Science and Justice 53 (2013) 409–414 Contents lists available at ScienceDirect Science and Justice journal homepage: www.elsevier.com/locate/scijus Technical note Rapid determination of scopolamine in evidence of recreational and predatory use Jorge Sáiz a,b, Thanh Duc Mai c,d, María López López a,b, Carmen Bartolomé e, Peter C Hauser c, Carmen García-Ruiz a,b,⁎ a Department of Chemistry I, Multipurpose Building of Chemistry, University of Alcalá, Ctra Madrid–Barcelona km 33.600, 28871 Alcalá de Henares, Madrid, Spain University Institute of Research in Police Sciences (IUICP), University of Alcalá, Ctra Madrid–Barcelona km 33.600, 28871 Alcalá de Henares, Madrid, Spain University of Basel, Department of Chemistry, Spitalstrasse 51, 4056 Basel, Switzerland d Centre for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Nguyen Trai Street 334, Hanoi, Viet Nam e Department of Life Sciences, University of Alcalá, Ctra Madrid–Barcelona km 33.600, 28871 Alcalá de Henares, Madrid, Spain b c a r t i c l e i n f o Article history: Received March 2013 Received in revised form 31 July 2013 Accepted August 2013 Keywords: Scopolamine Burundanga Predatory drugs Portable capillary electrophoresis Contactless conductivity detection a b s t r a c t In recent years, scopolamine has become a drug of common use for recreational and predatory purposes and several ways of administration have been devised A method for the rapid analysis of suspicious samples was developed, using a portable capillary electrophoresis with contactless conductivity detection The method allows the separation of scopolamine from atropine which has a similar structure and is present along with scopolamine in some samples The method was demonstrated to be useful for the fast analysis of several types of evidential items which have recently been reported to have been abused with fatal consequences or employed for criminal purposes An infusion of Datura stramonium L., in which scopolamine and atropine naturally coexist, was analyzed for being frequently consumed for recreational purposes A spiked moisturizing cream and six spiked alcoholic beverages were also analyzed In spite of the complexity of the specimens, the sample pre-treatment methods developed were simple and fast © 2013 Published by Elsevier Ireland Ltd on behalf of Forensic Science Society Introduction Scopolamine is a tropane alkaloid extensively used for clinical purposes because of its strong parasympatolytic, anticholinergic and anti-emetic actions [1,2] This alkaloid has an inhibitory effect on acetylcholine muscarinic receptors, having an influence on neurotransmission pathways concerning memory It can cause box lock amnesia by affecting the basal nucleus of Meynert, which is an important structure for amnesic functions, especially the retention of memory [3] Moreover, scopolamine can block free will For this reason scopolamine is used in drug-facilitated robberies and drug-facilitated sexual assaults Scopolamine is also known as “burundanga” within the circles of people who abuse it Scopolamine is tasteless and odorless and can be easily absorbed in the digestive tract; it can be delivered orally, dermally, or via inhalation [3] For these reasons, several ways of administration have been devised by attackers For example, it has been reported that scopolamine powder can be blown onto the face of the victim, who will be under the drug's effects within minutes Recently, in Madrid ⁎ Corresponding author at: Department of Chemistry I, Multipurpose Building of Chemistry, University of Alcalá, Ctra Madrid–Barcelona km 33.600, 28871 Alcalá de Henares, Madrid, Spain Tel.: +34 91 8856431 E-mail address: carmen.gruiz@uah.es (C García-Ruiz) URL: http://www.inquifor.com (C García-Ruiz) (Spain), one person was arrested after a complaint from a woman who had been drugged with scopolamine The suspect, who pretended to be a shaman, took the woman to his house and, after drugging her with a drink to which scopolamine had been added, he sexually assaulted her several times [4] After this case, 38 more women reported having been raped by the same person [5] Moreover, scopolamine has allegedly been added to moisturizing creams, which are applied by the ingenuous victims themselves causing them to be under the influence of the drug Scopolamine has also been added to drinks in night clubs to commit robberies or sexual assaults Approximately one in eight emergency room admissions by poisoning in Bogotá (Colombia) has been attributed to scopolamine [3] However, in spite of its characteristics, no mention of scopolamine can be found among information about predatory drugs, for which typical examples are ecstasy, ketamine, Rohypnol, gamma-hydroxybutyrate (GHB), and gamma-butyrolactone (GBL) [6–9] On the other hand, due to its hallucinogenic effects scopolamine is also used as a recreational drug Like other