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Analytical investigation of legal high products containing Salvia divinorum traded in smartshops and internet
Forensic Science International 242 (2014) 255–260
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Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Analytical investigation of legal high products containing Salvia divinorum traded in smartshops and internet Fernando Xavier Moreira *, Fe´lix Carvalho, Maria de Lourdes Bastos, Paula Guedes de Pinho * REQUIMTE, Laborato´rio de Toxicologia, Departamento de Cieˆncias Biolo´gicas, Faculdade de Farma´cia, Universidade do Porto, Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 April 2014 Received in revised form 9 July 2014 Accepted 11 July 2014 Available online 19 July 2014
Lately, the hallucinogenic plant Salvia divinorum has been considered a popular recreational product among adolescents, being legally sold in several countries in ‘‘smartshops’’ and in internet websites. Sellers frequently omit the safety information about the plant, encouraging its use for recreational purposes, without providing adequate qualitative and quantitative details. The principal hallucinogenic compound of the plant, salvinorin A, alongside with three other isomeric compounds, salvinorin B, C and D were evaluated in 10 products containing S. divinorum. These products were obtained in smartshops and from internet websites, and contained concentrated extracts of salvinorin A, with potencies labeled between ‘‘5x’’ and ‘‘60x’’. For that purpose a simple and rapid extraction protocol and a GC–MS methodology were developed and applied to the purchased samples. The analysis of S. divinorum samples allowed the identification of four salvinorins, salvinorin A being the most prevalent hallucinogen. In the tested samples, there were several unreliable data provided to consumers. Frequently, there was no information on salvinorin A concentration, but when it existed, it generally did not correspond to the true amount present in products. On the other hand, the concentration of salvinorin A in each product far exceeded the amount needed to produce hallucinogenic effects. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Salvinorin A Legal drugs Smartshops Salvia divinorum
1. Introduction Salvia divinorum is a psychoactive plant from the Labiatae family, originating from Oaxaca, Mexico, with recognized psychoactive properties [1,2]. It is one of the most important hallucinogenic plants of the Mesoamerican cultures, frequently used by earlier civilizations in religious ceremonies, allegedly enabling the contact between physical and spiritual worlds. This approach to the spiritual reality was believed to reveal disease diagnosis, to ensure good harvests and to predict the rains to come [1–3]. Mazatec Indians from Oaxaca, Mexico, commonly chewed the leaves or drank the infusion from the plant S. divinorum. Lately, the use of S. divinorum has been reemerging in recreational settings [3,4]. Although leaves of S. divinorum can be chewed or used to prepare oral tinctures, inhalation of the vaporized smoke of
* Corresponding authors at: REQUIMTE, Faculty of Pharmacy, Department of Biological Sciences, Laboratory of Toxicology, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Tel.: +351 220428599; fax: +351 226093390. E-mail addresses: [email protected] (F. Xavier Moreira), [email protected], [email protected] (P. Guedes de Pinho). http://dx.doi.org/10.1016/j.forsciint.2014.07.009 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
salvinorin A was proven to be the most efficient method for achieving its psychoactive effects in humans [2,5]. When consumed orally, S. divinorum has less expressive effects, probably due to reduced sublingual absorption of salvinorin A [6,7]. The inhalation of small amounts (200 mg) of salvinorin A (S. divinorum main compound), after heating in a piece of aluminum foil, proved to be enough to elicit strong psychotropic effects. Through this exposure route, the effects take only 30 s to be expressed [6]. Thus, the intensity and speed of the effects highlight the difference between the effects on indigenous populations (that normally chew the plant) and newer users in western countries (that normally inhale the vapors). The use of S. divinorum has spread through Europe and North America in the last few years, being purchased from internet websites and at smartshops [5]. The percentage of young adults in western countries, such as the United States of America and Canada that admitted to have used S. divinorum in their lifetime is about 5% [8,9]. Young wealthy males, that have already tried other drugs seem to be more prone to start using S. divinorum [10]. Although, no serious adverse effects (death, hospital stay or emergency room visit) have been attributed to S. divinorum
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consumption [11], a 21 year old person evidenced persistent psychosis and paranoia after smoking the drug [12]. The hypothesis of re-experiencing hallucinations and/or other neuropsychiatric consequences such as de´ja` vu experiences cannot also be ruled out [13]. Several websites selling S. divinorum products biase the safety information about these products, encouraging the use of S. divinorum for recreational purposes, without providing adequate qualitative and quantitative details on the product [14]. For example, more than 90% of websites from the United Kingdom, where the most prevalent commercialized legal drug is S. divinorum, have no information about the drug’s side effects [15]. Additionally, some sources wrongly associate analgesic effects and euphoria with S. divinorum consumption [14]. Salvinorin A is considered the most important active compound present in S. divinorum [16]. Although this plant has other identified chemical compounds, salvinorin A is present in much higher concentrations than the remaining salvinorins, which may be considered insignificant from the pharmacological point of view [17]. Salvinorin A was isolated for the first time in 1982 [18] and its psychoactivity was established in 1994 [16]. The relationship between salvinorin A and the kappa opioid receptors was established in 2002 [19], still being considered salvinorin A as the most potent naturally occurring hallucinogen, being chemically and structurally unique: it is the first known psychoactive diterpene and the first non-nitrogenous hallucinogen [20] (Fig. 1). Considering the inappropriate information given by sellers of S. divinorum products, the specific objectives of this work are to validate a protocol of extraction and quantification of salvinorin A and the other related molecules from concentrated extracts of S. divinorum using GC–MS. The developed methodology was further applied to quantify salvinorin A and estimate the amounts of salvinorins B, C and D; confronting the obtained results with previous similar researches on S. divinorum commercialized products and evaluating the reliability and quality of the information that is provided to consumers in the labeled packages purchased.
the purchased packages) according to the potency/amount of extracts used. Packages usually contained 1 g of concentrated extract of S. divinorum. Many of the packages did not explain what the classification (hereafter referred to as ‘‘labeled potency’’) meant. Only samples acquired from website E guaranteed that, for example the ‘‘5x’’ extract, had 12.5 mg of salvinorin A, while the ‘‘10x’’ extract contained 25 mg of salvinorin A. Packages from smartshop A had printed on the label that 1 g of the ‘‘5x’’ extract equals 5 g of S. divinorum leaves; 1 g of the ‘‘10x’’ extract equals 10 g of S. divinorum leaves, and so on. The remaining information (expiration date, batch, contraindications, side effects) provided to customers was generally scarce (Table 1). After the purchase, all the samples were kept in the laboratory in a humidity controlled environment, at room temperature (similar to the environment observed in ‘‘smartshops’’) until analysis. The standard of salvinorin A was purchased from Sigma– Aldrich Chemie Gmbh (Munich, Germany), and was used for the identification and quantification of salvinorin A in the samples. A semi-quantitative determination of other salvinorins (B, C and D), was performed by using the calibration curve of the available standard. The standard of salvinorin A was kept at 20 8C, in a humidity controlled environment, and protected from light. Acetonitrile and acetone were purchased from Fisher Chemical1, 99.99% and 99.98% of purity, respectively, according to GC assays. Chloroform was obtained from Fisher Scientific1 and was 99.99% pure, also determined by GC assay. Thymol, used as the internal standard, was purchased from Sigma Aldrich1, with a purity of 99.5%. In order to separate leaves from solvent, polytetrafluoroethylene (PTFE) hydrophobic acrodisc syringe filters were used.
