Agilent 1200 series high

Quantification of GAMG in the biological samples was performed using an Agilent 1200 series high-performance liquid chromatography with tandem Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). An RRHD Eclipse Plus C18 column (50 mm × 2.1 mm, i.d., 1.8 μm) was employed to separate GAMG and GA from complex biomatrices. Injection volume was 10 μL. Elution was fulfilled with an isocratic mobile phase (delivered at 0.2 mL/min) consisting of methanol (0.1% formic acid)-10% methanol (85:15, v/v). The autosampler was maintained at 4 °C. GAMG and internal standard (IS) were quantified via a positive electrospray ionization interface. They were monitored by multiple reaction monitoring (MRM) mode with transitions of m/z 647.4 → 453.5 for GAMG, m/z 471.3 → 189.3 for GA and m/z 514.4 → 189.4 for IS. The compound-dependent parameters, such as collision energy and fragmentor voltage, were optimized as 15 eV and 148 V for GAMG, 41 eV and 144 V for GA and 32 eV and 90 V for IS, respectively. High purity nitrogen served as both the nebulizing and drying gases. Other parameters of the mass spectrometer were also optimized and set, including spray voltage (3.2 kV), ion source temperature (340 °C), nebulizer pressure (30 psi), and capillary temperature (270 °C).

The liquid chromatography-mass spectrometry (LC-MS) analysis was performed on an Agilent 1200 Series high-performance liquid chromatography (HPLC) (Agilenttechnologies CO, USA) connected to a linear ion trap mass spectrometer 4000 QTRAP system (AB Sciex CO., Canada) equipped with an ESI TurboV source. Chromatography was carried out on a column ZORBAX Eclipse XDB-C18 (4.6 x 150 mm, I.D.-5 μm) (Agilent technologies CO., USA). The mobile phase consisted of 1 % Formic acid (Solvent A) and Acetonitrile (Solvent B) in the gradient mode as follows : 0–8 min 0–25 % B; 8–12 min 25–35 % B; 12–17 min 35–50 % B; 17–25 min 50–100 % B; 25–28 min 100–0 % B at flow rate of 1.0 ml/min at 30 °C. Conditions of the MS analysis were as follows: positive ion mode, spectra range from m/z 100 to 500, nebulizer; 70.0 psi. Molecular weight identification of purified fraction (PA-6) and berberine was carried out by electrospray ionization mass spectrometry (ESI-MS).

Racemic PCB149 (99.9%) was provided by Dr. Ehrenstorfer GmbH (Germany). The racemate was separated and prepared on a Lux Cellulose-2 column (250 × 4.6 mm, 5 μm, Phenomenex, Torrance, CA) using an Agilent 1200 series high performance liquid chromatography (HPLC) instrument (Wilmington, DE) with 100% n-hexane as the mobile phase at a flow rate of 1.0 mL/min. The enantiomers (−)-PCB149 and (+)-PCB14948 (link) were repeatedly collected separately, concentrated to dryness using a nitrogen-evaporator (Hangzhou Allsheng Instruments company, China) and then dissolved in acetone (Fisher). The purities and concentrations of the isomers were determined by gas chromatography-mass spectrometry (GC-MS).
An Agilent 7890A/5975C GC-MS system equipped with a Chirasil-Dex capillary column (25 m × 0.25 mm; I.D. 0.25 μm df) from Agilent was used for purity and concentration determinations, and the oven temperature was programmed as follows: 60 °C for 2 min, 60–150 °C at 10 °C·min⁻¹ (held for 5 min), 150–180 °C at 1 °C·min⁻¹ (held for 22 min). The SIM ions were m/z 360 (quantification ion), 362, and 3587 (link). The purities of (−)-PCB149 and (+)-PCB149 were higher than >98.0%.