Abstract
Purpose: :
Residual endotoxin in hyaluronic acid is one of causative factors of toxic anterior segment syndrome (TASS). Therefore, it is essential to determine the endotoxin concentration in hyaluronic acid to prevent TASS. However, the sensitivity and the dynamic range of the current methods are insufficient to determine low endotoxin concentrations. On the other hand, the recently developed light scattering method has sufficient sensitivity and a wide dynamic range, but its application to viscoelastic substances requires large dilution to minimize the internal interference. We investigated an optimal additive to determine endotoxin concentration in the viscoelastic substances with minimal dilution factor.
Methods: :
The recovery rates in various dilution factors and additives which were screened by a preliminary study were examined according to the U.S. Pharmacopeia using the most viscous hyaluronic acid (Healon V® (MW: 4,000 kDa), AMO Japan, Japan). The original solution comprising Healon V with 0.1 EU/mL of endotoxin was prepared by using either of the following endotoxin-free additives: distilled water, NaCl (0.1M), or phosphate-buffered saline (PBS, 0.01M, pH 7.4). To investigate additive effect of endotoxin-free human serum albumin (HSA), the recovery rates were compared between samples (n = 6 or more) with HSA (0.25% (w/v)) and without HSA. The final dilution factor in each sample was adjusted to 20, 30, 40, or 100 fold of the original solution. The endotoxin concentrations in the final test samples were determined by the light scattering method (EX-300, Kowa, Japan).
Results: :
The endotoxin concentrations could not be measured in any of the 20- and 30-fold diluted samples with and without HSA. In the 40-fold diluted samples without HSA, the recovery rates for distilled water, NaCl, and PBS groups were determined to be 56.3 ± 12.4, 78.8 ± 12.1, and 78.9 ± 8.8%, respectively. In the samples with HSA, the recovery rate in NaCl and PBS groups increased significantly to 103.4 ± 17.2% (p = 0.0085), and 103.5 ± 14.2% (p = 0.00048) (unpaired t-test), but not in the distilled water group. In the distilled water group, the recovery rate was 87.1 ± 17.4% for 100-fold diluted sample (n = 8).
Conclusions: :
In the light scattering method, a 40-fold dilution with either NaCl or PBS enables a determination of the endotoxin concentration in viscoelastic substances. In this condition, adding HSA can provide better recovery rates.
Keywords: anterior segment • cataract • detection