Hi All,

Has anyone done the assay of Glucosamine in plasma before? LCMSMS assay was performed but a puzzling result was obtained. Peaks were observed when Glucosamine is dissolved in methanol or acetonitrile. However, once it is obtained from plasma, the extract did not produce any peak correlated to Glucosamine. All the fluid obtained from each step of the sample cleanup were injected too but did not produce any peaks. Can anyone help out on this please. Any suggestion would be highly appreciated.

Thanks

I suspect that you saw the protonated molecule with STD while the molecule exists in Na adduct form from extraced plasma. Just a guess.

a blank plasma was extracted and the glucosamine was spiked into the extract at the final stage of the preparation, but there was still no peak observed. I don't know why.

You said your standards were in acetonitrile or methanol. What was the final solvent for your extracts? What were your LC conditions? Did you see any peaks at all in your spiked extracts, even at different retention times (with same MRM pair as glucosamine)?

Dear MG,

Thanks for your reply. Sorry for the delayed response. Anyway, I used Acetonitrile to precipitate the proteins in my plasma/urine sample. I injected the supernatant into the LCMS. I did not see any peaks with same MRM pair as glucosamine. Liq-liq or solid-liq sample prep is not possible. So only resorted to proyein precipitation. Like I said earlier though. Even if a blank filtrate after ACN treatment was used and Glucosamine was spiked into it immediately prior to injection, I could still not see any peak similar to MRM of Glucosamine. Please explain why this happen. IF it had been that there were peaks similar to MRM of Glucosamine, are you suggesting that they might be the one that I am looking for?

It is possible that your analyte co-precipitates with the proteins. It is also possible that you have ion suppression in MS that prevernts you to see the analyte. Protein precipitation is very dirty. I would attempt to do SPE with a cation exchange sorbent. If you do not have experience with this go to manufacturers websites and look for methods. I found fairly good methods on the Waters website.

Thanks, A.Mouse, I will look into your suggestions.

Original poster: What I was suggesting, was that if you had a solvent mismatch between your sample solvent and mobile phase, and injecting a large enough volume, you might observed different chromatographic behavior in samples vs. standards. Since you aren't seeing anything, I tend to agree with A.Mouse's ion suppression hypothesis. In addition to offline cleanup, it is sometimes possible to adjust your chromatography and get your analyte separated from the matrix trash, but this will slow down your analysis time. What is the k' value for glucosamine under your LC conditions? Are you using reverse phase or something else?