Developing a Quantitative Profiling Method for Detecting Free Fatty Acids in Crude Lanolin Based on Analytical Quality by Design

In this study, a quantitative profiling method for detecting free fatty acids in crude lanolin based on the Quality by Design (QbD) concept was developed. High-performance liquid chromatography (HPLC) equipped with a charged aerosol detector (CAD) and a Proshell 120 EC C18 column was employed for the separation of crude lanolin components. Initially, the analytical target profile and critical method attributes were defined. Potential critical method parameters, including column temperature, flow rate, isocratic run time, gradient end organic phase ratio, and gradient time, were identified using fishbone diagrams and single-factor experiments. The definitive screening design (DSD) was then utilized to screen and optimize these parameters. Stepwise regression was applied to establish quantitative models between the critical method attributes and the method parameters. Subsequently, the method operable design region (MODR) was calculated and was successfully verified. The analytical conditions established were configured with 0.1% formic acid in water and 0.1% formic acid in acetonitrile serving as the mobile phases. The flow rate was set at 0.8 mL/min, and the column temperature was maintained at 35 °C with the evaporation tube temperature also set at 35 °C. An injection volume of 10 μL was used for each analysis. The gradient elution conditions were as follows: from 0 to 30 min, 75% of solvent B was used, and from 30 to 60 min, the proportion of solvent B was increased from 75% to 79%. Ten components, including 12-hydroxystearic acid, 2-hexyldecanoic acid, and palmitic acid, were identified by mass spectrometry, and seven common peaks were found in the fingerprints. The contents of palmitic acid, oleic acid, and stearic acid in the crude lanolin were quantitatively determined. Both the fingerprint and quantitative analysis methods were validated. The method was applied to analyze 15 batches of crude lanolin from different sources. The new established quantitative profiling method for free fatty acids can be potentially used for industrial applications to enhance the quality control of crude lanolin.