Abstract
Fluorescence-coupled PCR technology was employed to quantify DNA segments specific for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. The PCR procedure is put forward as an alternative method for detecting microbial contaminations in pharmaceutical preparations and is compared to the tests for specified microorganisms described in European Pharmacopoeia (EP) 2, 2.6.13 and the USP, chapter 61. Data presented here describe the validation of this analytical method when used for proof of absence of specified microorganisms. The detection systems were specific for the microorganisms analyzed, and led to linear results over a wide range (more than 6–7 log intervals). The correlation coefficients lay above 0.99. The precision of replicate determinations within a single test was observed to be high, the relative standard deviation being between 0.39% and 1.53%. The precision between different tests was also high, with a relative standard deviation between 0.76% and 1.91%. The sensitivity without pre-enrichment amounted to 1–10 CFU. Since determination of the specified bacteria was performed following pre-enrichment, the limit of detection amounted to 1 CFU. Equivalent results were obtained in a study on nine batches of a milky hydrophilic cream (SH-No. M 440 A) with the conventional test for microbial contamination and the PCR procedure. The data presented here strongly indicate that the use of fluorescence-coupled PCR techniques can prove the absence of specified bacteria faster and more efficiently than conventional methods.
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