Agilent 6410 triplequad lc ms system

Extraction, purification and measurement of IAA were performed as described in Lu et al. [58 (link)] with minor modifications. Briefly, freeze-dried materials were homogenized by TissueLyser (Qiagen), then extracted with methanol containing 1% acetic acid. D2-IAA (Olchemim) was added to the samples prior to methanol extraction. Solid-phase extraction was performed with Oasis HLB cartridge columns. Samples were subjected to liquid chromatography equipped with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS, Agilent 6410 TripleQuad LC/MS system, Agilent). LC conditions and MS settings are described in Lu et al. [58 (link)].

A fully expanded leaf (5th leaf from the top) was sampled and immediately packed in Eppendorf tubes and frozen in liquid nitrogen before being stored in a −80°C freezer until processed for analysis. The ABA content was determined as described in Yan D. et al. (2016 (link)). The samples were centrifuged to remove debris, and the pellet was washed twice. The supernatant was evaporated in a SpeedVac, reconstituted in 1 ml of 1% (v/v) acetic acid, and purified by solid phase extraction using Oasis HLB, MCX, and WAX cartridge columns (Waters Limited, Mississauga ON, Canada). The solvent was removed under vacuum and subjected to the LC-ESI-MS/MS analysis (Agilent 6410 TripleQuad LC/MS system). A Liquid Chromatography (Agilent 1200 series) equipped with a 50 × 2.1 mm, 1.8 μm Zorbax SB-Phenyl column (Agilent) was used with a binary solvent system comprising 0.01% (v/v) acetic acid in water (Solvent A) and 0.05% (v/v) acetic acid in acetonitrile (Solvent B). The separations were performed using a gradient of increasing acetonitrile content with a flow rate of 0.2 ml min⁻¹. The gradient was increased linearly from 3 to 50% B over 15 min. The retention time for the ABA was 14.0 min.

The quantification of baicalin, baicalein, wogonoside, and wogonin was carried out using an Agilent 6410 Triple Quad LC/MS system. Root samples of S. baicalensis were collected and homogenized to obtain a fine powder, which was then subjected to ultrasonic extraction with 70% (v/v) ethanol at 60°C for 1 h. The obtained extracts were centrifuged at 10,000 g at 4°C for 15 min and subsequently filtered through a Millipore Millex-HV filter (Merck Millipore, Billerica, MA, USA). To determine the flavone content, an absolute quantification method with external standards was employed. Calibration curves were constructed using standard solutions of baicalin, baicalein, wogonoside, and wogonin at known concentrations. The analytical parameters of the Agilent 6410 Triple Quad LC/MS were as follows: column, Waters T3 column (2.1 mm × 100 mm, 3 μm); oven temperature, 350°C. The mobile phase contained D: water (containing 0.1% [v/v] formic acid); C: acetonitrile; flow rate, 0.3 mL/min; column temperature, 50°C; and injection volume, 5 μL.