High-performance gel permeation chromatography (HPGPC) was used to assess weight-averaged molecular weights of the enzyme products with an LC-10Avp system (Shimadzu Company, Japan) and a TSK-gel G-3000 PWXL column (7.8 × 300 mm). The UPLC-MS method was performed using a Waters Acquity H-Class UPLC system linked to an ESI-MS mass spectrometer (amaZon speed ETD, Bruker, Germany) in the negative ion mode. Operating parameters were as follows: capillary voltage, 4.5 KV; capillary temperature, 200°C; nebulizer gas, 2 bar; dry gas, 6 l/min; scan range, 100–1000. RG oligosaccharide separation was carried out on an Acquity UPLC BEH Amide column (1.7 μm, 2.1 mm × 150 mm) at 35°C with a flow rate of 0.3 ml/min mobile phase. The mobile phase consisted of ACN and H2O in a ratio of 20/80 (v/v) for mobile phase A, 80/20 (v/v) for mobile phase B, and pH 3.0 200 mM ammonium formate/50 mM formic acid buffer for mobile phase C. The run time for oligosaccharide separation was 60 min, and the elution procedure was as follows: concentration of C remained at 5% during the entire elution process for 0–30 min, 0%-20% A; 30–31 min, 20–35% A; 31–40 min, containing 35% A; 40–41 min, 35–0% A; 41–50 min, containing 0% A.
Acquity h class uplc system
Manufactured by Bruker
Sourced in Germany
The Acquity H-Class UPLC system is a high-performance liquid chromatography (HPLC) instrument developed by Bruker. It is designed to provide efficient and accurate separation and analysis of complex chemical samples. The system utilizes a unique column technology to achieve high-resolution, high-speed separations with improved sensitivity and reproducibility.
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2 protocols using acquity h class uplc system
1
Characterization of Enzyme Products by HPGPC and UPLC-MS
2
Inhibitor Stability in Mouse Plasma
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A 5 µL aliquot of compound solution (10 mM in DMSO) was added to 995 µL of Non Swiss Albino Mouse Plasma (DivBioScience, Breda, Netherlands) in sodium citrate to obtain a final concentration of 50 µM inhibitor in plasma. The mixture was gently shaken for 6 hours at 37°C.
Aliquots of 100 µL were taken at various time points (0, 30, 60, 120, 180 and 360 minutes) and diluted with 400 µL of cold methanol (Sigma-Aldrich, MO, USA) (stored at 4°C). The suspension was centrifuged at 400 x g for 5 minutes. Thereafter, 50 µL of the supernatant was diluted with 950 µL of methanol and analyzed with LC-MS/MS (Waters Acquity H-class UPLC system with ion trap mass spectrometer, Bruker Daltonics Esquire 3000 plus, and Agilent 1100
Series LC system). Samples were analyzed in triplicate and plotted against a standard curve (compound at 3.12-100 µM in mouse plasma). weeks. The osmotic minipumps were replaced after 2 weeks to obtain a total treatment period of 28 days. Mice that were not treated with oxaliplatin received an I.P. injection with saline at the same time. Body weight was measured twice a week and tumor growth was monitored three times a week with digital calliper measurements starting upon detection of palpable tumors.
Aliquots of 100 µL were taken at various time points (0, 30, 60, 120, 180 and 360 minutes) and diluted with 400 µL of cold methanol (Sigma-Aldrich, MO, USA) (stored at 4°C). The suspension was centrifuged at 400 x g for 5 minutes. Thereafter, 50 µL of the supernatant was diluted with 950 µL of methanol and analyzed with LC-MS/MS (Waters Acquity H-class UPLC system with ion trap mass spectrometer, Bruker Daltonics Esquire 3000 plus, and Agilent 1100
Series LC system). Samples were analyzed in triplicate and plotted against a standard curve (compound at 3.12-100 µM in mouse plasma). weeks. The osmotic minipumps were replaced after 2 weeks to obtain a total treatment period of 28 days. Mice that were not treated with oxaliplatin received an I.P. injection with saline at the same time. Body weight was measured twice a week and tumor growth was monitored three times a week with digital calliper measurements starting upon detection of palpable tumors.
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