2. Methods
2.3. Extraction procedure
2.1. Samples and chemicals
To determine the most effective solvent to extract salvinorin A from commercial S. divinorum leaves fortified with concentrated extracts, samples were ground until a homogeneous powder was obtained. Three solvents with different polarities were tested: acetonitrile, acetone, and chloroform. The main objective was to extract the greatest amount of salvinorin A, but it was also important to provide the extraction of salvinorins B, C, D. All the extractions were tested in triplicate, on a sample containing a mixture of 4 commercialized products in similar amounts.
Ten S. divinorum samples were purchased in Portuguese smartshops, from September of 2012 until April 2013, prior to the approval of the Decree 54/2013 [21], prohibiting the marketing of these products. Samples were purchased in four different ‘‘smartshops’’ (A–D), and from two websites (E, F). The samples consisted of S. divinorum crushed leaves fortified/soaked, by producers, with salvia concentrated extracts. They were classified as ‘‘5x’’, ‘‘10x’’, ‘‘15x’’, ‘‘20x’’, ‘‘40x’’ or ‘‘60x’’ (designation labeled on
Fig. 1. Chemical structures of salvinorins A–D.
2.2. Standard solutions Standard solutions of salvinorin A were prepared by sequential dilutions of the stock solution of salvinorin A (1 mg/ml) in acetonitrile. Concentrations ranging from 5 mg/ml to 1000 mg/ml were obtained and stored at 20 8C.
2.3.1. Extraction with acetonitrile and acetone In order to perform the extraction of salvinorins from purchased concentrated extracts with acetonitrile and acetone, samples were submitted to a methodology already performed by Tsujikawa et al. [22], being additionally introduced an internal standard to prevent systematic errors and random errors. All commercial samples were ground in a mortar into a homogeneous powder. Then, 50 mg of each sample were transferred to a plastic tube. In this tube, 2 mL of acetonitrile or acetone and 0.10 mL of the internal standard (thymol, 1 mg/ml) were added, followed by 1 min vortex shaking and 5 min of ultrasonication. The mixture was centrifuged at 1530 g for 3 min and the supernatant then transferred into a second tube. To the initial tube, 2 mL of acetonitrile or acetone were added, shaken, ultrasonicated, centrifuged and transferred to the second tube with both extracts combined. For each sample, extractions were performed in triplicate.
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Table 1 Information available in every package of concentrated extracts of Salvia divinorum. Samples
Seller
Labeled Potency
Batch
Expiration
Weight
Concentration
Additional information in packaging
A(i)
Smartshop A – Porto
5x
M
Feb-14
1g
38–40
A(ii)
Smartshop A – Porto
10x
NM
NM
1g
76–80
A(iii)
Smartshop A – Porto
40x
M
Feb-14
1g
304–320
A(iv)
Smartshop A – Porto
60x
M
Feb-14
1g
456–480
B(i)
Smartshop B – Porto
10x
M
Jun-13/Apr-13
1g
NM
B(ii)
Smartshop B – Porto Smartshop C – Aveiro Smartshop D – Espinho
40x
M
Jun-13
1g
NM
15x
NM
Aug-14
10x
M
Jun-13
1g
NM
‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Sensations similar to those obtained with marijuana’’; ‘‘Some people might experience slight headache, insomnia and bronchial irritation’’; ‘‘should be used in the presence of a sober person’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. Preparation: ‘‘Boil about 200 ml of water for a cup of tea and add the sage’’. How to use (‘‘smoke in pipe or bong, or prepare a tea’’); ‘‘product 100% natural’’. ‘‘Sensations similar to those obtained with marijuana’’; ‘‘Some people might experience slight headache, insomnia and bronchial irritation’’; ‘‘should be used in the presence of a sober person’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. Effects (‘‘hallucinations start in 10 min and last for 45 min’’); interactions (‘‘Must be avoided simultaneous administration with alcohol, other drugs and monoamine oxidase inhibitors’’. Preparation: ‘‘Boil about 200 ml of water for a cup of tea and add the sage’’.
C D
0.5 g
NM
F
Internet Website F
10x
NM
NM
1g
25
E
Internet Website E
20x
M
Jun-13
1g
NM
M = mentioned; NM = not mentioned. Information available in every package of concentrated extracts of Salvia divinorum.
2.3.2. Extraction with chloroform Samples (50 mg) were submitted to extraction with 4 mL of chloroform, as S. divinorum leaves supplemented with concentrated extracts. At this stage, 0.1 mL of the internal standard (thymol 1 mg/mL) was added. The mixture was vortexed for 1 min and ultrassonicated for 5 min. After extraction, leaves were separated from solvent by using a PTFE hydrophobic acrodisc syringe filter. 2.4. Equipment and quantitative analysis Gas-chromatography analysis was performed using a Varian CP-3800 gas chromatograph (USA) equipped with a Varian Saturn 4000 Ion Trap mass selective detector (USA) and Saturn GC/MS workstation software version 6.8. Stationary phase consisted of a capillary column VF-5 ms (30 m 0.25 mm 0.25 mm) from Varian (USA). Helium C-60 (Gasin, Portugal) at a constant flow rate of 1.0 mL/min, was used as mobile phase. Two microliters of the original 4 mL of each sample extract were injected using a split ratio of 1:40 at 250 8C. The column oven temperature was maintained at 100 8C for 1 min and then raised to 300 8C at 15 8C/min and held at 300 8C for 20 min totaling 34.3 min. Trap setpoint was at 180 8C, manifold setpoint was at 50 8C, and transfer line setpoint was at 280 8C. Electron energy was 70 eV. In order to avoid solvent overloading, ionization was maintained off during the first 4 min. Data were collected from m/z 40–1000. The emission current was 30 mA and the maximum ionization time was 25,000 ms. Mass spectra acquisition occurred between 4 and 34 min.
All determinations were performed in Full Scan Mode. For quantification purposes, full scan chromatograms were reconstructed through the selection of qualifier ions for each molecule. The selected ions used for the quantitative measurements were: m/ z 135 for thymol, m/z 94, m/z 273 and m/z 432 for salvinorin A; m/z 43, m/z 94, m/z 291, m/z 372 and m/z 390 for salvinorin B; m/z 94, m/z 313, m/z 372 and m/z 414 for salvinorin C; m/z 94, m/z 313, m/z 400 and m/z 432 for salvinorin D. The identification of salvinorin A in the samples was performed by comparison of retention times and mass spectra of the salvinorin A standard and salvinorin A in the samples, under the same chromatographic conditions. The remaining salvinorins B, C and D were identified comparing retention times and mass spectra of the peaks obtained upon the samples injection with previous published work and results obtained by Jermain and Evans [23] (Table 2).
