Peak purity, evaluation

A PDA detector provides UV spectra of eluting peaks in addition to monitoring the absorbance of the HPLC eluent like the UVA is absorbance detector. It is the preferred detector for testing impurities and for method development. PDA facilitates peak identification during methods development and peak purity evaluation during method validation. Detector sensitivity was an issue in earlier models but has improved significantly (more than ten-fold) in recent years. ... 

If reference materials are not available, the challenge study lives np to its name. Specificity may still be demonstrated by a comparison of test results containing the impurities of interest to a second, well-characterized procedure (e.g., USP method). If a secondary method is unavailable, peak purity evaluation may be used to further demonstrate specificity of the method. 

Peak purity tests are used to demonstrate that an observed chromatographic peak is attributable to a single component. Mass spectrometry is the most sensitive and accurate technique to use for peak purity evaluation because of the specific information derived from the analysis. However, a good number of HPLC methods use mobile phase conditions that are incompatible with mass spectrometry detection. In this case, PDA spectrophotometers using peak purity algorithms may be used to support the specificity of the method. Almost all commercially available diode array detectors are equipped with proprietary software that will perform these calculations. Although this technique is more universal in application to HPLC methods, the data provided is neither particularly... 

Ideally, one wants an orthogonal (nonoverlapping) separation technique for the alternate method. In this respect, CE is a good first choice. Other choices include TLC, NP-HPLC, and RP-HPLC using different columns, different pH conditions, and different mobile phases. Peak purity evaluations using photodiode array (PDA) UV analysis (to evaluate UV-homogeneity) as well as LC-MS analyses are recommended. The ICH supports this approach in suggesting, Peak purity tests may be useful to show... 

Peak purity evaluation, by comparing the upslope, apex and downslope spectra, assessment of UV spectral peak purity can give some limited assurance that there is not an impurity with different characteristics coeluting with the main peak... 

Figure 9.5. Diagram illustrating peak purity assessment using UV and MS. Panel (a) shows that since the UV spectra of the leading, apex, and trailing parts of the first peak look fairly similar visually, one might conclude erroneously that the first peak contains only one single component. Panel (b) shows that the first peak actually contains two components with masses at 311 and 287, respectively. These two components might have similar UV spectra, making UV spectroscopy an insensitive tool for peak purity evaluation. Diagram courtesy of Waters Corporation.

It is possible to carry out a chromatographic separation, collect all, or selected, fractions and then, after removal of the majority of the volatile solvent, transfer the analyte to the mass spectrometer by using the conventional inlet (probe) for solid analytes. The direct coupling of the two techniques is advantageous in many respects, including the speed of analysis, the convenience, particularly for the analysis of multi-component mixtures, the reduced possibility of sample loss, the ability to carry out accurate quantitation using isotopically labelled internal standards, and the ability to carry out certain tasks, such as the evaluation of peak purity, which would not otherwise be possible. 

The principles of diode-array detection in HPLC and their application and limitations to peak purity are described in the literature [25-27], Examples of pure and impure HPLC peaks are shown in Figure 1. While the chromatographic signal indicates no impurities in either peak, the spectral evaluation identifies the peak on the left as impure. The level of impurities that can be detected with this method depends on the spectral difference, on the detector s performance, and on the software algorithm. Under ideal conditions, peak impurities of 0.05-0.1% can be detected. 

Analogously to HPLC, photodiode array or multiwavelength fast scanning detectors can be used to increase the quantity and quality of information. These detectors allow the analyst to evaluate the on-line UV(-visible) spectra of the separated zones, and, by comparison with recorded reference spectra, to investigate peak purity and peak indentity. 

In addition to structural analysis and purity evaluation, Raman spectroscopy can also be used to estimate the crystal size of nanostructured solids. In most cases size characterization using Raman spectroscopy is based on the phonon confinement model (PCM), which uses changes in Raman frequency and Raman peak shape to estimate the crystal size. Although several attempts have been made to relate confinement-induced changes in the Raman spectrum of ND to the crystal size, the agreement between calculated and experimental data and the accuracy of the fitting procedure are still unsatisfactory. A detailed discussion of the limitations of the PCM and the accuracy of previous studies on ND powders is given in Ref [86]. 

The evaluation of a peak purity analysis is assisted by a well-organized visual display of the output of various peak purity analysis techniques. 

If the system has a spectral detector, spectral evaluation such as peak purity and compound identity should be part of the test. Additional tests should be developed if there are other software functions used in routine analysis, but they are not part of the sample or standard analysis test. If the system is used over a wide concentration range with multiple calibration points, the tests should be run over many concentrations to verify correct function of the calibration curve. 

Develop integration method, calibration method and procedure for spectral evaluation, for example, peak purity or/and identity checks. 

Example 1 Evaluation of peak purity study of the tryptamine peak... 

The evaluation of the peak purity as a selectivity criterion is a fundamental issue deserving thorough attention. If peaks are found to be heterogeneous, chemometric methods based on curve resolution can be used to isolate the pure analyte contributions from a mixture system, thus making possible an accurate quantification of components (16). 

---