A novel thin layer chromatography based method for the quantification of quillaic acid saponins

Saponins display medicinal properties such as enhancing the drug delivery of biologicals, and boosting immune responses within vaccinations. As plant derived molecules, their phytochemical analysis is key to ensure pharmaceutical quality. The quantification of saponins is typically carried out by high-performance liquid chromatography (HPLC). The aim of this study was the development of a simple thin-layer chromatography (TLC) based method for the quantification of the sapogenin quillaic acid (QA), the triterpenoid backbone of saponins such as QS21 that are used as immunological adjuvants. Following acidic hydrolysis of the saponins, the corresponding sapogenins were derivatized with 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H) to form fluorescent hydrazones, increasing both the sensitivity and selectivity of the method. The detection of sapogenins with NBD-H is reported here for the first time. The QA-containing TLC bands were identified by mass spectrometry and their quantification was subsequently performed by densitometry. After validation, the method was applied to two plant species from Caryophyllaceae. To verify the plant's QA contents determined by TLC, a complementary HPLC method was developed. This study presents a new cost-effective method to quantify QA, enabling laboratories with limited resources to monitor plant cultivation and perform phytopharmaceutical quality control.