RESUMEN
Vitamin D3, an essential micronutrient, often requires supplementation via medicines or food supplements, which necessitate quality control (QC). This study presents the development of a method for detecting and quantifying seven impurities of vitamin D3 in oily drug products using supercritical fluid chromatography-mass spectrometry (SFC-MS). Targeted impurities include two esters of vitamin D3 and five non-esters including four that are isobaric to vitamin D3. Firstly, a screening study highlighted the Torus 1-AA column and acetonitrile modifier as adequate for the separation, followed by optimization of the SFC conditions. Secondly, make-up solvent composition and MS settings were optimized to reach high sensitivity. For both the separation and MS response, the screening design of experiments proved useful. Lastly, a fast saponification and liquid-liquid extraction method was developed, enabling efficient sample cleanup and impurities recovery from the complex oily matrix. The SFC-MS method suitability was assessed in two validation studies. The first study employed the ICH Q2 guideline for impurity limit test to demonstrate method specificity and establish a limit of detection (LOD) and a limit of quantification (LOQ) at 0.2% and 0.5%, respectively, for ester impurities. The second study conducted a comprehensive quantitative assessment for three non-ester impurities using a total error approach, determining method validity through accuracy profiles. The validated method exhibited reliable performance across impurity concentrations from 0.1% to 2.0%, with estimated LODs ranging from 2 to 7 ng/mL. This study further promotes SFC-MS as a valuable, versatile, and green tool for routine pharmaceutical QC.