3. Results and discussion 3.1. Extraction procedure The extraction efficiency of acetone and acetonitrile for salvinorin A was very similar. Acetonitrile was slightly more effective, since the peak areas had consistently higher values in the acetonitrile extracts. Nevertheless, acetone extracts gave more precise results with lower standard deviations. Chromatographic resolution was similar for both solvents. With regard to the
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Table 2 Retention time (min) and characteristic ions (m/z) of active ingredients detected directly by GC–MS. Compound
Basis for comparison
Retention time (min)
Characteristic ions (m/z)
Salvinorin Salvinorin Salvinorin Salvinorin
Standard solution Findings from Jermain and Evans [23] Findings from Jermain and Evans [23] Findings from Jermain and Evans [23]
18.00 17.00 18.75 18.95
m/z m/z m/z m/z
A B C D
94, 43, 94, 94,
m/z m/z m/z m/z
273 and m/z 432 94, m/z 291, m/z 372 and m/z 390 313, m/z 372 and m/z 414 313, m/z 400 and m/z 432
Retention time (min) and characteristic ions (m/z) of active ingredients detected directly by GC–MS.
chloroform extraction, the mean peak areas of salvinorin A was lower than the results obtained with acetonitrile and acetone, but the peak resolution was acceptable. Chloroform also had the disadvantage of not allowing the detection of salvinorin D. In addition, although most crime laboratories extract salvinorin A with chloroform [23], the separation between plant residue and chloroform is difficult, and a filtration step is necessary. The filtration process is fairly lengthy because of the large amount of plant material that eventually saturates the filter, requiring its constant renewal. The extraction of salvinorins B, C and D was accomplished either using acetone or acetonitrile. Acetone was more efficient for the extraction of salvinorin B, while the extraction of salvinorins C and D were similar for both solvents. The recovery was determined by comparing the peak areas for each solvent. 3.2. GC–MS analysis of salvinorin A, B, C and D The selected ions used for the detection of salvinorin A were: m/ z 94, m/z 273 and m/z 432. The retention time of the compound was around 18 min, matching the results obtained with the standard (Table 2). In order to detect salvinorin B, ions m/z 43, m/z 94, m/z 291, m/z 372 and m/z 390, were selected. The retention time of the compound was around 17 min. Both selection of ions and comparison with retention time, were legitimized by previous mass spectrum standard published by Jermain and Evans [23], since the standard solution for this compound was not available. The same study allowed the definition of ions to be selected and retention time to be considered, concerning salvinorins C and D. The selected ions used for the detection of salvinorin C were: m/z 94, m/z 313, m/z 372 and m/z 414, being the retention time of the compound 18.75 min. The selected ions used for the detection of salvinorin: m/z 94, m/z 313, m/z 400 and m/z 432 and the retention time was 18.95 min (Table 2) (Fig. 2).
3.3. Method validation The identification and quantification of salvinorin A in samples were accomplished by comparing the mass spectrum obtained with the standard solution. In order to quantify salvinorins B, C and D, the calibration curve obtained from the salvinorin A standard was conveniently adopted and results presented as semiquantitative. The concentrations of the standard salvinorin A used for the establishment of a calibration curve to quantify salvinorin A in samples were 5 mg/mL, 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 200 mg/mL, 500 mg/mL and 1000 mg/mL. Given the much lower values of the signals of the other three salvinorins, to quantify salvinorin B the calibration curve was constructed with concentrations based on salvinorin A concentrations between 5 mg/mL and 200 mg/mL, while for salvinorins C and D the interval of salvinorin A between 5 mg/mL and 100 mg/mL was used. The correlation coefficients obtained from the 3 calibration curves were above 0.99, being therefore acceptable according to the Forensic Toxicology Laboratory Guidelines from Society of Forensic Toxicologists and the American Academy of Forensic Sciences (SOFT/AAFS) [24] (Table 3). The obtained results in intra-day and inter-day analysis revealed a high precision of the adopted procedures of extraction and detection of salvinorins A, B and C, since the intra-day (n = 5) coefficient of variation was between 3.6% and 8.6%, and the inter-day coefficient ranged from 6.6% to 14.9% (n = 3). Although the intra-day coefficient of variation of salvinorin D revealed an acceptable value of 9.2%, inter-day coefficient (18.2%) exceeded the 15% limit admitted by the US Food and Drug Administration Guidance for Industry Bioanalytical Method Validation. Extremely low concentrations of salvinorin D turned out to be a barrier to the semi-quantitative determination of the compound, revealing an inter-day precision of 18.22%. The LOQ was defined as the lowest concentration of the calibration curve and it was estimated after 3 injections based on a signal-to-noise ratio of 10. A signal-to-noise ratio of 3 was
Fig. 2. Example of full scan chromatographic profile of GC–MS detection of salvinorins A–D from one sample of Salvia divinorum extract, with respective retention times.
F. Xavier Moreira et al. / Forensic Science International 242 (2014) 255–260 Table 3 Analytical data experimentally determined for salvinorin A standard (LOD, LOQ, linearity) and acquired samples (coefficient of variation of intra-day precision, coefficient of variation of inter-day precision). Analytical data Limit of detection Limit of quantification Linearitya
Intra-day precisionb
Inter-day precisionb
1.25 mg/mg 2.5 mg/mg Salvinorin A: Linear range: 5–1000 mg/mL r2 = 0.9951 Salvinorin B: Linear range: 5–200 mg/mL r2 = 0.9958 Salvinorin C: Linear range: 5–100 mg/mL r2 = 0.9962 Salvinorin A coefficient of variation: 3.63% Salvinorin B coefficient of variation: 8.60% Salvinorin C coefficient of variation: 6.23% Salvinorin A coefficient of variation: 6.64% Salvinorin B coefficient of variation: 14.88% Salvinorin C coefficient of variation: 13.82%
Analytical figures experimentally determined for salvinorin A standard (LOD, LOQ, linearity) and acquired samples (coefficient of variation of intra-day precision, coefficient of variation of inter-day precision). a Calculated from salvinorin A standard. b Using a sample containing a mixture of 4 commercialized products in similar amounts.
