Impurity testing HPLC analysis

In order to identify any excipient- and packaging-related impurities in the formulation, placebo cores and hlm-coated placebos, were prepared using the same excipients as in the active tablets. The placebo cores and film-coated placebos, as well as active cores and hlm-coated active tablets were set up on a stability study in high density polyethylene (HDPE) bottles with and without desiccant and in foil-foil blisters. At the designated time intervals, the tablets were tested for purity by gradient HPLC analysis. 

Figure 6.7 shows an example of an HPLC analysis of impurity testing of a drug product stored for 6 months under accelerated stability conditions (40°C/75% RH). Note that to maximize sensitivity of the trace impurity peaks,... 

Impurity testing of pharmaceuticals is one of the most difficult HPLC method development tasks because of their requirements for both high-resolution and trace analysis in additional to stringent regulatory and reporting guidelines.20,21 There are several recent trends for these methods 22,23... 

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

Application of the analytical techniques discussed thus far focuses upon detection of proteinaceous impurities. A variety of additional tests are undertaken that focus upon the active substance itself. These tests aim to confirm that the presumed active substance observed by electrophoresis, HPLC, etc. is indeed the active substance, and that its primary sequence (and, to a lesser extent, higher orders of structure) conform to licensed product specification. Tests performed to verify the product identity include amino acid analysis, peptide mapping, N-terminal sequencing and spectrophotometric analyses. 

There are four basic system types. Type I are basic isocratic systems used for simple, routine analysis in a QA/QC environment often for fingerprinting mixtures or final product for impurity/yield checking. Type II systems are flexible research gradient systems used for methods development, complex gradients, and dial-mix isocratics for routine analysis and standards preparation. They fit the most common need for an HPLC system. Type III systems are fully automated, dedicated systems used for cost-per-test, round-the-clock analysis of a variety of gradient and isocratic samples typical of clinical and environmental analysis laboratories. Type TV systems are fully automated gra-... 

In assessing peptide purity, there were occasional discrepancies noted among the different analytical methods. MALDI-MS analysis of the peptide samples often revealed impurities not readily observed by ESI-MS and HPLC. In most of these cases, greater dilution of the sample prior to MALDI-MS yielded data more consistent with ESI-MS and HPLC assessment. This is illustrated by sample 1354 in Figure 3A-C and in Table I. These results underscore the importance of using more than one analytical method to evaluate peptide purity, and of testing samples under several different conditions. 

All phases of analytical development are ideally supported by chemical separation techniques such as HPLC, TLC, GC, SFC, and CE. HPLC continues to be the primary method of analysis throughout the pharmaceutical development process. Although HPLC is limited in its ability to separate more than 15-20 components in a single analysis, and variations in columns and instrumentation manufacturer to manufacturer complicate transfer of methods, HPLC can readily be implemented to meet ICH requirements for method performance. For early-phase methods, HPLC can be coupled dynamically to mass and nuclear magnetic resonance spectrometers to facilitate the identification of unknown impurities. In later phases, HPLC can be implemented in a fully automated format as a high-throughput method for release and stability testing. 

As with GC, HPLC can be used qualitatively for identification or quantitatively to determine how much of a compound is present. Some examples of the use of HPLC in qualitative work include identification of impurities and toxicity screening. Some examples of the use of HPLC in quantitative work are drug testing in athletes and in sports supplements, pharmacokinetic studies of drugs pharmaceutical assays and fatty acid analysis. ... 

In the field of industrial pharmaceutical analysis the situation is different, because TLC instrumentation has reached a relatively high level. In some special application areas, such as the analysis of the extracts of medicinal plants, fermentation mixtures, etc., modem TLC (precoated or HPTLC layers, densitometric evaluation) has a distinct role, because the interference of so-called unknown background materials can be more easily eliminated than with other chromatographic techniques. Many chromatographers working in the pharmaceutical industry prefer to use reversed phase HPLC in conjunction with normal phase TLC or HPTLC to analyze raw materials for purity and impurities as well as for stability testing. 

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