Kinetex phenyl hexyl column

FLA and FLE were separated and identified according to previous methods [23 (link),31 (link)]. The crude flaxseed oil was extracted as follows: 500 mL of crude flaxseed oil mixed with 100 mL of silica gel was first loaded onto a glass column and subjected to flash column chromatography with several solvents: 500 mL of 100% hexane; 300 mL of 20% ethyl acetate (EtOAc) in hexane; 300 mL of 50% EtOAc in hexane; 300 mL of 100% EtOAc (A); and 300 mL of 10% methanol in dichloromethane (B). Fractions A and B, concentrated with crude powder, were concentrated in a rotary evaporator at 40 °C, the dry mixture was then dissolved in 100 mL of methanol and centrifuged at 8000 rpm for 15 min at 25 °C afterward. The supernatant was collected, concentrated, and stored for further purification. High-performance liquid chromatography (HPLC) was performed on an Agilent 1260 LC system (Agilent Technologies, Wilmington, DE, USA) with a manual injector with a photo-diode array (PDA) detector set at 214 nm and equipped with a reversed phase Kinetex^® Phenyl-hexyl column (250 mm × 21.2 mm, 5 μm, Phenomenex Inc. Torrance, CA, USA). The HPLC elution conditions were as follows: initial acetonitrile (ACN) to water ratio of 40%, final ACN ratio of 80%, elution time of 21 min, flow rate of 16 mL/min, sample loading of 12.5 mg at a concentration of 80 mg/mL (methanol).

Drug quantification by LC–MS/MS was based on the principles of isotope dilution [28 (link)]. ORDX SD rats (n = 5–7) were treated with DHED (100 μg/kg, p.o.) and then euthanized 0.25, 0.5, 1, 2, 4, and 8 h after treatment. The hypothalami were dissected from the harvested brains, followed by tissue homogenization (20% w/v, pH 7.4 phosphate buffer) and adding ¹³C-labeled internal standard (IS) (¹³C₆-E₂, 100 pg) for E₂ quantitation, as reported previously [16 (link),19 (link),28 (link)]. Liquid–liquid extraction was performed with 4 volumes of methyl tert-butyl ether. The organic layers obtained from the extractions were removed and evaporated under a nitrogen stream to yield samples for derivatization and subsequent LC–MS/MS analysis using electrospray ionization. Derivatization of the analyte and the IS was done by dansyl chloride [28 (link)]. The dansylated samples were centrifuged at 4500 rpm for 3 min, transferred to autosampler vials, sealed, and assayed using an LC–MS-8050 triple quadrupole tandem mass spectrometer connected to an LC-20AD liquid chromatograph (Shimadzu, Tokyo, Japan). Separation was performed according to our earlier publication using a Phenomenex (Torrance, CA, USA) Kinetex phenyl-hexyl column (50 mm × 2.1 mm i.d., 2.6 µm particles with 100 Ǻ pores), and quantification relied on selected-reaction monitoring [28 (link)].

The high-resolution tandem mass spectrometry (HRMS/MS) analyses were carried out by the regional platform MALLABAR at Institut Méditerranéen de Biodiversité et d’Ecologie marine et continentale IMBE (Marseille, France). The samples were dissolved in 1 mL of methanol (LCMS grade, CarloErba, Val de Reuil, France) and filtered with 0.22 µm PTFE syringe filters (Restek, Bellefonte, PA, USA). The HRMS/MS analysis was performed on a Dionex Ultimate 3000 UHPLC system (Thermo Scientific, Waltham, MA, USA) coupled to a QtoF Bruker Impact II mass spectrometer (Bruker, Billerica, MA, USA). For the separation, a Phenomenex Kinetex phenyl hexyl column (1.7 µm, 150 × 2.1 mm) (Phenomenex, Torrance, CA, USA) was used for elution. A gradient solvent system with ACN (MS grade, CarloErba, Val de Reuil, France) (phase A) and Milli-Q water (phase B), each of them containing 0.1% formic acid (FA) (analytical grade 99% purity, CarloErba, Val de Reuil, France), was used for the analyses. The extract was eluted by a linear gradient from 0 to 100% B for 8 min at a flow rate of 0.5 mL/min. Data were acquired in positive mode (ESI⁺; 20–40 eV). The main MS data acquisition parameters were: the MS1 spectra acquisition range from 20 to 1200 Da, the collision energy was set to 40 eV and the acquisition speed was 4 Hz. The 5 major precursors in MS1 were selected for the recording of the MS2 spectra.

Both 8-hydroxy-2′–deoxyguanosine (purity ≥ 98%) and 5-hydroxymethyluracil (purity 97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and 8-hydroxy-2′-deoxyguanosine-15N5 (purity 95%) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). 8-hydroxyguanosine (purity ≥ 98%), 8-isoprostane (purity ≥ 99%), and 8-isoprostane-d4 (purity ≥ 99%) were obtained from Cayman Chemical (Ann Arbor, MI, USA). 8-hydroxyguanosine-13C,15N2 (purity 98%), 4-hydroxynonenal (purity ≥ 98%), and 4-hydroxynonenal-d3 (purity 97%) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Methanol (LC-MS grade) was purchased from Burdick and Jackson (Muskegon, MI, USA). Formic acid and ammonium hydroxide were obtained from Sigma-Aldrich (St. Louis, MO, USA). Strata-X-A cartridges (33 µm Polymeric Strong Anion, 200 mg/3 mL; 8B-S123-FBJ), analytical columns (Kinetex phenyl-hexyl column, 100 mm × 4.6 mm, 2.6 µm particle size; 00D-4495-E0), and Kinetex C18 column, (100 mm × 4.6 mm, 2.6 μm particle size, 00D-4462-E0) were purchased from Phenomenex (Torrance, CA, USA).

THC, THC-COOH, THC-OH, THC-D3, THC-COOH-D3, and THC-OH-D9 were purchased from Cerilliant (Round Rock, TX, USA). A second set of THC, THC-OH, and THC-COOH was purchased from Lipomed (Cambridge, MA, USA) to be used for quality control samples. Water and acetonitrile (LC–MS-grade) were obtained from Millipore (Burlington, MA, USA). Dansyl chloride, sodium bicarbonate, sodium carbonate, acetic acid, and formic acid (LC–MS-grade) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Captiva EMR-Lipid columns (1 mL, 40 mg) were purchased from Agilent Technologies (Santa Clara, CA, USA). Chromatography was performed with a Kinetex Phenyl Hexyl column (3.0 × 50 mm, 2.6 μm) purchased from Phenomenex Inc. (Torrance, CA, USA).