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Zorbax RRHD SB-C8 Carry Over of Domoic Acid

RoganVK

Hello Everyone,

I am currently trying to separate Domoic Acid in Mussel Tissue on a Zorbax SB-C8 column using a 1290 UHPLC and 6470 Triple Quad MS, the issue I am seeing as that the Domoic Acid is sitting in the column as no injections and MeOH solvent injections contain the peak in question for Domoic Acid.

I have run solvent (MeOH) multiple times as well as try and flush with mobile phase B for an hour as well as mobile phase A for an hour as well as flushing with pure MeOH and H2O with nothing working to remove Domoic Acid from the column. I have also tried conditioning/coating the column with Bovine Serum Albumin that has not worked either. 

Am I correct in saying that because Domoic Acid is an Amino Acid the compound is binding with the stationary phase and/or possible sitting in the pores of the column via hydrophobic interaction between the compound and the stationary phase itself? The pore size of the column in question is 300 Angstrom, so would running with an 80 Angstrom pore size column alleviate the issue?

I'm not sure what else the issue might be other than the pore size based on the literature review and research I have conducted and any assistance would be greatly appreciated.

Kind regards, Rogan Van Kerpel

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  • +1

    RoganVK-

    Maybe review this article.

    1. Validated HPLC-MS/MS Method To Quantify Low Levels of Domoic Acid in Plasma and Urine after Subacute Exposure
      Sara Shum, Jay S. Kirkwood, Jing Jing, Rebekah Petroff, Brenda Crouthamel, Kimberly S. Grant, Thomas M. Burbacher, Wendel L. Nelson, and Nina Isoherranen
      ACS Omega 2018 3 (9), 12079-12088
      DOI: 10.1021/acsomega.8b02115

    It may retain strongly and you may need ACN or 50/50 IPA/MeOH to elute the compound.

    Can you see it by infusion without a column in positive or negative mode?

  • 0 in reply to andyg

    Hi andyg

    Thank you for coming back to me so quickly, I have seen the paper in question during our extensive research and we are running very similar mobile phase just with ammonium formate added as well to both bottles and a bit more formic acid than what the paper mentions. I'll definitely try the ACN and 50/50 IPA/MeOH, thank you for the suggestion.

    I do see they mention in the paper that they use 100 Angstrom pore size column which makes me tend to think that this is the issue as I have seen a paper that compares 100 Angstrom to 300 Angstrom with peptides and amino acid screening, but would obviously like some reassurance that this is the case or could be the case rather as I don't want to peg my hopes on the new column being the resolution when it might not be. Will have to see when it arrives if this is the case or not.

    Thanking you. 

Reply
  • 0 in reply to andyg

    Hi andyg

    Thank you for coming back to me so quickly, I have seen the paper in question during our extensive research and we are running very similar mobile phase just with ammonium formate added as well to both bottles and a bit more formic acid than what the paper mentions. I'll definitely try the ACN and 50/50 IPA/MeOH, thank you for the suggestion.

    I do see they mention in the paper that they use 100 Angstrom pore size column which makes me tend to think that this is the issue as I have seen a paper that compares 100 Angstrom to 300 Angstrom with peptides and amino acid screening, but would obviously like some reassurance that this is the case or could be the case rather as I don't want to peg my hopes on the new column being the resolution when it might not be. Will have to see when it arrives if this is the case or not.

    Thanking you. 

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