Compound identification by retention time: Guidelines ?

[LCFan]

Hello,

we perform identification of the active substance in a drug product by normal-phase TLC as well as by reversed-phase HPLC. The rf value in TLC and the retention times in HPLC, respectively, have to be similar.

But what is similar? Retention time of drug substance in the drug product chromatogramm within 5% of the drug substance retention time in the standard chromatogram? Or within 2% ? Or 10%? Or dependent on the absolute value of RT and therefore better to set an absolute range?

Is there any guideline on this topic availale?

Florian

[Kristof]

I've been looking for an answer to very similar question a while ago. Best thing I've found was that RT window for detection & identification should be less or equal to peak width at 50% height. This was based on interpretation of equations for chromatographic resolution. It was explained in simple words as - if component RT would move more than that, then it would not resolve from its neighbour i.e. can't be distinguished from its neighbour.

[lmh]

No criticism whatsoever of the original poster or anyone else obliged to work this way but:

if you are identifying something by chromatography, then look at it this way: divide the length of your chromatogram by the acceptable window size. You will probably find that your chromatogram is not much longer than 100 windows, so you have a 1% chance that a purely randomly-chosen chemical will elute in the acceptable window of the thing whose identity you wish to confirm.

In fact the situation is far worse, because often the likely alternative things you would find are related to the target compound, and are more likely to elute in the same region than at a wildly different retention time. And the peaks tend to be clustered in any method anyway.

In effect, if you are identifying by retention time, you can only be sure you're right, if you know that all the alternative chemicals that might be present don't coelute. If that's the case, the big question is where do they elute? If none elute within 5 minutes of the target compound, you could actually have a 4 minute window. If one elutes only 2 seconds away, then you're going to need a very tiny window indeed.

What I'm trying to say is that Kristof's approach is correct in so far as he's saying "if the peak is closer than this, then its retention time doesn't differ with statistical significance; if it is further, it does, and must be something different". But the approach is inadequate in that lack of statistically signficantly different retention time is a truly terrible guarantee that the chemicals detected in two runs are identical. A statistical approach is attractive but inappropriate: in the end you do need to know whether the method can distinguish the target compound from the nearest mistakeable alternative that could happen, and no amount of clever statistics will tell you the retention times of the two.

Chromatography doesn't identify things, it only separates them. Chromatography with non-selective detection is, I'm sure, fundamentally unsafe unless you know what's in your matrix.

[tristanewalters]

In methods we run for HPLC we typically have a +/- 2% retention time requirement. For the TLC, I'm not totally sure what our rf value requirement is exactly, but I know that we typically include that the shape and color intensity should be visibly similar. This assumes that the concentrations of your standard and sample are the same.

[LCJEN]

We prepare a ID solution by mixing 1 mL of sample solution and 1 mL of standard solution, if the ID solution appear to be a single peak, then the ID is positive.