Chromatography Forum

Hello everyone,

I am working on the determination of neomycin with HILIC-MS/MS (bare silica column, 100 x 3 mm). I have tried lots of mobile phases, including the following, but did not obtain any peak for neomycin, even at 10 ppm:

20% ACN - 80% (20 mM ammonium acetate pH=4)
50% ACN - 50% (20 mM ammonium acetate pH=4)
50% ACN - 50% (150 mM ammonium acetate pH=4.5)

I have also tried a C18 and a bare silica column. I can see clearly neomycin when I infuse it into the mass spectrometer, but I obtain no signal at all when I use a column.

Any suggestions will be very helpful!

Thanks a lot!

20% water - 80% (20 mM ammonium acetate pH=4)
50% water - 50% (20 mM ammonium acetate pH=4)
50% water - 50% (150 mM ammonium acetate pH=4.5)

Am I reading this right? You're only using aqueous mobile phases on a HILIC column? If so, you shouldn't be. Your mobile phase should be more like 90% acetonitrile:10% aqueous buffer, with a gradient to about 50:50 acetonitrile:aqueous buffer.

Aminoglycosides are quite basic and require substantial amounts of buffer to have sharp peaks for recovery. An additional problem you may have is that you are using too small a pore silica so that there is too much surface to interact with.

Check out the chromatograms in Figure 5.1.8: (McGrane et al., EuroResidue IV (May 2000), Pg. 765 (van Ginkel & Ruiter, eds.), Bilthoven, The Netherlands.) which shows the separation achieved for aminoglycosides.
HPLC Conditions ; Column : PolyHYDROXYETHYL™ Aspartamide, 200 x 4.6 mm, 5 µm, 1000Å.
Mobile phase gradient ; 80 % acetonitrile, 20 % 250 mM NH4Ac, pH 4 at t=0,
@ 1.0 ml min-1, 25 % acetonitrile, 75 % 250 mM NH4Ac, pH 4 at t=5 min.
25 % acetonitrile, 75 % 250 mM NH4Ac, pH 4 at t=15 min.
80 % acetonitrile, 20 % 250 mM NH4Ac, pH 4 at=18 min.,
Injection volume ; 50 µl which I have posted at:
http://www.nestgrp.com/protocols/polylc ... tics.shtml .
Note they were more successful using a 1000Å pore, bonded HILIC column, which at pH 4 is neutral to a slightly basic surface chemistry. You might also want to consider using a basic surface as your HILIC column to reduce the amount of electrostatic attraction.

Best wishes

Nest Group provides some good recommendations.

I agree that a bonded HILIC phase is better than bare silica for this analyte. Luna HILIC could be a good option due to the high inertness of the cross-linked diol phase. This may allow the use of less buffer (5mM?) which will improve sensitivity.

I am assuming that you are trying 20% aqueous buffer with 80% acetonitrile or else there is very little point in using the HILIC series of columns.

If so then one of the issues I have observed is that in high acetonitrile content solutions ionisation decreases to nothing. Did you use the proposed mobile phase as your dilutent when you infused into the MS directly? In these type of situations I normally use a range of diluent compositions (10,25, 50, 75 % ACN) at differing buffer concentrations (2, 5, 10 & 20 mM) and pH's to verify that my compound has sensitivity in the solution in which it will elute from the column. Normally you don't have to infuse every type of condition, a little intuitive logic can shorten the investigation but it is always best to know you compound will ionise in the elutant before you start chromatography.

hope this helps

RJH

If you need to analyze only neomycin, I would suggest an amide HILIC column with the following mobile phase

A: 1% formic acid in water
B: Acetonitrile

I have tried TSK gel Amide 80 and it works great and pretty robust. If you need more info. I would suggest our article - Hydrophilic interaction chromatography for the analysis of aminoglycosides (http://onlinelibrary.wiley.com/doi/10.1 ... 100860/pdf)

Thanks to everyone for your helpful posts!

After bisnettrj2's correct remark, I made a minor correction to my post concerning the mobile phase's components.

here is something similar, done in mixed-mode reversed-phase cation-exchange mechanism.

http://www.sielc.com/Compound-Amikacin.html

let me know if you have questions.

Hi Stdev,
Do you have your ACN/buffer compositions reversed? In HILIC, the amount of ACN should be decreasing and amount of buffer increasing in order to elute the polar analytes. It might be worth checking the original method to verify the mobile phase. Did you check the void volume peak to see if the Neomycin is unretained?

If you're able, it might also be worth while to try a gradient from 10% buffer/90% ACN to 90% buffer/10% ACN and see at what point the Neomycin elutes.