Eclipse plus c18 column

Non deuterated DMSO solutions of validated hits were analyzed by HPLC(UV)-MS and NMR in order to determine the purity of the compounds.
High-performance liquid chromatography coupled to mass spectrometry (HPLC−MS) was performed with an Agilent 6520 QTOF LCMS system connected to an Agilent 1290 LC, equipped with a Zorbax Eclipse Plus C18 column (50 mm ×2.1 mm, particle size 1.8 μm). At a flow rate of 0.8 ml/min a stepwise gradient of Water (+ 0.01% HCOOH) (Eluent A) to Acetonitrile (+ 0.01% HCOOH) (Eluent B) was employed as follows: 0 min 5% B; 0.3 min 5% B; 4.5 min 99% B; 5 min 99% B; 5.1 min 5% B. Mass spectra were acquired in both ESI+ and ESI− mode, scanning from m/z 100 to 3200 Da and UV detection was done at 215 (or 220) and 265 (or 255) nm.
NMR Spectra were recorded on a Bruker Avance III 600 MHz spectrometer, equipped with a 5mm TCI Probe, at 25°C. Samples were diluted with DMSO-d6 to an end volume of 160 μl to be measured in 3mm NMR tubes. Double pre-saturation on the water and DMSO resonance frequency as well as ¹³C decoupling were employed in a gradient ¹H-NMR experiment.
The purity level of the compounds was >95% as determined by both methods.
The syntheses of fragments 1 and 4 were described.[49 , 50 (link)] The syntheses of fragments 2 and 3 have not been disclosed to the public.

The drugs were assayed by HPLC on a Waters system to assess the plasma concentration of diclofenac and ketorolac, as previously described (with slight modifications) [22 ]. Briefly, reversed phase chromatography was used for drug analysis. Ketorolac was examined in a Zorbax Eclipse Plus C18 column (150 × 4.6 mm, 4.5 μm) using a mobile phase of 0.04 M phosphate buffer:acetonitrile:methanol 60:20:20 (v/v/v) delivered at a flow rate of 1.4 mL/min. Diclofenac was evaluated in a Symmetry C18 Waters column (150 × 4.6 mm, 5 µm), with a mobile phase of 0.041 M phosphate buffer:acetonitrile:methanol 49:51 (v/v) at a flow rate of 1.4 mL/min. The lower limit of quantification was 0.5 µg/mL for diclofenac and 0.05 µg/mL for ketorolac. For diclofenac, the intra- and inter-day precision coefficients of variation (CV) were less than 3.86% and 5.97%, respectively. The accuracy of the intra- and inter-day determinations was 93.62–100.82% and 95.80–99.8%, respectively. For ketorolac, the intra- and inter-day precision CV were less than 8.45% and 10.03%, respectively. The accuracy of the intra- and inter-day determinations was 96.87–119.14% and 93.75–108.3%, respectively.

About 3 mg of oil was dissolved in 1 mL of n-hexane and passed through a 0.22 μm organic filter membrane for Q-TOF [24 (link)] analysis (high-performance benchtop quadrupole-orbitrap LC-MS/MS system). Liquid chromatography conditions were as follows: Zorbax Eclipse plus C18 column (2.1 mm × 50 mm × 1.8 μm) was used. Mobile phase A was an acetonitrile (60%) and water (40%) solution, mobile phase B was an acetonitrile (10%) and isopropanol (90%) solution, and mobile phases A and B were both added with 0.1% formic acid and 10 mmol/L ammonium formate. The elution conditions were 0–1 min 70% A, 1–31 min 87% A, 31–32 min 87% A, 32–33 min 70% A and 33–37 min 70% A. The flow rate was 0.2 mL/min, the injection volume was 2 μL, and the column temperature was 45 °C. Mass spectrometry conditions were an ESI ionization source, positive ion mode, collision energy of 20 v and fragmentation energy of 150 v.

LC-MS analysis was performed using an Agilent 1290 Infinity II UHPLC in line with an Agilent 6546 Q-TOF with a dual AJS ESI source. Two μl samples were injected onto a Zorbax Eclipse Plus C18 column (100 mm × 2.1 mm × 1.8 μM) and run on a 16-min linear gradient from 5 to 100% solvent B at 0.45 ml/min. Solvent A was 0.1% formic acid in water, Solvent B was 0.1% formic acid in acetonitrile. MS parameters in negative ionization mode were as follows: capillary voltage, 4000 V; nozzle voltage, 2000 V; drying gas temp, 300 °C; drying gas flow rate, 10 L/min; sheath gas temperature, 350 °C; sheath gas flow rate 12 L/min, nebulizer pressure, 45 psi; fragmentor voltage, 100 V. Parameters for positive ionization mode were the same, except capillary voltage, 3500 V and nozzle voltage, 500 V. MS/MS was collected on selected ions with 10, 20, and 40 V collision energies. Data were collected and analyzed using MassHunter Workstation Version 10 (Agilent Technologies).

For the assessment of SIRT6 deacetylation on RHKK-Ac-AMC, 5 μM WT SIRT6 was incubated in a 50-μL reaction mixture (75 μM RHKK-Ac-AMC, 2.5 mM NAD⁺, and assay buffer) with DMSO or the indicated concentrations of MDL-800/MDL-811 at 37 °C for 2 h. Reactions were terminated with 100 mM HCl and 160 mM acetic acid. After centrifugation at 12,000 r.p.m. for 10 min, the supernatant was analyzed by high-performance liquid chromatography (HPLC) using a Zorbax Eclipse Plus C18 column (4.6 × 100 mm, 3.5 μM). The solvents used for HPLC were water with 0.1% trifluoroacetic acid (solvent A) and acetonitrile with 0.1% trifluoroacetic acid (solvent B). Each experiment was independently repeated three times.

Aqueous solutions of MPs (50 μg ml⁻¹; pH 6.8) were incubated at 21 °C in amber HPLC vials (adapted from de Saint Germain et al., 2016 (link)). For sample preparation, 50 μl of an acetone solution (1 mg ml⁻¹) was diluted to the final concentration with methanol (425 μl), water (500 μl) and 25 μl of 1-indanol (1.0 mg ml⁻¹ solution in acetone) as internal standard. The time course of degradation was monitored by UHPLC analysis on a Dionex Ultimate 3000, using a Zorbax Eclipse Plus C₁₈ column (3.5 μm, 2.1 × 150 mm). The column was developed at a flow rate of 0.6 ml min^–1 at 35 °C with a linear gradient from 5% to 95% acetonitrile in water within 15 min, maintaining the final conditions for another 4 min. The column was operated at 35 °C with a flow rate of 0.25 ml min⁻¹. Compounds eluted from the column were detected with a diode array detector. The relative quantity of remaining (non-degraded) product was determined by comparison with the internal standard. Stability was monitored at 24 h intervals up to 3 weeks.