Waters 2690 hplc system

The gastrointestinal digest was re-dissolved in 10 mg/mL elution buffer (20 mM sodium phosphate buffer with 300 mM NaCl, pH 7.3) and filtered (0.45 µm), before being applied to a gel filtration Superdex^® Peptide 10/300 GL (30 cm × 10 mm, 13 μm, GE Healthcare Life Sciences, Chicago, IL, USA). Elution was carried out at room temperature on a Waters 2690 HPLC system (Waters Corporation, Milford, MA, USA), equipped with an automatic sample injector and 2998 UV photodiode array (PDA) detector, at a flow rate of 0.5 mL/min. The absorbance of each fraction was measured at a wavelength of 214 nm. After collection, fractions were concentrated, reconstituted in assay buffers, then the bioactivities were measured, as described above.

The FMS concentrations in biological samples were determined by a modification of the previously reported LC-MS/MS assay [22 ]. Briefly, 200 μL of acetonitrile and 50 μL of the internal standard solution (BR-A-563 100 ng/mL in acetonitrile) were added to 50 μL of the thawed biological samples and mixed on a vortex mixer for 1 min. The sample mixture was then centrifuged for 10 min at 15,000 × g at 4 °C. The supernatant was transferred to a polypropylene tube and diluted with the same volume of distilled water. A volume of 10 μL was injected into LC-MS/MS.
The LC-MS/MS comprised API 2000 mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) coupled with Waters 2690 HPLC system (Waters, Milford, MA). Fimasartan was separated on a Kinetex C₁₈ column 50 × 2.10 mm, i.d., 2.6 μm (Phenomenex, Torrence, CA). The isocratic mobile phase composition was a mixture of acetonitrile and 0.05 % formic acid in water (40:60, v/v). The flow rate of the mobile phase was set at 0.2 mL/min, and the column oven temperature was 30 °C. The mass spectrometer was operated using electron spray ionization (ESI) with positive ion mode. The transition of the precursors to the product ion was monitored at 502.3→207.0 for fimasartan, and 526.4→207.2 for the internal standard (BR-A-563).

The instruments used were a Waters 2690 HPLC system coupled to a 2996 PDA detector (Waters, Milford, MA). B-FAHF-2 tablets were ground, and a 22.5 mg/mL solution was prepared using 1:1 ratio of mobile phase mixture. The B-FAHF-2 solution was centrifuged at 10,000 rpm for 10 minutes. 10μL of the supernatant was injected into the HPLC system and separated on a ZORBAX SB-C18 (4.6 × 150 mm, 5 μm) column (Agilent, Santa Clara, CA). The mobile phase A was made of 0.1% of formic acid aqueous solution. Mobile phase B was acetonitrile. The separation was performed at 1 min/mL flow rate following a linear gradient elution of 2–25% mobile phase B in 45 min, 25–35% B in the following 25 min, 35–55% in the next 15 min, 55–75% in another 10 min. This mobile phase composition was maintained for 5 min and then rapidly switched to 2% mobile phase B. An equivalent amount of FAHF-2 formula, 99 mg/mL, was also prepared following the same procedure as for B-FAHF-2. 10μL of FAHF-2 supernatant was analyzed on the HPLC system. Data was collected and processed with Waters' Empower software.

cwMPs were isolated from C. neoformans cells as described previously (30 (link), 53 (link)). N-linked glycans were released from the purified cwMPs using PNGase F (New England Biolabs) and were purified over a Carbograph Extract-Clean (Grace) column. The N-glycans were labeled with 2-aminobenzoic acid (2-AA; Sigma) and purified using a Cyano Base cartridge (Bond Elut-CN-E; Agilent) (100 mg) to remove excess 2-AA. Purified N-glycans were reacted with α-1,2 mannosidase (α-1,2 MNS; Prozyme) and subsequently with α-1,6 mannosidase (α-1,6 MNS; New England Biolabs). To remove enzymes, the N-glycans were purified using a 30K Microcon device (Millipore). 2-AA-labeled oligosaccharides were analyzed with a Waters 2690 HPLC system and a 2475 fluorescence detector with excitation and emission wavelengths of 360 nm and 425 nm, respectively. Data were collected using Empower 2 software (Waters). For MALDI-TOF analysis, the matrix solution was prepared as previously described (53 (link)) and was mixed with samples of equal volume. Mixed glycan samples were spotted on a MSP 96 polished-steel target (Bruker Daltonics). Crystallized samples were analyzed using a Microflex mass spectrometer (Bruker Daltonics).