Maxis hd

Separation was performed by UPLC (Dionex UltiMate 3000 System, Thermo Scientific, USA) and screened with ESI-MS. The LC system was comprised of an Acclaim™ RSLC 120 C₁₈ column (2.2 µm, 2.1 × 100 mm; Thermo Scientific, USA). The mobile phase was composed of solvent A (0.1% formic acid-water) and solvent B (acetonitrile) with a gradient elution (0-1 min, 98–90% A; 1–9 min, 90–80% A; 9–16 min, 80–70% A; 16–20 min, 70–2% A). The flow rate of mobile phase was 0.3 mL/min. The column temperature was maintained at 40°C, and the sample manager temperature was set at 4°C.
Mass spectrometry was performed on a Quadrupole-Time of Flight Mass Spectrometer (QTOF-MS; maXis HD, Bruker, Germany) using an ESI source. The scanning mass range (m/z) was from 50 to 1500 with spectra rate of 1.00 Hz. The capillary voltage was set at 3500 V and 3200 V (positive and negative mode, resp.). The pressure of the nebulizer was set at 2.0 Bar, the dry gas temperature at 230°C, and the continuous dry gas flow rate at 8 L/min.
At the beginning of the sequence, we ran five quality control (QC) samples to avoid small changes in both chromatographic retention time and signal intensity. The QC samples were also injected at regular intervals (every six samples) throughout the analytical run.

Ishige okamurae was collected in April 2016 in Seongsan, Jeju Island, South Korea. IO extract was prepared and IPA isolated using a previously described method [12 (link)]. Briefly, 50% ethanolic extract of IO was fractionated using centrifugal partition chromatography (CPC 240, Tokyo, Japan) and further purified using semipreparative HPLC column (YMC-Pack ODS-A; 10 mm × 250 mm, 5µm) to obtain IPA. The identity of IPA (99% of purity) was verified using MS fragmentation of m/z 1986.26 using ultrahigh resolution Q-TOF LC-MS/MS coupled with an electrospray ionization (ESI) resource (maXis-HD; Bruker Daltonics, Breman, Germany) at the Korea Basic Science Institute (KBSI) in Ochang, South Korea. According to a previously validated method [12 (link)], the IO extract used in this study had 1.81% ± 0.362 IPA.

Chromatography was carried out on an UHPLC system (Dionex Ultimate 3,000; Thermo Scientific, Waltham, MA, United States) equipped with an Acclaim™ RSLC 120 C18 chromatographic column (2.1 mm × 100 mm, 2.2 μm) and the column temperature was maintained at 40°C. The mobile phase comprised 0.1% formic acid–water (v/v, phase A) and acetonitrile (phase B) and the flow rate was 0.3 mL/min. Injection of each sample (2 μL) was analyzed after equilibration. The gradient elution program for urine samples was: 0–3 min, 5%–17% B; 3–17 min, 17%–18% B; 17–20 min, 18%–95% B; 20–22 min, 95%–5%; 22–26 min, 5%. The gradient elution program for serum samples was: 0–4 min, 10%–75% B; 4–12 min, 75%–80% B; 12–14 min, 80%–95% B; 14–16 min, 95%–10%; 16–19 min, 10%.
MS with an eletrospray ionization source (ESI) was carried out on a Q/TOF mass spectrometer (maXis HD; Bruker, Billerica, United States) in positive and negative ion modes to analyze urine and serum samples, respectively. The main MS parameters were: spectra rate = 1 Hz; scanning range of m/z = 50–1,500; capillary voltage = 3,500/3200 V (positive/negative ion modes); pressure of nebulizer gas = 2.0 bar; dry-gas temperature = 230°C; flow rate = 8 L/min.