Analytical quality by design based HPLC method development validation and green assessment for tenofovir alafenamide quantification in ultradeformable vesicles

Abstract QbD makes it easy to develop an analytical method using a logical process to evaluate and control critical characteristics for robustness and ruggedness, providing more reliable methods than traditional trial-and-error approaches. It enables understanding of critical factors affecting product performance. The HPLC method was developed using a QbD approach on the Agilent 1260 Infinity II system equipped with a Shim-pack GIST-C18 (250 × 4.6 mm, 5 μm) for determination of TAF. The analytical target profile and critical performance attributes were set, followed by risk assessment using failure mode and effects analysis and fishbone diagram. Critical method parameters were optimized by experimental design, and validation was done based on ICH Q2 (R2) Guidelines. Flow rate, injection volume, column temperature, and percentage of methanol were identified as critical variables. The optimized parameters (0.7 mL/min, 15 µL, 50 °C, 70% methanol) gave a peak area of 258.85 mAU, tailing factor of 0.936, theoretical plates of 13791.15, retention time of 5.476 min, and desirability of 0.814 indicating satisfactory optimization. The calibration graph was linear in the concentration range of 50ng/ml to 40mcg/ml at 261 nm. Precision, robustness, and recovery studies indicated %RSD of less than 2% and mean recovery of 98–102%.