Atlantis dc18 column

For HPLC/MS measurements, an Agilent Technologies 1200 series (Santa Clara, CA) equipped with a G1379B degasser, G1312B binary pump SL, G1367C HiP-ALS SL autosampler, a G1314C VWD SL UV detector, G1316B TCC SL column oven, and a G1956B MSD mass selective detector was used. The mass spectrometer was operated in positive electrospray ionization mode. The analytes were separated on an Atlantis® dC18 column (5 μm, 4.6 × 250 mm, Waters) at 25 °C by using aqueous eluent (0.1% formic acid) and acetonitrile at a flow rate of 1.0 ml min⁻¹. The column was equilibrated with 7% acetonitrile in water (0.1% formic acid), and the following gradient was used for analysis: 0–2 min, 7% acetonitrile; 2–10 min, 7–100% acetonitrile; 10–12 min, 100% acetonitrile; 12–14 min, 7% acetonitrile. 10 μl of 300 μm HPLC purified 8f-FAD or 300 μm FAD solution for control, dissolved in water, were injected for each run.

Liquid chromatographic separation was performed using an Agilent 1260 autosampler (Agilent Technologies Inc, Santa Clara, CA, USA). The temperature of the autosampler was maintained at 7°C, and 5 μL of each reconstituted sample was separated into components using a Waters Atlantis dC18 column (50 × 2.1 mm, 3 μm; Milford, MA, USA) at 35°C. An isocratic mobile phase was used, containing 10 mM ammonium formate (pH 2.7) and methanol (20:80, [v/v]), at a flow rate of 0.3 mL per min.
The components eluted from the column were delivered into an API 4500 triple quadrupole mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, CA, USA) with electrospray ionization in positive ion mode for ion production. The ion spray voltage was set at 5.5 kV, and the source temperature was set at 550°C. Multiple reaction monitoring was performed using nitrogen as the collision gas. Analytes were detected by monitoring the transitions 436.2 (Q1) → 291.0 (Q3) and 439.2 (Q1) → 294.0 (Q3) m/z, with a declustering potential of 28 V and collision energies of 23 V, for VST and IS, respectively. Nebulizer gas (Gas 1) at 40°C and heater gas (Gas 2) temperatures were both set at 70°C. For quantifying VST in the plasma samples, each peak area of VST was divided by that of the internal standard, and the ratio was compared with a calibration curve obtained using VST standard solution in the same manner.

The contents albiflorin, paeoniflorin, z-ligustilide, decursin, and nodakenin in the DJS water extract were analyzed using an HPLC instrument (Agilent Technologies, USA) with a Atlantis dC₁₈ column (4.6 × 250 mm, 5 μm; Waters, USA). The mobile phase consisted of the solvents, distilled water (A) and acetonitrile with 0.1% formic acid (B). The following gradient was used: 0 min, A : B 90 : 10 (v/v); 20 min, A : B 75 : 25; 25 min, A : B 75 : 25; 30 min, A : B 50 : 50; 45 min, A : B 20 : 80; and 60 min, A : B 0 : 100. The mobile phase flow rate was 1.0 mL/min, the column temperature was 30°C, the injection volume was 10 μL, and UV detection was at 230 and 330 nm.

The HPLC system consisted of 1525µ binary HPLC pump and 2707 autosampler, both from Waters. Retinol and α-tocopherol were quantified with Jasco FP-920 fluorimetric detector (Jasco Co.). The samples were separated in an isocratic system with a 5-µm Atlantis dC18 column equipped with guard cartridge (5 µm, 150 mm × 3.0, Waters). The mobile phase, consisting of acetonitrile methanol, 80:20 (v/v), was added at a flow rate of 1.5 mL/min, with 5-µL injection volume. The effluent was monitored by means of fluorimetric detection (λ_ex. = 340 nm, λ_em. = 472 nm for retinol, and λ_ex. = 290 nm, λ_em. = 330 nm for α-tocopherol and internal standard), and analyzed with Empower software.

Hgd tm1a −/− (n = 4) and Hgd tm1a −/+ (n = 4) mice were injected with ¹³C₆-HGA into the lateral tail vein, adjusted to body weight to achieve a final blood concentration of ~1 mmol/L. Under anaesthesia, venous tail bleeds were collected at time points post-injection, ranging from 2 to 60 min. Whole blood was centrifuged, and the supernatant removed and immediately frozen.
Non-targeted metabolic flux analysis was performed to trace metabolism of ¹³C₆-HGA. Metabolic profiling was performed using a published mass spectrometric technique (44 (link)). Briefly, plasma was diluted 1:9 plasma:deionized water and HPLC performed on an Atlantis dC₁₈ column (3 × 100 mm, 3 μm, Waters, UK) coupled to an Agilent (Cheadle, UK) 6550 quadrupole time-of-flight mass spectrometer. An accurate-mass compound database with potential association to HGA was generated for data mining using Agilent Pathways to PCDL. Data were mined for these compound targets with an accurate mass window of ± 5 ppm using ‘batch isotopologue extraction’ in ProFinder (build 08:00, Agilent). Isotopologue extraction investigates association with the injected ¹³C₆-HGA by examining the relative abundances of the M + 0–M + 6 isotopologues for compound targets.