Validation of an Reverse phase high performance liquid chromatography Method for In Vitro Quantification and Degradation Analysis of Naphthol AS-E Phosphate in Bulk Drugs and Nanoparticles.

Abstract

This study presents the first validated High-performance liquid chromatography (HPLC) technique for quantifying naphthol AS-E phosphate (NASEP) in bulk drugs and nanoparticle formulation. A C18 HPLC cartridge (250 × 4.6 mm, 5 µm particle size) served as the stationary phase for quantification. The mobile phase consisted of Milli-Q water with 0.1% trifluoroacetic acid (TFA) in pump A and acetonitrile with 0.1% TFA in pump B, with a flow rate ranging from 0.8 to 1.2 mL/min. A 3² factorial design was employed to evaluate the robustness of the proposed method, using mobile phase composition (X₁), flow rate (X₂), and column temperature (X₃) as independent variables and peak area (R₁), retention time (R₂), and percent recovery (R₃) as response variables. The calibration range curve (10-500 µg/mL) was best fitted by quadratic regression. The linearity was reported in the above-mentioned range. The accuracy was 99.952% ± 0.961% at the 75% level, 99.58% ± 1.483% at the 100% level, and 99.789% ± 1.936% at the 125% level. The coefficient of variation was below 2% for both intraday and interday measurements, and the limits of detection and quantification were 0.038 and 0.115 µg/mL, respectively. The NASEP solution was stable (99.04% ± 0.0251%) for 48 h at 8°C. The forced degradation study also revealed that the NASEP solution remained stable in an acidic environment for 48 h at 40°C but degraded at 80°C (p < 0.046) in a time-dependent manner. In contrast, it was unstable in an alkaline medium, independent of temperature, and degraded in the presence of strong oxidizing agents (p < 0.039). Furthermore, NASEP encapsulated in a Gly-Arg-Gly-Asp-Ser pentapeptide and low-molecular-weight heparin functionalized metal-organic framework exhibited sustained drug release at acidic pH 5.4. The proposed NASEP quantification method was validated and is suitable for routine analysis in pharmaceutical formulations.