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On-line TOC analysis
Jonathan Yourkin, global pharmaceutical product manager GE Analytical Instruments (Boulder, CO)
Evaluating variables in a measurement system. A key challenge in any shift to on-line TOC analysis is ensuring that the measurement system that measures an analyte on-line provides the same or better data quality as the laboratory-based measurement system. Often, analytical methods used to produce on-line and laboratory data are different. These challenges can be solved when all analytical methods used are essentially the same or the analytical capabilities of the laboratory-based and on-line TOC methods have proven to be like-for-like. Once method comparability is established, any variability observed between laboratory and on-line TOC results can be attributed to different sampling processes.
When comparing TOC data from similar samples analyzed by on-line and laboratory TOC instruments, observed variation can be attributed to two major sources: the sampling process used to introduce the sample to the measuring device and the inherent analytical capabilities of the analytical method. Given the ubiquitous presence of organics in the environment, grab-sample collection of TOC samples is subject to numerous error sources. With the on-line method, sampling-process variability is minimized or eliminated because the analyzer is physically integrated within the process-water stream for continuous quality-assurance monitoring. In addition, this evaluation quantifies sampling-process variation at TOC concentrations produced by the pharmaceutical water system, which are typically an order of magnitude lower than the TOC standard concentrations used to validate the analytical method. Sampling process variation is generally much more pronounced at these lower TOC concentrations.
Equivalency testing. Equivalency testing demonstrates the sameness of two measurement systems based upon the analytical results the methods produce. This type of testing differs from instrument-validation testing, which typically assesses various attributes using standards of known concentration. Validation protocols are not designed to identify differences in the test-sample data quality. Therefore, it is possible to validate two methods with the same protocols using the same criteria, yet have the methods yield nonequivalent analytical results under actual conditions of use. It is particularly important in on-line TOC implementations to augment validation protocols with equivalency testing. The fundamental hypothesis supporting the justification for measurment-system equivalency testing is that, given the sameness of on-line and laboratory analytical methods, any lack of equivalency observed between the two data sets can be attributed to aspects of the measurement system rather than the core analytical method.
This type of analysis is typically performed using a Gage Repeatability and Reproducibility (Gage R&R) study to quantify measurement-system variation relative to variation of the process as a whole. The Gage R&R addresses several components affecting variability, including the gauge (the instrument), the operator, and the sample itself. Ordinarily, a full-scale Gage R&R would be used to assess variability contributed by all components. This example uses a simplified Gage R&R approach based on the degree of sameness of the analytical methods and the unique differences in the operational or environmental conditions associated with laboratory and on-line measurements. By pairing the on-line and laboratory data collection, process water variability remains common between the two systems. In addition, the inherent accuracy of the instruments remains common, based on like-for-like methods that have been validated appropriately.
TOC section reference
1. FDA, Guidance for Industry: PAT (Rockville, MD, 2004).
Ultra-fast liquid chromatography
Simon Robinson, HPLC product manager, Shimadzu Scientific Instruments (Columbia, MD)
Over-the-counter cold medicines often contain multiple active ingredients. These actives include combinations of decongestants, antihistamines, pain relievers, cough suppressants, and expectorants in addition to numerous vitamins and herbal extracts—all of which exhibit different chemical properties. A wide range of analyte polarities often makes developing chromatographic methods challenging. Separating ingredients in a cold medicine is important to understand the content of each medication and product quality. A high-speed liquid chromatographic system (Prominence UFLCxr with a SPD-M20A photodiode array detector, Shimazdu Scientific Instruments) was used to analyzed 20 common cold medcines.
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.
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.
Case study : in-process sterility testing . Because of the ubiquity of AK in living organisms, AK-amplified assays can detect bacteria, molds, and yeasts, and this microbial testing is used in sterility testing to determine product release (4, 5). A company was testing its sterile, in-process materials using a 14-day sterility test. A comparability test was performed to see if equivalent results could be obtained using a rapid method. Spiking studies were conducted by inoculating samples at a level approximating 1 colony-forming unit (CFU). Following inoculation, the samples were enriched for 24 h, and contamination was detected using a rapid microbial system (Celsis AKuScreen). Microbial hold times and the corresponding time for quarantined inventory was 48 h, a savings of 12 days over traditional microbial testing.
