6460 triple quadrupole system

The stereotaxic apparatus used for rat brain surgery was a Model 900LS Small Animal Stereotaxic Instrument, Lazy Susan (KOPF, TUJUNGA, CA, USA). The aCSF was filtered using a 1000 mL Corning Vacuum Filter/Storage Bottle System, 0.22 μm Pore 5 (Corning, NY, USA). Syringes containing aCSF were Hamilton 1.0 mL 1001RN SYR (22/2′′/2) syringes (Reno, NV, USA). EICOM (Kyoto, Japan) products, such as a syringe pump (ESP-64), guide cannula (AG-8, length 8 mm), dummy cannula (AD-8, length 8 mm), cap nut (AC-5), anchor screw (AN-3), joint Teflon tube (JT-10), and the microdialysis probe (FX-I-8-02, shaft length 8 mm, membrane length 2 mm) were used for microdialysis. Microdialysis samples were collected in a Hard-Shell^® High-Profile 96-Well Semi-skirted PCR Plate (BIO-RAD, HSS9601, Contra Costa County, CA, USA) and sealed using applied biosystems MicroAmp Optical Adhesive film (ThermoFisher, Waltham, MA, USA).
The HPLC instrument used was 1260 liquid chromatography, and the tandem mass spectrometer (MS/MS) was the 6460 triple quadrupole system, both manufactured by Agilent (Santa Clara, CA, USA). The analytical column and guard column were Atlantis™ T3 3 μm (2.1 × 100 mm²) and ACQUITY UPLC^® BEH HILIC 1.7 μm VanGuard™ Pre-column (2.1 × 5 mm²), respectively, and were purchased from Waters Corporation (Milford, MA, USA).

Pyridinic compounds were analyzed by TLC, HPLC (Agilent, series 1100) or UV2600 UV-Vis spectrophotometer. The samples were subjected to TLC analysis using silica gel 60 F254. The composition of the eluent was chloroform:methanol:acetic acid = 10:1:0.1 (vol/vol/vol). After the separation process, the plate was dried and observed at 254 nm. The pyridinic compounds in resting cell reactions were determined by HPLC using an Eclipse XBD-C₁₈ reverse-phase column (5 μm; 4.6 × 150 mm; Keystone Scientific, Bellefonte, PA) with a DAD detector. The mobile phase consisted of 85:15 (vol/vol) methanol: 1 mM H₂SO₄ at a flow rate of 0.5 mL/min at 30 °C. The mobile phase for the samples of NaaA catalyzed reaction consisted of 80% (vol/vol) 20 mM ammonium acetate and 20% (vol/vol) methanol at a flow rate of 1.0 mL/min at 30 °C. LC-MS analysis was performed on an Agilent 6460 triple quadrupole system equipped with electrospray ionization (ESI) sources in 20% (vol/vol) methanol and 80% (vol/vol) deionized water (18 MΩ/cm) (0.05% formic acid [vol/vol]) at a flow rate of 0.2 mL/min with the same Eclipse XBD-C₁₈ reverse-phase column. All samples were treated with the addition of 9 volumes of methanol at 4 °C for 10 min followed a centrifugation at 12,000 × g for 2 min. Then the samples were filtered through a 0.22-μm Sartorius filter prior to HPLC and LC-MS analysis.

Anthocyanins and its derivatives were determined using LC-ESI-QQQ (6460 Triple Quadrupole System, Agilent, CA, USA) in combination with a UV detector (1260 Infinity, Agilent, CA, USA). Both positive and negative ion electrode mass spectra and tandem mass spectra were recorded. The injection volume was 3.0 µL. Separations of anthocyanins and anthocyanidin aglycones were performed on analytical column Zorbaz Eclipse C18 (2.1 × 50.0 mm, 1.8 μm, Agilent, CA, USA). The mobile phase consisted of water (solvent A) and acetonitrile (solvent B) each containing 0.1% formic acid. The flow rate was 0.3 mL/min and the gradients between the time points were as follows: 0–5 min, 0–30%B; 5–8 min, 30–75%B; 8–25 min, 75–100%B. UV-Visible absorption spectra of anthocyanins were recorded at 535 nm. The MS conditions were as follow: gas temperature, 275 °C; gas flow, 8 L/min; Nebulizer, 45 psi; sheath gas temperature, 350 °C; sheath gas flow: 12 L/min; capillary: 3500 V (positive), 3500V (negative); Nozzle voltage: 500 V (positive), 500V (negative); and scan mass: 270–1000 (m/z) (positive).