Agilent zorbax eclipse plus c18

The content of obacunone, fraxinellone, and Dictamnine in the four Dictamni Cortex crude drug samples were quantified by LC/MS. Briefly, the crude drug samples dissolved in methanol (1 µL) was injected into an Agilent ZORBAX Eclipse Plus C18 column (1.8 µm, 3.0 × 100 mm) (Agilent Technologies, Santa Clara, CA, USA), equipped with Agilent ZORBAX Eclipse Plus C18 (1.8 µm, 3.0 x 10 mm) guard column (Agilent Technologies). Gradient elution with solvent system 0.1% formic acid in water and 0.1% formic acid in methanol was used. The mixture of obacunone (Chengdu Must Bio-Technology Co., Ltd., Chengdu, China) (retention time: 11.4 min) and fraxinellone (Chengdu Must Bio-Technology Co., Ltd.) (retention time: 11.9 min) was prepared as the standard and analyzed. Dictamnine (Chengdu Must Bio-Technology Co., Ltd.) (retention time: 5.7 min) was also used as a standard and analyzed. Chemical markers were identified based on the retention time and the UV absorbance that resemble the standards.

Hepatic NAD⁺ and NADH extraction and subsequent quantitative analysis were performed according to the methods of Goldman et al. [31 (link)]. Frozen liver samples (typically 180–200 mg) were homogenized in 2.0 mL of 0.6 M HClO₄ on ice and precipitated by the addition of 1 M NaOH with shaking for 2 min. The mixture was centrifuged at 12,000× g for 5 min at 4 °C and filtered using 0.45-μm membrane filters (Millipore, Bedford, MA, USA). After that, metabolites were analyzed using a HPLC system (UltiMate™ 3000; Thermo Fisher Scientific, Waltham, MA, USA) equipped with a UV detector and a reverse-phase column (Agilent ZORBAX Eclipse Plus C18; 5 μm, 250 × 4.6 mm; Agilent Technologies, Palo Alto, CA, USA). Chromatography conditions were set as follows: UV detection, 254 nm; injection volume, 10 μL; column temperature, 40 °C; mobile phase, consisting of 215 mM KH₂PO₄, 1.2 mM tetrabutylammonium bisulfate, and 10% methanol; flow-rate, 1.0 mL/min. Peaks were identified by their retention time using authentic standards, and the contents of NAD⁺ and NADH in the liver samples were normalized to the wet weight of each liver sample.

Tandem ultra high-performance liquid chromatography- mass spectroscopy (UHPLC-ESI-MS) analyses allowed us to determine the detection level and the amounts of zinniol in different A. dauci cultures extracts. Dried extracts of A. dauci cultures were extemporaneously dissolved in ethyl acetate/methanol (50∶50, v/v) at a working concentration of 6.67 mg mL⁻¹ and filtered through a 0.2 µm nylon membrane prior to immediate analysis by UHPLC. These analyses were performed using an Accela High Speed LC System (ThermoFisher Scientific) consisting of a quaternary pump with an online degasser, autosampler, PDA detector and a TSQ Quantum Access MAX triple stage quadrupole mass spectrometer with an ESI interface. The chromatographic analysis was achieved on a Agilent Zorbax Eclipse Plus C₁₈ reversed-phase analytical column (2.1×100 mm×1.8 µm). An elution gradient of water (Milli-Q quality) and acetonitrile (LC–MS grade) was used. Two microlitres of each A. dauci culture extract or standard zinniol solution were injected using the partial loop injection mode (10 µL loop size). The PDA detector was set in the 200–500 nm wavelength range with two selected channels at 210 and 233 nm. Data were acquired and processed using the Xcalibur 2.0 software package (ThermoFisher Scientific). Standard zinniol solutions were freshly prepared to obtain five concentrations in the 0.05–5 mg mL⁻¹ range.