The colorimetric assay is quantitative and relies on an extractable colored ion-pair complex of oseltamivir with Congo red or bromochlorophenol blue.
Because oseltamivir phosphate (Figure 1) possesses amine groups, the protonated form may act as a cationic site for anionic dyes such as Congo red and bromochlorophenol blue to produce colored ion-pairing complexes.
We purchased Congo red (dye content [approximately equal to] 97%), bromochlorophenol blue sodium salt (dye content [approximately equal to] 95%), potassium hydrogen phthalate, monobasic potassium phosphate, sodium bicarbonate, and sodium hydroxide from Sigma-Aldrich (St.
Congo red and bromochlorophenol blue salt were evaluated for the colorimetric assay and prepared at a concentration of 1 mg/mL in water.
We determined the optimum pH for complex formation between oseltamivir and Congo red to be 4; the optimum pH for bromochlorophenol blue and oseltamivir was 6-7.
The greater slope associated with the Congo red curve relative to the bromochlorophenol blue curve in Figure 3 demonstrates a more sensitive assay.