Reagent analysis of phytochemicals - application to Sceletium spp and Mesembryanthemums. Colourimetric reagent analysis has become more widely available to the general public, mostly marketed as a presumptive analytical technique for testing substances of abuse. This paper attempts to briefly explore the use of common colourimetric reagent assays such as Marquis reagent, Mandelin reagent, Simon's reagent, Ehrlich reagent etc in determining the presence of phytochemicals in plants, most notably attempting to find a suitable reagent for the colourimetric analysis of mesembrine-type alkaloids from plant material. Finding a suitable reagent may also open avenues for simple visualisation of such on TLC plates beyond the crude use of I 2 vapour, or less readily available reagents such as Dragendorff reagent or iodoplatinate used in prior literature. Most alkaloids give some colour change when reacted with the sulfuric acid-type reagents. This is explored in [1]. Due to the corrosive and/or toxic nature of these reagents, along with transport issues, they are often commonly available as extremely small quantities in glass ampoules for single use testing but other options are available. Marquis reagent - a common presumptive test, including for alkaloids This reagent uses formaldehyde and concentrated sulfuric acid to detect a wide variety of chemicals. The initial, and later stage colour changes can be used to guide a presumptive analysis Mandelin reagent - a common assay for alkaloids This uses concentrated sulfuric acid and metavanadate to give a wide variety of colours with different alkaloids The VO2+ ion formed by dehydration of metavanadic acid formed in situ can react with an array of organic molecules eg in the case of strychnine [2], insertion of a VO2 group affords a tertiary carbonium ion and organometallic complexes Simon's reagent - an assay for secondary amines Simon's reagent uses nitroprusside and acetaldehyde to detect the formation of a condensation product (enamine) between acetaldehyde and a secondary amine, which later forms a blue Simon-Awe complex. 1 Mecke reagent - alkaloids Uses sulfuric acid and selenous acid to detect componds. Frohde's reagent Sulfuric acid and molybdate to detect compounds. Ehrlich reagent - an assay for indolic constituents Ehrlich reagent uses 4-dimethylaminobenzaldehyde to detect a product that forms between indolic constituents and the benzaldehyde to form a blue coloured product 2 Experiment Reagent testing for mesembrine-type alkaloids from a plant material? As mesembrine is a tertiary amine lacking an indolic nucleus, both Ehrlich and Simon's reagent are presumed to be unsuitable. It is likely dimeric condensation products (requiring an aromatic nucleus) from Marquis reagent, or complexes with Mandelin reagent may be more promising. Structures sharing the unsaturated cyclic alkenol structure and tertiary amine of mesembrenol have particularly reacted with Marquis reagent, namely morphine giving a "very deep reddish purple" colouration. Saturated ketones sharing an aromatic nucleus and tertiary amine of mesembrine, such as methadone have given a "light yellowish pink" with Marquis and "dark greyish blue" with Mandelin. Structures related to a,β-unsaturated ketone functionality and aromatic nucleus, along with tertiary amine structures seem less explored. With Mandelin reagent, things like oxycodone, sharing a cyclic ketone and tertiary amine along with aromatic nucleus (although with an alcohol functional group), have given a dark greenish-yellow. Molecules sharing the 3,4-dimethoxy pattern on the aromatic nucleus and some distant similarity have given olive, to olive-brown colouration [3]. All these colour reactions are from quite distantly related molecules so should not be expected to apply to mesembrine-type alkaloids but offer the glimpse that these may be suitable reagents. A complicating factor is that the plant material only contains a small percentage total alkaloids and reagent testing requires a relatively concentrated sample. Also, other constituents, particularly coloured ones (chlorophyll etc), or ones likely to react with the reagent may interefere with accurate identification. While not selective for alkaloid extraction, the aim was to see if rapid identification was feasible and so a simple 95% ethanol extraction was selected. On evaporation, this significantly reduced the plant material to a very small quantity, which albeit coloured (brown-greenish), was likely a concentrated enough sample for reagent testing. Another complicating factor is the spectrum of alkaloids present, from the mesembrine, to mesembrenone and mesembrenol/mesembranol. As discussed elsewhere, there is 3 significant chemotypic variation, along with variations in profile induced by fermentation etc, among different Sceletium plants, meaning different alkaloids present in different ratios may give different colours on reagent testing. Also, traces of non-related alkaloids, such as hordenine, have been detected. Results: Sceletium EtOH extract tested with: a) Marquis: yellow/orange -> light brown b) Mandelin: olive brown to deep black-brown with some reddish hues Using Mandelin as a TLC stain on Sceletium highlighted the characteristic constituents well, turning a deep brown Using Mandelin reagent as TLC stain. Brown colouration slowly faded. This was followed up on D. bosseranum extract, similarly used as a kanna substitute. The following was obtained 4 D. bosseranum alcohol extract tested with: a) Marquis - yellow/olive brown b) Mandelin - dark olive brown Discussion: The colour changes were reasonably pronounced, particularly with Mandelin reagent and in line with the colour changes noted for some 3,4-dimethoxy substituted aromatic molecules. As for whether the colour change is distinct enough vs a typical plant residue devoid of mesembrine-type alkaloids to guide initial analysis is unclear. A distinct brown colouration was noted on TLC visualisation with Mandelin reagent at regions corresponding to hypothesised mesembrine-type alkaloids References [1] Dingjan, H. A., Dreyer-van der Glas, S. M., & Tjan, G. T. (1980). Colour tests for the identification of alkaloids (and related compounds). Pharmaceutisch Weekblad Scientific Edition, 2(1), 445–467. doi:10.1007/bf02273053 https://link.springer.com/article/10.1007/bf02273053 [2] The chemistry of Mandelin’s test for strychnine Francisco Sánchez-Viesca and Reina Gómez Open Access Research Journal of Chemistry and Pharmacy, 2023, 03(01), 001–004. https://doi.org/10.53022/oarjcp.2023.3.1.0040 [3] https://www.ojp.gov/pdffiles1/nij/183258.pdf 5