HPLC Quantification of Salvinorin A from Tissues of Salvia divinorum

[Paradoxic]

Title: High Performance Liquid Chromatographic Quantification of Salvinorin A from Tissues of Salvia divinorum Epling & Játiva-M (Summary)
Authors: John W. Gruber, Daniel J. Siebert, Ara H. Der Marderosian and Rick S. Hock
Journal: Phytochemical Analysis
Date: 1999

Link to original paper: http://sagewisdom.org/phytochemical.html

Abstract
A reversed‑phase high performance liquid chromatographic method for the determination of salvinorin A, a psychotropic diterpene isolated from the Mexican sage Salvia divinorum, has been developed. Extracts from several plant collections were examined on a C‑18 column with UV detection and isocratic elution with acetonitrile: water (45:55). This assay allowed quantification of salvinorin A in extracts of leaves and stems of S. divinorum and has also been applied to the screening of related species for the production of salvinorin A. Levels of salvinorin A in leaves range from 0.89 to 3.70 mg/g dry weight.

Introduction
No clearly wild populations of S. divinorum have been identified, which suggests that all existing stands of the plant have been intentionally cultivated by humans. It is readily propagated through asexual reproduction by rooting stem cuttings and it rarely, if ever, produces seed in nature. It has been suggested that S. divinorum is a hybrid, which resulted in its substantially reduced fertility.
 
Methods
General experimental procedures: Salvinorin A was extracted from S. divinorum leaves and authenticated by nuclear magnetic resonance (NMR) spectroscopy and by thin layer chromatographic (TLC) comparison with authentic salvinorin A. All solvents used were HPLC grade and all water used was distilled and filtered.
 
Plant materials: Live plants were purchased from Logee's Greenhouses and to multiply them cuttings were taken and then rooted in a greenhouse.
 
Extraction of samples: Plant tissue was harvested and lyophilized and then ground into powder. 0.5g samples of powder were taken and extracted in flasks by steeping in 25 mL of chloroform for 30 min. The extract was then filtered and the flask was rinsed with more chloroform to remove the remaining extract. Next, the solution was evaporated in a rotary evaporator. Then the dry solids were redissolved in a mixture of 20.0 mL methanol and 5.0 mL acetone using sonication to assist in dissolving of all solid material.
 
Chromatography conditions: HPLC was performed using a Milton Roy HPLC system with a Zorbax column. All analyses were performed with a coupled C‑18 guard column to protect the integrity of the column. Aetonitrile:water (45:55) was used for the mobile phase and salvinorin A was detected by UV absorption at 208 mn.
 
Composite standard curve: A standard curve for salvinorin A was produced by dissolving a 0.051 mg/mL solution of standard into the HPLC mobile phase. Six different amounts of salvinorin A were chromatographed by varying the injection size. Three injections were made for each of the six amounts of salvinorin A so three separate determinations of the standard curve were performed. The data from all three was averaged and replotted to yield a composite standard curve.
 
Quantification of salvinorin A in plant tissue samples: For each sample 20 microL of reconstituted methanol:acetone extract was injected into the HPLC and the area of the peak due to salvinorin A was calculated. By applying area of the peak to the linear equation obtained from the composite standard curve, the amount of salvinorin A was calculated.

Results/Discussion


Figure 1. HPLC chromatograms of an authentic sample (0.2551Ag) of salvinorin A (A), and of representative Salvia divinorum tissue extracts obtained from "Palatable" clone (Bret Blosser) (B), from Cerro Rab6n clone (L. J. Valdés) (C), and from a seed grown plant DS03 (D. J. Siebert) (D). In each case the retention time of the peak representing salvinorin A is indicated. (For chromatographic protocol see Experimental section).



Figure 2. The composite standard calibration for the quantification of salvinorin A by HPLC (the error bars indicate ±1 standard deviation; the linear regression equation for the calibration curve is y= 759,334x ‑44,127).

The coefficient of variation among the three individual determinations of the standard curves was 5.9%. The coefficients of variation for each injected amount across all three determinations ranged from 1.7 to 5.7%.
 
In order to determine the range of salvinorin A concentration found in various populations of S. divinorum, 20 different samples of leaves were collected from cultivated plants in private collections as well as endemic populations in Oaxaca. The chromatogram representations for each population, based on the average of two separate injections, are shown in Figure 1. Salvinorin A content varied significantly among samples; values ranged from 0.89 to 3.70 mg/g dry weight. Salvinorin A concentration in stems was equivalent to approximately 4% of the level found in leaves.


 
The genetic heterogeneity is unknown for the various populations of S. divinorum so the variation of salvinorin A concentration cannot be attributed to specific environmental or genetic conditions. The results show that the variation of salvinorin A concentration is just as great among clones as it is among plants of the same clone grown in different locations. These results contrast other findings that found no significant variation in salvinorin A concentration in plants from Michigan, Louisiana, and Mexico.
 
There was a report that S. divinorum was most closely allied to S. concolor Lamb ex. Benth so S. concolor was screened for salvinorin A content, but results did not show that any was present. S. blepharophylla, S. chiapensis, S. gregii var. San Isidro, S. leucantha, S. membranacea and S. recurva were also screened and not found to contain any salvinorin A.
 
The HPLC methodology used was a successful method for quantifying salvinorin A in S. divinorum leaves and can be used for other parts of the plant as well. Further screening of additional sage species for salvinorin A content may help to determine the origins and taxonomic position of S. divinorum.

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