LUIS JIMENEZ MicroChem2000, 13-26 20th Street, Fairlawn, NJ 07410 ABSTRACT: An Adenosine Triphosphate (ATP) bioluminescence system was compared and validated against standard methods for rapid microbiological monitoring of several non-sterile pharmaceuticalformulations such as creams, tablets, and capsules. Results obtained using 1%, 2.5%, and 10% of product suspensions indicated that most samples that did not contain non-microbial ATP neither inhibited the bioluminescence reaction nor did something else. Ten percent product suspensions were inoculated with different concentrations of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli,Salmonella typhimurium, Candida albicans, and Aspergillus niger. Samples were incubated for 24 –120 h at 35 °C with shaking. Results indicated a strong inhibitory effect of microbial growth, as no microorganisms were detected by using the ATP bioluminescence assay. However, when 1% and 2.5% product suspensions were spiked with the same microorganisms, positive detection was conﬁrmed. After incubation,all microorganisms were detected by the bioluminescence system within 24 –72 h. All positive samples were conﬁrmed by using standard plating media. However, to optimize detection of all microorganisms, different enrichment media were developed. Introduction For non-sterile pharmaceuticals, microbiologists have relied on standard methods for detecting microbial contamination in ﬁnished product andraw material samples (1–3). These methods are based upon incubation times that range from 5 to 7 days. In addition to be labor intensive, these procedures require different types of media. Approximately 12 different types of growth media are used in a regular microbial limit test (3). This is because standard microbiological testing is based upon transferring and incubating sample aliquots fromdifferent types of media for bacteria, yeast, and mold (3). However, new molecular methods are available that can rapidly detect microorganisms in contaminated samples within 24 –27 h without compromising efﬁcacy and sensitivity (4, 5). In the 21st century, pharmaceutical manufacturing requires faster and accurate quality control evaluation of processes and products (6). Therefore, there is a need forrapid quality control testing. A rapid screening method will improve quality control testing by releasing all the clean batches in a shorter time period than standard methods allow. One rapid technology is based upon detecting essential molecules such as Adenosine Triphosphate (ATP) (4). ATP is a high-energy phosphate compound found in microorganisms that is essential for microbial growth andviability. ATP is present in all living microorganisms and can be used to ascertain the presence or absence of microbial contamination in pharmaceutical products. Rapid microbiological analysis of clinical, food, and pharmaceutical samples has been reported by using ATP bioluminescence analysis (4, 7, 8). Recently, pharmaceutical water samples were also analyzed using a quantitative ATPbioluminescence assay (9). Samples were enumerated within 24 h. Microbial viability in pharmaceutical clean room environments has also been analyzed using a qualitative ATP bioluminescence assay (10). The bioluminescence assays are based upon the light-producing enzyme luciferase, which will hydrolyze ATP to produce light. Light production is detected by a luminometer and recorded as relative light units(RLU). The purpose of this investigation was to evaluate and validate the use of a qualitative ATP bioluminescence assay for detect159
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Vol. 58, No. 3, May–June 2004
ing microbial contamination in non-sterile pharmaceutical formulations with different...