How do you perform purity analysis?

[Madscientist]

It would be interesting to know how purity analysis of organic compounds (Mw200-800 Da) is performed within the pharmaceutical industry.

Detector issue: Determination of purity is not straight forward due to different response factors depending of the chemical structure of the analytes. Unfortunately, there is no established LC detector (UV, MS, ELSD, RI, CLN etc) with universal response. However, there is a new Charged Aerosol Detector that looks very promising. For the time being the most used detector in the pharmaceutical industry for determination of purity is the UV detector. The best compromise for using a UV detector is probably to look at a wavelength span between 200-400 nm. A combination of different detectors can of course also be used.

Another issue is the chromatographic system.

Is it enough to use one column and one set of mobile phases (pH) for the gradient elution or should one have 2 different pH’s or maybe 2 columns with different chemistry?

There are a lot questions to think about when a purity analysis system is set up.

How do you do your purity analyses??

[DR]

Actually, ELSD detectors (Corona Discharge units are a variant on this theme) are "universal" but they aren't as good as UV when it comes to following Beer's law and they lack dynamic range.

Assuming your API has a chromophore in the UV range (anything with double bonds generally does), your task is to develop a method that separates the API from degradation & process impurities (and any other stuff in the formulation for finished dosage forms).

To see if you've that well, you can employ PDA detection to analyze peak purities, MSDs and NMR to help with identification of the unknowns (usually after stressing samples or doing some semi-prep LC to enrich some samples).

The underlying assumption is that the related substances you don't want will, like your API, have some double bonds and also be detectable via UV. Of course, the extinction coefficients may be way off so quantification often depends on some structural determination.

We generally try to get it all done with one column and a pair of mobile phases that vary the organic concentration more than the pH.

[Uwe Neue]

On the method development side, you are likely to get a lot of different opinions. I for my part have successfully used a combination of different column chemistries, two pH values, and two solvents to screen the selectivity effects that one can get from this. Usually one or two results are then sufficiently promising to do some finetuning of the method by manipulating the gradient slope or mixing solvents. I know that very similar screening methods are used in many labs, but everybody will have his own set of screening tools.

I am using a standard C18, one with an embedded polar group, and a phenyl column. The pH's are 3 or 3.75 with formic acid or ammoniumformate, and pH 10 with ammonium bicarbonate. Solvents: acetonitrile and methanol. This makes 12 blind runs, usually with a run time of 15 to 20 minutes per run.

[syx]

Is it about how to read chromatographic purity using DAD?

[yogesh#14]

It would be interesting to know how purity analysis of organic compounds (Mw200-800 Da) is performed within the pharmaceutical industry.

Detector issue: Determination of purity is not straight forward due to different response factors depending of the chemical structure of the analytes. Unfortunately, there is no established LC detector (UV, MS, ELSD, RI, CLN etc) with universal response. However, there is a new Charged Aerosol Detector that looks very promising. For the time being the most used detector in the pharmaceutical industry for determination of purity is the UV detector. The best compromise for using a UV detector is probably to look at a wavelength span between 200-400 nm. A combination of different detectors can of course also be used.

Another issue is the chromatographic system.


Is it enough to use one column and one set of mobile phases (pH) for the gradient elution or should one have 2 different pH’s or maybe 2 columns with different chemistry?

There are a lot questions to think about when a purity analysis system is set up.

How do you do your purity analyses??

Hi,

The purity of a substance can be evaluated by ELSD or UV or even by RI detector depending upon the fact that weather the molecule under evaluation has UV chromophore or not. It can also be that a UV detector can be connected in line with RI for better evaluation of purity analysis. The chromatographic method (isocratic or gradient) can be set depending on how the parameters are set. Also gradient is not useful for RI detector.

Regards,

Yogesh