A systematic study of the retention behavior of isomeric triacylglycerols (TGs) in silver-ion HPLC on a ChromSpher Lipids column has been performed between 10 to 40°C using the most widespread hexane- and dichloromethane-based mobile phases. The randomization of mono-acyl TG standards and the random esterification of glycerol with fatty acids are employed to produce mixtures of TG isomers. The mobile phase composition has no influence on the general retention pattern, but significant differences in the retention order of double bond (DB) positional isomers in hexane and dichloromethane mobile phases are described and compared with the previous literature data. Saturated TGs with fatty acyl chain length from C7:0 to C22:0 are partially separated using the hexane mobile phase but not at all with the dichloromethane mobile phase. The hexane mobile phase enables at least partial resolution of TG regioisomers with up to seven DBs, while the resolution of only ALA/AAL and ALnA/AALn isomers is achieved with the dichloromethane mobile phase. The effect of temperature differs significantly depending on the mobile phase composition. Retention times of TGs increase with increasing temperature in the hexane mobile phase, while an opposite effect is observed for the dichloromethane mobile phase.

Rationale Narcissus cv. Hawera has been found to biosynthesize some Sceletium‐type alkaloids with antidepressant and anxiolytic activities. This ornamental plant has been poorly studied as a source of bioactive alkaloids including some contraversive reports on in vitro and intact plants. In this study, a detailed GC‐MS characterization of its alkaloid fractions is presented. Methods GC‐MS was used for the identification of compounds in the alkaloid fractions. Both underivatized and silylated samples were analyzed simultaneously. Elevated plus maze and tail suspension tests were used to assay the anxiolytic and antidepressant activities. Ellman’s and MTT‐dye reduction assays were used to evaluate the acetylcholinesterase (AChE) inhibitory and cytotoxicity activities, respectively. Results Of the 29 alkaloids, 13 of Sceletium‐type were detected. Two new alkaloids were identified as 2‐oxo‐mesembrine and 2‐oxo‐epi‐mesembrenol. Lycorine was found as a major compound (43.5%) in the crude silylated methanol extract. After the elimination of lycorine by pre‐crystallization, the major alkaloids were 40.8% 6‐epi‐mesembranol, 16.2% 6‐epi‐mesembrenol, and 13.8% sanguinine. This fraction showed anxiolytic and antidepressant‐like activities as well as potent AChE inhibitory and antineoplastic activities. Conclusions Silylation of the alkaloid fractions from Narcissus cv. Hawera provides better separation, structural information, and improved sensitivity for compounds with two and more hydroxyl groups. The lycorine‐free alkaloid fraction shows a great potential for further pharmacological studies.

Rationale Gas chromatography–mass spectrometry (GC–MS) is the most frequently applied technique for analyzing Amaryllidaceae alkaloids in plant extracts. Having these compounds, known for their potent bioactivities, is a distinctive chemotaxonomic feature of the Amaryllidoideae subfamily (Amaryllidaceae). The Amaryllidaceae alkaloids of homolycorine type with a C3–C4 double bond generally show molecular and diagnostic ions at the high‐mass region with low intensity in the EIMS mode, leading to problematic identification in complex plant extracts. Methods Eleven standard homolycorine‐type alkaloids (isolated and identified by 1D and 2D nuclear magnetic resonance) were subjected to separation with GC and studied with electron impact mass spectrometry (EIMS) including single quadrupole (GC–EIMS), tandem (GC–EIMS/MS), and high‐resolution (GC–HR‐EIMS) detectors, as well as with chemical ionization mass spectrometry (GC–CIMS). Alkaloid fractions from two Hippeastrum species and Clivia miniata were subjected to GC–EIMS and GC–CIMS for alkaloid identification. Results GC–EIMS in combination with GC–CIMS provided significant structural information of homolycorine‐type alkaloids with C3–C4 double bond, facilitating their unambiguous identification. Based on the obtained typical fragmentation, other 11 homolycorine‐type compounds were identified in extracts from two Hippeastrum species by parallel GC–EIMS, GC–CIMS, and liquid chromatography–electrospray ionization time‐of‐flight mass spectrometry and in extracts from C. miniata by GC–EIMS. Conclusions GC–MS can be successfully applied for the identification of new and known homolycorine‐type alkaloids, among others within the Amaryllidoideae subfamily, as well as for chemotaxonomical and chemoecological studies.