A novel HPLC method for simultaneous estimation of natamycin and voriconazole for ocular drug delivery applications

Natamycin and Voriconazole are commonly used antifungal agents to treat fungal keratitis. Recently, the combination of these drugs has been proven synergistic in treating fungal keratitis. However, no simultaneous estimation method has been reported for Natamycin and Voriconazole using reverse-phase high-performance liquid chromatography (RP-HPLC). Hence, this study aims to develop an accurate, precise, reproducible, sensitive, and rapid HPLC method. The developed HPLC method was validated according to United States Food and Drug Administration (USFDA) guidelines. Chromatographic separation was performed using a Zorbax RX-C8 5 μm column with a dimension of 4.6 × 150 mm², employing a photodiode array (PDA) detector at 304 nm wavelength for Natamycin and 256 nm for Voriconazole. Water acidified with 0.025 % TFA (pH 2.4) and Acetonitrile were used as the mobile phase with a flow rate of 0.8 mL/min. The retention time for Natamycin and Voriconazole was 3.1 min and 8.4 min, respectively. The calibration curve was obtained from (0.031 μg/mL to 25 μg/mL), which showed good linearity with an R² value of 0.9997 for Natamycin and 0.9997 for Voriconazole, respectively. The coefficients of variation of the inter and intra-day precision were within the limits. Drug content and in vitro drug release studies were performed, and the samples were analyzed using the developed HPLC method.