1. Chromatographic Methods (Highly Recommended)

Chromatography is often the gold standard for quantification because it physically separates the molecule of interest (HA) from other components in the sample (including free metal ions) before detection.

a) Size Exclusion Chromatography (SEC) with Refractive Index (RI) Detection

Size Exclusion Chromatography (SEC), also known as Gel Permeation Chromatography (GPC), is arguably the most powerful and reliable method for your situation.

• How it Works: SEC separates molecules based on their hydrodynamic volume (size in solution). A column is packed with porous beads. Larger molecules (like HA) cannot enter the pores and thus travel a shorter path, eluting from the column first. Smaller molecules (like salts, free metal ions, and small contaminants) enter the pores, travel a longer path, and elute later.
• Why it Solves Your Problem:Physical Separation: It physically separates the HA from the metal ions in your sample matrix during the analysis. Insensitive Detection: The most common detector for SEC of polymers is a Refractive Index (RI) detector. The RI signal is directly proportional to the concentration of the polymer and is not affected by the chelation state of the HA or the presence of metal ions in the mobile phase.
• Procedure Outline:Sample Preparation: Dissolve your HA-metal sample in the mobile phase. Filter it through a syringe filter (e.g., 0.22 or 0.45 µm) to remove any particulates. Calibration: Create a calibration curve by running a series of known HA concentration standards (prepared in the same mobile phase). Plot the area under the HA peak against the concentration. Analysis: Inject your unknown sample into the SEC system. Quantification: Identify the peak corresponding to HA (it will be the large, early-eluting peak). Calculate the area under this peak and use your calibration curve to determine the concentration.
• Considerations:You will also get molecular weight distribution data (Mw, Mn, PDI), which is a significant bonus. You can couple SEC with other detectors like Multi-Angle Light Scattering (MALS) for absolute molecular weight determination, but for simple quantification, RI is sufficient and robust. 2. Turbidimetric (Precipitation) Methods

These methods rely on the precipitation of HA out of solution, followed by measuring the resulting turbidity (cloudiness). They are less sophisticated than chromatography but can be effective and are generally insensitive to metal chelation.

a) Cetyltrimethylammonium Bromide (CTAB) Turbidimetric Assay

• How it Works: HA is an anionic polysaccharide. In an acidic buffer, the cationic surfactant CTAB forms an insoluble complex with HA, causing the solution to become turbid. The degree of turbidity, measured by a spectrophotometer as absorbance at a specific wavelength (e.g., 400 nm), is proportional to the HA concentration.
• Why it Solves Your Problem: The strong electrostatic interaction between the negatively charged HA backbone and the positively charged CTAB is the dominant force. While chelated metals add positive charge, the overall anionic nature of the HA polymer chain remains, allowing for precipitation. The bulk precipitation is less sensitive to the fine conformational changes that affect fluorescence.
• Procedure Outline:Calibration: Prepare a series of HA standards of known concentration. Reaction: To each standard and your unknown sample, add an acidic buffer followed by a CTAB solution. Mix and incubate for a set time (e.g., 10-30 minutes) to allow the precipitate to form. Measurement: Read the absorbance (turbidity) of each solution at 400 nm using a spectrophotometer. Quantification: Plot absorbance vs. concentration for your standards to create a calibration curve and determine the concentration of your unknown.
• Considerations: Other anionic polymers or biomolecules (like chondroitin sulfate) can also precipitate, so this method is best for purified or semi-purified HA samples.

3. Gravimetric Analysis (Direct and Simple)

This is the most straightforward method but requires a relatively pure, concentrated sample and the ability to isolate the HA.

• How it Works: You precipitate the HA from the solution, isolate the precipitate, dry it completely, and weigh it.
• Why it Solves Your Problem: This method measures mass directly. As long as the metal ions do not co-precipitate in significant amounts, their presence in the original solution is irrelevant.
• Procedure Outline:Precipitation: Take a known volume of your HA-metal solution. Add a large excess of a non-solvent for HA, such as cold ethanol or acetone, to precipitate the polymer. Isolation: Collect the precipitated HA by centrifugation or filtration. Washing: Wash the precipitate with the non-solvent (e.g., ethanol) to remove any co-precipitated salts or metal ions. Drying: Dry the isolated HA thoroughly in a vacuum oven until a constant weight is achieved. Calculation: The concentration is the final dry mass divided by the initial volume of the solution. Concentration (mg/mL)=Mass of dry HA (mg) / Initial sample volume (mL)
• Considerations: This method is not very sensitive (requires milligrams of material) and assumes that only HA is precipitated. It’s best used as a validation tool or for bulk material analysis rather than for dilute solutions.

For your situation, where accuracy and reliability are paramount and previous methods have failed due to metal interference, I strongly recommend investing the time to develop a Size Exclusion Chromatography (SEC-RI) method. It directly addresses the root of your problem by separating the analyte from the interfering substance before quantification. If an HPLC system is not available, the CTAB Turbidimetric Assay is a viable and commonly used alternative that should be much more robust to metal ion presence than fluorescence or immunoassays.

Thanks a lot, Amin. I feel that the SEC you mentioned in your answer is very likely to solve my problem. I am currently trying to solve this problem using isotope labeling. If that fails, I will try your method.

Thank you very much and wish you all the best.

To quantify hyaluronic acid in the presence of metals, use chromatographic methods like HPLC-SEC-MALS or enzymatic assays that specifically target HA, as these are not affected by metal interference. Avoid fluorescence or dye-based methods since metals can quench signals or disrupt dye binding.