Journal of Analytical Toxicology, Vol. 34, June 2010
Use of High-Resolution Accurate Mass Spectrometry to Detect Reported and Previously Unreported Cannabinomimetics in “Herbal High” Products Simon Hudson1,*, John Ramsey2, Les King3, Sarah Timbers1, Steve Maynard1, Paul I. Dargan4, and David M. Wood4 1HFL Sport Science, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, United Kingdom; 2TICTAC Communications, Ltd., St. George’s University of London, Cranmer Terrace, London, SW17 0RE, United Kingdom; 3Home Office Advisory Council on the Misuse of Drugs, United Kingdom; and 4Guy’s and St. Thomas’ NHS Foundation Trust, London, United Kingdom
Abstract A range of “Herbal High” products were tested for synthetic cannabinoids (cannabinomimetics) to qualitatively determine and compare their individual and relative content. Liquid chromatography–high resolution accurate mass spectrometry was used to rapidly screen samples for a range of cannabinomimetics using mono-isotopic masses derived from the elemental composition of target analytes. A screening database of over 140 compounds was rapidly created. This approach, combined with further tandem mass spectrometric experiments, also facilitated the detection and identification of compounds for which reference materials were not available. Previously reported cannabinomimetics, including JWH-018 and CP47,497 and its homologues, were detected in varying relative proportions along with several tentatively identified unreported cannabinomimetics. In some countries, the decision has been made to include these substances within their drug control legislation, and other countries are considering similar action. The currently applied drug screening techniques are unlikely to be effective in providing scientific evidence to support their identification in seized products. The application of high-resolution accurate mass spectrometry offers a solution. In addition, the technology provides a relatively simple and quick method for screening products, building substance databases, and even identifying novel substances using a combination of accurate mass derived elemental composition and fragment ions combined with fragmentation prediction software.
Introduction Herbal incense mixtures, of which “spice” is perhaps the best known example, have been available globally for the past two or three years. Although declared as incense and not for human consumption, spice-type products are smoked as an * Author to whom correspondence should be addressed: Simon Hudson, HFL Sport Science, Newmarket Road, Fordham, Cambridgeshire, CB7 5WW, U.K. E-mail:
[email protected].
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apparently legal alternative to cannabis to deliver a so-called “herbal high”. The listed constituents of such spice products often include the following plant/herbal ingredients: baybean, blue lotus, lion’s tail, lousewort, Indian warrior, dwarf scullcap, maconha brava, pink lotus, marshmallow, red clover, rose, Siberian motherwort, vanilla, and honey. The intial assumption was that it was a botanical ingredient that was giving the effect. More recently, however, based on the reported effects of smoking these products, studies have focused on identifying any unlisted components that they may contain. To date, several synthetic cannabinoid receptor agonist (cannabinomimetic) compounds have been identified in spicetype products. The compounds detected to date include JWH018 (1–3), JWH-073 (4), JWH-250 (5), CP47,497 and its homologues (2,3), and HU-210 (6). The JWH compounds were developed as test compounds in the investigation of drugreceptor interactions in the study of the endocannabinoid system. The CP47,497 series of compounds and HU-210 have been reported in the scientific literature as having been tested successfully for cannabinoid action. These compounds can be subdivided into groups as many share the same core structure. Figure 1 highlights the groups relevant to this study, including the analytes detected both by previous workers and those considered within this study. A few of these have been designated as controlled drugs in some countries. It is apparent, however, that as one set of compounds is made illegal, more variants that fall outside current legislation take their place. There is a considerable volume of anecdotal evidence on various internet sites regarding the efficacy of spice products with different variants being said to deliver different effects to the user. There is also a documented case where a user reported that he had been smoking “spice gold” daily for eight months. He said that he had developed a tolerance to the product, which led to him increasing the amount he smoked each day. He also felt a conscious desire for the product. On treatment, the physical
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Journal of Analytical Toxicology, Vol. 34, June 2010
withdrawal symptoms were very similar to those seen with cannabis dependence (7). The reported symptoms included profuse sweating leading to internal unrest, a strong desire for spice, nightmares, nausea, tremor, and headaches. These symptoms led to a diagnosis of a dependency syndrome, according to both the International Statistical Classification of Diseases and Related Health Problems, 10th revision (ICD-10), and the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV). On admission to hospital, a urine sample had been taken. This was subjected to the “normal” immunological tests performed for cannabinoids, benzodiazepines, amphetamines, cocaine, opiates, and methadone. The results were all negative. It is unclear whether any portion of spice gold was subjected to scientific analysis at that time, either for routine drugs of abuse or for cannabinomimetics. The results from spice gold analyzed for this study are shown in Table I. In another study, a self-experiment was conducted by two authors (1). One cigarette containing 0.3 g of “spice diamond” was smoked. After approximately 10 min, the first noticeable effects occurred. These included considerably reddened conjunctivae, a significant increase in pulse rates, xerostomia, and an alteration in mood and perception. In psychomotor tests, no abnormalities were detected, although the subjects had the impression of being moderately impaired. These effects slowly subsided over a period of 6 h. The following day, some minor after-effects were reported. Further analytical work on the administered spice diamond identified the presence of JWH-018 and the C8 homologue of CP47,497. It is likely that different spice variants have different active ingredients. It is also likely that the active ingredients may differ in relative amounts from one variant to another and also from batch to batch of the same variant. In this investigation, 16 different spices products were analyzed using high-resolution accurate mass liquid chromatog-
raphy–mass spectrometry (LC–MS) to determine which cannabinomimetics, from a database of over 140 compounds, may be present. In some cases, multiple examples of the same product were available for analysis. Suspicious findings were then re-analyzed using MSn methodologies with accurate mass measurement of the product ions to help elucidate and confirm the structures of the detected compounds. The aim of this study was to carry out a rapid qualitative assessment of a range of these products, simply looking to identify the presence of any of these compounds. Quantitative analysis of individual compounds was not intended to be part of this initial study. This was due in part to difficulties in obtaining certified reference materials for these compounds within the study timeframe, even though several are advertised as being commercially available. It appears that rapidly changing legislation governing these compounds in different countries has complicated the manufacturing and shipment of reference materials across national borders, particularly where the compound is legally controlled in one, but not the other, of the source and destination countries.
