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Abstract

Background

Synthetic cannabinoids (NOIDS) are novel psychotropic drugs (NPS) currently freely sold in the United Kingdom as ‘research chemicals’. Detection of NOIDS use is not available in current routine methods. Here we describe a marker which helps determine which patients have used these substances.

Methods

In a test case, ultra-performance liquid chromatography mass spectrometry (UPLC-Tof) was used to screen the legal high Herbal Haze II, the contents of hand-rolled cigarettes and five patient samples for NOIDS and their metabolites.

Results

Analysis of legal high Herbal Haze II and cigarettes identified the third generation adamantyl-type NOIDS N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), 5F-AKB-48 and N-adamantyl-1-fluoropentylindole-3-carboxamide (STS-135). Out of 18 potential metabolites, 1-adamantylamine (C₁₀H₁₇N) was detected in all five urine samples. This adamantyl-type NOID marker was incorporated into our routine LC-MS/MS urine screen. Out of 14,436 random urine samples screened over eight months, 296 (2.05%) tested positive for the adamantyl-type NOID marker.

Conclusion

We have discovered a urine marker for identifying patients smoking legal high products containing the third generation adamantyl-type NOIDS such as AKB-48 and its fluoropentyl analogue 5F-AKB-48, which are among the most popular NOIDS currently available in legal high products sold in UK. This marker can be incorporated into routine LC-MS/MS drug screening alongside classic drugs of abuse. Positive detection rates for this new legal high marker are greater than for established classic drugs that are routinely screened such as amphetamine. This work highlights the need for a flexible toxicology screening service capable of adapting to changes in drug use such as the growing popularity of legal highs/NPS.

Keywords

Legal highs synthetic cannabinoids 1-adamantylamine

Introduction

Synthetic cannabinoids (NOIDS) sometimes referred to as Black Mamba, spice, K₂ or herbal incense are novel psychotropic drugs (NPS) which mimic the effect of cannabis. Typically legal high products are prepared by spraying the NOIDS onto herbs which users then incorporate into roll-ups and smoke usually together with tobacco. Sold as research chemicals ‘not for human consumption’, the 1968 medicines act does not apply. Consequently, these products are freely available to buy on the high street and on-line. Like Δ9-tetrahydrocannabinol (THC) found in cannabis, the psychotropic effects of NOIDS are a result of binding to cannabinoid receptors located in the brain.^1–3 Unlike THC, most NOIDS are full rather than partial agonists making them far more potent.⁴ Although clinical presentation varies, reported cases frequently describe catatonic states, dystonia, aggression, respiratory depression, hypotension and tachycardia.^5–9 As with cannabis abuse there is evidence that regular NOID abuse is linked to an increased risk of psychosis.^10–12 A number of deaths have been attributed to NOIDS.^13,14

Based on their chemical structure, first and second generation NOIDS have been broadly classified into six groups: classical, napthoylindoles, napthoylnapthalenes, phenylacetylindoles, benzoylindoles and cyclohexylphenols^15–17 and by 2013 all were recognized under UK Misuse of Drugs Act 1971 as class B controlled drugs.¹⁸ Shortly afterwards third generation NOIDS not covered by the 2013 legislation, started to appear in herbal type legal highs sold in the UK, and many incorporate an adamantyl group (adamantyl-type NOIDS).

Adamantyl-type NOIDS were first identified in legal high products sold in Japan in 2012; N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (SDB-100) and N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48).¹⁹ Further adamantyl-type NOIDS have since been identified, including N-adamantyl-1-fluoropentylindole-3-carboxamide (STS-135) and a flouropentyl version of AKB-48 (N-(adamantan-1-yl)-1-(5-fluoropentyl)-1H-indazole-3-carboxamide (5F-AKB48)).²⁰ The rationale behind the addition of an adamantyl group may be linked to reports that it can improve drug potency, by increasing the affinity for receptor binding and impeding degradation by hydrolytic enzymes.^21,22 The latest 2014 Forensic Early Warning System (FEWS) report identified adamantyl-type NOIDS as the most popular third generation NOID currently on sale in UK, with 5F-AKB-48 present in 45% of 345 products seized from head shops in Aberdeen and Plymouth.²³

Detection of NOID abuse presents a particular challenge, both to the clinician and testing laboratories. NOIDS lack the distinct pungent smell of cannabis. The information provided on legal high products is limited, inaccurate, and often users are unsure of what they have actually taken. Whilst it is relatively easy to test and identify parent drugs in legal high products, detection in biological samples is more challenging. NOIDS are not detected by conventional drugs of abuse screening methods.²⁴ NOIDS undergo extensive metabolism, and as they are more potent than cannabis, concentrations of both parent drug and metabolites are likely to be low in biological fluids.

