Validation of a New LC-MS/MS Method for Rapid Quantification of Neferine: Application to Lipid Nanocarriers

Neferine, a natural alkaloid, is known for its multifaceted properties, including antioxidant, neuroprotective, anti-inflammatory, and autophagy-regulating effects. A simple, robust, specific, and sensitive analytical method is a key requirement for quantifying an analyte. Although a few methods have been reported for simultaneous estimation, none are available for the quantification of neferine alone in pharmaceutical or nutraceutical formulations. Whereas, the quantification of drugs alone is considered as a valuable requirement in the preformulation and formulation studies. In this research work, we report the development and validation of a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the accurate quantification of neferine in bulk and formulations. The chromatographic separation was achieved using a UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) under an isocratic elution method. The mobile phase consisted of acetonitrile (80%) and 10 mM ammonium acetate with 0.1% formic acid (20%). The flow rate of the mobile phase was set at 0.3 mL/min. The method exhibited good linearity (R ² = 0.9944) over a range of 25-600 ng/mL. High sensitivity of the method was demonstrated by the limit of detection (LOD) and limit of quantification (LOQ) values of 1.29 and 3.90 ng/mL, respectively. Results of the accuracy and precision study were in complete alignment with the ICH guidelines. The analytical method was found to be robust under varied analytical conditions. Furthermore, the method was successfully applied for the quantification of neferine in nanostructured lipid carriers in the diffusion studies. In conclusion, the newly developed and validated LC-MS/MS method is specific, sensitive, rapid, accurate, and precise for the quantification of neferine in bulk and formulated products.