Glucosamine - Science topic

Hello! I‘m a student and the topic of my research is developing a method for quantitive analysis of glucosamine and chondroitin (in tablets after dissolution) via HPLC-DAD.

We‘ve been using tosyl chloride for derivatisation and NaOH for hydrolysis of chondroitine. After analyzing glucosamine and chondroitin (after hydrolysis and derivatisation) separately and together, we got just one peak. It seems that those are derivatized glucosamine and galactosamine together.

We tried using gradient mode, changing pH and mobile phases, but it didn’t make things better at all. I also tried ion-paired additives (octanesulfonate), but I was told that it can destroy the column so I stopped. Is it even possible to separate these two compounds with a C18 column?

I would be very grateful for your advice!

Thank you in advance!

I am trying to separate glucosamine and N-acetyl glucosamine from a filter-sterilized biological sample containing salt, amino acids, and other impurities. I used a standard solution containing an equimolar (2.5mM) solution of glucosamine+ N-acetyl glucosamine in milli-Q water. However, I am only getting one single peak of N-acetyl glucosamine (retention time 2.5 minutes). Can anyone help me with the separation process? I tried the following conditions:

Mobile phase A: 10mM sodium acetate+10 mM sodium octanesulfonate in water at pH 5.1

Mobile phase B: MeOH

Injection volume: 5 µl

Flow rate: 0.7ml/min

Column temperature: ambient

Column: Nucleodur C18 Gravity, 250x4.6 mm, 3 µm

I need how to made standard curve for   N acetyl D glucosamine in A582 you published it in paper "Chitinolytic assay of indigenous Trichoderma isolates collected from different geographical locations of Chhattisgarh in Central India"2012 

i need a method to prepare the dilution of NAGA with  1% DNS "3,5  dinitrosalycilic acid " 

how to prepare the NAGA ?? in this Method

Hi,

I need help understanding qPCR analysis, i have calculated the mean CT, ∆Ct, ∆∆Ct and 2^-∆∆Ct is this called the livak method? Where can i see where if there is downregulated or upregulated?

I have also dona ANOVA multiple comparisons (tukeys) test on SPSS and i have a control and two treatment groups, where the significance is <.001 what does that mean? please help!! I need some guidance.

Gram-negative bacterial pathogens interact with the host cells by using type III secretion systems (T3SS) to inject virulence proteins into cells. NleB effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected, due to the activity of chaperones . El Qaidi, et al (2020) reported that the bacterial glutathione synthetase (GshB) is glycosylated by NleB resulted in enhanced GshB activity, leading to an increase in glutathione production, and promoted bacterial survival in oxidative stress conditions. Is it possible that true role of T3SS effectors in bacteria was overlooked?

El Qaidi, S., Scott, N.E., Hays, M.P. et al. An intra-bacterial activity for a T3SS effector. Sci Rep 10, 1073 (2020). https://doi.org/10.1038/s41598-020-58062-y

(4) (PDF) An intra-bacterial activity for a T3SS effector (researchgate.net)

Dear all,

I'm currently interested in the hexosamine-pathway and especially in the enzyme Glutamine fructose-6-phosphate amidotransferase (GFAT).

The end product of the enzymatic reaction of GFAT is D-glucosamine 6-phosphate. It is known that glucosamine can enter the cells when added to cell culture via glucose transporters, but there is no data in the literature whether D-glucosamine 6-phosphate can be transported by these transporters as well. Does somebody know whether D-glucosamine 6-phosphate can enter the cell through glucose-transporters or maybe via alternative ways? And my second question is, whether D-glucosamine 6-phosphate is stable in cell culture conditions at all?

Kind regards,

Robert

Hi,

I have been trying on a USP method regarding Glucosamine Sulfate Potassium Chloride. The assay is rather simple. It requires the use of amino-phase column to be used in reversed phase mode (which some may call HILIC?). The column i am using is Phenomenex Luna NH2 column (150 x 4.6mm, 5micron). The mobile phase used is premixed 75% acetonitrile: 25% phosphate buffer.

After about 20 injections, i discovered that the retention time shifts forward (earlier elution). Initially, it was around 10min (as indicated in the monograph), later shifted to about 5min and stabilised. This is on of the issues. However, a second issue later arises. After about 140 injections, the theoretical plate numbers fell steadily from 2400 to 1200. Then, i tried washing the column as instructed by the manufacturer with EtOH, followed by Hexane for 20 column volume. Unfortunately, the efficiency fell sharply to 700 (i might have killed the column?).

Here are some of the things that i have been doing:

There are actually a few questions on my mind right now:

Your help and sharing is much appreciated! Please let me know if i have left out any info to allow you to better understand my situation.

Thanks!!

Please suggest me a standardized method to prepare DNS reagent for the determination of reducing sugar by using N acetyl D glucosamine.