considered acceptable for estimating the LOD. Results are shown in Table 3. 3.4. Salvinorins concentrations in samples Although there were some exceptions, in most cases, the products that had higher labeled potencies, had higher concentrations of salvinorin A. Concentrations of salvinorin A ranged from 2.6 mg/g to 521.2 mg/g. The literature presently reports two studies concerning the identification and quantification of salvinorin A in concentrated extracts of S. divinorum. These works from Wolowich et al. [25] and Tsujikawa et al. [22] only contemplated samples with labeled potencies ranging from ‘‘1x’’ to ‘‘20x’’ and from ‘‘2x’’ to ‘‘25x’’, respectively. Our study was the first to analyze commercialized products with labeled potencies as high as ‘‘40x’’ and ‘‘60x’’, demonstrating a range of concentrations between 2.6 and 521.2 mg/g (Table 4). Although the labeled potencies of the analyzed products in the mentioned studies were both approximately in the same range, the results obtained after quantitative analysis were different. While Wolowich et al. [25]
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presented a range concentrations of salvinorin A between 0.126 and 0.951 mg/g, Tsujikawa et al. [22] presented a range concentrations between 4.1 and 38.9 mg/g. In order to compare the range of concentrations of salvinorin A found in the present study with previous similar studies, it was only taken into account the seven samples in which the labeled potency was between ‘‘5x’’ and ‘‘20x’’ (Table 4). The obtained concentrations range in the present study is similar to the one presented by Tsujikawa et al. [22]: 2.6 mg/g and 44.5 mg/g, thus, quite different from the study of Wolowich et al. [25]. Most of the purchased samples analyzed in the present study did not provide information about the concentration of salvinorin A, simply referring the ‘‘potency’’ of the products. The Website of the sample ‘‘E’’ was the only one that objectively indicated salvinorin A content. According to the company’s website, the sample with a ‘‘10x’’ labeled potency had 25 mg of salvinorin A. After the analysis of the sample, it was found a concentration of 34 mg of salvinorin A. Therefore, in this case, the seller sustained a concentration 26% lower than its real concentration. The other seller – indicating the contents in salvinorin A – was the smartshop ‘‘A’’. However, in this case, the concentration of salvinorin A was not objectively mentioned. Instead, the company ensures that 1 g of ‘‘S. divinorum 5x’’ corresponds to 5 g of S. divinorum leaves; 1 g of ‘‘S. divinorum 10x’’ corresponds to 10 g of S. divinorum leaves; 1 g of ‘‘S. divinorum 40x’’ corresponds to 40 g of S. divinorum leaves; and 1 g of ‘‘S. divinorum 60x’’ corresponds to 60 g of S. divinorum leaves. In accordance with Medana et al. [26] and Kennedy and Wiseman [27], the amount of salvinorin A present in leaves of S. divinorum ranges from 0.76% to 0.80% (w/w). In order to clarify the reliability of information provided by smartshop ‘‘A’’, samples were submitted to comparison between the experimentally determined concentrations of salvinorin A and the concentrations advertised by the seller. Three of four samples had a concentration much lower than the expected (Table 4). Only the highest labeled potency sample (‘‘60x’’) gave after analysis a concentration approximate to the expected one (8–12% above the expected). Besides the lack of information about salvinorin A concentration, several samples revealed insufficient information about other items such as batch number (not mentioned in 3 samples) and expiration date (not mentioned in 2 samples, and with two different expiration dates in one sample). Some samples provided scarce information about how to use the drug (only stating that it must be smoked in a pipe or bong, and sometimes explaining that it must be used to produce an infusion). Surprisingly, the information most often exhibited in the packages were the
Table 4 Most relevant results on analyzed samples of Salvia divinorum commercialized extracts. Sample
A(i) A(ii) A(iii) A(iv) B(i) B(ii) C D E F
Concentration of SalA publicized (mg/g)
38–40 76–80 304–320 456–480 NM NM NM NM 25 NM
Concentration (mg/g)
Amount estimated to reach hallucinogenic effects (mg)
salA
salB
salC
18.2 2.64 155.6 521.2 35.8 44.6 20.9 3.5 33.9 44.5
nq nq 25 117.9 5.9 6.4 3.1 nq 6.8 9
nq nq 2.9 11.9 nq nq nq ni nq nq
11.0 75.8 1.3 0.4 5.6 4.5 9.6 57.7 5.9 4.5
NM = not mentioned; nq = not quantified (lower than LOQ). Most relevant results on the analyzed samples of Salvia divinorum commercialized extracts. Legend: M = mentioned; NM = not mentioned; NE = not expired; DM = double mentioned (with two different data); nq = not quantified (lower than LOQ); ni = not identified (lower than LOD).
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expressions ‘‘Do not cause dependence’’ and ‘‘Not dangerous’’ (both mentioned in 6 samples) (Table 1). The estimated concentrations of salvinorin B and salvinorin C, ranged between 3.08 and 117.86 mg/g and 2.91–11.87 mg/g, respectively, being the salvinorin B the most prevalent compound in S. divinorum, besides salvinorin A. As earlier mentioned, the estimated quantification of salvinorin D was not possible, due to residual amounts of the compound present in the analyzed S. divinorum samples. 3.5. Amount of S. divinorum concentrated extracts expected to induce psychoactivity Given the concentration of salvinorin A in each sample, and admitting that 200 mg of salvinorin A are enough to induce hallucinogenic effects [6], the amount of the concentrated extract needed to obtain the pretended effects was estimated (Table 4). Results revealed, in all analyzed samples, that the consumption of a very small amount of the extract is enough to produce hallucinogenic effects. Since most of the consumers do not have this knowledge, when they consume S. divinorum extracts with the highest labeled potencies (‘‘60x’’, for example), they are consuming excessive amounts of the drug, which may elicit severe hallucinogenic effects. In fact, exposed individuals describe an intensely but short-lived experience, appearing in less than 1 min and lasting 15 min or less, psychedelic-like changes in visual perception, mood and somatic sensations. Depending on the dose and the route of administration, the effects are sometimes extremely negative and potentially leading to risk behavior (overly-intense experiences; fear, terror, and panic; depersonalization and possible difficulty integrating experiences) [28]. Although there have been only few documented negative occurrences associated with S. divinorum use and the eventual long-term dependence seems to be unlikely, the coordination loss, failure on speech, interpersonal impairments and loss of self-care should be valued as an increased risk to users and those around them (risks of injury, interpersonal conflict and property damage) [29]. The analysis of S. divinorum samples allowed the identification of the four salvinorins described in this plant, being salvinorin A the most prevalent hallucinogen in all the analyzed samples. In the tested samples, there were several unreliable data provided to consumers. Frequently, there was no information on salvinorin A concentration, but when it existed, generally did not correspond to the true amount present in the products. On the other hand, the concentration of salvinorin A in each sample, far exceeded the amount required to produce hallucinogenic effects. Conflict of interest The author(s) confirm that this article content has no conflicts of interest. Acknowledgment This work received financial support from ‘‘Fundac¸a˜o para a Cieˆncia e Tecnologia’’ (FCT) through grant no. PEst-C/EQB/LA0006/ 2013.
