Nicotinamide Riboside and Hypercarb

[trammells]

Hello,

I am currently trying to separate nicotinamide riboside on a Thermo Scientific Hypercarb column with the following conditions: A: .05% ammonium hydroxide in 10 mM ammonium acetate and B: .05% ammonium hydroxide in acetonitrile, a flow rate of .2 mL/min, and a gradient from 5% B to 20% from 2 to 15 minutes followed by 20% to 60% from 15 to 27 minutes. I would like to stick with a pH of 8 or above to avoid conversion of other metabolites of interest to their oxidized forms. The problem is that upon going to a more basic pH the retention time of NR shifted to very high organic percentile (greater than 40%) and the peak shape became intolerable with a width of greater than two minutes and incredible tailing. I have tried varying the salt content without any effect as increasing the eluting strength of solvent B by adding more isopropanol (also with no effect). I know that the pH has greatly affected retention here as I originally separated compounds with a pH of 4 to 4.5. This led to a much early and neater retention time.

Does anyone have advice for an ion pairing agent that may help or general LC advice with this problem?

[Andy Alpert]

In some cases, HyperCarb exhibits behavior similar to an anion-exchange material. For example, it's significantly more retentive of phosphopeptides than is C-18 silica. This retention is antagonized by high salt concentrations. To elute a peptide with more than one phosphate, organic solvent alone does not suffice. You also need a significant amount of base. 200 mM NH4OH works pretty well, but 200 mM pyrrolidine is somewhat more effective. In your case, then, try a higher concentration of NH4OH or else use pyrrolidine. Incidentally, none of this behavior is explained by the ionization state of nicotinamide (pKa = 3.3).

[Vlad Orlovsky]

your compound is hydrophilic quaternary amine. You can use mixed-mode cation-exchange similar to analysis of these quaternary compounds:
http://www.sielc.com/Application-HPLC-S ... isc-R.html
http://www.sielc.com/Application-Separa ... mines.html

[Andy Alpert]

Vlad is correct. I was mistaken about the pKa of this compound; it's 12, not 3.3. That said, it still wouldn't hurt to assess the results of using a much higher concentration of NH4OH in the eluting solvent.

[Vlad Orlovsky]

i don't think that it is correct to state pKa of quats as they are always charged, I usualkly call it indefinite pKa >14

[trammells]

Hello,

I appreciate the advice regarding that column but we are trying to stay with the same column because we have acceptable separation of other analytes. I have tried increasing the concentration of NH4OH to 200 mM; however, the signal disappeared at that point on the mass spectrometer I am using. I tried running in both positive and negative ion mode and could not detect NR. I am trying other methods I have located in the literature and should have the results of those by Monday. I really appreciate your interest in my problem and value your insights.

[trammells]

Update: I have essentially given up hope of eluting NR in a reasonable amount of time and with good peak shape in basic conditions and have therefore moved on to a two separation scheme. All but NR will be analyzed with the basic pH and NR, plus some other metabolites that I know I can add to the list will be separated with the other.

I agree wholeheartedly that increasing the salt concentration to 200 mM would likely solve the LC problem. However, I am running in-line with a mass spectrometer. The amount of ion suppression would be far too high and the system would dirty quickly. Thank you all for your advice.