01, 0 10, 1, and 10 EU/mL One hundred microliters of the blank w

01, 0.10, 1, and 10 EU/mL. One hundred microliters of the blank was used according to standard endotoxin concentrations (ie, 0.01, 0.10, 1, and 10 EU/mL), and 100 μL of the samples was added in a 96-well microplate with respective PPC. All reactions were achieved in duplicate to validate the test. The test procedure and calculation of the endotoxin

level were performed following the manufacturer’s instructions. The absorbencies of endotoxin were individually measured Osimertinib purchase by using an enzyme-linked immunosorbent assay plate-reader (Ultramark, Bio-Rad Laboratories) at 340 nm. The spike procedure was performed according to the manufacturer’s instructions by the addition of a known concentration value of endotoxin for each LAL method in order to detect any Natural Product Library nmr possible inhibition or enhancement from the samples in relation to the LAL substrate. To verify

the lack of product inhibition, an aliquot of test sample (or a dilution of test sample) is spiked with a known amount of endotoxin (0.4 EU/mL). The spiked solution is assayed along with the unspiked samples, and their respective endotoxin concentrations are determined. The difference between these two calculated endotoxin values should be equal to the known concentration of the spike ±25%. For the kinetic tests (chromogenic kinetic assay and turbidimetric assay), the WinkQCL Software (LONZA, Walkersville, MD) was used to calculate the amount of endotoxin recovered in the positive product control (PPC) in the comparison with the known amount of endotoxin spiked. The endotoxin recovered should be equal to the known concentration of the spike or within 50% to 200% as determined by the pharmacopeia. If positive, Palmatine the test was considered validated because a good interaction between the samples and LAL substrate was shown without interfering with the recovery of endotoxin. The linearity of the standard curve within the concentration range used

to determine the endotoxin values were verified for all tests according to the manufacturer’s instructions. The absolute value of the correlation coefficient (r value) of the calculated standard curve had to be ≥0.980. Replicates were run in order to assess the technique and coefficient of variation. The percentage of the coefficient of variation for replicates of a sample had to be less than 10%. Reproducibility between 3% and 4% was considered the best as indicated by the manufacturer’s instructions. After the measurement of endotoxin, if the levels of endotoxin were out of the standard curve or if any possible interference with LAL method by the root canal samples was detected, serial dilutions were considered and reassayed. The endotoxin values were statistically analyzed by using SPSS for WINDOWS, version 12.0 (SPSS Inc, Chicago, IL). The comparison between the chromogenic endpoint and chromogenic and turbidimetric kinetic methods was performed by using the Friedman test (p < 0.05).

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