Chromatography Forum

Dear all:

I need your help! Any suggestions are welcome, please!

I am working on the quantitation of an oligosaccharide in plasma. The analyte has 24 carbon and MW around 600 Da. The instrument is LC/MS/MS. This compound is too hydrophilic to retain by the RP-HPLC. I use Luna amino column (150x2.1). I start with 75% B (ACN), hold for 0.3 mins and drop to 30% ACN for 2.4 mins and back to 75% B to 6.5 mins. Right now the retention time is 4.3 mins. I have two major problems right now:

1. The reproducibility is bad, even in solvent standard. For solvent standard of 20 ng/mL, the peak height was shift from 270 to 190 during three continue injections. However, at higher concentration like 1000 ng/mL, the variation is smaller. The RT has no significant shift (maybe 0.02 mins).
Here are some tests I performed:

I. Extent each run form 6.5 to 8.0. No luck
II. Change the gradient like 90-30, 85-30 and 75-30. Same problem.
III. Change the mobile phase, A from 0.1% FA in water to 5 mM NH4OAC (pH=5.0). Same problem.
IV. I also try CN column. Similar problem was noticed.

One concern is my Massspec is not stable. But it is fine when I use same mobile phase to analyze other compound. I still feel that this is a LC problem. Whenever the sensitivity was change, the peak shape was change too. The lower sensitivity, the more significant tailing was observed.

I wonder if I can try something else. Shall I try ion pair reagent? Which ion pair reagent you can recommend for the LCMS system?

2. Significant matrix effect was observed. After inject 10 or more matrix samples, the signal of solvent was significantly dropped (about 70%). Even after long time washing and inject solvent standard, the sensitivity recovered slowly.


Any suggestions?

Thanks

Any other positive suggestions? I still waiting.

A large amount of your problems could be caused from using an amino column. Amino columns bleed a lot. A HILIC silica column is likely to be better, but retention could be lower.

Thanks for your answer!
The column I used was Luna Amino (150x 2.1, 5 u, 100A). I was told that it was HILIC column. Am I correct ? What is the difference between HILIC column and amino column? If you think HILIC column may help, which HILIC column you may recommend? I appreciate your help!

HILIC is the standard name for hydrophilic interaction column, as RP is for reversed phase. Since there are other options to do RP than C18, there are better ways to do HILIC than amino columns.

Above, I suggested a HILIC silica column. Specifically, I recommend the Atlantis HILIC silica column from Waters.

You can also try an Hypercarb (from Thermo) column (porous graphitic carbon) as it is able to retain some oligosacharides...

We also have a silica column:

Unison UK-Silica

I will not go on offering more alternatives, instead I'd suggest you go to www.sequant.com, where you can leave your contact details and a copy of the Pratical Guide to HILIC will be sent to you. It is a booklet that gives a thorough introduction to HILIC as well as it exemplifies areas of use via applications. It will give you a good explanation to your reproducibility problems with the amino column. It will also illustrate the difference among silica columns and more dedicated HILIC columns.

We don't have hypercarb. But I did try ultracarb (100x3.2 mm). The RT could hold as long as 3.0min. Believe or not the solvent front came out at about 1.5 mins. But the matrix effect was more severe. I think I will try some HILIC column too. Why I didn't get any advice on ion pair reagent?

The reason that you did not get any suggestions on an ion-pair reagent is that you said that you are working with an oligosaccharide. Not knowing more about it, I do not expect such a beast to have ionic functions...

I have been surprised by how many oligosaccharides, especially biologically interesting ones, have charged modifications, both anions and cations. If you expect your molecule to be charged, perhaps ion exchange would be a viable option.

Thats my suggestion as well. If it's really a mixture of charged and uncharged modification of the oligosaccharide first choice is indeed an ion exchange column for this specific problem. Another nice benefit would be an adjustable selectivity between the two groups, thus a nice resolution might be the observed effect.

If I understand westlake than he has one oligosaccharide Does it have ionizable groups which are succeptible to pH manipulation?