Here is a method I came up for Inulin. It uses size exclusion to do the job.
SUGGESTED USAGE:
Method used to measure the total amount of Inulin in any raw material or finished product.
REAGENTS:
Deionized water
Concentrated NH4OH (Ammonium Hydroxide)
Formic Acid (90%)
Inulin (Chicory Root) Sigma Chemical Company Cat. no. I-2255
SOLUTIONS:
Mobile Phase: Add 40 mL of concentrated NH4OH to 6L of DI water. By using the pH meter, adjust the pH to 9.00 +/- 0.05 with formic acid (90%). Initially 10ml of 90% formic acid can be added to the flask and then it should be added drop by drop until pH range is met. If range missed, simply readjust drop by drop with NH4OH and 90% formic acid as needed. Filter the mobile phase through a 0.45µm filter, however, do not degas for more than 3 minutes because NH3 is volatile and the pH can be effected by a long degassing. Mobile phase can be used for 1 month from preparation date.
STANDARD PREPARATION:
For the stock standard, using an analytical balance, accurately weigh 0.35g of standard into a 200 mL volumetric flask and dissolve by adding 150 mL of boiling DI water. Allow the volumetric flask to cool to room temperature and bring to volume with DI water. Then make 4 dilutions of that stock standard using a 10 mL auto-pipette to add 5, 10, 15 and 20 mL of the stock solution to four separate 25 mL volumetric flasks. Then bring each volumetric flask to volume using DI water and record the values of each standard. Filter each through 0.45m nylon syringe filter. Standard concentration is used as g/200mL in the chromatographic software.
A standard curve check sample should be prepared by weighing 0.2g of standard into a 200 mL volumetric flask.
Standards should be kept in the refrigerator and are good for 1 month.
SAMPLE PREPARATION:
Raw Materials: Using an analytical balance accurately weigh 0.2 – 0.22g of Inulin into a 200 mL volumetric flask and dissolve by adding 150 mL of boiling DI water. Allow volumetric flask to cool to room temperature and bring to volume with DI water. Filter each through 0.45m nylon syringe filter.
Finished Products: Using an analytical balance accurately weigh an amount of ground tablet containing 0.2 – 0.22 g of Inulin into a 200 mL volumetric flask and dissolve by adding 150 mL of boiling DI water. Allow volumetric flask to cool to room temperature and bring to volume with DI water. Filter each through 0.45m nylon syringe filter.
IMPORTANT - In order to dissolve Standards, Raw Materials or Finished Products it is important that after the addition of boiling DI water a slight amount of swirling will be necessary to help dissolve the products.
HPLC COLUMNS:
Columns: PolymerLabs Aquagel-OH 30 and PolymerLabs Aquagel-OH 40 both 8 µm and 30cm x 0.75cm
Precolumn: PolymerLabs Aquagel-OH Precolumn 5cm x 0.75cm
Use in this order Pump to Detector: Pre-column, Aquagel-OH 30 Column, Aquagel-OH 40 Column
HPLC EQUIPMENT:
Suitable HPLC equipment including auto-sampling, pumping, solvent degassing and column heating is used for the analysis. The detector is a Polymer Labs ELS-1000 Evaporative Light Scattering Detector or any evaporative light scattering detector available.
CONDITIONS:
For HPLC Equipment
Solvent Flow Rate: 1 mL / min
Column Temperature: 40C
Sample Injection Volume: 60L
Standard Curve Type in Chromatographic Software: Log-Log Linear
Sample Collection Time: 22 min
For ELS-1000
Evaporator Temperature - 110C
Nebulizer Temperature - 105C
Gas Flow Rate – 0.5 SLM (standard liters per minute)
ANALYSIS:
The five standards should be injected first followed by samples. No more than six samples should be injected per standard curve. Following the injection of the samples, the control sample should be injected. Then for further samples a new standard curve should be injected.
The control sample, when calculated, should be ±8% of the value weighed out. This insures that the auto-sampler is operating correctly.
RESULTS:
All samples are measured as Inulin g / 200mL. Because the samples and standards are prepared and calculated as g / 200mL then all that is necessary is to do direct comparison with raw materials and number of tablet weights for finished products.
Inulin elutes at approximately 15.5 minutes. The automated chromatographic software can integrate the standards, build a standard curve and deliver values of Inulin.
For all standard curves R should be between 0.99 and 1.0.
Raw Material Calculations
The following dilution factor can be entered into the chromatographic software in order to yield a final value in Percent Inulin in the Raw Material:
Finished Product Calculations
A dilution factor entered into the chromatographic software for each finished product will yield the amount to Inulin present in each tablet. The following factor can be used:
Once the value for mg/tablet has been determined, calculate the percent label claim for each tablet sample measured using the following equation:
