Analytical Applications - Pharmaceutical Technology

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PharmTech Europe

Analytical Applications
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.


Pharmaceutical Technology
Volume 34, pp. s36-s30


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.


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