tropane alkaloids such as atropine, scopolamine is produced by plants of the Solanaceae family such as Hyoscyamus albus L., Datura stramonium L., Mandragora autumnalis Bertol., Scopolia carniolica Jacq., Brugmansia candida Pers., and other plants belonging to the same plant genera Many species of this family are used as food, medicinal, or ornamental plants These plants are naturally distributed throughout the world, but occur mainly in temperate regions, and several of them are used in gardening To produce 1355-0306/$ – see front matter © 2013 Published by Elsevier Ireland Ltd on behalf of Forensic Science Society http://dx.doi.org/10.1016/j.scijus.2013.08.001 410 J Sáiz et al / Science and Justice 53 (2013) 409–414 the hallucinogenic effects, they can be smoked and much information can be found in Internet forums about how to prepare an infusion from the plants, from either the roots, leaves, stems, flowers, or fruits and seeds [10,11] Abuse of scopolamine is very risky since its active dose is very close to the lethal dose Recently, in Madrid (Spain), two 18 year-old men died and one more was hospitalized in serious condition after drinking an infusion made with seeds [12,13] Additionally, the Security Forces of Spain have been removing plants of the Datura genus and other related species from the land surrounding some cities during the last years [14] However, plants of the Datura genus coexist with corn crops where the plants have found the optimal medium for living Therefore, the distribution of these plants increases year by year [15] Due to the increasing use of the drug, analytical methods for its determination are required A difficulty is the rapid elimination of scopolamine from the body, which precludes the detection of its presence in the organism after 24 h [3] Moreover, the victims usually not report the assaults out of shame, or complain too late, when the drug has already been eliminated from the organism Then, it becomes necessary to analyze the evidence used to administer the drug A variety of techniques have been used for the analysis of samples with scopolamine, such as gas chromatography [16,17] and high performance liquid chromatography [18–29] Most of these studies have been focused on the determination of scopolamine in plant material [16,17,25,28,29] Scopolamine has also been determined in serum and plasma [18,19,27] and other biological samples, such as urine [22] or hair [21,26] Additionally, scopolamine has been determined in pharmaceuticals [20,23,24] Capillary electrophoresis (CE) has also been used for the determination of scopolamine in plant samples [1,29–37] and in pharmaceutical compositions [2,38] Different methods have been devised for the simultaneous determination of scopolamine and related compounds For example, atropine and scopolamine have been separated in Datura metel L samples [34] Scopolamine and atropine derivatives have been separated in pharmaceutical formulations [38] With regard to their use as drugs, scopolamine can exist together with atropine in certain specimens and so the availability of methods allowing their separation is cardinal since both substances show different properties Among other differences, scopolamine is much more likely to produce sedation and amnesia than atropine Therefore, both substances could, in principle, be used for different purposes and, accordingly, their simultaneous determination in samples in which scopolamine and atropine are present is necessary, and easily possible by CE due to its high separation power For CE separations of these compounds, a variety of detection methods have been employed, such as time of flight-mass spectrometry and ion trap-mass spectrometry [33], electrochemiluminescence [1,30,37], electrochemistry [1], and DAD or UV detectors [2,31,32,34–36,38] However, the sensitivity in absorbance measurements is restricted by a short optical pathlength Moreover, some species such as inorganic anions, metal ions, and non-aromatic organic compounds, can only be determined by indirect UV-detection, a detection approach which is relatively insensitive and has a limited linear range [39] Capacitively coupled contactless conductivity detection (C4D) is a sensitive and universal tool in CE, which has been widely used in recent years for many applications (see, for example, the following review articles [39–41]) C4D detectors are small, cheap, lightweight, and consume only little electrical power Since the electrodes are not in contact with the solution inside the capillary, they not corrode and, moreover, for this reason the placement of the capillary is easy and removal of the polyimide coating is not required The aim of this work was the development of a CE-C4D method for the rapid analysis of samples suspicious to contain scopolamine A portable CE (P-CE) instrument was used and a special effort was made