| Assay Method Information | |
| | Inhibition of E. faecalis Decarboxylation Activity In Vitro |
| Description: | Table 2: A vial of 200 μL of Enterococcus faecalis v583 was removed from the −80° C. freezer and thawed in an anaerobic chamber containing an atmosphere of either 95/5 N2/H2 (v/v) or 90/5/5 N2/H2/CO2 (v/v). 200 μL was inoculated into 10 mL of sterile, anaerobic BHI broth, pH 5 (adjusted with NaOH). The culture was grown overnight at 37° C. under anaerobic conditions.After overnight incubation, 40 μL of the saturated starter culture was mixed with 744 μL of sterile, anaerobic BHI broth, pH 5 that had been supplemented with 1.5 mM levodopa. To this was added 16 μL of a 50-fold concentrated stock solution of inhibitor that had been dissolved in either DMSO, H2O, or DMSO:H2O (1:1 v/v). The final concentration of the inhibitor in each condition was 0, 0.001, 0.01, 0.1, 1, 10 or 100 μM. The contents of each incubation were mixed, and then 100 μL was transferred into a fresh 96-well plate. A standard curve of levodopa (0-1.5 mM) in BHI broth, pH 5.5 was likewise prepared on a 100 μL scale and aliquoted into the plate. The plate was sealed and incubated for 24 h at 37° C. under an atmosphere of either 95/5 N2/H2 (v/v) or 90/5/5 N2/H2/CO2 (v/v) in an anaerobic chamber.After 24 h incubation, the seal was removed, and the contents of each plate was mixed with 400 μL acetonitrile containing 0.1% (v/v) formic acid and 200 nM tolbutamide as an internal standard. The samples were mixed and then centrifuged (4,000 g, 10 min). 200 μL of each supernatant was transferred to a separate plate.The samples were analyzed by using an Agilent 6470 triple quadrupole mass spectrometer equipped with an Acquity UPLC. Mobile phase A consisted of H2O containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. Mobile phase B consisted of acetonitrile containing 10 mM ammonium formate, pH 3.0 and supplemented with 0.1% (v/v) formic acid. 5 μL of each sample was injected onto a BEH Amide column (Waters Corporation, 2.1×50 mm, 1.7 μm). The gradient was set to: 100% mobile phase B at 0 min, decreasing linearly to 65% mobile phase B by 1.5 min, held constant at 65% mobile phase B until 2.5 min, ramped back up to 100% mobile phase B by 2.6 min, and held constant at 100% mobile phase B until 4.2 min. The flow rate was 0.6 mL/min. The levodopa was detected by using the mass spectrometer in multiple reaction monitoring (MRM) mode, quantifying the transition 198.1 to 151.9 m/z in positive mode. The fragmentor setting was 78, the collision energy was 13, and the cell accelerator voltage was 4, and the dwell time was 20. Tolbutamide was monitored using MRM and quantifying the transition of 271.1 to 91.0 m/z in positive mode. The fragmentor setting was 88, the collision energy was 37, and the cell accelerator voltage was 4, and the dwell time was 20.The amount of levodopa was quantified by comparing normalizing the area to the area of tolbutamide internal standard within each sample. This relative response was then compared to that of the standard curve to obtain the residual levodopa within each sample. |
| Affinity data for this assay | |
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