UPLC-MS/MS Analysis of Salvinorin A from Salvia Divinorum

This application note demonstrates the use of the ACQUITY UltraPerformance LC (UPLC) System coupled with tandem mass spectrometry to provide positive identification and of Salvia divinorum via the analysis of salvinorin A using a simple extraction technique. Amounts of salvinorin A in the plant material were also calculated using the same chromatographic run.

Benefits

• The extraction method was straightforward and the UPLC separation was rapid
• Methanol/water blanks showed no carryover, reducing the likelihood of false positives

Salvia divinorum is a perennial herb native to certain small areas of southern Mexico. It is a large plant with large green leaves, and can be grown outside of its native habitat. Salvia divinorum is known to be a psychoactive herb and is used by the native Mazatec Indian Shamans to create altered states of consciousness during spiritual healing and other spiritual ceremonies and rituals. The active hallucinogenic component of Salvia divinorum has been identified as salvinorin A, Figure 1, (Siebert, 1994).

Currently, neither Salvia divinorum nor any of its constituents, including salvinorin A, are controlled under the federal Controlled Substances Act (CSA) within the United States, although the U.S. Drug Enforcement Agency (DEA) does consider Salvia divinorum to be a drug of concern. Several states (Louisiana, Missouri, Tennessee, Oklahoma, Delaware, Maine, and North Dakota) have laws prohibiting possession of Salvia divinorum and salvinorin A and other states are considering legislation regarding this plant. A number of countries (Australia, Denmark, Iceland, Finland, Norway, Sweden, Italy, Japan, Brazil, South Korea, United Kingdom, Estonia, Belgium, Spain) have laws that prevent the sale, distribution, importation, and possession of Salvia divinorum and salvinorin A.

When dried plant material becomes crushed or powdered many of the unique identifying anatomical features of the plant can become obscured.

This makes chemical identification of unique plant phytochemical constituents one of the few ways to confirm a plant’s true identity. Because there may be legal implications for the possession of Salvia divinorum and salvinorin A, the confirmation of identity must be reliable and irrefutable.

Salvia divinorum can be identified by analyzing for the presence of the active component, salvinorin A, which is unique to Salvia divinorum. Mass spectrometry, in combination with liquid chromatography (LC-MS) can provide positive, unambiguous confirmation of the presence of salvinorin A in the sample matrix, and just as importantly, provide evidence that plant material does not contain salvinorin A and therefore is not Salvia divinorum.

This application note demonstrates the use of the ACQUITY UltraPerformance LC (UPLC) System coupled with tandem mass spectrometry to provide positive identification and of Salvia divinorum via the analysis of salvinorin A using a simple extraction technique. Amounts of salvinorin A in the plant material were also calculated using the same chromatographic run.

Extraction

Dry Salvia divinorum (leaf and stems) samples and non-Salvia divinorum material were finely ground with a mortar and pestle. Portions (10 mg) were added to a 1.5 mL polypropylene snap cap centrifuge tube. To each tube was added 1 mL of methanol/water (9:1) and the tube and contents sonicated in a heated (35 °C) water bath for 1 hour. Following centrifugation, sample extracts were diluted 1/10 with extraction solvent and injected directly. Analyses were performed in triplicate.

Salvinorin A was separated from any interfering substances quickly using UPLC. Utilizing UltraPerformance Liquid Chromatography enables assays to be performed in a rapid manner without sacrificing separation quality. In this method, the UPLC run time was 90 seconds.

Three Multiple Reaction Monitoring (MRM) transitions were monitored from salvinorin A (Figure 2). The primary transition (433.4 > 373.2) was used for quantification and confirmation, while the other two transitions were only used for peak confirmation purposes. Peak height ratios of the primary and secondary MRM transition calculated using an authentic standard of salvinorin A were determined to be 6.1 ±0.8.

The presence of salvinorin A in the ground plant material samples was considered to be confirmed by the following criteria:

1. All three monitored MRM ions were present.

2. A chromatographic peak was present at a retention time of 0.8007 ±0.0040 minutes (~0.5% RSD)

3. Peak height ratios of the primary and secondary MRM transition (373:323) were within ±20% of the calculated ratio which was determined to be 4.9–7.3.

A total of eight samples were analyzed. Three were dry, whole plant Salvia divinorum from different internet suppliers, two others were described by the suppliers as “enhanced” leaf (prepared by extracting salvinorin A, and adding it back into the leaves), and three were non-Salvia divinorum material (common sage, whole mint leaf, and ground mint).

Injections of sample extracts were bracketed by injections of authentic salvinorin A (Sigma-Aldrich Chemical Co.). Methanol/ water blanks were also injected throughout the sample set to assess carryover.

As can be seen from Table 1, salvinorin A was confirmed to be present in the five samples claimed to be Salvia divinorum. Ratios of the two MRM transitions fell between the prescribed levels, and retention times of the peak of interest were within the defined limits. Salvinorin A amounts were calculated from a five-point linear calibration curve (R² = 0.9975) based on the primary MRM transition (Figure 3).

Standard concentrations ranged from 0.002 mg/mL to 0.050 mg/mL. Salvinorin A amounts in the leaf material ranged from 3.9 to 5.6 mg/g (dry weight) for unaltered material and 14.6 and 27.1 mg/g for the two enhanced leaf samples. Variability between different lots of plant material has been noted previously (Gruber et al, 1999). As expected, salvinorin A was not found in the common sage, mint leaf, and mint flake samples.

Using a weak wash solvent of 90:10 water/methanol and a strong wash solvent of 90:10 methanol/water; extraction solvent blanks run directly after the most concentrated standard and the final enhanced leaf extract showed no measurable carryover (Figure 4) proving the methodology has a low probability of false positives.

• Quantitation and confirmation of salvinorin A from Salvia divinorum using an ACQUITY TQD System was demonstrated in a single analysis
  
• The extraction method was straightforward and the UPLC separation was rapid (run time of 1.5 minutes)
  
• Unambiguous confirmation of the presence of salvinorin A was demonstrated using a combination of exceptional retention time reproducibility, three MRM transition ions present, and stable ion ratio between the primary and secondary MRM transitions
  
• Methanol/water blanks showed no carryover, reducing the likelihood of false positives

1. Siebert DJ. Salvia divinorum and salvinorin A: New Pharmacologic Findings. Journal of Ethnopharmacology. 1994 June; 43(1): 53–6.
   
2. Gruber JW, Siebert DJ, Der Marderosian AH, Hock RS. High Performance Liquid Chromatographic Quantification of Salvinorin A from Tissues of Salvia divinorum. Epling & Játiva-M. Phytochem. Anal. 1999; 10: 22–25.