Inertsustainswift c18 column

Samples were analyzed using a quantitative HPLC system (LaChrom, Hitachi Ltd., Ibaraki, Japan), and the measurement conditions have been previously described [16 (link)]. The samples were analyzed using a quantitative HPLC system (LaChrom, Hitachi Ltd.) by comparison with standard compounds using an InertSustainSwift C18 column (4.6 × 150 mm) (GL Sciences Inc. Tokyo, Japan). The analysis conditions were as follows: UV detection, 280 nm (0–7.5 min) and 370 nm (7.5–60 min); column oven temperature, 40 °C; flow rate, 1.0 mL/min; mobile phases, (A) 0.1 % formic acid/water and (B) 0.1 % formic acid/acetonitrile; and gradient condition, 0 min → (100:0) → 2 min (90:10) → 15 min (65:35) → 20 min (65:35) → 20.10 min (5:95). For HPLC analysis, two hot water extractions were performed, and two injections were made on the same extract, for a total of four replicates of data.

LC–MS/MS analysis was performed using an Agilent Technologies (Boeblingen, Germany) Model 1100 series LC system and an Applied Biosystems (Foster City, CA, USA) API 4000 triple quadrupole mass spectrometer, with LC separation on an InertSustain swift C18 column (100 mm × 2.1 mm, particle size 5 μm; GL Sciences, Tokyo, Japan). The LC conditions included a column temperature of 30 °C, a mobile phase consisting of distilled water/acetonitrile (55/45, v/v), and a flow rate of 0.2 mL min⁻¹. Electrospray ionization (ESI)–MS/MS conditions included: a turbo ion spray voltage of –4500 V; a turbo ion spray temperature of 450 °C; ion source gas GS1 and GS2 flows of 20 and 11 L min⁻¹, respectively; a curtain gas (CUR) flow of 10 L mL⁻¹; and a collision gas (CAD) flow of 4.0 L min⁻¹. Multiple reaction monitoring (MRM) transitions in negative ion mode and other parameters, including dwell time, declustering potential (DP), entrance potential (EP), collision energy (CE), and collision cell exit potential (CXP), are shown in Supplementary Table S2. Quantification was performed by MRM of the deprotonated precursor molecular ions [M−H]⁻ and the related product ions for each compound. Quadrupoles Q1 and Q3 were set at unit resolution (Table S2). Analyst Software 1.6.2 (Applied Biosystems) was used for LC–MS/MS data analysis.