Case study: mold detection. Pharmaceutical companies use microbial specifications that are based on United States Pharmacopeia, European Pharmacopoeia, and Japanese Pharmacopoeia guidelines, product history, and risk profile. With respect to mold, this typically means the ability to detect Aspergillus brasiliensis. Slow-growing, molds are often the rate-determining step for product release. AK-amplified assays (e.g., Celsis AKuScreen) can detect yeast and mold in 24 h compared with 48 h if standard bioluminescence is used or 4–7 days if using traditional methods.
Rapid microbial testing section references
1. B.S. Riley, Am. Pharm. Rev, 7 (2), 28–31 (2004).
2. D.J. Squirrel et al., Anal. Chim. Acta 457 (1), 109–114 (2002).
3. D.J. Squirrel and M.J. Murphy, "A Practical Guide to Industrial Uses of ATP-Luminescence" in Rapid Microbiology (Cara Technology, Lingfield, Surrey, UK, 1997).
4. L. Noda, Adenylate Kinase: The Enzymes (Vol. 8) ( Academic Press, New York, 1973), p. 279–305.
5. G.E. Shultz, Cold Spring Harbor Symposia on Quantitative Biology, 52 , 429–439 (1987).
6. L. Dane, "Study Results: Modifications to Detect P. expansum in 24 Hours Using AKuScreen Reagents" (Celsis, Chicago, 2006).
Kevin P. Menard, PhD, business manager of thermal analysis at PerkinElmer (Shelton, CT)
Differential scanning calorimetry (DSC) and Raman spectroscopy are well-known techniques in pharmaceutical analysis. DSC determines glass transitions, amorphous content, melting temperatures, and enthalpy and estimates the degree of crystallinity. In its fast-scanning mode, DSC also suppresses kinetic changes (i.e., polymorphism and decompositions). This allows measurements to be made before kinetic changes can occur, allowing one to determine the initial polymorphic form as well as measure melting and heat capacity on materials before they decompose. Raman spectroscopy is able to detect changes in chemical composition as well as positional and stereochemical changes in a sample; this allows it to identify specific polymorphic states. Raman spectroscopy, however, has some difficulties in measuring temperature-dependent reactions such as dehydrations and polymorphic rearrangements. These reactions are sensitive to the temperature applied, and care must be taken so that the energy added by the Raman's laser does not affect the data by causing changes in the sample temperature (1). A combination of dual furnace (i.g., power-compensated) DSC and a shuttered laser in an Eschelle Raman spectrometer were found to give minimal increases in bulk sample temperatures (2).
As a hyphenated technique, however, DSC–Raman spectroscopy works well in detecting the changes in polymorphic materials as a function of temperature (3). For example, running a sample of acetaminophen in heating showed a series of peaks corresponding to changes in polymorphic forms, but the changes in the material corresponding to those thermal events are inferred (2). For example, an endothermic peak in the DSC themogram can be a melt, a water loss, or a polymorphic change. Raman spectroscopy allows one to confirm what the transition is; the chemical and structural changes detected from the Raman spectra clarify the DSC data. Because the DSC can performed under a wide range of thermal conditions, including heating and cooling rates from 0.1 to 750 ° C/min, the combined techinques can be used to characterize the materials' kinetic behavior under a wide variety of conditions. For example, crystallization processes can be studied using DSC–Raman during cooling experiments. Other work has been reported on hydrates and pseudo polymorphs as measuring the reaction of materials by tracking changes in assigned bands in the Raman spectra and the energetic changes (4, 5).
DSC–Raman spectroscopy section references
1. R. Alexander et al., Proceedings of the North American Thermal Analysis Society (NATAS) Annual Conf. (Atlanta, 2008), pp. 131–136.
2. A Dennis, K. Menard, and R. Spragg, Proceedings of the Annual Technical Conf. of the Society of Plastic Engineers (Chicago, 2009), pp 647–653.
3. K. Menard et al., Am. Lab. 42 (1), 21–23 (2010).
4. N. Redman et al., Furey, , Proceedings of NATAS Annual Conf. (Albuquerque, NM, 2003), pp. 120–129;
5. A. Bigalow-Kern et al., J. ASTM, 2 (7), 42–61 (2005).