Abstract
Our recent studies suggest that aliphatic β-nitroalcohols (BNAs) may represent a useful class of compounds for topical therapeutic corneoscleral cross-linking agents. Thus, this study was undertaken in order to standardize a simple method for nitroalcohol quantitation based on a denitration step followed by colorimetric Griess nitrite assay. Conditions necessary for denitration included a pH of 7-9 and heating for 1 hour at 100°C. Standard curves for two mono-nitroalcohols (2-nitroethanol and 2-nitro-1-propanol), a nitro-diol (2-methyl-2-nitro-1,3-propanediol), and a nitro-triol (2-hydroxymethyl-2-nitro-1,3-propanediol) showed excellent linearity in the 100-500 M range with absorbance values <1.0 and R2 values >0.98. The lower limit of detection was ~20 M. Recovery from tissue homogenates (10mg/mL wet weight) of rabbit cornea and sclera as well as solutions of gelatin B (1mg/mL) ranged from 89-103% for scleral tissue, 68-106% for corneal tissue, and 90-99% for gelatin B. The Griess colorimetric nitrite assay can be successfully used for the quantitative determination of BNAs and is simpler to use than conventional chromatographic techniques.