Brazilian jalap root, Operculina hamiltonii (G. Don) D.F. Austin & Staple, Convolvulaceae, belongs to the morning glory family and is used as a purgative or laxative medicinal plant. After hydrolysis and peracetylation of the EtOH-soluble extract, the known operculinic acids A and B and turpethic acid C, in addition to three undescribed glycosidic acids, operculinic acids L-N with a tetrasaccharide or pentasaccharide moieties with unusual 12-hydroxy fatty acid aglycones of different chain lengths (C₁₇ and C₁₈), were isolated from a commercial sample of pulverized roots by preparative recycling HPLC. Analysis of sixteen samples, including crude drugs and commercial phytopharmaceuticals based on jalap root, led to the generation of distinctive chromatographic profiles for each sample. The major isolated glycosidic acids were used as diagnostic peaks for HPLC-ESIMS fingerprinting by selected ion monitoring mode using target ions at [M + Na]⁺.