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Several industry experts describe applications in pharmaceutical applications, including on-line total organic carbon analysis, ultra-fast liquid chromatography, rapid microbial testing, and differential scanning calorimetry-Raman Spectroscopy.
Table I (UFLC): Analytical conditions. (TABLE 1 (UFLC) IS COURTESY OF SHIMAZDU SCIENTIFIC INSTRUMENTS)
Results. Figure 1 (UFLC) shows the results of five serial ultra-high-speed analyses of the cold medicine. Using UFLC, the autosampler
cycle time becomes a critical factor. The injection speed of the autosampler was 10 s, and the total time for all five analyses
took no more than 5 min. This cycle time, in conjunction with a reduction in carryover, increased sample throughput. The run
time of a single analysis was shortened, and the total cycle time of the injection sequence and run time was optimized.
Rapid microbial testing
Lori Daane, PhD, vice-president of Celsis Rapid Detection, and Judy Madden, vice-president of Celsis International (Chicago)
Microbiological tests may fall into several categories. Final product-release testing includes microbial limits testing of
nonsterile drug products and sterility testing of sterile products. Environmental monitoring tests air, water, surfaces, and
personnel for viable microorganisms. Bulk drug products, including active pharmaceutical ingredients and excipients, may be
tested for bioburden (1). Rapid microbial screening is used to detect the presence or absence of microbial contamination within
24 h compared with 3–7 days using traditional microbial testing. For example, Celsis AKuScreen is a rapid microbial method
that can detect contamination from bacteria, yeast, or mold definitively within 24 h.
ATP bioluminescence. Rapid microbial methods can be based on adenosine triphosphate (ATP) bioluminescence, which is commonly known as the firefly
luciferase reaction as follows, where AMP is adenosine monophosphate and PPi is inorganic phosophate (2):
ATP drives the reaction, which emits light that is measured in a luminometer. ATP is present in all living cells. If microbial
contamination is present, the reaction moves forward, and light is emitted. Because definitive detection is based on having
a sufficient quantity of microbial ATP present to generate a light signal that is distinguishable from sample background,
detection becomes more difficult if the sample contains nonmicrobial ATP. Enzyme and molecular-based technologies, such as
adenylate kinase (AK) or AK-amplified assays (i.e., Celsis AKuScreen, Celsis), allow bacteria to be detected in 18 h and yeast
and mold in 24 h. The AKuScreen assay uses microbial adenylate kinase (AK) to generate ATP in a reaction that is not constrained
by the finite amount of metabolic ATP available in the standard bioluminescence assay. The generation or amplification of
ATP beyond that inherent in the assayed sample is accomplished via the enzyme-catalyzed and reversible reaction as follows:
The AK reaction uses adenosine diphosphate (ADP) and microbial AK to catalyze the production of ATP. The produced ATP is detected
and measured using the traditional firefly luciferase reaction. The longer the AK reaction is allowed to proceed in the presence
of an adequate supply of ADP, the more ATP is generated and the greater the bioluminescence signal and the resulting assay
sensitivity. Assuming the ratio of AK and ATP in a typical bacterium and a reaction turnover number of 40,000, approximately
40 times as much ATP as originally represented by microbial ATP can by generated per minute by the AK reaction (2, 3). Similarly,
if the reaction is allowed to continue for 25 min, the amount of ATP can be 1000 times more than what was originally present,
allowing the test-enrichment period and time-to-result to be reduced.
Lori Daane is vice-president of Scientific Affairs, Celsis, Inc., 600 West Chicago Avenue, Suite 625, Chicago, IL 60610-2422. tel. 312.476.1200, fax 312.476.1201, ldaane@celsis.com
Articles by Lori Daane
