Hypersil gold pfp column

The rice bran oil extracts were dissolved in methanol, were further filtered with a 0.45 μm filter and were analyzed by HPLC. δ-Tocotrienol standard sample was purchased from Sigma (Sigma Co., Ltd., St. Louis, MO, USA). HPLC analysis was performed on a Shimadzu Prominence series apparatus with a fluorescence detector (Prominence RF-20A/Axs, Shimadzu Co., Ltd., Kyoto, Japan). The excitation wavelength was 296 nm and the emission wavelength was 325 nm, which were operated on fluorescence detector. The Hypersil Gold PFP column (250 mm × 4.6 mm i.d., 5 μm, ThermoFisher Scientific, Waltham, MA, USA) was used as an analytical column. The eluents were methanol/H₂O (85:15, vol/vol) at a flow rate of 0.8 mL/min. All the results were recorded, and the peaks were integrated by the chromatography software Labsolution LC (Shimadzu, Kyoto, Japan). δ-Tocotrienol was purified by Shim-pack PREP-ODS (20.0 mm × 250 mm, 15 µm, Shimadzu, Kyoto, Japan). The eluents include acetonitrile, tetrahydrofuran, methanol, 1% ammonium acetate (684:220:68:28) and the flow rate was 8 mL/min. The δ-tocotrienol standard that was purchased from Chromadex, Inc. (Irvine, CA, USA) was used to determine the absorption peak of δ-tocotrienol.

For tricarboxylic acid metabolite isotope quantification, we used a derivatization method followed by UPLC-MS analysis using a LTQ-Orbitrap XL mass spectrometer [32 (link)]. Briefly, tumor samples were homogenized before adding acetonitrile and valproic acid (internal standard). After 1h at −20 °C, samples were centrifuged and supernatant recovered. The derivatization reaction was then conducted using 3-nitrophenylhydrazine, N′-ethylcarbodiimide hydrochloride, and pyridine. Following the reaction, the derivatized products were recovered by liquid-liquid extraction and the samples resuspended in methanol before injection on the LC-MS system. Separation was conducted on a hypersil gold PFP column (Thermo Fisher Scientific, Waltham, MA, USA) maintained at 40 °C using a gradient between water-acetonitrile (A) and acetonitrile (B), both acidified with acetic acid. Tricarboxylic acid metabolites (as well as their isotopic forms) were analyzed using the LTQ-Orbitrap XL analyzer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an APCI probe operated in positive mode. For each analyte, the signal (AUC) was normalized with the signal of the internal standard and further normalized with the sample weight. Based on glucose metabolism, for pyruvate and lactate we analyzed M+1, M+2, and M+3 isotopes, and for the other metabolites we analyzed the M+1, M+2, M+3, and M+4 isotopes.

Under isoflurane inhalation anesthesia, the spinal cords were collected from mice at ZT10 and then homogenized with 100 µL of 0.1 M HClO₄. The lysates were centrifuged at 12,000 ×g for 15 min, and the supernatants were used as the sample for LC/MS/MS analysis. NE was resolved using a Hypersil GOLD PFP column (150 mm × 2.1 mm, 50 mm, part no. 25402-152130, Thermo Fisher Scientific) and an AQUITY UPLC H-Class XHCLQT0100 system (Waters), consisting of a vacuum degasser, binary pump, thermostatically controlled column compartment, thermostatically controlled autosampler, and a diode array detector. Mobile phase A consisted of 0.1% formic acid/water; mobile phase B consisted of 0.1% formic acid/acetonitrile. A linear gradient was generated at 0.5 mL/min: 0.0 min, 0% A and 100% B, 2.0 min, 5% A, and 95% B; 3.0 min, 5% A, and 95% B; 7.0 min, 100% A, and 0% B (Waters application note). The injection volume was 10 μL. The column temperature was controlled at 35 °C and the autosampler compartment was set to 4 °C. An AQUITY TQ mass spectrometer controlled by MassLynx software (Waters) was operated in a selected reaction monitoring mode. The multiple reaction monitor was set at a mass-to-charge ratio (m/z) of 152–107 m/z (cone voltage: 50 V, collision voltage: 20 V).