Lysine contamination on HILIC-Z for LC-MS

[foodtesterchemist]

Hello,

I am trying to bring a version of an Agilent underivatized amino acid method in-house: https://www.agilent.com/cs/library/appl ... gilent.pdf

This uses an Agilent HILIC-Z column, and the mobile phases are (A) 100% water + 0.1% formic acid + 10 mM ammonium formate, and (B) 90% acetonitrile 10% water + 0.1% formic acid + 10 mM ammonium formate. The gradient starts at 100% B, ramps down to 20% B, then re-equilibrates at 100% B. The Q1 and Q3 masses I'm using are 147.1, and 130.1/84.1.

So far, all I've done is (1) condition the column per Agilent's instructions, and (2) test the handful of amino acids we happened to have in-house. These are not analytical standards; they're generally 98+ or 99+% grade. I made a concentrated aqueous solution, then diluted into MPB from 1 ppb up to 2.5 ppm, following the Agilent paper. My calibration curves all look nice, with one exception: I get a large and constant integrated area for lysine (regardless of concentration), the peak shape isn't very Gaussian, and the peak even shows up with a zero-volume injection.

I've tried disconnecting the column and pumping through a union directly into the MS. When I did this, I saw no peak when injecting a blank, but I did see a peak when injecting a standard. So I am pretty sure that I have somehow contaminated my column with lysine, even though I've only ever injected a small number of relatively dilute standards.

I should also mention: the lysine I used was rather old. It was yellow and the top layer of powder had hardened.

I have tried cleaning the column overnight with 50-50 acetonitrile-water + 0.1% formic acid + 200 mM ammonium formate. I followed this with an extended 80% aqueous flush, then re-equilibrated with full organic. This did not help. The first blank after this procedure showed about 2-3X as much lysine signal as before (and even worse peak shape), but then the second and third blanks looked exactly like the ones before cleaning. Same shape, same area.

Is this actually lysine/lysine byproducts contamination? Did I maybe fail to properly condition the column? Is there something else / more aggressive I can do to clean the column that's still safe for HILIC-Z? How high on aqueous content can I safely go with this column? I would appreciate any help at all. Thank you!

[itspip]

I am thinking that the free amines on lysine are interacting with the residual silanols of the column. Agilent claims that it's a zwitterionic column, but there are probably some silanols still around. Their data does show nice gaussian peaks for all of the amino acids so I would expect that you should be able to get a decent peak for lysine as well.

For your question about washing the column, water is the strong solvent in HILIC. Go ahead and up your water concentration for the wash, and since the column temperature max is 80 C I would also make the temperature higher. 60 C should be sufficient to help coax some sticky compounds off the stationary phase. If the column were bare silica I'd suggest 100% water, since there is a phase bonded to it I would try 90 or 95 % water and near 60 C. If you have a UV-Vis attached I would monitor the baseline that way during the wash.