I am currently processing a peritoneal dialysis solution with 7.5% icodextrin and would like to verify whether its concentration remains the same post-processing or decreases, whether slightly or significantly. How can I quantify icodextrin in an aqueous solution?
As icodextrin is a polysaccharide/glucose biopolymer, could I use HPLC-RID for this purpose? The intended mobile phase is 80% acetonitrile water, and the column proposed is Lichrospher® 100 NH2 Purospher® STAR NH2 5um, suitable for carbohydrates (mono-, di-, and oligosaccharides).
May 2, 2024
Dear Fatima,
I'll try to answer your question.
Icodextrin is a polysaccharide that is polydisperse, i.e., its molecular weight ranges from ~12,000 to ~20,000 daltons. HPLC will not work for measuring its concentration. Assuming you could get it to pass through the HPLC column (not likely), you won't get a single peak, but a "smear" of peaks that will exit the column during the entire elution process.
I suggest using a colorimetric method for measuring total carbohydrate, specifically the Phenol-Sulfuric Acid method. This is an old method, but it works well, is sensitive, is easy to do and gives good results. It is still in use today. The reagents for the assay are 5% phenol solution (w/v) in water and concentrated sulfuric acid. You also need a carbohydrate (CHO) standard. For this you can use a glucose solution (or even icodextrin) of known concentration, e.g., 0.1% (1 mG/mL).
Assay Procedure:
1. Combine sample + DI-water to 1.0 mL
2. Add 1.0 mL 5% phenol
3. Add 5.0 mL concentrated H2SO4
4. After H2SO4 addition, mix samples on a vortex mixer. The tubes will initially be very hot (See note below.). Wait ~20-30 min for the tubes to cool before reading on the spectrophotoeter. Color is stable for hours.
(If you wish, you can save on chemicals by using proportionally smaller volumes (25-50%) of the reagents listed above.)
When the acid mixes w/ sample + phenol, an orange color is produced. The absorbance of this color can be measured on a spectrophotometer at 490 nm. In addition to your icodextrin samples, you will also need to run a range of standards from which a calibration curve can be prepared. The absorbance values of your icodextrin samples can be compered to the curve to determine concentrations. The assay is linear from ~5 to 100 ug of CHO.
Several safety concerns! H2SO4 is highly corrosive. Nasty Stuff! Be sure to wear gloves, lab coat and (especially!) eye protection.
Also, when H2SO4 mixes w/ the sample + phenol, the mixture immediately gets VERY HOT, so don't grasp the bottom of the test tube during the H2SO4 addition step or you may get a burn. Hold the tube at the top, near the mouth of the tube. Be sure to wait ~20-30 min for samples to cool to room temperature before reading them.
For reading the absorbance, you can use glass cuvets if you have them. If you use disposable cuvets, DO NOT use polyacrylic plastic cuvets, as the H2SO4 start to dissolve the plastic after a few minutes. Instead, use POLYSTYRENE cuvets.
The link below is a video that shows how the assay is done.
https://youtu.be/q-CwrO6cU_o
I hope this information helps you. Good luck w/ your research:).
Bill Colonna Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA [email protected]
I prefer the Ca-type ligand exchange (ion exclusion) polymeric column instead of NH2-type ligands column to get high dp oligosaccharides as a single response rather than polydispersed peaks...
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