The methods of Saha et al. formed the basis for the advent of a modified method as described in Table 2. In the modified method, 25 μL of 5% (m/v) phenol was added to 25 μL of sugar solution previously aliquoted into the microplate, followed by mixing with a pipettor. Next, 125 μL of H2SO4 was added to each well, followed by rapid mixing with a pipettor. Solutions were incubated for 30 min at room temperature (18–25 °C) before the absorbance was read at 485 nm in the microplate reader. Where applicable, samples were diluted this website in reverse osmosis-purified, distilled water. Except for the comparative study performed with glucose

as a test sample, all PHS measurements were made with the modified method. All mixing was performed via 5 aspiration cycles with a pipette. Standard curves were run in triplicate with absorbance values corrected for the blank. The final yellow colour was found to be stable for 1 h, although slight development occurred with prolonged Carfilzomib in vivo incubation following the reaction. Phenol solution was stored in the dark when not in use. In certain circumstances with the modified PHS assay, a glass microplate (Zinsser,

Germany) was evaluated. A pyrogen assay (PyroGene™, Lonza, inhibitors Maryland, USA) based on recombinant Factor C for endotoxin was qualified. The instructions provided by the assay kit manufacturer (version: 08299P50-658U/NV-612/07) were followed except where noted. Pyrogen-free consumables including reagent reservoirs, pipette tips, conical tubes, LAL Reagent Water, and serological pipettes were purchased from Lonza Walkersville. Samples were diluted into LAL Reagent Water. Standard curves were prepared and run in triplicate. For assay interference testing and positive product controls, 10 μl of a 10 EU/mL standard solution was added to 90 μL of sample, yielding a 1 EU/mL reference standard concentration. Endotoxin

samples and standards were vortexed vigorously for the prescribed amount of time. Except where noted otherwise, only microplates were incubated for 1 h at 37 °C inside the plate reader prior to reading. The measurement parameters were: excitation wavelength set to 380 ± 20 nm, emission wavelength set to 440 ± 20 nm, and an integration time of 40 μs. The log amount of endotoxin present was proportional to the log change in the relative fluorescent unit (RFU), with second order polynomial fits offering the most accuracy. The methodology employed differed from the manufacturer’s recommendations in two significant ways. A single measurement was taken approximately 60 min after the start of incubation at 37 °C instead of the recommended two-point measurement. In addition, incubations at 22 °C, 26 °C, and 37 °C were evaluated for varying durations during one experiment. Several permutations of the original PHS method for sugar quantitation have been described.