for the development of simple sample pre-treatments, in order to make possible the fast analysis of suspicious samples Materials and methods 2.1 Reagents and samples All chemicals used were of analytical grade Tris(hydroxymethyl) aminomethane (Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium hydroxide (NaOH), atropine and scopolamine hydrobromide were obtained from Sigma-Aldrich (St Louis, MO, USA) Methanol was from Scharlau (Barcelona, Spain) Ultrapure water was obtained from a Millipore Milli-Q water system (Bedford, MA, USA) Soft drink and alcoholic beverages, as well as a moisturizing cream, were purchased in a local supermarket Samples of D stramonium L were collected in San Andrés del Congosto (Guadalajara, Spain) in mid-January and stored at room temperature 2.2 Instrumentation and experimental procedure CE experiments were performed on a purpose-built P-CE The P-CE was previously described by Sáiz et al [42] The instrument was fitted with a contactless conductivity detector (eDAQ, Deninstone East, NSW, Australia) The detector was modified to work at 12 V with the batteries of the P-CE The detector excitation frequency was set to 1200 kHz and the amplitude to 100% Bare fused silica capillaries of 50 μm I.D and 365 μm O.D (Polymicro Technologies, Phoenix, AZ, USA) with a total length of 80 cm and an effective length of 65 cm were employed New capillaries were conditioned by flushing with M NaOH for 40 min, water for min, and running buffer for 30 The running buffers consisted of 10 mM HEPES/Tris at different pH values between 7.2 and 7.6 and were prepared daily The pH-values were determined with a pH-meter (Crison GPL-21, Crison Instruments, Barcelona, Spain) Optimized conditions were achieved when the buffer at pH 7.6 was used The injection was carried out by sample splitting in the interface for s and the separations were carried out by applying −25 kV at the outlet of the capillary After each analysis, the capillary was rinsed with the running buffer for to maintain the reproducibility of the analyses When a new buffer was used, the capillary was rinsed with water for and then with the running buffer for 2.3 Evidence preparation Stock solutions of mg/mL of atropine and scopolamine were prepared in methanol For the qualitative determination of scopolamine and atropine, the samples were spiked with standards 2.3.1 Infusion specimen Fresh seeds of D stramonium L were homogenized in a domestic grinder g (fresh weight) of the homogenate was added to 50 mL of water and heated on a hot plate until it begins to boil After cooling down, the infusion was filtered by gravity through a Whatman filter grade GF/A (1.6 μm) in a conical funnel Samples were then directly injected into the P-CE system without dilution 2.3.2 Moisturizing cream specimen Samples were prepared by dissolving mg of scopolamine (MW 438.31) in 500 μL of methanol employing a vortex mixer The solution was added to g of the moisturizing cream in a polypropylene tube, which was vortex-mixed for The extraction of scopolamine from the spiked moisturizing cream was carried out by adding mL of methanol to g of the moisturizing cream evidence The mixture was vortex-mixed for and then centrifuged for 10 at 4000 g After centrifugation, three phases were clearly visible and the middle liquid phase was recovered by puncturing the tube with a syringe Finally, the sample was diluted 10-fold (v/v) in water and injected in the P-CE system without filtration J Sáiz et al / Science and Justice 53 (2013) 409–414 2.3.3 Beverage specimens The beverages studied were prepared using 1/3 (v/v) of alcoholic drink and 2/3 (v/v) of soft drink because these proportions are usually utilized in mixed alcoholic beverages The beverages prepared were the following: whisky with cola, rum with cola, rum with lemon, gin with tonic water, vodka with tonic water, and vodka with lemon The total volume of the drink was considered to be 150 mL Scopolamine was added to the beverages in a concentration of 1.3 mM Then, samples were vortex-mixed for min, diluted 5-fold (v/v) in water, and injected into the P-CE system without filtration 2.4 Data treatment The electropherograms were processed in Origin (OriginLab Corporation, USA) Limits of detection (LODs) were estimated as three times the signal to noise ratio in the electropherograms obtained for the spiked beverages and the moisturizing cream The noise value was measured as the maximum deviation of the baseline around the migration time of the analyte signal Corrected peak areas were calculated by dividing the peak area by the migration time of the corresponding peak to correct for the area deviation produced when peaks are delayed depending on the sample Results and discussion The first aim was to develop sample pre-treatments that can be readily and quickly carried out Then the separation conditions were optimized in