Chromatography Forum

Hi all..

I have a problem in conducting system suitability test (SST) of tretinoin.
I use acetonitrile/water:90/10 as solvent.
I also use gradient separation with acetonitrile/phosphoric acid 0.1% (90% acetonitrile to 75% acetonitrile).

The problem is, the area of tretinoin become lower each time I inject the standard, so the accuracy is less than 98%.
I have check the pressure profile, and i think there's nothing wrong with my HPLC.

Do you have any suggestions?
Thanks a lot.

My experience from working with tretinoin is that it is fairly photosensitive, so I would lean towards degradation of your standard as it sits in the vial. Are you using amber HPLC vials? Is your autosampler protected from light? Is low actinic glassware being used during preparation? Tretinoin has a lot of known degradation products, do you notice an increase in other peaks from injection to injection as your standard peak area decreases?

hi Blazer..

Yes, you are right. There is increasing in another peak area from injection to injection of standard. I have used amber glassware, but it still happen.
Do you have any idea how to minimize this degradation?

thanks a lot

At the company where I was working with tretinoin, we had actually installed a second set of lights in one laboratory. When we were doing any testing with tretinoin, we would turn off the usual laboratory lighting and turn on this secondary lighting which was a kind of yellow lighting. I'm not entirely certain what it was, I was simply told it was meant to minimize photodegradation. We would also close all the doors and pull down the blinds to all windows in that lab. This might be have been a little extreme, but it worked for us.

You probably can't make these changes to your laboratory, at least not very quickly. You could try wrapping your volumetric flask in aluminum foil, even if they are low actinic. If your low actinic glassware is old, the coloring on the flasks can flake off, thus allowing light to get to your solutions. Is your autosampler also protected from light? If not, find a way to get the autosampler tray protected from light. On Agilent systems, you can again use foil to cover the opening to the tray if you don't have the dark plastic doors that come with some modules. On Waters Alliance systems, the autosamplers are already enclosed so it is less of an issue.

There are degradation products in tretinoin that result from other sources, not just photodegradation. If your method is not designed to separate these from one another, it's possible that you're also seeing these in the increasing peak, too. You can also try to keep your autosampler chilled to help minimize some of these.

How much are your areas decreasing from injection to injection? Keep in mind that there is no realistic way to completely eliminate all degradation while working with this compound, it's just too tall of a task to do so. If you could post some real data, I am curious to see if the drops in area count are as significant as I remember.

Actually, I do the injection manually..because i don't have any autosampler.
Your advice makes me think how to minimize the sample from being exposured to light.
I used 1 ml syringe, and now i changed it into 50 ul syringe..and that's work. So, i can do the SST with acceptable recovery result.

Thank you very much for your help Blazer.

Glad I could be of help. I've learned so much from these forums over the past few years, I'm happy to help out where I can.