A Review of the Methods of Determination and Quantification of Salvinorin A within Biological Fluids Karyn Crawford The Plan

• My first introduction to Salvia • Actual background information • The methods • Compare and contrast!

My First Introduction

• Random sketchy head shop in San Luis Obispo in CA • No idea what Salvia was – “It’s a legal way to get high!” • My friends were too nervous to ask for it outright so we wandered around like the awkward high schoolers we were • One finally manned up enough to ask, got a look but was offered a price • He was broke so he couldn’t buy any anyway Actual Background Information

• Salvia Divinorum – Active ingredient Salvinorin A • Agonist to κ opiod receptor

– Rather than affecting the serotonin 5-HT2A receptors like classic hallucinogens (LSD) – Only known non-nitrogenous opiod agonist – Full metabolism is not known

More Actual Background Information

• Historically used by Mazatec peoples – Ska pastora – Ska Maria pastora • Street names: Magic mint, Diviner’s sage, or Salvia • Then: Chewed or brewed into a tea – Now: dried to smoke or chew

Legislation

• Not federally controlled – Attempt made in 2002 to make it schedule I but it did not go through • States have made legislation against it – Illegal in: Alabama, Delaware, Illinois, Louisiana, Michigan, Missouri, Ohio, Texas and more! – Other states were not successful in getting it passed • Controlled in some manner: Australia, Belgium, Canada, Denmark, Estonia, Finland, Italy, Japan, Russia, Spain, Sweden Legal and ‘natural’ ≠ Safe

• Symptoms – Loss of contact with reality, short-term loss of consciousness, panic crisis, hearing voices, nausea, unrealistic visions, sense of death, potential self-injuries without pain – No deaths linked to overdoses recorded • Growing concern – Need robust, reliable methods of detection Experiment 1

• 2005, Schmidt et al – SPE followed by HPLC – First study to detect Salvinorin A within biological fluids • Human and rhesus monkey plasma, rhesus monkey cerebrospinal fluid, and human urine • Sal. A was extracted from Salvia Div. to >98% purity • Samples were then spiked Experiment 2

• Later in 2005, Pichini et al – Quantification of Sal. A in biological fluids using LLE then GC-MS • Urine, serum, saliva and sweat from a sweat patch • Serum was not able to be tested as planned – Using samples from volunteers who smoked a controlled amount of Salvia • ~0.58 mg of Sal. A smoked between the two (7.7mg/g dry leaves) • Some could have been lost through vaporization

Experiment 3

• 2008, McDonough et al – Wanted to detect and quantify Sal. A using SPE and LC-MS – Sal. A purchased in crystalline form (>98% purity) – Spiked urine and blood with the Sal. A Experiment 4

• 2012 – Barnes et al – Analyze the Sal. A content in different parts of the plants, water, and urine using SPE and LLE and two dimensional GC-time of flight MS – Sal. A stock was purchased, but extractions of S. divinorum leaves, stems and roots were performed separately • Quantitation of Sal. A in each part of the plant was performed Experiment 5

• 2013 – Margalho et al – Detection and quantification in pericardial fluid, vitreous humor, whole blood and plasma using SPE and GC-MS – Sal. A standards purchased and used to spike the blood, plasma, vitreous humor, and pericardial fluid. – To help if Sal. A was part of the reason a person could have died MS of Salvinorin A LOD and LOQ

Author and Instrument Method Matrix LOD (ng/mL) LOQ (ng/mL)

Schmidt et al (LC-MS) SPE Plasma 2 n/a Pichini et al (GC-MS) LLE Plasma 5 15 LLE Urine 5 15 LLE Saliva 5 15 LLE Sweat 5 10 McDonough et al (LC-MS) SPE Urine 2.5 2 Barnes et al (GCxGC-MS) LLE Water 230 n/a SPE Water 6 n/a LLE Urine 200 n/a SPE Urine 4 n/a Margalho et al (GC-MS) SPE Vitreous Humor 5 n/a SPE Pericardial Fluid 5 n/a SPE Blood 5 n/a SPE Plasma 5 n/a Linear Range

Author Method Linear Range Schmidt et al LC-MS 2 – 1000 ng/mL Pichini et al GC-MS 15 – 5000 ng/mL McDonough et al LC-MS 5 – 100 ng/mL Barnes et al GCxGC-MS 8 – 500 ng/mL Margalho et al GC-MS 5 – 100 ng/mL Plant Extraction

• Has been well established • Barnes et al focused on extraction from separate parts of the plant – Leaves have the highest amount of Sal. A • Approximately 60 ng/g of dry leaves • Typical Euphoric dose is between 200 and 500 µg • Suggested that cultivation practices in their lab were not optimized in any way – Stems had a detectable amount – Roots did not have a detectable amount

Where to go from here

• Quantification of Sal. A – In a few of the studies, the amount of Sal. A increased after extraction – Over 100% yields • Degradation studies • Sampling time studies References

1. Mark S. Schmidt, Thomas E. Prisinzano, Kevin Tidgewell, Wayne Harding, et al. Determination of Salvinorin A in Body Fluids by High Performance Liquid Chromatography – Atmospheric Pressure Chemical Ionization. J. of Chrom B. 818: 221-225 (2005) 2. Simona Pinchini, Sergio Abanades, Magi Farre, Manuela Pelligrini, et al. Quantification of the Plant-Derived Hallucinogen Salvinorin A in Conventional and Non-Conventional Biological Fluids by Gas Chromatography/Mass Spectrometry after Salvia divinorum Smoking. Rapid Commun. Mass Spect. 19: 1649-1656 (2005) 3. Pamela C. McDonough, Justin M. Holler, Shawn P. Vorce, et al. The Detection and Quantitative Analysis of the Psychoactive Component of Salvia divinorum, Salvinorin A, in Human Biological Fluids Using Liquid Chromatography – Mass Spectrometry. J. of Analytical Tox. 32: 417-421 (2008) 4. Matthew W. Johnson, Katherine A. MacLean, Chad J. Reissig, et al. Human Psychopharmacology and Dose-Effects of Salvinorin A, a Kappa Opiod Agonist Hallucinogen Present in the Plant Salvia divinorum. Drug and Alcohol Dependence. 115: 150-155 (2011) 5. Brian B. Barnes, Nicholas H. Snow. Analysis of Salvinorin A in Plants, Water, and Urine using Solid- Phase Microextraction-Comprehensive Two-Dimensional Gas Chromatography-Time of Flight Mass Spectrometry. J. of Chrom A. 1226: 110-115 (2012) 6. Claudia Margalho, Eugenia Gallardo, Alice Castanheira, Duarte Nuno Vieira, et al. A Validated Procedure for Detection and Quantitation of Salvinorin A in Pericardial Fluid, Vitreous Humor, Whole Blood and Plasma using Solid Phase Extraction and Gas Chromatography-Mass Spectrometry. J. of Chrom A. 1304: 203-210 (2013)