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Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Analytical investigation of legal high products containing Salvia divinorum traded in smartshops and internet Fernando Xavier Moreira *, Fe´lix Carvalho, Maria de Lourdes Bastos, Paula Guedes de Pinho * REQUIMTE, Laborato´rio de Toxicologia, Departamento de Cieˆncias Biolo´gicas, Faculdade de Farma´cia, Universidade do Porto, Porto, Portugal
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 April 2014 Received in revised form 9 July 2014 Accepted 11 July 2014 Available online 19 July 2014
Lately, the hallucinogenic plant Salvia divinorum has been considered a popular recreational product among adolescents, being legally sold in several countries in ‘‘smartshops’’ and in internet websites. Sellers frequently omit the safety information about the plant, encouraging its use for recreational purposes, without providing adequate qualitative and quantitative details. The principal hallucinogenic compound of the plant, salvinorin A, alongside with three other isomeric compounds, salvinorin B, C and D were evaluated in 10 products containing S. divinorum. These products were obtained in smartshops and from internet websites, and contained concentrated extracts of salvinorin A, with potencies labeled between ‘‘5x’’ and ‘‘60x’’. For that purpose a simple and rapid extraction protocol and a GC–MS methodology were developed and applied to the purchased samples. The analysis of S. divinorum samples allowed the identification of four salvinorins, salvinorin A being the most prevalent hallucinogen. In the tested samples, there were several unreliable data provided to consumers. Frequently, there was no information on salvinorin A concentration, but when it existed, it generally did not correspond to the true amount present in products. On the other hand, the concentration of salvinorin A in each product far exceeded the amount needed to produce hallucinogenic effects. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Salvinorin A Legal drugs Smartshops Salvia divinorum
1. Introduction Salvia divinorum is a psychoactive plant from the Labiatae family, originating from Oaxaca, Mexico, with recognized psychoactive properties [1,2]. It is one of the most important hallucinogenic plants of the Mesoamerican cultures, frequently used by earlier civilizations in religious ceremonies, allegedly enabling the contact between physical and spiritual worlds. This approach to the spiritual reality was believed to reveal disease diagnosis, to ensure good harvests and to predict the rains to come [1–3]. Mazatec Indians from Oaxaca, Mexico, commonly chewed the leaves or drank the infusion from the plant S. divinorum. Lately, the use of S. divinorum has been reemerging in recreational settings [3,4]. Although leaves of S. divinorum can be chewed or used to prepare oral tinctures, inhalation of the vaporized smoke of
* Corresponding authors at: REQUIMTE, Faculty of Pharmacy, Department of Biological Sciences, Laboratory of Toxicology, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Tel.: +351 220428599; fax: +351 226093390. E-mail addresses: [email protected] (F. Xavier Moreira), [email protected], [email protected] (P. Guedes de Pinho). http://dx.doi.org/10.1016/j.forsciint.2014.07.009 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
salvinorin A was proven to be the most efficient method for achieving its psychoactive effects in humans [2,5]. When consumed orally, S. divinorum has less expressive effects, probably due to reduced sublingual absorption of salvinorin A [6,7]. The inhalation of small amounts (200 mg) of salvinorin A (S. divinorum main compound), after heating in a piece of aluminum foil, proved to be enough to elicit strong psychotropic effects. Through this exposure route, the effects take only 30 s to be expressed [6]. Thus, the intensity and speed of the effects highlight the difference between the effects on indigenous populations (that normally chew the plant) and newer users in western countries (that normally inhale the vapors). The use of S. divinorum has spread through Europe and North America in the last few years, being purchased from internet websites and at smartshops [5]. The percentage of young adults in western countries, such as the United States of America and Canada that admitted to have used S. divinorum in their lifetime is about 5% [8,9]. Young wealthy males, that have already tried other drugs seem to be more prone to start using S. divinorum [10]. Although, no serious adverse effects (death, hospital stay or emergency room visit) have been attributed to S. divinorum
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consumption [11], a 21 year old person evidenced persistent psychosis and paranoia after smoking the drug [12]. The hypothesis of re-experiencing hallucinations and/or other neuropsychiatric consequences such as de´ja` vu experiences cannot also be ruled out [13]. Several websites selling S. divinorum products biase the safety information about these products, encouraging the use of S. divinorum for recreational purposes, without providing adequate qualitative and quantitative details on the product [14]. For example, more than 90% of websites from the United Kingdom, where the most prevalent commercialized legal drug is S. divinorum, have no information about the drug’s side effects [15]. Additionally, some sources wrongly associate analgesic effects and euphoria with S. divinorum consumption [14]. Salvinorin A is considered the most important active compound present in S. divinorum [16]. Although this plant has other identified chemical compounds, salvinorin A is present in much higher concentrations than the remaining salvinorins, which may be considered insignificant from the pharmacological point of view [17]. Salvinorin A was isolated for the first time in 1982 [18] and its psychoactivity was established in 1994 [16]. The relationship between salvinorin A and the kappa opioid receptors was established in 2002 [19], still being considered salvinorin A as the most potent naturally occurring hallucinogen, being chemically and structurally unique: it is the first known psychoactive diterpene and the first non-nitrogenous hallucinogen [20] (Fig. 1). Considering the inappropriate information given by sellers of S. divinorum products, the specific objectives of this work are to validate a protocol of extraction and quantification of salvinorin A and the other related molecules from concentrated extracts of S. divinorum using GC–MS. The developed methodology was further applied to quantify salvinorin A and estimate the amounts of salvinorins B, C and D; confronting the obtained results with previous similar researches on S. divinorum commercialized products and evaluating the reliability and quality of the information that is provided to consumers in the labeled packages purchased.
the purchased packages) according to the potency/amount of extracts used. Packages usually contained 1 g of concentrated extract of S. divinorum. Many of the packages did not explain what the classification (hereafter referred to as ‘‘labeled potency’’) meant. Only samples acquired from website E guaranteed that, for example the ‘‘5x’’ extract, had 12.5 mg of salvinorin A, while the ‘‘10x’’ extract contained 25 mg of salvinorin A. Packages from smartshop A had printed on the label that 1 g of the ‘‘5x’’ extract equals 5 g of S. divinorum leaves; 1 g of the ‘‘10x’’ extract equals 10 g of S. divinorum leaves, and so on. The remaining information (expiration date, batch, contraindications, side effects) provided to customers was generally scarce (Table 1). After the purchase, all the samples were kept in the laboratory in a humidity controlled environment, at room temperature (similar to the environment observed in ‘‘smartshops’’) until analysis. The standard of salvinorin A was purchased from Sigma– Aldrich Chemie Gmbh (Munich, Germany), and was used for the identification and quantification of salvinorin A in the samples. A semi-quantitative determination of other salvinorins (B, C and D), was performed by using the calibration curve of the available standard. The standard of salvinorin A was kept at 20 8C, in a humidity controlled environment, and protected from light. Acetonitrile and acetone were purchased from Fisher Chemical1, 99.99% and 99.98% of purity, respectively, according to GC assays. Chloroform was obtained from Fisher Scientific1 and was 99.99% pure, also determined by GC assay. Thymol, used as the internal standard, was purchased from Sigma Aldrich1, with a purity of 99.5%. In order to separate leaves from solvent, polytetrafluoroethylene (PTFE) hydrophobic acrodisc syringe filters were used.
