AQbD-Based HPLC Method Development, LC-MS/MS Characterization, and Bivariate Conformity Assessment for Estradiol Valerate and Norethisterone Enanthate in Injectable Contraceptives

Abstract This study aimed to develop and validate an Analytical Quality by Design (AQbD)-based HPLC method for the simultaneous determination of estradiol valerate (EV) and norethisterone enanthate (NE) in injectable contraceptives, integrating LC–MS/MS characterization of degradation products, measurement uncertainty, and bivariate conformity assessment. Chromatographic conditions were systematically investigated using experimental design and statistical modeling, followed by validation according to ICH Q2(R2). Forced degradation studies were used to evaluate chromatographic selectivity toward degradation products, and major degradation products were characterized by LC–MS/MS. The optimized method showed satisfactory linearity (r² ≥ 0.9938), precision (RSD ≤ 3.0%), and accuracy (mean recoveries of 98.4–101.6%). Combined standard uncertainties were 2.1% for NE and 2.3% for EV. Incorporating these uncertainties into conformity assessment showed that conventional univariate guard-bands controlling the risk for each analyte individually may not adequately control the overall risk when both active ingredients are assessed simultaneously. A bivariate guard-band approach therefore provided more restrictive acceptance regions to maintain the total consumer’s risk at approximately 5%. The main contribution of this study is the integration of AQbD-based analytical development, degradation-product characterization, measurement uncertainty, and multiparametric decision rules within a single analytical framework. This strategy extends analytical validation beyond conventional performance assessment and provides a risk-based basis for conformity decisions in multicomponent pharmaceutical products.