Drug Testing Research article and Analysis Received: 2 August 2015 Revised: 20 August 2015 Accepted: 21 August 2015 Published online in Wiley Online Library: 12 October 2015 (www.drugtestinganalysis.com) DOI 10.1002/dta.1884 Return of the lysergamides. Part I: Analytical and behavioural characterization of 1- propionyl-d-lysergic acid diethylamide (1P-LSD) Simon D. Brandt,a*PierceV.Kavanagh,b Folker Westphal,c Alexander Stratford,d Simon P. Elliott,e Khoa Hoang,f Jason Wallachg and Adam L. Halberstadth 1-Propionyl-d-lysergic acid diethylamide hemitartrate (1P-LSD) has become available as a ‘research chemical’ in the form of blotters and powdered material. This non-controlled derivative of d-lysergic acid diethylamide (LSD) has previously not been described in the published literature despite being closely related to 1-acetyl-LSD (ALD-52), which was developed in the 1950s. This study describes the characterization of 1P-LSD in comparison with LSD using various chromatographic and mass spectromet- ric methods, infrared and nuclear magnetic resonance spectroscopy. An important feature common to LSD and other serotonergic hallucinogens is that they produce 5-HT2A-receptor activation and induce the head-twitch response (HTR) in rats and mice. In order to assess whether 1P-LSD displays LSD-like properties and activates the 5-HT2A receptor, male C57BL/6 J mice were injected with vehicle (saline) or 1P-LSD (0.025–0.8 mg/kg, IP) and HTR assessed for 30 min using magnetometer coil recordings. It was found that 1P-LSD produced a dose-dependent increase in HTR counts, and that it had ~38% (ED50 = 349.6 nmol/kg) of the potency of LSD (ED50 = 132.8 nmol/kg). Furthermore, HTR was abolished when 1P-LSD administration followed pretreatment with the selective 5-HT2A receptor antagonist M100907 (0.1 mg/kg, SC), which was consistent with the concept that the behavioural response was mediated by activation of the 5-HT2A receptor. These results indicate that 1P-LSD produces LSD-like effects in mice, consistent with its classification as a serotonergic hallucinogen. Nevertheless, the extent to which 1P-LSD might show psychoactive effects in humans similar to LSD remains to be investigated. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: new psychoactive substances; LSD; 5-HT2A receptor; lysergamides; psychedelics Introduction LSD-like effects in animals that originated from academic research and have been available for purchase in powdered and blotter It is perhaps fair to consider that the synthesis[1] and discovery of form. Another closely related derivative with modification at the the psychoactive properties of d-lysergic acid diethylamide (LSD)[2] (Figure 1) in 1943 triggered an avalanche of investigations * Correspondence to: Simon D. Brandt, School of Pharmacy and Biomolecular that continue to capture the imagination of researchers across all Sciences, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK. [3–10] disciplines. Although the pharmacology and properties of E-mail: [email protected] LSD have been investigated in many studies, major questions still remain unanswered and are expected to occupy the attention of a School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK researchers in the future.[11–16] Reports have been published indicating LSD may possess b Department of Pharmacology and Therapeutics, School of Medicine, Trinity therapeutic efficacy in patients suffering from disorders such as Centre for Health Sciences, St. James Hospital, Dublin 8, Ireland anxiety, alcoholism, cluster headaches, and autism, but unfortu- nately most of this evidence is anecdotal in nature or confounded c State Bureau of Criminal Investigation Schleswig-Holstein, Section Narcotics/ Toxicology, Mühlenweg 166, D-24116, Kiel, Germany by methodological shortcomings.[17–19] Importantly, although most clinical work with LSD ceased in the late 1960s, human trials have d Synex Ltd, 483 Green Lanes, N13 4BS, London, UK cautiously resumed during the last few years.[20–23] A range of lysergamide derivatives have been prepared to e ROAR Forensics, Malvern Hills Science Park, Geraldine Road, WR14 3SZ, UK [24–32] explore their molecular pharmacology but the extent to f Department of Chemistry and Biochemistry, University of the Sciences, 600 South which these show psychoactive properties in humans is not always 43rd Street, Philadelphia, PA, 19104, USA clear.[7] In recent years, several lysergamide derivatives have been distributed as new psychoactive substances (NPS) or ‘research g Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, chemicals’ in the UK and Europe.