Experimental Chemicals and reagents
HPLC-grade methanol and acetic acid were obtained from ThermoFisher (Waltham, MA). Sample preparation and extraction
Fifty milligrams of product was placed in a 20-mL glass vial, and 2 mL of methanol was added. This was sonicated for 10 min, then left for 10 min to settle. One milliliter of extract was transferred to a glass tube and evaporated to dryness. The sample was reconstituted in 10 µL methanol followed by 90 µL of ultrapure water before transferring to an autosampler vial for analysis. Instrumental analysis
Figure 1. Structures of the cannabinomimetic compounds relevant to this study. The monoisotopic M+H+ mass is included.
The samples were analyzed on a ThermoFisher Accela UPLC system interfaced to a ThermoFisher LTQ Orbitrap Discovery. The chromatographic separation was performed using a Waters Symmetry C8 column (3.5 µm × 2 mm × 100 mm) (Milford, MA) linked to a Phenomenex Security Guard C5 pre-column (4 × 2 mm, Torrance, CA) operating in gradient mode at 40°C. The mobile phases were 0.1% acetic acid with 300 µg/L uracil and 0.1% acetic acid in methanol with 300 µg/L uracil. The uracil was added to the mobile phase as a lock mass for the Orbitrap. The gradient was formed from starting conditions of 90% aqueous mobile phase.
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Organic mobile phase was increased through the run to give 40% organic at 1.0 min and 99% at 2.5 min. The 99% organic step was held for 3.5 min before reverting to starting conditions at 6.01 min. The flow rate throughout was 400 µL/min. The LC–MS interface was an Ion Max API source (ThermoFisher) fitted with an electrospray probe. A sheath gas flow rate and auxiliary gas flow rate of 30 and 10 arbitrary units, respectively, were used together with a source voltage of 4.5 kV
and a heated capillary transfer temperature of 200°C. A 10 µg/mL solution of ephedrine was introduced at a rate of 3 µL/min into a solvent flow of 400 µL/min consisting of 50% organic mobile phase and 50% aqueous mobile phase to optimize the remaining source parameters. This was achieved using the autotune functionality within the Xcalibur operating software using m/z 166 from ephedrine (Thermo Scientific). The Orbitrap was calibrated for positive ion analysis using
Table I. Summary of Results for All Products Tested*
Spice Product
Summed Analyte Response for Each Product as % of Strongest Product
EX-SES PLATINUM BLUEBERRY EX-SES PLATINUM BLUEBERRY EX-SES PLATINUM BLUEBERRY EX-SES PLATINUM BLUEBERRY EX-SES PLATINUM CHERRY EX-SES PLATINUM STRAWBERRY EX-SES PLATINUM STRAWBERRY EX-SES PLATINUM VANILLA MAGIC SILVER SPICE ARCTIC SYNERGY SPICE ARCTIC SYNERGY SPICE ARCTIC SYNERGY SPICE DIAMOND SPICE DIAMOND SPICE DIAMOND SPICE DIAMOND SPICE GOLD SPICE GOLD SPICE GOLD SPICE GOLD SPICE TROPICAL SYNERGY SPICE TROPICAL SYNERGY SPICE TROPICAL SYNERGY SPICE TROPICAL SYNERGY SPICEY REGULAR XXX BLUEBERRY SPICEY REGULAR XXX BLUEBERRY SPICEY REGULAR XXX BLUEBERRY SPICEY REGULAR XXX STRAWBERRY SPICEY ULTRA STRONG XXX VANILLA SPICEY ULTRA STRONG XXX VANILLA SPICEY ULTRA STRONG XXX VANILLA SPICEY ULTRA STRONG XXX VANILLA SPICEY ULTRA STRONG XXX VANILLA SPICEY ULTRA STRONG XXX STRAWBERRY SPICEY ULTRA STRONG XXX STRAWBERRY SPIKE 99 ULTRA BLUEBERRY SPIKE 99 ULTRA BLUEBERRY SPIKE 99 ULTRA CHERRY SPIKE 99 ULTRA STRAWBERRY SPIKE 99 ULTRA STRAWBERRY
14 9 34 8 28 10 10 17 29 31 35 33 12 100 22 31 10 19 10 9 30 30 25 26 10 21 10 7 7 10 21 13 32 23 37 7 8 13 9 48
Analyte by % Response JWH007/019 JWH JWH18 047/122
JWH073
JWH081 related
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