after quenching with acetonitrile, were centrifuged at 11,000× g for 5 min and the supernatants were analyzed by HPLC, in order to quantify compound Oxa12.

HPLC analysis of anthocyanins was carried out on a Dionex HPLC system equipped with a Dionex PDA-100 photodiode array detector (wavelength 260–650 nm) (Thermo Fisher, Dreieich, Germany) and a Dionex STH 585 column oven according to Würth et al. [41 ] and modified by Hey et al. [42 ]. Separation was performed on a reversed phase LiChrospher 100 RP-18 (250 mm × 3 mm, 5 µm, Merck, Darmstadt, Germany) at 20 °C. A gradient consisting of solvent A (water/acetonitrile/o-phosphoric acid (85%) (94/4/2, v/v/v) and solvent B (water/acetonitrile/o-phosphoric acid (85%) (48/50/2, v/v/v) was applied at a flow rate of 500 μL/min. An aliquot of 20 µL of red wine, previously filtered through a 0.45 μm RC syringe filter (Durafill, Duratec GmbH, Hockenheim, Germany), was injected onto the column. Quantification was carried out by peak area measurements at 520 nm. The standard report was performed using the chromatography software Dionex™ Chromeleon™ (Version 6.8, Thermo Scientific™, Dreieich, Germany). All analyses were operated in duplicate. The concentration of each anthocyanin was expressed as the equivalent of malvidin-3-O-glucoside.

Cortisol, cortisone and EGCG were purchased from Sigma-Aldrich (St. Louis, MO). The following chemicals were procured from Carl Roth GmbH + Co. KG (Karlsruhe, Germany): Ethylacetate, NADP, NADPH, non-fat dry milkpowder, monosodium phosphate and potassium chloride, HPLC column LiChrospher® 100 RP-18, methanol, sodium chloride and monopotassium phosphate were obtained from Merck (Darmstadt, Germany). (−)-Epicatechin gallate, (−)-catechin and (−)-gallocatechin were purchased from Biomol GmbH (Hamburg, Germany), Cayman Chemical (Ann Arbor, MI) and LKT Laboratories Inc. (St. Paul, MN), respectively. The different types of commercially available teas consisting of green, oolong and black teas were bought from supermarkets in Kiel, Germany. The anti-HSD11B1 antibody (ab83522) was obtained from Abcam (Cambridge, UK).

ATP, ADP and AMP levels were measured by HPLC. For adenine nucleotide extraction, after one rinse in cold PBS, 0.5 ml PBS and 0.5 ml perchloric acid/EDTA were added to each dish. Cells were scraped from the dishes, placed in 1 ml micro-centrifuge tubes and centrifuged for 2 minutes at 14,000 × g. Pellets were then re-suspended in 50 μl of 1 M NaOH and later used for protein quantification by the BCA protein assay (23227, Thermo Scientific; Rockford, IL, USA). The supernatant was neutralized with 3 M KOH in 1.5 M Tris and centrifuged again. The supernatant was again collected and stored at −80°C until analyzed by reverse-phase high performance liquid chromatography. The chromatographic apparatus was a Beckman-System Gold, consisting of a 126 Binary Pump Model and a 166 Variable UV detector, controlled by computer. The detection wavelength was 254 nm, and the column was a Lichrospher 100RP-18 from Merck (Darmstadt, Germany). An isocratic elution with 100 mM phosphate buffer (KH₂PO₄), pH 6.5 and 1% methanol was performed with a flow rate of 1 ml/min. The time required for each analysis was 5 minutes.

All samples were harvested directly from the system outflow using a peristaltic pump (BVP Z, ISMATEC SA, Switzerland). The water was pumped through a flow cell, and the redox potential (SenTix ORP Pt/Ag+/AgCl/Cl‐, WTW, Germany), pH (pH 539, WTW, Germany) and temperature (Checktemp1, Hanna Instruments, Germany) were recorded. To measure the dissolved oxygen concentration, an optical trace oxygen mini sensor (FTC‐TOS7‐PSt3, PreSens GmbH, Germany) and a Fibox‐3‐Trace single channel fiber‐optic trace oxygen meter were used (PreSens GmbH, Germany).
Ibuprofen concentrations were analyzed in an HPLC prominence line (Shimadzu, Japan), equipped with LC Solution Postrun Analysis software, a degasser DGU‐20A3, a binary gradient pump LC‐20AB, an autosampler SIL‐20A, a column oven CTO‐20AC and a UV‐detection system SPD‐M20A. The column and security guard used were both LiChrospher 100 RP‐18 (5 µm) (Merck KGaA, Germany). The column was operated at a constant temperature of 30°C. The mobile phase consisted of 0.1% v/v formic acid in water (Eluent A), and methanol (Eluent B) and the gradient program was selected to be 20% B to 80% B (25 min).