LC-DAD and LC-MS-MS Analysis of Piperidine Alkaloids of Lobelia inflata L. (In Vitro and In Vivo)

Abstract

An LC-DAD method was developed for determination of lobeline from in vitro and in vivo cultures of Lobelia inflata. Samples were extracted with 0.1 N HCl-acetonitrile (1: 1, v/v), and purified by solid-phase extraction. Optimized conditions resulted in high recovery. LC separations were performed on an Eurosphere C8 reversed-phase column using 30:70 (v/v) acetonitrile-0.1 % trifluoroacetic acid as a mobile phase. Quantitative determination of lobeline was performed by external standard method at 250 nm, in the range of 2.4-80 mu g mL(-1). Validation studies proved that the repeatability of the method was good and the recovery was satisfactory. In vitro organized cultures contained considerable amount of lobeline (herb: 175 mu g g(-1), root: 100 mu g g(-1)). When these cultures were transplanted into the open field, the lobeline content increased significantly (herb: 323 mu g g(-1), root: 833 mu g g(-1)). Plants obtained from seed propagation contained 382 mu g g(-1) lobeline gin the herb. For direct characterization of di-substituted piperidine alkaloids in extracts of L. inflata, tandem mass spectrometric method was developed using electrospray ionization. Analysis was performed in the positive ion mode on a triple quadropole LC-MS system. LC separations were achieved on Eurosphere C8 column with a modified mobile phase (acetonitrile-30 mM ammonium Formate, pH 2.80) to ensure proper molecular ionization. The identification and structural elucidation of the alkaloids were performed by comparing their changes in molecular mass (AM), full-scan MS-MS spectra with those of lobeline, norlobelanine and lobelanidine. These alkaloids and ten other derivatives were identified in the plant extracts. Three piperidine alkaloids were reported in L. inflata for the first time.