2. Methods
2.3. Extraction procedure
2.1. Samples and chemicals
To determine the most effective solvent to extract salvinorin A from commercial S. divinorum leaves fortified with concentrated extracts, samples were ground until a homogeneous powder was obtained. Three solvents with different polarities were tested: acetonitrile, acetone, and chloroform. The main objective was to extract the greatest amount of salvinorin A, but it was also important to provide the extraction of salvinorins B, C, D. All the extractions were tested in triplicate, on a sample containing a mixture of 4 commercialized products in similar amounts.
Ten S. divinorum samples were purchased in Portuguese smartshops, from September of 2012 until April 2013, prior to the approval of the Decree 54/2013 [21], prohibiting the marketing of these products. Samples were purchased in four different ‘‘smartshops’’ (A–D), and from two websites (E, F). The samples consisted of S. divinorum crushed leaves fortified/soaked, by producers, with salvia concentrated extracts. They were classified as ‘‘5x’’, ‘‘10x’’, ‘‘15x’’, ‘‘20x’’, ‘‘40x’’ or ‘‘60x’’ (designation labeled on
Fig. 1. Chemical structures of salvinorins A–D.
2.2. Standard solutions Standard solutions of salvinorin A were prepared by sequential dilutions of the stock solution of salvinorin A (1 mg/ml) in acetonitrile. Concentrations ranging from 5 mg/ml to 1000 mg/ml were obtained and stored at 20 8C.
2.3.1. Extraction with acetonitrile and acetone In order to perform the extraction of salvinorins from purchased concentrated extracts with acetonitrile and acetone, samples were submitted to a methodology already performed by Tsujikawa et al. [22], being additionally introduced an internal standard to prevent systematic errors and random errors. All commercial samples were ground in a mortar into a homogeneous powder. Then, 50 mg of each sample were transferred to a plastic tube. In this tube, 2 mL of acetonitrile or acetone and 0.10 mL of the internal standard (thymol, 1 mg/ml) were added, followed by 1 min vortex shaking and 5 min of ultrasonication. The mixture was centrifuged at 1530 g for 3 min and the supernatant then transferred into a second tube. To the initial tube, 2 mL of acetonitrile or acetone were added, shaken, ultrasonicated, centrifuged and transferred to the second tube with both extracts combined. For each sample, extractions were performed in triplicate.
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Table 1 Information available in every package of concentrated extracts of Salvia divinorum. Samples
Seller
Labeled Potency
Batch
Expiration
Weight
Concentration
Additional information in packaging
A(i)
Smartshop A – Porto
5x
M
Feb-14
1g
38–40
A(ii)
Smartshop A – Porto
10x
NM
NM
1g
76–80
A(iii)
Smartshop A – Porto
40x
M
Feb-14
1g
304–320
A(iv)
Smartshop A – Porto
60x
M
Feb-14
1g
456–480
B(i)
Smartshop B – Porto
10x
M
Jun-13/Apr-13
1g
NM
B(ii)
Smartshop B – Porto Smartshop C – Aveiro Smartshop D – Espinho
40x
M
Jun-13
1g
NM
15x
NM
Aug-14
10x
M
Jun-13
1g
NM
‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Prohibited to be purchased by minors’’; ‘‘causes hallucinations, mindbody experience, travel to the past, becoming an object, presence in several places at once, and uncontrollable laughter’’; ‘‘do not smoke extracts stronger than 5x for the first time’’; ‘‘smoke in a bong or pipe’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. ‘‘Sensations similar to those obtained with marijuana’’; ‘‘Some people might experience slight headache, insomnia and bronchial irritation’’; ‘‘should be used in the presence of a sober person’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. Preparation: ‘‘Boil about 200 ml of water for a cup of tea and add the sage’’. How to use (‘‘smoke in pipe or bong, or prepare a tea’’); ‘‘product 100% natural’’. ‘‘Sensations similar to those obtained with marijuana’’; ‘‘Some people might experience slight headache, insomnia and bronchial irritation’’; ‘‘should be used in the presence of a sober person’’; ‘‘does not cause dependence’’; ‘‘not dangerous’’. Effects (‘‘hallucinations start in 10 min and last for 45 min’’); interactions (‘‘Must be avoided simultaneous administration with alcohol, other drugs and monoamine oxidase inhibitors’’. Preparation: ‘‘Boil about 200 ml of water for a cup of tea and add the sage’’.
C D
0.5 g
NM
F
Internet Website F
10x
NM
NM
1g
25
E
Internet Website E
20x
M
Jun-13
1g
NM
M = mentioned; NM = not mentioned. Information available in every package of concentrated extracts of Salvia divinorum.
2.3.2. Extraction with chloroform Samples (50 mg) were submitted to extraction with 4 mL of chloroform, as S. divinorum leaves supplemented with concentrated extracts. At this stage, 0.1 mL of the internal standard (thymol 1 mg/mL) was added. The mixture was vortexed for 1 min and ultrassonicated for 5 min. After extraction, leaves were separated from solvent by using a PTFE hydrophobic acrodisc syringe filter. 2.4. Equipment and quantitative analysis Gas-chromatography analysis was performed using a Varian CP-3800 gas chromatograph (USA) equipped with a Varian Saturn 4000 Ion Trap mass selective detector (USA) and Saturn GC/MS workstation software version 6.8. Stationary phase consisted of a capillary column VF-5 ms (30 m 0.25 mm 0.25 mm) from Varian (USA). Helium C-60 (Gasin, Portugal) at a constant flow rate of 1.0 mL/min, was used as mobile phase. Two microliters of the original 4 mL of each sample extract were injected using a split ratio of 1:40 at 250 8C. The column oven temperature was maintained at 100 8C for 1 min and then raised to 300 8C at 15 8C/min and held at 300 8C for 20 min totaling 34.3 min. Trap setpoint was at 180 8C, manifold setpoint was at 50 8C, and transfer line setpoint was at 280 8C. Electron energy was 70 eV. In order to avoid solvent overloading, ionization was maintained off during the first 4 min. Data were collected from m/z 40–1000. The emission current was 30 mA and the maximum ionization time was 25,000 ms. Mass spectra acquisition occurred between 4 and 34 min.