High resolution mass analysis, for example time of flight mass spectrometry (Tof), has been used to identify NPS in legal high products.^25,26 Tof has also been used to investigate NOID metabolism using in-vitro studies to identify potential markers for screening, with hydroxlated and carboxylated metabolites a common finding.^27,28 This has led to development of specific screening methods using immunoassay^29,30 and mass spectrometry.^31–35 These specialist tests exclusively screen for NOIDS and metabolites and do not include other drugs.

We are a UK NHS laboratory that provides a specialist clinical toxicology service, including screening for classic drugs of abuse and NPS. In 2014 we were referred samples from a clinical incident of multiple patients suspected of using NOIDS, for which the legal high product, hand-rolled cigarettes and patient urine samples were sent to us for analysis. We have used ultra-performance liquid chromatography mass spectrometry (UPLC-Tof) to identify a novel marker for the third generation adamantyl-type NOIDS in urine. We have then incorporated this marker into our routine drugs of abuse ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) screen, which includes other classic drugs of abuse and legal highs. Here we present data for the positive detection rate of this marker for adamantyl-type NOID use over eight months of routine screening (July 2014 to February 2015).

Experimental

Chemicals and solutions

All solvents and water were purchased from Fisher Scientific® (Leicestershire, UK) and were LC-MS/MS grade. Leucine encephalin, 1-adamantylamine and internal standard 1-(1-adamantyl)ethylamine hydrochloride (rimantadine) were brought from Sigma-Aldrich (TX, USA). Reference drugs used for UPLC-Tof were purchased from Cerillant® Sigma-Aldrich, LGC® (Luckenwalde, Germany) and Purechemicals.net (London, UK).

Validation samples

Herbal legal high Herbal Haze II (1 g, London WC1N 3XX), two hand-rolled cigarettes and five random urine samples (test patient samples) were received from a secure mental health facility for NPS screening by UPLC-Tof (January 2014). Drug free urine used as blanks for method development, validation, calibrators and controls were provided by laboratory volunteers.

Routine urine samples for drugs of abuse screening

Random urine samples (14,477) collected into plain white universals were received for routine urine drug screening by LC-MS/MS for both clinical and medico-legal purposes from locations across the UK.

Preparation of non-biological samples

For legal high and tobacco products 1 mg/mL stocks were prepared in methanol by sonicating for 10 min and centrifuged for 5 min. For analysis, supernatants were then further diluted in methanol to give ∼1000 μg/L extracts.

Preparation of urine samples

For both LC-MS/MS and UPLC-Tof analyses urine drugs were extracted and concentrated using a method developed in-house. Into a 2 mL vial was added 400 µL of urine, 10 µL of IS mix and 600 µL of a reagent containing acetonitrile, ethyl acetate and 500 mmol/L ammonium carbonate buffer (pH 8). After mixing, the bottom layer was removed by centrifugation and the upper drug-containing layer transferred to a fresh vial and dried under a stream of nitrogen. The dried extracts were then reconstituted in 100 µL of loading buffer.

UPLC-Tof analysis

The UPLC-Tof system used was a Waters ACQUITY UPLC® (binary solvent manager, sample organizer, sample manager and column manager) interfaced with a hybrid Waters Xevo G2 QTof detector, with electrospray ionization in positive ion mode (Waters, Co., Milford, MA). Two methods were used for NPS qualitative screening, a NOID specific method and a general drug screen that includes over 1300 drugs and metabolites.

The NOID specific method includes a collision-induced dissociation (CID) mass spectral library containing over 100 first, second and third generation NOIDS and their metabolites and was developed in-house. It uses a Waters ACQUITY UPLC® BEH C18 1.7 µm 2.1 × 100 mm column protected with a BEH C18 1.7 µm 2.1 × 5 mm VanGuard pre column as a stationary phase, maintained at 50℃. Drug separation was performed over a total of 12 min using a gradient of 5 mmol/L ammonuim formate pH 3 (solvent A), and 0.1% (v/v) formic acid in 100% acetonitrile (solvent B) at a flow rate of 500 µL/min. Data acquisition was performed using Waters MassLynx software at a cone voltage of 25 V, capillary voltage 3.0 kV, desolvation gas flow of 900 L/h at 500℃, with a scan rate of 0.2 per sc in centroid mode from m/z 30 to 600 (MS^E function).

