Measurement uncertainty evaluation and risk of false conformity assessment for microbial enumeration tests.

Microbial enumeration tests are widely used to assess the microbiological quality of non-sterile pharmaceutical products. Despite of all efforts to guarantee the reliability of microbial enumeration tests, there will always be an uncertainty associated with the measured values, which can lead to false conformity/non-conformity decisions. In this work, we evaluated the measurement uncertainty using a bottom-up approach and estimate the consumer's or producer's risk due to the measurement uncertainty. Three main sources of uncertainty were identified and quantified: dilution factor, plated volume, and microbial plate counts. The contribution of these sources of uncertainty depends on the measured value of microbial load in pharmaceutical products. The contribution of dilution factor and plated volume uncertainties increase with an increase of measured value, while the contribution of microbial plate count uncertainty decreases with an increase of measured value. The overall uncertainty values were expressed as uncertainty factors, which provide an asymmetric 95% level confidence level of microbial load in pharmaceutical products. In addition, the risk of false conformity/non-conformity decisions due to measurement uncertainty was assess using Monte Carlo method. When the measured value is close to the upper specification limit and/or the measurement uncertainty is large, the risk of false conformity/non-conformity decisions may be significantly high. Thus, we conclude that the use of uncertainty factor in the conformity/non-conformity assessment is important to guarantee the reliability of microbial enumeration test results and to support decision-making.

Top-down evaluation of the matrix effects in microbial enumeration test uncertainty.

Microbial contamination may compromise the efficacy and safety of pharmaceutical products. Microbial enumeration tests are required by most of the pharmacopeial compendia and consist in conventional pour-plate inoculation of a sample aliquot followed by incubation under appropriate conditions. Despite of this, the measurement uncertainty evaluation for microbial enumeration tests is rarely considered. Thus, the aim of this paper was to assess the matrix effects in microbial enumeration tests and their top-down uncertainty evaluation. Microbial counting methods for eighteen pharmaceutical products were validated concerning the trueness (mean recovery) and precision (repeatability and intermediate precision) using seven test microorganisms. Uncertainty factors values were found to be between 1.1 and 3.3, based on trueness and precision results. Trueness uncertainty component was the most relevant in 59% of the cases. This issue can be explained due to the matrix interference cause by preservatives or antimicrobial agents, particularly for lower dilutions when compared to higher dilutions. Thus, we concluded that the higher the matrix interference the higher the uncertainty factor, and consequently, the higher the asymmetry for the interval around the measurement.