What derivaties were you using for your GC anslysis and what is your internal standard? MS isn't going to help you other than to determne the mass. Also, what is your extraction procedure prior to your GC analysis?

Your best first bet is to try and solve this with GC, try a set of standards that is likely with your tubers in mind. I would personally start with a GC based monosaccharide analysis, to find out what kind of monosaccharides you have. Then you can later determine whether your huge peak is a mono- or disaccharide. YOu could use MS to determine the mass and see whether it is mono- or di.

Agree with Sander and Allen as approaches and questions. Extraction is a very important question along with are you sure it is a sugar?

1. What sort of derivatisation have you done before GC? and can you attach the trace and methodology of derivatisation

2. What mass is the unknown? disaccharide, tri, - I am assuming the GC had an MS on the back not FID which may not be correct. If it was FID was the unknown in the di, mono or tri area?

3. LC-MS won't be the magic bullet to identify your unknown. LC MS/MS and classical GC MS/MS may be useful additions to the tools you will need to apply if the oligosaccharide is intransigent

4. you may have to isolate/purify a small amount to undertake monsaccharide analysis and the other techniques for linkage and sequence analysis which is where the fun really begins. But do the easy approaches first eg think through a wider variety of standards such as Sander has suggested.

Thanks a lot all of you for taking your time to share my problem with me.

Paul, I used the stox (hydroxylamine hydrochloride in pyrudin), HMDS (hexamethyldinsilizaan, and TFA , trifloroacetic acid for my derivatisation. the GC has FID and we know that the unknown cpd is a disaccharide.

I think the next step is Sander's suggestion. A full monosaccharide composition analysis will help decide which disaccharide standards to try. If you've got a disaccharide fraction to use for the monsaccharide analysis after hydrolysis that would even be better. Even co-elution won't be 100% identification but it is a good start in the characterisation

I would still like to know what the extraction procedure is and what tissue is being extracted? If an acid step is used is it strong enough to cleave glycosidic bonds and if so why should there be a disaccharide present? This is unless the suspected disaccharide requires extraordinary measures to achieve hydrolysis which I have run in to on one occasion.

may be you can try ICP-MS, may be add a metal in your extract, this metal make bound with your disaccharide, you can calculate concentration on the glycolitic bound. If you want identificate this disaccharide, you must have standart about this according to retention time and uv spectrum, you can identify with LC-MS.

Thank you Ozan, Interesting. Please can you elaborate it a bit.,

Knowledge of the tissue and extraction procedure would still be very helpful as well as knowing what your internal standard is.  I would suggest HPAEC.

Hi Gifty

I would be interested to know the answer to Allen's question too. Tubers are usually more polysaccharide than disaccharide so you may be dealing with a hydrolysis product due to the extraction process.

This could be construed as a silly question (don't hit me!!).....do you see the same peak each time you do an extraction?

Do you have access to HPAEC-PAD?

Cheers

Paul

of course i see the same peaks all the time from GC varian FID. However I don't have access to HPAEC-PAD

You can not identify reliably sugars by MS, or more exactly you can not distinguish them: you will know whether you have an hexose or a pentose of a dihyhyxose; but not which hexose or pentose (there may be difference sin the MS spectra but they are faint (differences in relative fragments heights) and machine-dependant thus claibratin is necessary anyway) . So you need calcibration for that NMR would be a bette roption, there the signals are distinctive.

thank you Catherine for your input

How is the analysis going Gifty? you made progress? Just interested to see

Gifty,

I don't understand why you say you do not have access to HPAEC-PAD since there are several groups at your university publishing HPAEC-PAD data.  Have you approached any of those groups?

Thanks for the information.I Would  have approached them if i knew that group. Would you mind if i ask you which group? 

Gifty,

Just go to Google Scholar and use the search terms HPAEC and Gent.  You will get a whole page of listing of papers from Gent including the Laboratory for Glycobiology.  There are probably more on the following pages as well.  That should give you several people to contact.

Thanks a lot indeed. 

Gent has a glycobiology dept. As well as a beer brewery lab with pilot plant. Both have at least ons HPAEC-PAD 

Thank you so much for your information  i found out that and will communicate them sooner

GC/MS using the aldonitrile method.

BTW how are you doing sugars? I'm looking to do GC of sugars. So far my conclusions from the lit review is aldonitrile acetate method (hydroxylamine then acetic anhydride) method works best for monosaccarides as there is just one peak per sugar especially reducing aldoses but it only produces derivatives of monosaccarides that are volatile enough for GC. The reagents are also very clean and easy on the GC and liner.

Straight silylation produces as many as 4 peaks per sugar. Also silyation is very water sensitive and the reagents tend to fould the GC.

Oximation followed by silyation produces a maximum of 2 peaks per sugar syn and anti at the aldehyde.