Chromolith performance rp 18e column

The HPLC analysis was performed with a VWR Hitachi Chromaster 600 chromatograph with a spectrophotometric detector (DAD) on a Chromolith^® Performance RP-18e column (100 × 4.6 mm) (Merck, Darmstadt, Germany). The chromatographic parameters were evaluated using EZChrom Elite Software (Merck, Darmstadt, Germany). All chromatographic experiments were replicate tree times. Figures presenting interaction between variables were created using Statistica 10 software (StatSoft, Polska).

Leaf tissue (100 mg) was extracted overnight at -20°C with 1.5 mL 100% methanol. Doubly deionized water (100 μL) was mixed with 90 μL extract in a GC-MS vial microinsert. Diluted sample (20 μL) was injected onto an Agilent HPLC-1100 with a flow rate of 1 mL/min of a gradient of 0.05% aqueous trifluoroacetic acid (TFA) and acetonitrile (ACN) through a 100 x 4.6 mm Chromolith Performance RP-18e column, 100 x 4.6 mm (Merck; Darmstadt, Germany). Acetonitrile concentration was increased linearly from 10% to 34% over 8 min, followed by a 2 min column wash with 100% ACN and a 2 min re-equilibration to a 9:1 ratio of 0.05% aqueous TFA:ACN. Rutin was identified by spectral comparison with a commercial standard (Sigma-Aldrich, St. Louis, MO) and quantified by absorbance at 330 nm based on an external standard curve.

U87MG and PC GSCs were maintained under standard culture conditions (37 °C, 5% CO₂) for 4 days. Then, GSCs were washed with PBS 1X two times and incubated in 500 μL of Tyrode’s buffer for 1 h at 37 °C. Later, 200 μL of this incubation medium was mixed with 100 μL of citrate buffer (pH 4). Following derivatization with 2-chloroacetaldehyde (Merck^®, Darmstadt, Germany) adenosine levels were determined by HPLC fractionation in a Chromolith Performance RP-18e column (Merck^®) and fluorescent detection [19 (link)]. Adenosine concentrations (nM) were normalized to the total protein levels (μg).

An Agilent HP 1100 system (Waldbronn, Germany) with a 20 µL loop, coupled to a Kontron Ultra-Violet (UV) detector (Zurich, Switzerland) and piloted by EZchrome Elite workstation software was used. LC separation was performed on a Chromolith Performance RP-18e column (100 mm × 4.6 mm ID, 2 µm) provided by Merck (Darmstadt, Germany), with a gradient system containing water as solvent A and Methanol as solvent B, using solvent B from 40% to 51% over 9 min, at 3 mL·min⁻¹. To avoid any peak tailing and samples precipitation, they were injected in a mixture of water:methanol of 80:20. Detection was done by UV at 220 nm, and by ELSD giving the profile shown in Figure 3 where we can see the detection of the three main compounds present in the plant: andrographolide 1, didehydroandrographolide 2 and neoandrographiside 3.

The 6-OH-BTA-0 precursor was obtained from either ABX advanced biochemical compounds GTmbH (Radeberg, Germany) or Pharmasynth AS (Tartu, Estonia), standard reagents were obtained from Sigma-Aldrich/Merck, sterile solutions were obtained from either Sygehus Apotek Fyn (Odense, Denmark) or Region Hovedstadens Apotek (Herlev, Denmark), and sterile Cathivex-GV 0.22 µm filters (SLGV0250S) were obtained from Merck Millipore. [¹¹C]CO₂ was produced by the bombardment of the target gas, 99.5% N₂ + 0.5% O₂ (Strandmøllen A/S, Klampenborg, Denmark), using a GE PETtrace cyclotron (Table 3).
The automated [¹¹C]PiB production and product purification processes were performed on a Tracerlab FXc (GE HealthCare, Uppsala, Sweden) fitted with a S1021 HPLC pump (SYKAM, Eresing, Germany), Chromolith Performance RP-18e column (100 × 10 mm, Merck, Soeborg, Denmark), and K-2001 UV detector (Knauer, Berlin, Germany). The radiosynthesis process and semi-preparative HPLC are described further in Section S3 (Supplementary Materials) and Table 2.

GC/MS analysis was performed as described previously [17 (link)].
Non-chiral LC/MS measurements were performed on a LC/MS-2020 (Shimadzu, Duisburg, Germany) equipped with a Chromolith^® Performance RP-18e column (100 × 4.6 mm, Merck, Darmstadt, Germany). A solvent gradient of methanol and 0.1 % formic acid at a flow rate of 0.5 ml min⁻¹ was applied as follows: starting from 20 to 35 % methanol in 5 min, hold for 5 min, increase to 70 % methanol within 15 min, then to 90 % methanol within 1 s, hold for 1 min, re-equilibration with 20 % methanol. UV/Vis spectra were monitored in the range between 190–800 nm. The interface temperature was 350 °C, the desolvation line temperature was 275 °C, and the heat block temperature was 400 °C. The nebulizing gas flow and the drying gas flow were set to 1.5 and 15 l min⁻¹, respectively.
For determination of the enantiomeric composition of pinoresinol 3, lariciresinol 4, and secoisolariciresinol 5 reaction mixtures were analysed by chiral HPLC (Shimadzu, Duisburg, Germany) equipped with a CHIRALPAK^®IB column (250 × 4.6 mm, Chiral Technologies Europe, Illkirch Cedex, France). The solvents n-hexane/ethanol were used under isocratic conditions (pinoresinol 3: 50/50; lariciresinol 4: 80/20, secoisolariciresinol 5: 75/25) at a flow rate of 0.7 ml min⁻¹.