FMOC-Cl cleanup for LC-MS

[Triple Quad]

I'm doing a project with FMOC-Cl derivatization of glyphosate. We started off using a 500ul of 2mg/ml FMOC-Cl concentration which is added to 500ul of extracted glyphosate and borate buffer. We would filter then inject the sample on a 2.1x150mm XBridge column with 5mM Ammonium Acetate in water Water and Acetonitrile as the mobile phase.

In reading the literature, I see a wide variation in the recommended FMOC-Cl concentrations for derivatizing. Thus, I was compelled to investigate how the results would change if I increased the FMOC concentration. The highest concentration that I tried is giving satisfactory results. I'm now using 500ul of 25mg/ul FMOC-Cl. I'm still injecting the derivatized samples without further cleanup after derivatization.

I've seen where a researcher working with peptides published that they do a 80%pentane-20%ethylacetate partition to remove the FMOC by products. Surely this is also applicable to FMOC-glyphosate, and I've tried this but my peaks look horrible after the partitioning.

I added 2mls of the pentane:ethylacetate to my 1ml of sample, borate buffer, and FMOC-Cl mixture, then vortex for 5 seconds. Let sit for 10 miunutes then use a pasture pipette to remove the lower aqueous portion and put in a vial, then cap and inject. Do I need to centrifuge step after doing the vortex?

Or, since the chromatography still looks ok, should I just not worry about the FMOC by products on the column. I've been increasing the acetonitrile in the mobile phase to 100% for 10 minutes to flush the column after each injection, and so far so good.

Any recommendations?

[ericf13]

Try to use a polymer supported primary amine to scavenge Fmoc Cl. Then you would just filter to remove the beads and inject your sample as previously.

[Triple Quad]

Try to use a polymer supported primary amine to scavenge Fmoc Cl. Then you would just filter to remove the beads and inject your sample as previously.

Thanks Ericf, I'll give that a try.

A piece of equipment is down so I had time to do this comparison. I went ahead and did a comparison between injecting some low range standards 50-200 ppb after a single pentane:ethylacetate partition. Compared with no clean-up of the derivatized sample.

My straight injection results are probably acceptable, however, after the pentane:EA clean-up, I had nice peak shape and significantly improved signal to noise ratios; s/n's increases were 10-30%.

I like the sound of the amine terminated beads. That might be easier.