Simultaneous Analysis of Six Bioactive Lignans in Phyllanthus Species by Reversed Phase Hyphenated High Performance Liquid Chromatographic Technique

An online-hyphenated high-performance liquid chromatography-photodiode array-mass spectrometry (HPLC-PDA-MS) analytical method was developed for the simultaneous determination of six lignans of therapeutic importance in four Phyllanthus spp. (P. amarus, P. maderaspatensis, P. urinaria, and P. virgatus). HPLC with monolithic reverse phase silica column (4.6 x 100 mm) and simple isocratic elution of methanol-water mixed with dioxane facilitated the separation of lignans of diverse nature such as diarylbutyrolactone, tetrahydrofuran, isomeric aryltetralin, and diarylbutane type for quantitative analysis. Targeted lignans viz. heliobuphthalmin lactone (1), virgatusin (2), hypophyllanthin (3), phyllanthin (4), nirtetralin (5), and niranthin (6) were confirmed unambiguously in four Phyllanthus species by their abundant molecular adduct ions, retention time, UV, and mass spectra as compared with those of reference compounds. Advantages and limitations of both detection techniques for qualitative (fingerprinting) and quantitative analysis of the above mentioned lignans in four Phyllanthus spp. are discussed. The method was validated following international guidelines. The described method can be utilized for assays and stability tests of P. amarus extracts as well as traditional Indian medicine based on Phyllanthus herb.