sugar phosphate clean up using Qasis cartridge

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

5 posts Page 1 of 1
Hi all,

Recently, I came across a problem of cleaning up sugar phosphate using oasis MAX. The protocol was developed based on Waters brochure. The sugar phosphate standards (included Ribose 1-5-p, Fructose 1-6-p, 6-phosphogluconic acid, ribose 5-p) was loaded on the cartridge at neutral pH and wash with H2O. I collected the fraction from loading and washing and didn't see any sugar phosphate, which meant MAX can trap the sugar-p very well. However, I couldn't elute them out well. I 've tried to make elution pH drop close to 0.2 by adding HCl and tried to use 70% ACN instead of 100% MeOH. But whatever I changed eluting condition, the best recovery I got was just around 55%.

Anyone have any idea to make sugar-p come out the cartridge. Can Oasis WAX work for them as sugar-p first pka is close to 1.0. Or any other SPE cartridge can suit better for sugar phosphate?


Thanks any commends to help me out!

Based on my SPE experience, you have to condition the cartridges before you use them to maximize recovery. For that, you can use large excess of whatever sugar phosphate compound that is not going to interfere with your assay. Bind it to the cartridge, then elute it completely, the excess amount of this compound will block any non-specific binding sites. Then pre-condition the cartridge with your loading solvent and do your clean up procedure with your target compounds. You should be able to get your recoveries in the 80-90% range. Generally, if you want a quantitative assay, you have to use internal standard anyway, in which case absolute recovery is not a big issue.

The cartridge was activated by 1 ml MEOH and conditioned by 1 ml water before the loading of sugar-p mixed standards (1ml at 10uM of each one). The cartridge size is 1cc, 30mg.

Thanks for any suggestion!

I suggest to ignore the proposal by Alera.

What are the details that the Waters protocol suggests for elution? Maybe we can take it from there. Also, what is the recommended internal standard?

It really depends on the quantities you are working with, SongJack. I used to work with a formulation that had low ug amount of the target compound and a large excess of excipient (mgs). Without pre-conditioning that I came up with, my recoveries were 50-60%. After pre-conditioning that I described, my recoveries were up to 90%. If you are working with much larger quantities though it might not be an issue, as obviously you are losing a much smaller fraction to the non-specific binding.
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