All determinations were performed in Full Scan Mode. For quantification purposes, full scan chromatograms were reconstructed through the selection of qualifier ions for each molecule. The selected ions used for the quantitative measurements were: m/ z 135 for thymol, m/z 94, m/z 273 and m/z 432 for salvinorin A; m/z 43, m/z 94, m/z 291, m/z 372 and m/z 390 for salvinorin B; m/z 94, m/z 313, m/z 372 and m/z 414 for salvinorin C; m/z 94, m/z 313, m/z 400 and m/z 432 for salvinorin D. The identification of salvinorin A in the samples was performed by comparison of retention times and mass spectra of the salvinorin A standard and salvinorin A in the samples, under the same chromatographic conditions. The remaining salvinorins B, C and D were identified comparing retention times and mass spectra of the peaks obtained upon the samples injection with previous published work and results obtained by Jermain and Evans [23] (Table 2).
3. Results and discussion 3.1. Extraction procedure The extraction efficiency of acetone and acetonitrile for salvinorin A was very similar. Acetonitrile was slightly more effective, since the peak areas had consistently higher values in the acetonitrile extracts. Nevertheless, acetone extracts gave more precise results with lower standard deviations. Chromatographic resolution was similar for both solvents. With regard to the
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Table 2 Retention time (min) and characteristic ions (m/z) of active ingredients detected directly by GC–MS. Compound
Basis for comparison
Retention time (min)
Characteristic ions (m/z)
Salvinorin Salvinorin Salvinorin Salvinorin
Standard solution Findings from Jermain and Evans [23] Findings from Jermain and Evans [23] Findings from Jermain and Evans [23]
18.00 17.00 18.75 18.95
m/z m/z m/z m/z
A B C D
94, 43, 94, 94,
m/z m/z m/z m/z
273 and m/z 432 94, m/z 291, m/z 372 and m/z 390 313, m/z 372 and m/z 414 313, m/z 400 and m/z 432
Retention time (min) and characteristic ions (m/z) of active ingredients detected directly by GC–MS.
chloroform extraction, the mean peak areas of salvinorin A was lower than the results obtained with acetonitrile and acetone, but the peak resolution was acceptable. Chloroform also had the disadvantage of not allowing the detection of salvinorin D. In addition, although most crime laboratories extract salvinorin A with chloroform [23], the separation between plant residue and chloroform is difficult, and a filtration step is necessary. The filtration process is fairly lengthy because of the large amount of plant material that eventually saturates the filter, requiring its constant renewal. The extraction of salvinorins B, C and D was accomplished either using acetone or acetonitrile. Acetone was more efficient for the extraction of salvinorin B, while the extraction of salvinorins C and D were similar for both solvents. The recovery was determined by comparing the peak areas for each solvent. 3.2. GC–MS analysis of salvinorin A, B, C and D The selected ions used for the detection of salvinorin A were: m/ z 94, m/z 273 and m/z 432. The retention time of the compound was around 18 min, matching the results obtained with the standard (Table 2). In order to detect salvinorin B, ions m/z 43, m/z 94, m/z 291, m/z 372 and m/z 390, were selected. The retention time of the compound was around 17 min. Both selection of ions and comparison with retention time, were legitimized by previous mass spectrum standard published by Jermain and Evans [23], since the standard solution for this compound was not available. The same study allowed the definition of ions to be selected and retention time to be considered, concerning salvinorins C and D. The selected ions used for the detection of salvinorin C were: m/z 94, m/z 313, m/z 372 and m/z 414, being the retention time of the compound 18.75 min. The selected ions used for the detection of salvinorin: m/z 94, m/z 313, m/z 400 and m/z 432 and the retention time was 18.95 min (Table 2) (Fig. 2).
3.3. Method validation The identification and quantification of salvinorin A in samples were accomplished by comparing the mass spectrum obtained with the standard solution. In order to quantify salvinorins B, C and D, the calibration curve obtained from the salvinorin A standard was conveniently adopted and results presented as semiquantitative. The concentrations of the standard salvinorin A used for the establishment of a calibration curve to quantify salvinorin A in samples were 5 mg/mL, 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 200 mg/mL, 500 mg/mL and 1000 mg/mL. Given the much lower values of the signals of the other three salvinorins, to quantify salvinorin B the calibration curve was constructed with concentrations based on salvinorin A concentrations between 5 mg/mL and 200 mg/mL, while for salvinorins C and D the interval of salvinorin A between 5 mg/mL and 100 mg/mL was used. The correlation coefficients obtained from the 3 calibration curves were above 0.99, being therefore acceptable according to the Forensic Toxicology Laboratory Guidelines from Society of Forensic Toxicologists and the American Academy of Forensic Sciences (SOFT/AAFS) [24] (Table 3). The obtained results in intra-day and inter-day analysis revealed a high precision of the adopted procedures of extraction and detection of salvinorins A, B and C, since the intra-day (n = 5) coefficient of variation was between 3.6% and 8.6%, and the inter-day coefficient ranged from 6.6% to 14.9% (n = 3). Although the intra-day coefficient of variation of salvinorin D revealed an acceptable value of 9.2%, inter-day coefficient (18.2%) exceeded the 15% limit admitted by the US Food and Drug Administration Guidance for Industry Bioanalytical Method Validation. Extremely low concentrations of salvinorin D turned out to be a barrier to the semi-quantitative determination of the compound, revealing an inter-day precision of 18.22%. The LOQ was defined as the lowest concentration of the calibration curve and it was estimated after 3 injections based on a signal-to-noise ratio of 10. A signal-to-noise ratio of 3 was
Fig. 2. Example of full scan chromatographic profile of GC–MS detection of salvinorins A–D from one sample of Salvia divinorum extract, with respective retention times.
F. Xavier Moreira et al. / Forensic Science International 242 (2014) 255–260 Table 3 Analytical data experimentally determined for salvinorin A standard (LOD, LOQ, linearity) and acquired samples (coefficient of variation of intra-day precision, coefficient of variation of inter-day precision). Analytical data Limit of detection Limit of quantification Linearitya
Intra-day precisionb
Inter-day precisionb
1.25 mg/mg 2.5 mg/mg Salvinorin A: Linear range: 5–1000 mg/mL r2 = 0.9951 Salvinorin B: Linear range: 5–200 mg/mL r2 = 0.9958 Salvinorin C: Linear range: 5–100 mg/mL r2 = 0.9962 Salvinorin A coefficient of variation: 3.63% Salvinorin B coefficient of variation: 8.60% Salvinorin C coefficient of variation: 6.23% Salvinorin A coefficient of variation: 6.64% Salvinorin B coefficient of variation: 14.88% Salvinorin C coefficient of variation: 13.82%
Analytical figures experimentally determined for salvinorin A standard (LOD, LOQ, linearity) and acquired samples (coefficient of variation of intra-day precision, coefficient of variation of inter-day precision). a Calculated from salvinorin A standard. b Using a sample containing a mixture of 4 commercialized products in similar amounts.
