Accela hplc system

Analytical HPLC was performed on a Shimadzu LC-10Avp series HPLC system consisting of an autosampler, high-pressure pumps, column oven and photodiode array detector. HPLC conditions were as follows: C18 column (Eurospher 100-5, 250 × 4.6 mm) and gradient elution (MeCN/0.1% (v/v) trifluoroacetic acid (TFA) 0.5/99.5 in 30 min to MeCN/0.1% (v/v) TFA 100/0, MeCN 100% for 10 min), flow rate 1 ml min⁻¹. Preparative HPLC was performed on a Shimadzu LC-8a series HPLC system with photodiode array detector. LC-MS measurements were performed using an Exactive Orbitrap High Performance Benchtop LC-MS with an electrospray ion source and an Accela HPLC system (Thermo Fisher Scientific, Bremen). HPLC conditions were as follows: C18 column (Betasil C18 3 μm 150 × 2.1 mm) and gradient elution (MeCN/0.1% (v/v) HCOOH (H₂O) 5/95 for 1 min, going up to 98/2 in 15 min, then 98/2 for another 3 min; flow rate 0.2 ml min⁻¹). For tandem mass spectrometry measurements, a Q Exactive Orbitrap mass spectrometer with an electrospray ion source (Thermo Fisher Scientific) was used. NMR spectra were recorded on a Bruker AVANCE III 600 MHz instrument equipped with a Bruker cryo platform. Spectra were normalized to the residual solvent signals. The infrared spectra were recorded on a JASCO FT/IR-4100 type A.

An Accela HPLC system interfaced to an Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used for the liquid chromatographic separations. ZIC-pHILIC (150 × 4.6 mm, 5 µm) and ACE C4 (150 × 3.0 mm, 3 µm) HPLC columns supplied by HiChrom (Reading, UK) were used. Samples were run on LC-MS under the following conditions: the ZIC-pHILIC mobile phase consisted of 20 mM ammonium carbonate in HPLC-grade water (A) and acetonitrile (B); the solvent gradient used was 80% B (0 min), 20% (30 min), 8% (31–36 min), and 80% (37–45 min) at a flow rate of 0.3 mL/min. For the ACE C4 column, the mobile phase was 1 mM acetic acid in water (A) and 1 mM acetic acid in acetonitrile (B). The solvent gradient used was 40% B (0 min), 100% (30–36 min) and 40% (37–41 min) at a flow rate of 0.4 mL/min. The nitrogen sheath and auxiliary gas flow rates were maintained at 50 and 17 arbitrary units. The electrospray ionisation (ESI) interface was employed in a positive/negative dual polarity mode, with a spray voltage of 4.5 kV for positive mode and 4.0 kV for negative mode, while the ion transfer capillary temperature was set at 275 °C. Full scan data were obtained in the mass-to-charge ratio (m/z) between 75 and 1200 amu for both ionisation modes. The data were collected and processed using Xcalibur 2.1.0 software (Thermo Fisher Scientific, Bremen, Germany).

Tissues were homogenized in CHCl₃ (10 ml), then MeOH (5 ml), H₂O (2.5 ml) and HCl 2 N were added and the lipids extracted by vigorous mixing. The organic layer was recovered and dried under N₂. The resulting lipid fraction was pre-purified by solid-phase extraction over silica, and NAPEs were eluted with CHCl₃-MeOH (6:4, v/v). The resulting lipid fraction was analyzed by HPLC-MS using a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to an Accela HPLC system (Thermo Fisher Scientific). Analyte separation was achieved by using a C-18 Kinetex C-18 column (5 μm, 4.6 × 150 mm; Phenomenex, Utrecht, Netherlands) and a C-18 pre-column. Mobile phases A and B were composed of MeOH-H₂O-NH₄OH (75:25:0.1, v/v/v) and MeOH-NH₄OH (100:0.1, v/v), respectively. The gradient (0.5 ml min⁻¹) was as follows: from 100% A to 100% B in 15 min, followed by 10 min at 100% B and subsequent re-equilibration at 100% A. Mass spectrometry analysis in the negative mode was performed with an ESI source. The measurement of eCB were generated as previously described32 (link), and the data were normalized to tissue sample weight.

Cleavage of colistin and production of the concomitant metabolites in the aforementioned reaction were analyzed by high-performance liquid chromatography (HPLC) using a Kinetex C-18 column (Phenomenex, USA) and an Alltech 3300 Evaporative Light Scattering Detector (ELSD) (BUCHI, Switzerland). The mobile phase comprised a gradient of 24 to 29% acetonitrile (J.T. Baker) and 0.021 to 0.024% trifluoroacetic acid (Sigma-Aldrich) with a flow rate of 1.0 mL/min for 10 min. Detection was performed at 60 °C and 1.5 mL/min nitrogen gas flow by ELSD. A linear ion trap mass spectrometer (LTQ-Velos, Thermo Scientific, USA) with a nano sprayer coupled to the Accela HPLC system (Thermo Scientific) was used for LC–MS/MS analysis [48 (link)]. The Xcalibur software v. 2.1 (Thermo Scientific) was used for tandem mass spectral data analysis. A chemically synthesized authentic compound of the cyclic peptide moiety of colistin (Peptron, Korea) was used to confirm the chemical structure of the metabolite using MS/MS fingerprinting.

7α,25OHC levels were analyzed using a validated HPLC-MS method^{37 (link)}. Briefly, serum samples or tissue homogenates were placed in glass vials containing deuterated internal standards and dichloromethane, methanol and water in the presence of butylated hydroxytoluene (10 µg) and ethylenediaminetetraacetic acid (20 ng) to prevent oxidation. Following extraction, the lipid fraction was purified by solid phase extraction to remove cholesterol. The oxysterol fraction was analyzed by HPLC-MS using an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher) coupled to an Accela HPLC system (Thermo Fisher). Chromatographic separation was performed using an Ascentis Express C-18 column (2.7 µm, 150 × 4.6 mm, Sigma), kept at 15 °C. Mobile phase was a gradient of methanol and water containing acetic acid. Calibration curves were prepared in the same conditions.