(M1330-08-52) Optimization of USP Method for Quantitation of Beta Glucan Using a High-Performance Liquid Chromatography and Adapting to a Low Strength Semisolid Dosage Form

Purpose: Beta Glucan (β-Glucan) is a biological polysaccharide of yeast cells, which activates the body’s own macrophages and stimulates an immune response that leads to accelerated wound healing. β Glucan is a large compound with a molecular weight of about 1.2MDa. The API is insoluble in water and only soluble in DMSO at 80°C or above. Apart from the huge molecular weight, absence of solvent for dissolution, absence of UV absorption of API, low strength of 0.1% w/w presents extreme challenges for the development of a reliable analytical methodology. USP method using K-YBGL kit from Megazyme was applied to hydrolyze β Glucan to D-Glucose and D-glucose to a colored dye. Unlike USP which uses Visible Spectrophotometer for quantitation, a QC-friendly RP-HPLC method was developed to quantify the API in Gel formulation by overcoming the above-mentioned issues. The method provides a superior alternative to the USP method.

Methods: The USP β-Glucan assay method is tedious and a time-consuming biochemical reaction. Some steps were optimized to cut down analysis time. A boiling water bath was chosen instead of 50°C to reduce 18 hours heating to 2 hours as well as an RP-HPLC method was developed for more accurate quantitation of D-Glucose. Since the product has a label claim of 0.1% w/w, it was required to make the method sensitive enough to be capable of determining levels of D-Glucose at ~0.31 ppm for adequate extraction of drug. Since that level of sensitivity is not possible, sample solution was therefore concentrated to ~9.3 ppm D-Glucose by evaporation and reconstitution. This also minimized the enzyme blank interference and helped in achieving a pure peak. A C18, 4.6×250 mm, 5µm column successfully detected D-Glucose derivative using a gradient chromatographic separation at 485 nm, wherein 10 mM Phosphate buffer pH 7.4 and acetonitrile were used mobile phases. The run time is 10 min and the column and autosampler temperature was maintained at 30°C, and 25 °C, respectively.

Results: The retention time for D-Glucose was found to be approximately 5.3 minutes. The system suitability and peak characteristics met the ICH requirements. The RSD for n=5 standard injections was ≤ 2.0%, the capacity factor was ≥ 2.6, and the tailing factor was 1.2. The purity profile was evaluated with a PDA detector and the λ_max determined to be 485 nm instead of 510 nm per USP. The peak was found to be pure. The calibration curves were found to be linear in the concentration range of 70% (~6.5 ppm) to 130% (~12.1 ppm) level. The correlation coefficient (R²) was found to be 0.99. The drug product extraction method was confirmed to be precise with % RSD of ~3.3% and accurate with average recovery of ˃90%. A full qualification of the method is being performed including robustness and forced degradation to obtain peak purities.

Conclusion: Optimized USP method under reversed-phase VIS-LC condition was developed for quantification of β- Glucan as D-Glucose derivative in gel formulations. The method has been determined to be linear, accurate, precise, and specific and more QC friendly.

Figure 1: Representative chromatogram of Gel formulation sample for Total Glucan

Figure 2: Representative chromatogram of Gel formulation sample for α Glucan