order to obtain a fast complete baseline separation of atropine and scopolamine Next, both compounds were determined in an infusion prepared with D stramonium L seeds, normally used for recreational purposes Finally, two types of spiked samples for evidence specimens (a moisturizing cream and six commonly consumed beverages), used for predatory purposes, were also analyzed 3.1 Preparation of the specimens preparation of the beverage samples This concentration was chosen because high concentrations of scopolamine are required to induce the symptoms of amnesia, sedation, and loss of free will without risk of death 3.2 Selection of evidence treatment When dealing with complex samples, it is important to rely on treatment procedures which allow the recovery of the majority of the analyte in the shortest time possible, without many steps in the procedure This prevents contamination of samples and allows for quick results For this reason, evidence treatments as fast and simple as possible were designed The infusion sample was filtered by gravity through a membrane filter in a conical funnel and then directly injected into the P-CE system This step does not require specific instrumentation, such as vacuum filters, and was carried out manually within Beverage samples were simply diluted 5-fold because of the high concentration of scopolamine in the drinks and no further sample treatment was needed before the analysis This sample treatment was carried out within In the case of the moisturizing cream sample further treatment was needed due to the complexity of the evidence item, which was viscous and non-aqueous However, an easy step of centrifugation with methanol followed by a 10-fold dilution in water of the middle phase was enough to detect the scopolamine contained in the moisturizing cream and to have a clean sample that can be injected into the P-CE The whole pre-treatment process for the moisturizing cream was accomplished in less than 15 Therefore, the time needed for the treatment of the infusion and the beverage specimens was insignificant The moisturizing cream needed more time to be treated, but considering the complexity of the sample, the time spent was reasonably short 3.3 Optimization of the separation conditions Atropine and scopolamine are two tropane alkaloids present in Solanaceous plants Both tropanes have very similar structures (Fig 1) 3.1.1 Infusion specimen Nowadays, plenty of information can be found in certain Internet forums on the preparation of scopolamine infusions from some plants [10,11] These drinks have increasing popularity for their recreational use According to the information retrieved from the Internet forums, an infusion of seeds was prepared Seeds of D stramonium L were homogenized and directly boiled to prepare the infusion 3.1.2 Moisturizing cream specimen The possibility of administering scopolamine by dermal contact is well known Novartis™ is marketing Transderm Scōp® patches for people who are suffering from motion sickness These patches contain 1.5 mg of scopolamine and are designed to deliver in vivo approximately 1.0 mg of scopolamine over days [43] Considering that this is a therapeutic dosage, a concentration of approximately mg/mL of scopolamine was added to the moisturizing cream (5 mg of scopolamine to g of moisturizing cream) This dose can be used for predatory purposes, as it is a higher dose compared to that in the Transderm Scōp® patches 3.1.3 Beverage specimens The beverages studied were chosen for being commonly consumed in night clubs The amount of scopolamine added to the beverages was chosen considering the available data about lethal and active dosages of scopolamine As an example, the ingestion of 10 mg has been reported to be lethal in children, whereas adults survived more than 100 mg of scopolamine [44–46] However, exact data on lethal and active doses of scopolamine are lacking [44,47] For this reason, a concentration of 1.3 mM of scopolamine (MW 438.31), representing a total amount of 85.5 mg in 150 mL of drink, was chosen for the 411 Fig Structures of atropine and scopolamine 412 J Sáiz et al / Science and Justice 53 (2013) 409–414 Hence their electrophoretic separation could be difficult at certain pH values However, the separation of scopolamine and atropine is important in evidence in which both compounds are present, such as plant samples The pKa-values of atropine and scopolamine are 9.8 and 7.6, respectively Therefore, a pH close to the pKa-value of scopolamine is expected to allow separation of atropine and scopolamine because atropine then has a higher net charge and should therefore migrate faster than scopolamine Then, the main consideration for the selection of a suitable running buffer in this work was based on the ionic characteristics of the basic analytes studied (Fig 1) and the conductivity of the buffer and the analytes Buffers