Chromatography Forum

what is the condition for derivtizing of alendronate sodium for detection on fluorescence detector(hplc)?
with FMOC CHLORIDE (DERIVETIZING AGENT)

There are several posts on this forum dealing with alendronate. However, after a brief search there was no on derivatization, but for sure there should be in the literature.

Nevertheless, since this is a primary amine it should be possible use post-column derivatisation with OPA (o-phthaldialdehyde) [also more info on the forum] forming a fluorescent isoindole derivatives. For retaining alendronate you could use ion-exchange or HILIC at alkaline conditions.

I haven't done alendronate with FMOC, but I have done some other things that way. First, you need to estimate enough FMOC-Cl to derivatize every amine in your sample at least 5x excess, not just the alendronate, everything. Use anhydrous acetonitrile to dissolve the FMOC-Cl; make the reagent solution so that you use about 9:1 sample:reagent ratio.

You need to buffer the sample to pH 9 with a borate buffer before adding the FMOC-Cl.

If you are doing this manually, use a capped vial for the reaction. Pipette the FMOC-Cl reagent into the buffered sample, immediately cap the vial and mix it vigorously. Your precision depends on good mixing. The reaction is complete is a minute. The derivative will be stable for several hours.

If the reaction byproduct, FMOC-OH interferes, you can extract it with toluene or heptane.

If you can, use a robotic autosampler. Again, good mixing is key.

PS. 'dronates are chelating agents, and will react badly to traces of metal ions in your system. You can use a competetive chelator (EDTA, pyrophosphate, etc.). Alternatively, you can put calcium in the mobile phase and sample and do the analysis as the Ca complex. I've done the second for ion-exchange.

I've also done OPA/thiol post-column for alendronate, and it works.

i had tryed this method
in ph9.0 borate buffer,added excess FMOC CHLORIDE
and the mobile phase was tried different ph like 9.0,8.5,6.5
but theproblem is here that the peak which was observed ,it was also observed in blank sample(without drug)and the response of both were same so it was conclued that the peak is obeserved by FMOC CHLORIDE
SO please tell me what's the problem in this procedure
has it any specific condition?

I had found in literature search:

http://www.sciencedirect.com/science?_o ... 0003c6e407

Min-Hyuk Yun and Kwang-il Kwon

High-performance liquid chromatography method for determining alendronate sodium in human plasma by detecting fluorescence: Application to a pharmacokinetic study in humans

Journal of Pharmaceutical and Biomedical Analysis
Volume 40, Issue 1 , 23 January 2006, Pages 168-172

yes i did follow this method but the problem remains the same .
in this method i obtain only FMOC CHLORIDE PEAK.
but the analyte peak was not detected.the peak was observed in also a blank sample (without drug)
and the area of peak are same in both sample

FMOC-Cl is an acid chloride. It reacts with water to make FMOC-OH, but it reacts with amines more rapidly to make the FMOC-amine derivative. What you are seeing is the FMOC-OH peak which is going to be huge. This can be extracted with a non-polar solvent. The derivative is polar enough that it will mostly stay in the aqueous phase.

Derivatize some of your alendronate stock solution and see if you can find that peak.

With amino acid one also always has traces of NH3 which gives a strong peak, maybe here also? Anyway, any remaining FMOC-CL can be removed with a suitable amine containing substance (adamantyl amine for very late elution, glycine for early elution).... it has been done millions of times.

The beauty with using OPA is that only the derivate is fluorescent.

The back-side is the low stability of the derivative, meaning that the reaction has to be done online and post-column.

You can minimise the excess regent problem as suggested above. By using OPA is another way, but then you get other things to keep in mind.

I just happen to have my system set up for automated pre-column OPA/thiol derivatization. I'll see what happens.

Yes, "at line" is more proper than "online", as Mark point out.

Mark,
please tell us how many derivatives you get per analyte and how you prevent the stabilty problem. As I stated before, I deeply admire anybody who gets practical and useful results with OPA.

This is from the group that developed the FMOC derivatization

J Pharm Biomed Anal. 1989;7(12):1719-27. Related Articles, Links


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