[33] For example, lysergic acid University of the Sciences, 600 South 43rd Street, Philadelphia, PA, 19104, USA [31] 2,4-dimethylazetidide (LSZ) and N6-allyl-6-norlysergic acid h Department of Psychiatry, University of California San Diego, 9500 Gilman Drive, 891 [24,30] diethylamide (AL-LAD) are two lysergamide derivatives with La Jolla, CA, 92093-0804, USA Drug Test. Analysis 2016, 8, 891–902 Copyright © 2015 John Wiley & Sons, Ltd. Drug Testing and Analysis S. D. Brandt et al. MO, USA), and LSD tartrate was from Ultrafine Chemicals (Manchester, UK). One blotter labelled to contain 100 μg1P-LSD was purchased from an Internet vendor. Instrumentation Nuclear magnetic resonance spectroscopy NMR spectra were recorded in d6-DMSO using a Bruker Avance 300 1 13 Figure 1. Chemical structures of lysergamides d-LSD, 1-acetyl-LSD spectrometer ( H at 300.1 MHz; C at 75 MHz) and suggested (ALD-52) and 1-propionyl-LSD (1P-LSD). assignments were aided by 1-D and 2-D experiments. Internal chemical shift references were based on residual solvent peaks. indole nitrogen is 1-acetyl-LSD (ALD-52) (Figure 1). Synthesis of Gas chromatography-mass spectrometry ALD-52 was first reported in 1957[34] and it was found to be psycho- – active in humans[35 38] but it is not clear whether ALD-52 was also Electron ionization (EI) mass spectra (70 eV) were recorded using a sold in the UK. Recent changes in UK legislation, however, pre- Finnigan TSQ 7000 triple stage quadrupole mass spectrometer cludes the open sale of several lysergamides, including ALD-52, coupled to a gas chromatograph (Trace GC Ultra, Thermo Electron) LSZ and AL-LAD.[39] In response to these legal restrictions, 1-propi- using a CTC CombiPAL (CTC Analytics, Zwingen, Switzerland) onyl-LSD (Figure 1), also known as 1P-LSD, became available as a autosampler. The emission current was 200 μA and the scan time ‘research chemical’ either as powdered material or on blotters. was 1 s spanning a scan range between m/z 29 and m/z 600. The Although an assortment of LSD derivatives substituted at the ion source temperature was maintained at 175°C. Samples were – 1-position have been described,[34,37,40 42] it is noteworthy that introduced via gas chromatography with splitless injection using chemical, analytical, or pharmacological data related to 1P-LSD a fused silica capillary DB-1 column (30 m x 0.25 mm, film thickness appear to be absent from the literature. 0.25 μm). The temperature program consisted of an initial temper- Because of the difficulty associated with studying hallucinogens ature of 80 °C, held for 1 min, followed by a ramp to 280°C at in humans, animal behavioural models are often used to investigate 15 °C/min. The final temperature was held for 21 min. The injector the pharmacology of hallucinogenic drugs. One behavioural model temperature was 220°C. The transfer line temperature was main- that has been widely adopted is the head-twitch response (HTR), a tained at 280 °C and the carrier gas was helium in constant flow paroxysmal side-to-side head movement induced by 5-HT2A mode at a flow rate of 1.0 mL/min. Approximately 2 mg were agonists in rats and mice. HTR is considered to be a rodent behav- dissolved in 1.5 mL methanol. For analysis, 1 μL sample solutions ioural proxy for human hallucinogenic effects because it can distin- were injected into the GC-MS system. guish between hallucinogenic and non-hallucinogenic 5-HT2A agonists.[43–46] Although HTR has traditionally been assessed using High-resolution electrospray mass spectrometry direct observation, new methods have been developed to detect The ultrahigh-performance liquid chromatography quadrupole HTR in a semi-automated fashion using a head-mounted magnet time of flight single and tandem mass spectrometry (UHPLC- [46,47] and a magnetometer coil. QTOF-MS/MS) conditions were used as described previously.[48,49] The present report details the analytical characterization of Briefly, mobile phases used for UHPLC separation consisted of 1P-LSD using various chromatographic, mass spectrometric, and 100% acetonitrile (1% formic acid) and an aqueous solution of 1% spectroscopic methods relevant to clinical and forensic investiga- formic acid. The column temperature was set at 40°C (flow rate tions. Supplementary analytical data derived from the characteriza- 0.6 mL/min) and data were acquired for 5.5 min. The elution was a tion of LSD have also been included for comparative purposes. In 5–70% acetonitrile gradient ramp over 3.5 min, then increased to addition, in vivo studies were conducted to assess the potential 95% acetonitrile in 1 min and held for 0.5 min before returning to similarity between 1P-LSD and LSD in regard to their 5-HT2A receptor 5% acetonitrile in 0.5 min. QTOF-MS data were acquired in positive pharmacology and behavioural effects. Previous investigations using mode scanning from m/z 100 to m/z 1000 with and without auto magnetometer-based measurements have shown that LSD and MS/MS fragmentation. Ionization was achieved with an Agilent other serotonergic hallucinogens induce HTR in a dose-dependent JetStream electrospray source and tuned using internal reference [47] manner in male C57BL/6 J mice consistent with the mechanism masses.