Characterization and colorimetric analysis of semi-synthetic Salvia divinorum analogues
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Salvia divinorum is a hallucinogenic herb from the mint family, Lamiaceae. An estimated 1.8 million people over the age of 12 have used S. divinorum in their lifetime as of 2008. The abuse of S. divinorum is attractive to teens and young adults who wish to experiment with psychoactive materials. The plant material and extracts are widely available via the Internet, and it is known that S. divinorum will not show up on common drug screens. The active component in S. divinorum is salvinorin A, which is a non-nitrogenous diterpene that is a highly selective kappa opioid receptor (KOR) agonist, reported to be the most potent naturally occurring hallucinogen. Since salvinorin A is such a selective and potent agonist of the KOR, there is interest in researching analogues in efforts to develop and understand therapeutic drugs for depression, schizophrenia, and other mental illnesses, resulting in the discovery of analogues with increased potency. These semi-synthetic salvinorin analogues have been abused by spraying the drug on innocuous plant material or on cigarette papers as a substrate for smoking. This practice poses a significant health risk, as most new analogues will have little safety and toxicity data associated with common abuse routes. Chemical characterization of the potent analogue, salvinorin B ethoxymethyl ether (SB-EME) was performed in order to develop methods of differentiation from Salvia divinorum and salvinorin A. These characterization techniques include HPLC, UV/Vis, NMR, and a colorimetric assay with Ehrlich's reagent. Adulteration of other plant materials with salvinorin A and analogues was performed and analyzed to determine if fortified materials can be detected by colorimetric assay. The validation studies of the HPLC method for SB-EME were found to be accurate (%RE < 12%), precise (RSD = 12%), and linear (R2 = 0.9993) over the mass range of 0.038 µg - 4.8 µg. The LOD was determined to be 0.038 µg, and the LOQ was determined to be 0.113 µg. Significant matrix effects were observed when using Salvia officinalis as a blank matrix, affecting the accuracy and selectivity of the method. However, the purified solutions of SB-EME had baseline resolution from salvinorin A and salvinorin B, which allows for easy qualitative distinction if adulterated samples are suspected. UV/Vis analysis provided a rapid and facile SB-EME characterization method. The UV/Vis trace for SB-EME was distinguishable from both salvinorin A and salvinorin B. NMR analysis confirmed the structures of salvinorin A, salvinorin B and SB-EME, with resonances specific to each compound. The colorimetric assay with Ehrlich's reagent provided a red-orange result with salvinorin B and SB-EME, similar to salvinorin A. While this does not provide differentiation in the field, it does allow all materials related to Salvia divinorum to be identified and collected for further analysis in the lab, as this colorimetric analysis allows easy distinction from common kitchen herbs such as mint, basil, and sage. Characterization of the colored species in the assay with Ehrlich's reagent was performed with UV/Vis, HPLC, and NMR. The UV/Vis analysis showed a new peak at 500 nm in the aqueous layer, which would correspond to a red-orange color. HPLC analysis revealed a new, highly retained peak from the DCM layer of the assay. 1H NMR analysis indicated that the backbone of the salvinorins was not stable in acid, and the molecule that creates the color was likely a degraded analogue. The analysis of the adulterated plant materials by colorimetric assay was inconclusive, as color intensity decreased as concentration of spiked standard increased. HPLC analysis of the vial remnants after the colorimetric assay confirm recoveries of the spiking compounds up to an average of 22% for salvinorin A, 96% for salvinorin B, and 41% for SB-EME over all matrices, indicating incomplete deposition of standard material onto the plant material. In conclusion, salvinorin B ethoxymethyl ether can be detected in the field through the use of Ehrlich's reagent as a colorimetric assay. Further laboratory tests, including HPLC and UV/Vis, were shown to easily distinguish the ether derivative from salvinorin A and B.