Silica-Based Monolithic Coupled Column for the Simultaneous Determination of Echitamine, N^b-Demethylalstogustine, and Loganetin in Alstonia scholaris by RP-HPLC and Optimization of Extraction Method

Abstract

A simple, rapid, and sensitive high performance liquid chromatography (HPLC) method for the simultaneous quantification of echitamine (1), N b -demethylalstogustine (2), and loganetin (3) in Alstonia scholaris was developed using silica-based monolithic coupled column. The samples were analyzed on Chromolith RP-18 coupled column (100 þ 50 ¼ 150 mm  4.6 mm) using isocratic elution with acetonitrile: 0.01 M buffer (KH 2 PO 4 ) containing 0.1% trifluoroacetic acid (20:80, v=v) at 0.5 mL=min flow rate and detection at 254 nm. The limits of detection and quantitation were 0.64 and 2.15 mg=mL for echitamine, 0.72 and 2.43 mg=mL for N b -demethylalstogustine, and 0.78 and 2.62 mg=mL for loganetin, respectively. Standard curves were linear in the range of 3-15 mg=mL (r 2 ¼ 0.999). The method showed good repeatability (%RSD < 1.14), reproducibility (%RSD < 1.16), and recovery (mean% > 99.48). This is the first report on loganetin isolation from A. scholaris. Furthermore, various extraction methods and parameters were investigated to optimize markers extraction from the sample matrix. Finally, the optimized extraction method was used in the extraction of markers from different parts of the plant. The validated method was successfully used in the quantification of markers in various parts of the plant, which can be applied in the overall quality assessment of A. scholaris.