considered acceptable for estimating the LOD. Results are shown in Table 3. 3.4. Salvinorins concentrations in samples Although there were some exceptions, in most cases, the products that had higher labeled potencies, had higher concentrations of salvinorin A. Concentrations of salvinorin A ranged from 2.6 mg/g to 521.2 mg/g. The literature presently reports two studies concerning the identification and quantification of salvinorin A in concentrated extracts of S. divinorum. These works from Wolowich et al. [25] and Tsujikawa et al. [22] only contemplated samples with labeled potencies ranging from ‘‘1x’’ to ‘‘20x’’ and from ‘‘2x’’ to ‘‘25x’’, respectively. Our study was the first to analyze commercialized products with labeled potencies as high as ‘‘40x’’ and ‘‘60x’’, demonstrating a range of concentrations between 2.6 and 521.2 mg/g (Table 4). Although the labeled potencies of the analyzed products in the mentioned studies were both approximately in the same range, the results obtained after quantitative analysis were different. While Wolowich et al. [25]
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presented a range concentrations of salvinorin A between 0.126 and 0.951 mg/g, Tsujikawa et al. [22] presented a range concentrations between 4.1 and 38.9 mg/g. In order to compare the range of concentrations of salvinorin A found in the present study with previous similar studies, it was only taken into account the seven samples in which the labeled potency was between ‘‘5x’’ and ‘‘20x’’ (Table 4). The obtained concentrations range in the present study is similar to the one presented by Tsujikawa et al. [22]: 2.6 mg/g and 44.5 mg/g, thus, quite different from the study of Wolowich et al. [25]. Most of the purchased samples analyzed in the present study did not provide information about the concentration of salvinorin A, simply referring the ‘‘potency’’ of the products. The Website of the sample ‘‘E’’ was the only one that objectively indicated salvinorin A content. According to the company’s website, the sample with a ‘‘10x’’ labeled potency had 25 mg of salvinorin A. After the analysis of the sample, it was found a concentration of 34 mg of salvinorin A. Therefore, in this case, the seller sustained a concentration 26% lower than its real concentration. The other seller – indicating the contents in salvinorin A – was the smartshop ‘‘A’’. However, in this case, the concentration of salvinorin A was not objectively mentioned. Instead, the company ensures that 1 g of ‘‘S. divinorum 5x’’ corresponds to 5 g of S. divinorum leaves; 1 g of ‘‘S. divinorum 10x’’ corresponds to 10 g of S. divinorum leaves; 1 g of ‘‘S. divinorum 40x’’ corresponds to 40 g of S. divinorum leaves; and 1 g of ‘‘S. divinorum 60x’’ corresponds to 60 g of S. divinorum leaves. In accordance with Medana et al. [26] and Kennedy and Wiseman [27], the amount of salvinorin A present in leaves of S. divinorum ranges from 0.76% to 0.80% (w/w). In order to clarify the reliability of information provided by smartshop ‘‘A’’, samples were submitted to comparison between the experimentally determined concentrations of salvinorin A and the concentrations advertised by the seller. Three of four samples had a concentration much lower than the expected (Table 4). Only the highest labeled potency sample (‘‘60x’’) gave after analysis a concentration approximate to the expected one (8–12% above the expected). Besides the lack of information about salvinorin A concentration, several samples revealed insufficient information about other items such as batch number (not mentioned in 3 samples) and expiration date (not mentioned in 2 samples, and with two different expiration dates in one sample). Some samples provided scarce information about how to use the drug (only stating that it must be smoked in a pipe or bong, and sometimes explaining that it must be used to produce an infusion). Surprisingly, the information most often exhibited in the packages were the
Table 4 Most relevant results on analyzed samples of Salvia divinorum commercialized extracts. Sample
A(i) A(ii) A(iii) A(iv) B(i) B(ii) C D E F
Concentration of SalA publicized (mg/g)
38–40 76–80 304–320 456–480 NM NM NM NM 25 NM
Concentration (mg/g)
Amount estimated to reach hallucinogenic effects (mg)
salA
salB
salC
18.2 2.64 155.6 521.2 35.8 44.6 20.9 3.5 33.9 44.5
nq nq 25 117.9 5.9 6.4 3.1 nq 6.8 9
nq nq 2.9 11.9 nq nq nq ni nq nq
11.0 75.8 1.3 0.4 5.6 4.5 9.6 57.7 5.9 4.5
NM = not mentioned; nq = not quantified (lower than LOQ). Most relevant results on the analyzed samples of Salvia divinorum commercialized extracts. Legend: M = mentioned; NM = not mentioned; NE = not expired; DM = double mentioned (with two different data); nq = not quantified (lower than LOQ); ni = not identified (lower than LOD).
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expressions ‘‘Do not cause dependence’’ and ‘‘Not dangerous’’ (both mentioned in 6 samples) (Table 1). The estimated concentrations of salvinorin B and salvinorin C, ranged between 3.08 and 117.86 mg/g and 2.91–11.87 mg/g, respectively, being the salvinorin B the most prevalent compound in S. divinorum, besides salvinorin A. As earlier mentioned, the estimated quantification of salvinorin D was not possible, due to residual amounts of the compound present in the analyzed S. divinorum samples. 3.5. Amount of S. divinorum concentrated extracts expected to induce psychoactivity Given the concentration of salvinorin A in each sample, and admitting that 200 mg of salvinorin A are enough to induce hallucinogenic effects [6], the amount of the concentrated extract needed to obtain the pretended effects was estimated (Table 4). Results revealed, in all analyzed samples, that the consumption of a very small amount of the extract is enough to produce hallucinogenic effects. Since most of the consumers do not have this knowledge, when they consume S. divinorum extracts with the highest labeled potencies (‘‘60x’’, for example), they are consuming excessive amounts of the drug, which may elicit severe hallucinogenic effects. In fact, exposed individuals describe an intensely but short-lived experience, appearing in less than 1 min and lasting 15 min or less, psychedelic-like changes in visual perception, mood and somatic sensations. Depending on the dose and the route of administration, the effects are sometimes extremely negative and potentially leading to risk behavior (overly-intense experiences; fear, terror, and panic; depersonalization and possible difficulty integrating experiences) [28]. Although there have been only few documented negative occurrences associated with S. divinorum use and the eventual long-term dependence seems to be unlikely, the coordination loss, failure on speech, interpersonal impairments and loss of self-care should be valued as an increased risk to users and those around them (risks of injury, interpersonal conflict and property damage) [29]. The analysis of S. divinorum samples allowed the identification of the four salvinorins described in this plant, being salvinorin A the most prevalent hallucinogen in all the analyzed samples. In the tested samples, there were several unreliable data provided to consumers. Frequently, there was no information on salvinorin A concentration, but when it existed, generally did not correspond to the true amount present in the products. On the other hand, the concentration of salvinorin A in each sample, far exceeded the amount required to produce hallucinogenic effects. Conflict of interest The author(s) confirm that this article content has no conflicts of interest. Acknowledgment This work received financial support from ‘‘Fundac¸a˜o para a Cieˆncia e Tecnologia’’ (FCT) through grant no. PEst-C/EQB/LA0006/ 2013.
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