Chromatographic Peak Purity interpretation

 1.0 OBJECTIVE                    

To provide the procedure for the Chromatographic Peak Purity interpretation 

2.0 SCOPE

This procedure is Applicable to the Chromatographic Peak Purity interpretation 

3.0 RESPONSIBILITY 

3.1 QC-Chemist 

4.0 ACCOUNTABILITY         

4.1 Executive – Quality control

4.2 Head – Quality control

5.0 PROCEDURE       

5.1 Peak purity is critical factor in stability analysis. This is because the the spectrum obtained during the stability analysis is from only compound of interest. The spectrum should be pure and there should not have any co-eluting peaks at the same retention time. 

5.2 UV spectra are taken at various points across a chromatographic peak and compared. If the spectra are sufficiently alike, the peak is considered pure; if the differences in spectra are large enough, the peak is not pure.  

5.3 With some peak-purity outputs, all you get is an indication of the peak purity, sometimes called a match angle or purity angle.  

5.4 With sophisticated peak-deconvolution software, the peaks can be measured separately, in much the same manner as an LC-MS detector can distinguish between two co-eluting peaks of different molecular weights. 

5.5 Peak purity analysis must be performed for both Drug substance as well as for Impurities. Both must pass the test. 

5.6 For Empower 3 /2 

5.61 When an impurity is detected, the purity plot rises above the threshold line. This indicates spectral differences beyond noise contributions and implies the presence of more than one compound in the chromatographic peak. In the example below, the coeluting impurity is in the leading edge of the peak.

 

5.6.2 The above figure is the peak purity plot for a chemically pure chromatographic peak. Notice there are no valleys or shoulders on the peak. The thick solid line represents the spectral differences across the peak. The reference point is the apex spectrum. Most of the spectra in the peak are close to zero difference, which is characteristic with a pure compound. At low absorbances the baseline noise contributes to spectral differences

5.6.3 The purity of the entire peak is determined by the purity angle and the threshold angle. 

5.6.4 Purity angle: 

The average value of the angle between each spectrum of the peak and the spectrum at the top of the peak. In other words, the purity of the entire peak can be determined by this value.

・ If <0.2

The detection limit of this method is about 0.1 to 0.2. Therefore, it can be concluded that the peak for which the purity values below 0.2 consists of almost the same spectral components.

The measurement precision of this is better than the visual check (up to 1 angle).

・If > 1

Although there are differences in shape that can be observed by the visual check, the purity angle alone does not mean that components with other spectra have co-eluted.

This is because other than the co-elution of other components, the noise included in the spectra can also cause the differences. This should be noted especially with low-concentration components.

 5.6.5 Threshold angle: An index value indicating the effect of the noise (evaluated by S/N ratio) over the entire peak. This  is evaluated by comparing the values of the purity angles.

・If purity angle < purity threshold angle

 Even when the purity angle is large, it can be determined that there is no obvious co-elution within the range of the threshold angle that indicates the effect of the noise.

 ・If purity angle > purity threshold angle

If the purity angle is larger than the threshold angle indicating the shape differences of the spectra caused by noise, there is a spectra difference exceeding the effect of noise.

It is highly likely that components with different spectra are co-eluting.

6.0 ANNEXURES 

Nil