Development of a QAMS method based on analytical quality by design and greenness evaluation: a case study of crude cholesterol product

To enhance greenness and efficiency in qualitative analysis by a single marker (QAMS), this study proposed an improved method integrating environmental impact as a critical method attribute (CMA) and chromatographic column specifications as a critical method parameter (CMP). Using one-factor-at-a-time and split-plot designs, relationships between CMPs and CMAs were evaluated. The method operable design region (MODR) incorporating column variability was determined <i>via</i> Monte Carlo risk-based quantification. A QAMS method was developed for quantifying 24-dehydrocholesterol and lathosterol in crude cholesterol product using cholesterol as the internal standard. Results aligned with external standard method outcomes, validating reliability. The optimised approach improved analytical greenness and development efficiency while accommodating column variations, demonstrating potential for broader application in analytical method development. This systematic strategy addresses prior limitations in sustainability and robustness during QAMS implementation.