I am developing a LCMS method to profile metabolites in sample that contain sucrose buffer (250mM). I do methanol extraction prior to injection and use the divert value. I am wondering if there is an efficient way to remove sucrose from the samples. Some of the metabolites of interest are in the m/z range of sucrose but the data looks good. However, I wish to have a cleaner sample prep to sustain spray needle and column life. Please let me best ways to eliminate sucrose/Tris.
Thanks!
I think that adding the enzyme sucrase could be a viable solution. Sucrase has substrate specificity and It will break down your sucrose into a smaller range of m/z compound. As the enzyme itself will also be outside your lecture range (its basically huge). You will need to check for the pH of your samples. As sucrase has activity within the range of pH 3.5-8.3, with high activity at 3.5-5.2 (https://www.researchgate.net/publication/274776472/figure/fig3/AS:670709957263360@1536921123009/a-Effect-of-pH-35-83-on-sucrase-activity-in-hepatopancreas-of-N-granulata-The_W640.jpg)
My first approach would be used a chromatography column packed with gel spheres with the immobilised enzyme, but I think it wouldn't be suitable for a quantitative analysis of a sample. I would rather use a volumetric flask, adding a small amount of the enzyme along the sample, completing the volume with water R and ensuring that pH is somewhere near 5,0 and the temperature is between ambiental to 30ºC. I would use a 250mM sucrose buffer blank as test for ensuring that both the enzyme is working and that the amount of sucrose after the enzymatic digestion is below detection limits.
If you use reversed phase column for LC separation, sucrose would elute quickly so it will not affect your results.
As Zhiqian Liu indicated, if you are performing a reversed-phase analysis, the sucrose ingredient won't be a problem for analysis. It will wash out through the column. Furthermore, since its solubility in methanol is limited, I hope that complete of 250 mM will not be in extracted phase. If you are analyzing the MeOH phase, I assume you are going to analyze the mid-polar metabolome therefore you are going to choose RP mode. In this case, you don't need to concern due to the pinpointed reasons.
On the other hand, sucrase will not solve the problem. When the disaccharide (sucrose) content will be cleaved by a certain specific enzyme, it will conclude as isomeric monosaccharide products. These substances can also foul on the ESI needle/spray cone and suppress ionization in time. They will co-elute at RP conditions so, by using an embedded valve on the spectrometer you may get rid of these contaminants just before the metabolome elution time frame. In HILIC mode the effluent that comes from the LC column should be well operated and directed to MS considering the elution time of the sucrose and fructose/glucose to get monosacharide-free metabolome coverage.
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