Lcq deca

Mass spectrometric analysis was performed on an ion trap mass spectrometer equipped with an electrospray ion source ESI (LCQ-DECA, Thermo Fischer Scientific, San Jose, CA, USA). The mass spectrometer was coupled online with a and autosampler (Thermo Fischer Scientific, San Jose, CA, USA) and a LC-pump (Surveyor MS Pump, Thermo Fischer Scientific, San Jose, CA, USA). Samples were dissolved in methanol ((25 µg/µL) and 5µL were loaded onto a Waters Symmetry RP-C18 column (150 mm × 1 mm i.d., 100 Å, 3.5 µm). Separation was achieved thermosetting the column at 25 °C with a linear gradient of H₂O + 1% FA and ACN + 1% FA at 50 μL/min. Elution was performed, increasing solvent B from 5% to 15% in 25 min, 25% in 40 min, 30% in 45 min, and 55% in 55 min. Full scan mass spectra were acquired in negative ion mode in the m/z range 150–2000. ESI ion source operated with 220 °C capillary temperature, 30 a.u. sheath gas, −3.5 kV source voltage and −18 V capillary voltage. Mass spectrometric analysis was performed by the data-dependent method with normalized collision energy of 30 a.u. and activation Q set as 0.250. Mass calibration was achieved with a standard mixture of caffeine (Mr 194.1 Da), MRFA peptide (Mr 524.6 Da), and Ultramark (Mr 1621 Da). Data acquisition and data analyses were performed with the Xcalibur v. 1.3 Software.

The MS analyses were performed at a Thermo LCQ-Deca. Synthetic LMWH constructs and intermediates were directly diluted in 200 μl of 9:1 MeOH/H₂O. A syringe pump (Harvard Apparatus) was used to introduce the sample by direct infusion (35 μl/min). Experiments were carried out in negative ionization mode with the electrospray source set to 5 KV and 275 °C. Sulfated oligosaccharide (1 μl) was diluted in a different working solution containing 200 μl of 70% acetonitrile and 10 mM imidazole. Experiments for sulfated oligosaccharides were carried out in negative ionization mode with the electrospray source set to 2 KV and 200 °C. The automatic gain control was set to 1 × 10⁷ for full scan MS. The MS data was acquired and processed using Xcalibur 1.3.

Optical rotations were determined on a Perkin-Elmer 341 polarimeter at 20 °C. UV spectra were performed on a Shimadzu UV-2450 spectrophotometer, and ECD spectra were performed on an Applied Photophysics Chirascan spectrometer. Infrared spectra (IR) were measured on a Bruker Tensor 37 infrared spectrophotometer. NMR experiments were carried out on a Bruker AM-400 spectrometer at the temperature thermostatically controlled at 25 °C. Exact mass measurements and molecular formulas were obtained from ESIMS and HR-ESI-MS using a Finnigan LCQ Deca and a Thermo Scientific LTQ Orbitrap XL spectrometers, respectively. Semi-preparative reversed-phase (RP) HPLC was performed with a YMC-pack ODS-A column (10 × 250 mm, S-5 μm) or a Phenomenex Lux cellulose-2 chiral column (10 × 250 mm, 5 μm) under Shimadzu LC-20 AT equipped with a SPD-M20A PDA detector. Column chromatography (CC) was performed on RP-C₁₈ silica gel (S-50 μm, 12 nm, YMC Co., Ltd.), MCI gel (CHP20P, 75–150 μm, Mitsubishi Chemical Industries Ltd.), Sephadex LH-20 gel (Amersham Biosciences), and silica gel (300–400 mesh, Qingdao Haiyang Chemical Co., Ltd.). For RP-HPLC and CC, the analytical grade solvents (Guangzhou Chemical Reagents Company, Ltd.) were employed.

Analysis of aSL‐C22:0₁₃ was done with a Thermo LCQ Deca by the use of RP18 solid phase (150×2 mm, Phenomenex Luna) and solvents 1) methanol with 0.1 % formic acid and 2) water with 0.1 % formic acid as gradients. LC–MS analysis was done with a Shimadzu LC‐10‐AD HPLC system (Shimadzu Europe GmbH, Germany) connected to a quadrupole mass spectrometer (Waters, Milford, MA). Molecules were identified by their native molecular masses after ESI (electrospray ionization) without collision. The results were consistent with previous reports.23, 39