Chromatography Forum

I have a Question

Purity Peak test is necessary for Degradation products?

Thanks!

I guess you mean a spectral purity test of a chromatographic peak. This test is not a good way to demonstrate the specificity of a method. It cannot confidently confirm that there is no impurity hidden under the main component peak when the concentration of the impurity is small (ca. 0.1-1%) and the spectra of the compounds are similar (this is not uncommon for related substances). Apparent spectral purity of a peak is not a sufficient condition to say that the peak is chemically pure and the method is specific.

I guess you mean a spectral purity test of a chromatographic peak. This test is not a good way to demonstrate the specificity of a method. It cannot confidently confirm that there is no impurity hidden under the main component peak when the concentration of the impurity is small (ca. 0.1-1%) and the spectra of the compounds are similar (this is not uncommon for related substances). Apparent spectral purity of a peak is not a sufficient condition to say that the peak is chemically pure and the method is specific.

Hi vmu

And then? What?

Best regards

Fernando

And then? What?

Then one has to try to vary the chromatographic conditions and to compare the results:
- test several "similar" columns;
- use a more efficient column (higher length or lower particle size);
- change the mobile phase or use a gradient instead of an isocratic separation (provide enough retention for the components of interest; an isocratic method with k = 1-2 for the main peak is not the best choice);
- vary the column temperature (in a wider range than the one usually used for a robustness check);
- reduce the sample load (injection volume or sample concentration);
- try to use orthogonal separation conditions or even 2D-LC to confirm that the original simple method is selective enough.

All these tests do not give one a 100% guarantee that the method is specific but they greatly enhance the confidence in the method.

Also one can try to use MSD to confirm the results obtained with a UV detector.

One example about the sample load: related substances in methionine according to USP41.
Column: 250 mm x 4.6 mm, 5 um, C18.
Mobile phase: water/ACN/H3PO4, 1 mL/min, 30 °C, isocratic for 6 min, followed by a long linear gradient.
Detection: UV 205 nm.
Injection: 50 uL of a 30 mg/mL sample solution.
SST: Rs NLT 5.0 between methionine (RRT 1.0) and methionine sulfoxide (RRT 0.5) in a solution containing 0.1 mg/mL of each component.
Disregard limit: 0.05%.

The large injected amount of the sample results in a huge overloaded peak of methionine. The sensitivity of the method is so high that one can safely reduce the injection volume (allowed by pharmacopeias) 10-fold without compromising the LOQ. This volume reduction leads to a narrower and less tailing main peak and can uncover a previously hidden peak of an unspecified impurity eluted after the main peak.