How to completely remove FMOC-OH from the aqueous phase afte

[bo]

I ran into some trouble when using FMOC-Cl to derivatize alendronate sodium. After derivatization I cannot separate the alendronate derivative and FMOC-OH. Both have almost the same retention time and especially, FMOC-OH has a bad tailing. Is there some way to get rid of FMOC-OH? I tried methylene chloride and it doesn't extract all FOMC-OH.

[HW Mueller]

Generally: Most people move it to either "end" of the amino acid "window". If excess FMOC is reacted with aminoadamantane the derivative elutes behind the amino acids. We used glycine (there was none in the sample) to move the excess FMOC (as glycine derivative) ahead of the amino acid of interest. It shouldn´t be difficult to move it away from alendronate using this trick.

[bo]

Yes. It's true FMOC-Cl elutes far behind and it does not cause any problem itself. The problem is FMOC-OH left in the aqueous phase interferes with the desired derivative.

I think anilray1155 posted a similar post stating he had the same situation the blank also shows the peak which I believe is FMOC-OH. The tailing of FMOC-OH is terrible.

[HW Mueller]

Looks like you misunderstood me, probably because I wrote FMOC instead of FMOC-CL. Anyway, unless your alendronate reacts very slow under the conditions you use, one should be able to keep the FMOC-OH low enough to handle. Also, it´s important not to let sample stand around with excess FMOC-CL. I had more trouble with FMOC-NH2 than with FMOC-OH.

[bo]

Thanks for the clarification. The concentration of FMOC-OH is low, but it still contributes ~10% of the drug derivative area since the drug content is also low. Tried many columns and conditions, can't separate FMOC-OH. Any idea to get rid of FMOC-OH by extraction or reaction?

[HW Mueller]

Darn, just noticed that I made a mistake also: I originally read your question as FMOC-CL instead of what you wrote, namely FMOC-OH. Again I have only seen methods to reduce the latter, not to eliminate it. If you can not move (can´t imagine that) relatively low amounts away from your analyte I can think of one more posibility: Do a two step (two dimensional) chromatography.

[Mark Tracy]

When I tried alendronate, I found that adding a small amount of calcium to the mobile phase moved the retention time and improved symmetry.

[bo]

Mark, would you please be more specific about adding how much calcium in the mobile phase?

[Mark Tracy]

I guess I should tell you that I was doing the separation by anion exchange. I added about 5 millimolar CaCO3 to my KOH eluent. For an RP separation, I would use Ca(OAc)2, at no more than 10 mM. The idea is that since the 'dronate drugs are chelating agents, they will interact with traces of random metals in the system, and by converting the drug to the calcium complex it will be less prone to that problem. Also the net charge on the complex is lower (at least when my eluent is KOH) and that is what changed the retention time. Anyway, it did work for alendronate and clodronate.

[JMB]

My advice would be to not re-invent the wheel.........

This is from the group that developed the FMOC derivatization for alendronate

J Pharm Biomed Anal. 1989;7(12):1719-27.


The determination of 4-amino-1-hydroxybutane-1,1-diphosphonic acid monosodium salt trihydrate in pharmaceutical dosage forms by high-performance liquid chromatography.

De Marco JD, Biffar SE, Reed DG, Brooks MA.

Merck Sharp and Dohme Research Laboratories, West Point, PA 19486.

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay is reported for the determination of 4-amino-1-hydroxybutane-1, 1-diphosphonic acid (AHBuDP) monosodium salt trihydrate, a new inhibitor of bone resorption. The compound does not demonstrate any intrinsic UV properties and thus pre-column derivatization of the primary amino group of the drug with 9-fluorenylmethyl chloroformate (FMOC) at pH 9 in the presence of sodium citrate is required to facilitate UV detection of the analyte. Excess derivatization reagent is extracted with methylene chloride and an aliquot of the aqueous portion is assayed on a polymeric phase (Hamilton PRP-1) at 35 degrees C by reversed-phase HPLC. A mobile phase of 0.05 M citrate and 0.05 M phosphate buffer (pH 8.0)-acetonitrile-methanol (75:20:5, v/v/v) is utilized with UV detection at 266 nm. Application of the method to the analysis of AHBuDP in I.V. solution, tablet and capsule formulations is presented.

Good luck,

JMB

[bo]

Thank you guys. Actually, I was following the method in the article JMB mentioned and had the problems.

[HW Mueller]

Since I garbled it above I am repeating what I tried to say: Optimize your reaction so as to reduce the reaction time of FMOC-CL with H2O. Get rid of all FMOC-CL via reaction, immediatly after completion of the alendronate derivatization. Try to optimize your HPLC for FMOC-OH as well as the alendronate derivative. There is no reason as to why small amounts of FMOC-OH should have a large tail.