of low conductivity must be chosen for conductivity detection to minimize Joule heating whereas buffers with very high conductivities result in baseline instabilities In this work, buffers consisting of HEPES/Tris at pH 7.6 were prepared as background electrolytes at three different concentrations (10, 15, and 20 mM) As expected, higher concentrations led to larger peaks, but also to higher noise levels on the baseline Finally, the buffer consisting of 10 mM HEPES/Tris was chosen for showing the best signal-to-noise ratio A capillary with a total length of 80 cm and 65 cm to the detector was needed to achieve the initial separation of atropine and scopolamine When shorter capillaries were used, a single peak was observed Then, three pH-values close to the pKa of scopolamine, namely 7.2, 7.4, and 7.6 were studied The electropherograms for a mixture of 100 μM of scopolamine and 100 μM of atropine using the three above-mentioned buffers are shown in Fig At the working pH-values, scopolamine and atropine have a low charge and their equivalent conductivities are lower than the conductivity of the running buffers Note that for this reason, the detector signals have been inverted in this work in order to show the positive peaks for scopolamine and atropine As expected, scopolamine migrated after atropine and the detection of both compounds at the three selected pH-values was possible In Fig it can also be observed that, when the pH-values were increased from 7.2 to 7.6, scopolamine was losing its net charge and getting closer to the peak attributed to the EOF signal migrating after the scopolamine peak The buffer at pH 7.6 allowed Fig Electropherogram for a standard mixture of 100 μM of scopolamine and atropine in (a) 10 mM HEPES/Tris pH 7.2; (b) 10 mM HEPES/Tris pH 7.4; and (c) 10 mM HEPES/Tris pH 7.6 CE conditions: capillary length, 80 cm (65 cm to the detector); applied voltage, −25 kV; hydrodynamic injection for s; pressure in the system set at 1.5 bar SCO, scopolamine; ATR, atropine; *, system peak the complete baseline separation of scopolamine from atropine Moreover, at this pH the scopolamine peak did not overlap with the peak attributed to the EOF signal On the other hand, although the concentrations of scopolamine and atropine were the same, there were differences in their peak heights This is due to the different net charges and limiting equivalent conductivities of the analytes, which are lower for scopolamine at the given pH-values For this reason, the peak of scopolamine was higher than the peak of atropine Also, the peak heights of scopolamine and atropine increased with the pH-value of the buffer because scopolamine and atropine have lower limiting equivalent conductivities at pH 7.6 than at pH 7.4 or 7.2 Therefore, C4D was shown as a very good alternative to UV detection for the detection of atropine and scopolamine at the working conditions, since the increase of the pH value allowed not only the obtainment of the baseline separation of atropine and scopolamine, but also the increase in the detection sensitivity for both analytes Finally, the buffer at pH 7.6 was chosen as it allowed the baseline separation of scopolamine and atropine and showed the highest signal-to-noise ratio for scopolamine The strong electro-osmotic (EOF) flow created at the working pH allowed the determination of scopolamine and atropine within only 3.5 3.4 Evidence analysis Prior to the application of the developed method to the evidence analysis, its analytical performance in terms of LODs and precision, measured as repeatability and reproducibility, was studied The LODs for scopolamine were calculated to be 2.6 μg/mL in beverages and approximately 0.6 μg/mg of cream in the moisturizing cream sample A repeatability test of 10 consecutive beverage injections provided RSD values of 1.8% for migration times and 6.5% for corrected peak areas The inter-day reproducibility for the same samples during different days gave RSD values of 3.3% (n = 18, 3–10 injections per day) for migration times and 15.0% (n = 18, 3–10 injections per day) for corrected peak areas First, an infusion of seeds of D stramonium L was analyzed because it may be consumed for recreational purposes Then a moisturizing cream and different alcoholic beverages, all spiked with commercially available scopolamine, were also analyzed These evidential items were chosen for being commonly used for the administration of the drug for predatory purposes Fig shows the electropherogram for an infusion of seeds of D stramonium L In this evidence, atropine and scopolamine peaks were detected, as positive peaks as expected, for showing lower conductivity than the baseline A group of fast compounds having higher conductivities than the baseline migrated before atropine and scopolamine Therefore they are shown as negative peaks After scopolamine, a large positive