Samples of 24kDa-FSHβ and 21kDa-FSHβ were reduced and carboxymethylated (51 (link)), the buffer exchanged with 0.2 M ammonium bicarbonate, pH 8.5, by ultrafiltration, and subjected to overnight PNGaseF digestion at 37°C (9 (link)). Oligosaccharides were separated from deglycosylated protein using the Acquity UPLC system employing a Phenomenex (Torrance, CA) reverse-phase Kinetex C8 column. The column was equilibrated at 50°C with 0.01% TFA containing 5% acetonitrile at a flow rate of 0.7 mL/min. Oligosaccharides emerged in the void volume peak, which was collected manually, and carbohydrate recovered by evaporation in a Speed Vac.
Uplc system
Manufactured by Phenomenex
The UPLC (Ultra Performance Liquid Chromatography) system is a high-performance liquid chromatography (HPLC) instrument designed to separate and analyze complex mixtures of compounds. The core function of the UPLC system is to provide rapid, high-resolution separations with increased sensitivity and improved throughput compared to traditional HPLC systems.
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Lab products found in correlation
4 protocols using uplc system
1
Glycan Analysis of Recombinant FSH Isoforms
2
Quantitative UPLC-MS/MS Analysis of Quisinostat
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Quisinostat and the internal standard, panobinostat, were prepared via protein precipitation using ten times volume of cold acetonitrile. Upon adding acetonitrile, samples were vortexed for 5
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minutes followed by centrifugation at 14,000 rpm for 5 minutes at 4℃. The supernatant was transferred into a new tube and dried using the SpeedVac concentrator (Savant Instruments, Inc., Holbrook, NY). Samples were reconstituted in the mobile phase prior to the injection into a Synergi TM Polar-RP column (75 × 2 mm, 4 µm, 80 Å; Phenomenex, Torrance, CA) connected to an Acquity UPLC system coupled with a Quattro Ultima. An isocratic elution method with 68% distilled water with 0.1% formic acid and 32% acetonitrile with 0.1% formic acid was employed.
The m/z ratios were 394.93 → 143.89 and 350.04 → 157.91 for quisinostat and panobinostat, respectively (positive-ionization mode). Standards and quality controls were prepared in parallel.
The LOQ was 10 ng/mL in all specimens and the standard calibration curve was linear within the range of 10 -1000 ng/mL (weighted by 1/Y 2 ). For the analysis to be accepted, the %CV and %accuracy at each concentration was less than 15% (less than 20%CV for the lowest concentration, 10 ng/mL).
This article has not been copyedited and formatted. The final version may differ from this version.
minutes followed by centrifugation at 14,000 rpm for 5 minutes at 4℃. The supernatant was transferred into a new tube and dried using the SpeedVac concentrator (Savant Instruments, Inc., Holbrook, NY). Samples were reconstituted in the mobile phase prior to the injection into a Synergi TM Polar-RP column (75 × 2 mm, 4 µm, 80 Å; Phenomenex, Torrance, CA) connected to an Acquity UPLC system coupled with a Quattro Ultima. An isocratic elution method with 68% distilled water with 0.1% formic acid and 32% acetonitrile with 0.1% formic acid was employed.
The m/z ratios were 394.93 → 143.89 and 350.04 → 157.91 for quisinostat and panobinostat, respectively (positive-ionization mode). Standards and quality controls were prepared in parallel.
The LOQ was 10 ng/mL in all specimens and the standard calibration curve was linear within the range of 10 -1000 ng/mL (weighted by 1/Y 2 ). For the analysis to be accepted, the %CV and %accuracy at each concentration was less than 15% (less than 20%CV for the lowest concentration, 10 ng/mL).
3
Chemical Analysis of THS Extracts
4
Metabolite Analysis via LC-MS/MS
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The samples were analyzed on a Thermo Scientific Vantage LC-MS/MS with an Accela UPLC system using a 3×150 mm 2.6 micron Phenomenex Kinetex PFP column as detailed in [20] .
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