Impurities peak profiling

Figure 4.8. Comparison of impurity profiles for the same chemical intermediate from two different suppliers. The impurity peak-areas for each chromatogram were tallied in 0.02 area-% bins for each vendor, the data was normalized by dividing by the number of chromatograms. Vendor A s material has many more peaks in the 0.05-0.2% range, which drives the total impurity level to =5.2% (vs. 1.9 for Vendor B) for <0.2% the number of excess peaks above 0.2% does not appear as dramatic, but greatly adds to the total impurity level = 13.3 v.v. = 2.3% ...

Most samples may be prepared by dissolution in water. The final concentration should be optimized according to the aim of the analysis, counterion or impurity analysis. For the control of impurities, the main counterion may be fairly overloaded. This may have an impact on the ionic strength of the sample and will produce a disturbed peak profile for the main compound. When solubility problems are encountered, up to 30% of methanol, ethanol, or acetonitrile may be added to improve solubility. However, the presence of too much organic solvent may produce an instrumental error, because the conductivity of the sample plug will differ too much from BGE conductivity, leading to current leakage. Or, when the sample is insoluble in water, it may be suspended, vortexed, and then centrifuged. The analysis is then performed on the supernatant as the ions are water soluble. 

The success of the analysis is dependent on the selection of a suitable internal standard. Some important criteria for such selection are as follows (a) that it is not chemically reactive towards any of the components of the mixture (b) that it has a retention time which gives a base line separation from the other components, including any impurities (c) that the retention time is comparable with that of the components of the mixture (d) that the peak profile is symmetrical and therefore does not exhibit either fronting or tailing and (e) that the detector response to the internal standard is such that neither an excessive, nor a minute, weight of standard compared to the weight of mixture needs to be used. 

Fig. 23 Final observed (O) and calculated (solid line) synchrotron X-ray powder (A=0.79980 A) diffraction profiles for Sm2.75C6o at 5 K. The lower solid line shows the difference profile and the tick marks show the reflection positions. Some impurity peaks were excluded from the refinement...

The impurity exhibits the same peak profile as does the main compound that is, it completely coelutes with the main peak, across the entire peak. 

One way of uncovering contributions because of impurities in an HPLC peak is to overlay peak profiles acquired at several wavelengths. As two different com-... 

Analytical chemists generally run a series of different assays (orthogonal assays) to confirm that they have sufficiently characterized the sample and have identified all impurity peaks found in the sample. Furthermore, they ensure that peaks which may co-elute using one technique can be detected with an orthogonal method (see Chapter 10). Analytical HPLC is the most common technology employed for assay and impurity profiling of pharmaceuticals. When the sample has been analyzed, the impurity may need to be isolated (for structural characterization, for instance). While techniques such as LC-MS may give an indication of the compound identification from the bulk assay, the definitive proof is always obtained from an independent analysis of the isolated compound. 

One way of uncovering contributions because of impurities in an HPLC peak is to overlay peak profiles acquired at several wavelengths. As two different compounds are unlikely to exhibit identical absorption over multiple wavelengths, the presence of an impurity is revealed by the deviation of the profiles. To compensate for the differences in spectral intensity at different wavelengths, the signals to be compared are first normalized to the maximum absorbance value or to equal areas. Peaks free of impurities exhibit good overlap, but the presence of an impurity is indicated by a shift in the retention time maximum at different wavelengths (Fig. 2). 

After spectra are acquired and processed, they are overlaid for visual evaluation (Fig. 4). Although significant deviations between the profiles can indicate the presence of an impurity, the converse is not necessarily true, and spectral profiles that match quite well may still mask the presence of an impurity. Factors that may contribute to nondetection of impurity include large concentration differences between analyte and impurity and either highly similar spectral profiles or identical chromatographic peak profiles and retention times for both analyte and impurity. As a rule of thumb, impurity concentrations in the 0.1-1% range may be detected when the spectra are dissimilar. However, if the spectra of the different components are highly similar and the HPLC peaks are not well resolved, the impurity detection limit is on the order of 5%. 

Figure 4.18. Peak-size correlation in an HPLC-chromatogram. The impurity profile of a chemical intermediate shown in the middle contains peaks that betray the presence of at least two reaction pathways. The strength of the correlation between peak areas is schematically indicated by the thickness of the horizontal lines below the chromatogram. The top panel gives the mean and standard deviation of some peak areas (n = 21) the two groups of peaks immediately before and after the main peak were integrated as peak groups.

With capillary electrophoresis (CE), another modern primarily analytically oriented separation methodology has recently found its way into routine and research laboratories of the pharmaceutical industries. As the most beneficial characteristics over HPLC separations the extremely high efficiency leading to enhanced peak capacities and often better detectability of minor impurities, complementary selectivity profiles to HPLC due to a different separation mechanism as well as the capability to perform separations faster than by HPLC are frequently encountered as the most prominent advantages. On the negative side, there have to be mentioned detection sensitivity limitations due to the short path length of on-capillary UV detection, less robust methods, and occasionally problems with run-to-run repeatability. Nevertheless, CE assays have now been adopted by industrial labs as well and this holds in particular for enantiomer separations of chiral pharmaceuticals. While native cyclodextrins and their derivatives, respectively, are commonly employed as chiral additives to the BGEs to create mobility differences for the distinct enantiomers in the electric field, it could be demonstrated that cinchona alkaloids [128-130] and in particular their derivatives are applicable selectors for CE enantiomer separation of chiral acids [19,66,119,131-136]. 

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