I am developing HPLC method for measuring netilmicin and glucosamine drug in serum.
Both drugs have no chromophore (as I know). I am trying to develop condition for derivatization of netilmicin first. I choose FMOC as an derivatizing agent because it is said to be more stable than OPA. I use C18 column (Luna 5u c18(2) 250x4.60mm), acetronitrile/water as a mobile phase (have tried %acetronitrile as 90% and 70%) and ex260nm em315nm for fluorescence detector.
Condition for derivatization have FMOC, boric acid and glycine (the ratio and concentration are on the developing process).

In chromatographic profile, I can find a peak of FMOC reagent and this peak is decreased when there is netilmicin or gentamicin (internal standard) in standard sample, so it means the derivaization process occured. And the FMOC peak is almost disappeared when I increase concentration of glycine. The problem is there is no peak for netilmicin-FMOC derivative or gentamicin-FMOC derivative. Now I can’t guess where it has gone?

Please help me for FMOC derivatization condition or any suggestion. Thanks anyway.

Sorry for the above question! I think I should post it in LC message board.

Hi,

I have tried myself using Fmoc-Cl for drug derivatization and the biggest issue I got was the degradation of Fmoc-Cl to Fmoc-OH (i think it is called 9-fluorenemethanol). When Fmoc-Cl is placed in basic aqueous media it very quickly degrade so you obtain a lot of side products. Even if sounds stupid, you should try to check for hidden peek in your main reagents/products by testing different conditions/columns.

Good luck (you'll need it)

Dear Anonymus 1

The FMOC derivatization is one that has been tried many times with good results. I personally have used over and over, and it never failed me.

I have no idea of the metilmicin structure and I can not tell how easy the reaction would be. FMOC reacts with primary and secondary amine groups, and with some hydroxyls, and depending on the molecule it can derivatize more than one amine or hydroxyl group. If the expected derivative has two FMOC groups, then it would be difficult to elute from many columns.

Make sure you have enough reagent in the solution, I recommend you use at least a 3X excess. Another problem you may have is the pH. This reaction is pH dependent, and in general it will not run well below pH 9.

The fact that FMOC-Cl reacts with water is something of a detriment, but I have never found it to be an impossible problem. In most cases, the FMOC-OH peak can be easily separated from the rest of the products.

Another suggestion I can make is to use UV detection for the development of reaction conditions (256nm). UV is a lot easier to handle and is less suceptible to problems such as fluorescence quenching, reagent purity, etc. Once you know how the reaction proceeds and the conditions to use, then switch to FL.

FMOC derivatives are stable for a few hours at least. Therefore you should have no problems.

Good Luck

Benjamin

We can second Benjamins experience, FMOC is one of the best derivatizing reagents. Your amino group would have to be very inactive to produce a large H2O problem, we needed to be more concerned with trace NH3 amounts in the sample. My guess is also that you have way too little reagent and/or wrong conditions. Also, because FMOC is used as a blocking agent in peptide syn. there are all kinds of amino-bearing FMO-derivatives available on the market, no problem to get a good internal standard.
On the other chain of this: If you have a wavelength shift in the derivtive you are lucky. Unfortunately the chromofore of this reagent is not strongly influenced by surroundings. Remember: an ideal fluorescence derivatization reagent is one that fluoresces only as derivative.

Thanks for all comments.

Anyway, does anyone know the following matter,

- The important of boric acid in FMOC derivatization, such as how much of the concentration of boric acid in derivatization condition?

- The important of ACN in FMOC derivatization, such as how much of the ratio of ACN to water in derivatization condition?

-If the derivative is so difficult to be eluted from column, what chemical we should add in mobile phase? Should it be THF (tetrahydrofuran) to be added in mobile phase (I guess by myself, and I will try, but any comment should be good) ? Or anyone know why some mobile phase added THF?

Now I am still trying for the condition.

Thanks in advance

Hi, I work at Stable Isotope Lab, Science Faculty, Lisbon and I need to use FMOC-Cl to derivatize glyphosate so I can analyse it using HPLC (UV detection).
It´s the first time I´m trying this and I don´t have the recipe to perform it here in the lab.
Can anyone send it to me?

I´m not s chemistry so forgive me if this is a stupid question but, what kind of safety concerns should I have dealing with FMOC-Cl?

Thanks.

Carla Rodrigues

For toxicity of FMOC-Cl, go to website,
http://hazard.com/msds/

I don't know so much, but as I read information on website, I think we should not contact or inhale it and FMOC-Cl is rather toxic.

Hi, I'm doing studies on FMOC derivative with carnitine(OH) but I can't found any information on the stability of this derivative.
May anyone help me?
Thanks in advance