peak migrated, which was attributed to the EOF In this case, changes in the migration times of scopolamine and atropine, compared with those shown in Fig 2, were attributed to the changes in mobility caused by the sample matrix because direct injection of the sample was performed without any dilution or purification However, since scopolamine and atropine were the only positive peaks, despite the large peak attributed to the EOF, they were clearly detected in the electropherogram The electropherogram for the moisturizing cream spiked with scopolamine is shown in Fig As in the previous case, scopolamine is shown as a characteristic positive peak between the group of negative peaks belonging to the sample matrix and the large peak attributed to the EOF signal In this case, a good resolution of scopolamine from other peaks was achieved, making possible its determination The electropherograms for the six spiked alcoholic beverages analyzed are depicted in Fig All beverage samples showed similar electropherograms and the scopolamine peak migrated after the sample matrix peaks and before the peak attributed to the EOF Once again, scopolamine was detected in all the samples as a very distinctive peak, J Sáiz et al / Science and Justice 53 (2013) 409–414 Fig Electropherogram for the determination of scopolamine and atropine in an infusion of seeds of Datura stramonium L The reduced image shows an electropherogram in which the large peaks produced by the sample matrix are also shown Other experimental conditions as in Fig SCO, scopolamine; ATR, atropine since it was the only positive peak besides the large peak attributed to the EOF As can be seen in Figs 3, 4, and 5, the method has been proved to be highly selective for scopolamine and atropine due to the characteristic peaks shown in the electropherograms The determination of both tropane alkaloids was fast in all the evidential items analyzed This, together with the rapid evidence treatments, allowed a very fast analysis of samples Moreover, the employment of a P-CE may shorten even more the obtaining of results, since sample preservation and transportation are not required and the data are immediately available 413 Fig Electropherograms for alcoholic beverages spiked with 1.3 mM of scopolamine (a) Whisky with cola, (b) rum with cola, (c) rum with lemon, (d) vodka with lemon, (e) vodka with tonic water, and (f) gin with tonic water CE conditions: buffer, 10 mM HEPES/Tris pH 7.6; samples diluted 5-fold in water Other electrophoretic conditions as in Fig SCO, scopolamine Conclusions Nowadays, scopolamine has become a drug of increasing use in Europe, and several ways of administration and consumption have been conceived In this work, a P-CE method for the rapid screening of evidence with scopolamine was devised A HEPES/Tris buffer at pH 7.6 allowed the baseline separation of atropine and scopolamine within 3.5 min, being able to distinguish both compounds in those evidential items in which they coexist Moreover, this buffer provided a more sensitive C4D detection than the other buffers investigated The studied evidential items containing scopolamine were chosen because they are commonly used for recreational or predatory purposes (an infusion of seeds of D stramonium L., a moisturizing cream and alcoholic beverages, the last two having been spiked with scopolamine) The sample pre-treatment methods were easy and allowed fast evidence analysis C4D has been proven to be an excellent tool for the detection of atropine and scopolamine in the samples analyzed, under the experimental conditions, as they were always shown as highly characteristic peaks in the electropherograms, allowing the detection of down to 0.6 μg/mg of scopolamine in creams and 2.6 μg/mL in beverages Therefore, the method can be used for the fast analysis of evidential items in the search for scopolamine Acknowledgments J Sáiz thanks the Ministry of Science and Innovation for his contract associated to the project CTQ2008-00633-E The authors thank Alfonso Vega for his contribution to this work References Fig Electropherogram for the extract of the moisturizing cream spiked with scopolamine diluted 10-fold in water Other experimental conditions as in Fig SCO, scopolamine [1] B Yuan, C Zheng, H Teng, T You, Development and validation of a capillary zone electrophoresis method for the determination of atropine, homatropine and 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atropine, the samples... and atropine and showed the highest signal-to-noise ratio for scopolamine The strong electro-osmotic (EOF) flow created at the working pH allowed the determination of scopolamine and atropine... difficult at certain pH values However, the separation of scopolamine and atropine is important in evidence in which both compounds are present, such as plant samples The pKa-values of atropine and scopolamine