help in develop of glucosamine metod

[edwfel]

Hi!!!

I´m developing a new method for Glucosamine.

I have been testing with c8 and c18 with bad results. but with amino column (Buffer fosfate pH 7.5: ACN (25:75)) the peak of the active, eluting beside a negative peak, due to water that I use to dilute the sample.

Is possible eliminate the negative peak diluiting tha sample in the same Movil Phase??.

Do you now other way to test this active?

thanks a lot for your information!!!!

I´m not speak very well English, so, please excuseme grammar mistakes.

[Noser222]

Yes, you can minimize that negative peak by diluting the sample in mobile phase. Is the peak shape of the glucosamine ok?
Without some kind of derivitization and/or ion-pairing, you won't get much retention at all with a reverse phase column. If you are interested in that, here is a method I've seen:

http://www.uni-trier.de/uni/fb6/umweltc ... l-Chem.pdf

[SIELC_Tech]

Glucosamine is not retained on regular RP columns even at low organic concentration. Here is application for similar amino sugar. It is well retained by mixed mode approach. We are working on separation of glucosamine and chondroitin for one of our customers and I can update you when we have a method.
You can use ACN/water-buffer for retention of glucosamine on Primesep column. You will need to use ELSD or LC/MS to detect glucosamine.
Choices of buffer: TFA, ammonium formate or acetate, formic acid:

http://www.sielc.com/compound_003.html

Regards,

Vlad

[edwfel]

ok, thanks !!! Noser222


But, I have some questions about the derivatization method!!!

To perform a derivatization method is necessary to buy a new precolumn, or is a step made before inject the sample?

To work that develop I have a standard HPLC with UV or PDA detector, and regular columns. Can I develop that method of the article whit this tools? Or is necessary to buy more specific tools?

I´m just train to develop a easy method that be useful with the tools that I have, or will be great if you can show me some links about How develop a good Derivatization method, because I never made something like that.
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SIELC_Tech thanks, but in this moment our laboratory is not interested in buy more equipments, so, I have to tray to find de way to do this develop with the tools that I have.
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the USP method appear be easy but when try, the R.T. was too small, in C8 and C18 and for that reason I change the column for a NH2 column, and in that column there is a big treasure at pH 3 (195 nm) and at pH 7.5 is better but there is a negative peak that I couldn’t separate of the main peak by changes in the eluent and pH.

I run an inject with only diluent (water), and the signal was the negative peak.

[SIELC_Tech]

You can use different derivatization agents. They can convert your glucosamine to UV active compound and increas hydrophobicity at the same time.You can use dinitrobezoyl chloride to convert amino group to amide. Two nitrogroups will give you a very good increase in UV activity and increase your hydrophobicity. Treat you glucosamine with 3,5-dinitrobeonzoyl chloride in the presence of tertiary amine. Use 1 eq. of benzoyl chloride.
Here also some other derivatizing agents for amines:

http://www.sigmaaldrich.com/img/assets/ ... 2_new_.pdf

Regards,

Vlad

[malaka50]

this is one of my favourites, works very well.

http://www.nsf.org/business/ina/glucosa ... rogram=INA

i think chromadex sell a complete kit with the glucosamine standards, hplc column and method. not 100% sure of this, best to check with chromadex.

[Smilla]

Hi edwfel!

I’m a student who is about to finish my master thesis very soon.
The task has been to optimize a Luna NH2 column (Phenomenex) (250x4.6 mm) (5µ), for separation of N-acetyl-D-glucosamine and D-glucosamine.

Things haven´t worked as I wanted (just like it uses to be in a lab... ), but I got the best results when I used a mobile phase consisting of acetonitrile:ammoniumacetate 5 mM, 80:20, with a flow rate of 1.0 mL/min, and a column temperature of 50 oC. The detector was an ELS-detector.

The retention time for the glucosamine was ~9 minutes.

Hope this can help you in some way.

Regards,
Smilla

[Noser222]

The method I posted is precolumn derivitization, so you shouldn't need anything except for chemicals. The HPLC column that they use has embedded polar groups, so I don't know if an ordinary C18 would perform quite as well, but would be worth a shot.
Flourescence detector is best for this method, but a UV detector gives good sensitivity as well.

[Einar Ponten]

You only need to consider derivatization if detectability is an issue.

Else, this is a compound that will be nicely retained in HILIC mode as you already have tested.

Firstly, try to avoid phosphate buffer since the solubility in acetonitrile is low. Secondly, follow the recommendation above to dilute the sample in mobile phase or a solvent containing less water/buffer than the mobile phase.

Then, of cause, a ZIC®-HILIC column could be an alternative for optimised performance.... if you like to get a a hardcopy of our "Practical Guide to HILIC" please send us an e-mail.

[Chris Pohl]

edwfel,

Another option not mentioned is to retain and separate glucosamine at high pH via anion exchange. Dionex sells a range of columns designed specifically for such applications. In this case, detection is via electrochemical detection so no derivatization is necessary. You can find more information at this link: http://www1.dionex.com/en-us/columns_ac ... s4774.html

[Tony_Montanari]

This is from a prvious post of mine:

I have developed a method that uses Polymer Labs Aqua-Gel-OH columns (30 and 40) and any type of universal detection (RI, ELSD). Using Ammonium Formate at pH of 9 with these columns gives a broad uniform peak separated away from the Chondroitin Peak that comes out first. This mixed mode separation allows you to measure Glucosamine and ID Chondroitin at the same time. Glucosamine is separated by Ion Exchange and Chondroitin by size.

PL came up with a method using 50:50 Methanol:Potassium Nitrate (pH 9) and RI detection. Just play with the pH to get the retention time that works the best and gets Glucosamine away from Sodium. My mobile phase, which is detected by ELSD, is 100% water with 60mM Ammonium Formate at pH 9. The columns are expensive, but we must have well over 10000 injections on these with no sign of wear.

[sotsam]

the best choice for glucosamine (it worked perfectly in my case) is a HILIC (Hydrophilic Interaction) column. it is a special column for highly hydrophilic compounds and it works like a normal phase column. I used this column with a mobile phase acetonitile:buffer pH=2.5 in isocratic mode. I had tried previously C8, C18 columns with RT~2-3 min, derivatization methods and so on. Nothing worked expept the HILIC column. there is even a paper for glucosamine and HILIC separation. you hould really try it!

[Tony_Montanari]

The reason I use the Polymer Labs Aqua-Gel OH columns for the messurement of glucosamine is that it supplies a mixed mode separtion. Not only do I see glucosamine in this separation, I also get to see the Chondroitin which is in the same tablet. I can not measure Chondroitin because it is too polydisperse and the peak too broad. The nature of the mixed mode separation is a good diagnostic tool.