RGB Trichromatic Whiteness Assessment of Bio Analytical Chromatographic Tool Using Fluorescence for Quantitation of Semaglutide: Application to Pharmaceutical Preparations and Spiked Plasma

Semaglutide (SEMG) is one of the most widely used and trending medications to treat type II diabetes and obesity. This work aimed to develop a liquid chromatography with spectroflourimetric detection (HPLC-flourimetry) analysis of SEMG in both its tablet dosage form and plasma. The power of fluorescence detection coupled with HPLC proved its capability as a bioanalytical tool to assay SEMG in plasma samples owing to its simplicity and sensitivity which reached below the C_max of SEMG. Separation was done using a C₁₈ column with mobile phase of acetonitrile and water acidified with orthophosphoric acid (pH 3.5) (1.41 × 10^-5 M) in isocratic mode in ratio 57:43 and 1 mL/min flow rate after extraction using protein precipitation. Detection was carried out at λ excitation of 238 nm and λ emission of 416 and 307 nm for SEMG and the internal standard, respectively. Evaluation of greenness of the proposed method was done using AGREE (Analytical GREEnness Metric Approach), ComplexGAPI (Complementary Green Analytical Procedure Index) & the new algorithm RGB 12 model (Red-Green-Blue). They showed that these methods can be a greener alternative with acceptable sensitivity for analysis of SEMG. The developed seven min-assay was validated per ICH as well as FDA bio analytical methods' guidelines to prove its applicability for routine sample analysis and future pharmacokinetic studies.