Thank you

Hi everyone. I am currently working on HPLC assay of glucosamine, in accordance to USP method. Below are some of the specs of the method:

There are several problems I am facing currently, such as the retention times are not at around 10min (as stated in USP). Also, I am having a problem of delaying of retention time after each injection. For example, a sequence of 6 injections that I have ran gave such retention times, from injection 1 to 6: 5.265, 5.279, 5.294, 5.303, 5.313, 5.322.

The equilibration of the column took about 2hour before the initiation of sequence.

Did anyone encounter such situation before? Your advice is much appreciated.

I have been trying to synthesise it using Glucosamine HCl and Succinic Anhydride along with triethylamine. But I am unable to get a good yield of the product. Kindly provide valuable suggestions

Osteoarthritis is characterised as the deterioration of the articular cartilage which then causes bone-to-bone contact. Glucosamine is a primary constituent of cartilage proteoglycans, which is why there are claims that glucosamine could possibly be an anti-arthritic. Despite this, there has been no current and impressive evidence supporting this claim. Has there been any current advances on this claim and if so, what are they?

Dear Colleagues,

Currently, I am making Phospholipase D (PLD) catalyzed transphosphatidylation between Phosphatidylcholine(PC) and Glucosamine in order to get phosphatidyl-glucosamine '( A glycophospholipid). As the phosphatidyl-glucosamine is a new molecule therefore no standard for it, I would like to know how can I quantify the amount of phosphatidyl-glucosamine ?

Your king answers are highly appreciated

Hi, I have extracted glucosamine from lobsters shell, however my extracts are not soluble in water,Could you please tell me why and give me a suitable way to dissolve them in water?

Thank you

Hello,

 I am using EDC and NHS to attach fatty acids (oleic and linoleic acid) onto chitosan (5000 Da) backbone. The final product I am getting is extremely cytotoxic. I am wondering if EDC is the cause of this cytotoxicity, and it is not getting removed properly.

None of the other components I am using for this reaction are toxic to the cells (HEK-293).

 The procedure I am following is a widely used/published protocol. I dissolve chitosan oligosaccharide in distilled water at pH 5. Separately dissolve EDC (1 mol/mol of fatty acid), NHS (1 mol/mol of fatty acid), and fatty acids (0.3 mol/ mol of glucosamine unit of chitosan).

I add the ethanol solution dropwise into the chitosan solution while stirring, and let it stir at room temperature for 12-24 hours. I dialyze this solution against deionized water, repeatedly changing water 2 times a day, upto 4-5 days (protocol says 2 days, I have tried longer). Freeze dry the product, purify with ethanol to remove unreacted fatty acid, freeze dry again.

The final chitosan-fatty acid polymer sample is extremely cytotoxic (5-30% cell viability), while chitosan alone gives me almost 100% cell viability (using MTT assay). I am wondering if it is the EDC/NHS that hasn’t been removed properly by dialysis.

 Can someone suggest something that is wrong with this reaction, with the EDC, or the way I am using the EDC?

I'm trying to convert glucosamine into glucose. I've found one paper online explaining how this can be done, but it's over 100 years old, and requires about 10 steps ( CXIX.—The conversion of d -glucosamine into d -glucose ) . Does anyone know if there is a quicker way to do it? The only thing I have to go on right now is that the internal alcohols need to be protected before a deamination agent can be added to the mixture.

Thanks for all the help :)

During chitin hydrolysis, non-crystalline glucosamine also obtained whether is it due to caramelisation. If anyone worked on that please give me some references

How to generate a SER dipeptide with O-GlcNAc (beta- glucosamine) attachment at side chain with OH group of SER. I have genetared the dipeptide with O-GlcNAc , It shows complete dipeptide when visualized in pymol but not in Chimera (shows GlcNAc is not attached to serine). Thus I can not submit it to charmm-gui for further input file generation. Please Help.

Alginate is a water-soluble linear polysaccharide composed of alternating blocks of 1– 4 linked α -L-guluronic and β-D-mannuronic acid residues ,whereas chitosan is a co polymer of D-glucosamine and N-acetyl glucosamine.

They have favourable properties of biocompatibility, biodegradability, pH sensitiveness and muco adhesiveness.

What are the compatible columns of HPLC for analysis of N-acetyl glucosamine?

Hello all,

The molecule is a derivative of N-acetylated glucosamine with 3 -OBz groups, the glucosamine is also attached with an aliphatic chain which contains methyl ester.

Please suggest me on substituting the -OBz groups with -OAc groups in one or minimum number of steps. The methyl ester also later to be hydrolyzed.

I tried this in 3 steps, debenzoylation, hydrolysis of ester and acetylation of hydroxyl groups. But the impurity formation is high

 I am trying to read about this but I could not get a final answer and clear one 

Leaves dry powder

I am trying to develop a HPLC method for

Glucosamine is a chitin-derived mucopolysaccharide and putative disease modifying agent; anti-rheumatic agent.