The second general drug screening method was used as described by Rosano et al.³⁶ with additional mass spectra library additions performed in-house. It has a CID mass spectral library containing over 1300 drugs and metabolites, 10% of which are NPS. For routine screening the adamantyl-type NOID marker was incorporated into this second general drug screening method (C₁₀H₁₇N RT 2.88 min f:135.1174), and was used for the routine confirmation of all urine UPLC-MS/MS screen positive results.

For both methods leucine encephalin (2 ng/µL in 50/50 (v/v) acetonitrile/water containing 0.1%(v/v) formic acid) was used as a lock-mass calibrant and analysed for two ions at 556.2271 Da and 120.0813 Da every 30 s at a flow rate of 5 µL/min throughout the run. Sample injection volumes were 10 µL. Criteria used for drug identification was a mass accuracy of 5 ppm for parent compound (function one) and qualifier fragment (function two), with an average isotope fit within 20% of the calculated ratio and a target RT ± 0.3 min.³⁶

UPLC-MS/MS urine drug screen

The UPLC-MS/MS system comprised a Waters ACQUITY® UPLC system (binary solvent manager, sample organizer, sample manager and column manager) and Xevo TQD detector with positive electrospray ionization. Chromatographic separation was performed using a Waters ACQUITY® UPLC HSS C18 1.8 µm 2.1 × 150 mm column, and HSS C18 1.8 µm 2.1 × 5 mm VanGaurd pre column maintained at 30℃ with a total run time of 6 min using a gradient of 5 mmol/L ammonuim formate with 0.025% (v/v) formic acid (solvent A), and 0.025% (v/v) formic acid in 100% methanol (solvent B) and injection volume of 15 µL. The drug testing panel includes 26 drugs and metabolites, and for detection it uses multiple reaction monitoring (MRM). Creatinine is included as an integrity checker to identify adulterated samples.³⁷ For 1-adamantylamine (adamantyl-type NOID marker) quantifier and qualifier ions were 152.3/135.1 m/z and 152.3/107.2 m/z, respectively, with a RT of 3.19 min. Rimatadine was used as IS with monitoring of a single transition 180.3 > 163.2 m/z (RT 3.67 min). Criteria used for positive identification were a transition ion ratio (>10) of ±50% and target RT ± 0.2 min.^38–42 Included in each analysis was a five-point adamantyl-type NOID marker calibration curve (0–80 μg/L) and two controls ±25% of the positive cut-off threshold of 5 μg/L, all prepared in blank urine collected from a drug free volunteer.

Results

Validation samples

Legal high product Herbal Haze II came in a green metal foil sachet. The contents (1 g) were a light green plant like material with a pungent sweet smell. UPLC-Tof screening using the NOID specific method detected third generation NOIDS AKB-48 (C₂₃H₃₁N₃O RT 10.66 min f:135.1174), 5F-AKB-48 (C₂₃H₃₀FN₃O RT 9.56 min f:135.1175), 5F-PB-22 (C₂₃H₂₁N₂O₂F RT 6.19 min f:232.1135) as well as traces of STS-135 (C₂₄H₃₁FN₂O RT 8.7 min f:135.1164). AKB-48, 5F-AKB-48 and STS-135 are all adamantyl-type NOIDS. UPLC-Tof screening using the general drug screen was negative.

Examination of the contents of the two hand-rolled cigarettes revealed in addition to tobacco a small amount of light green material identical in appearance to the contents of legal high product Herbal Haze II (see Figure 1). UPLC-Tof screening using the NOID specific method detected AKB-48, 5F-AKB-48 and 5F-PB-22. UPLC-Tof screening using the general drug screen detected nicotine only.

Figure 1.

Contents of hand-rolled cigarettes from validation test case containing synthetic cannabinoid herb material. 1. Hand-rolled cigarette. 2. Tobacco contents containing light green herb material. 3. Contents legal high herbal haze II.

UPLC-Tof screening of the five patient urine samples failed to detect the NOIDS AKB-48, 5F-AKB-48 and 5F-PB22 found in Herbal Haze II and the hand-rolled cigarettes. Using the UPLC-Tof NOID specific and the general drug screening methods in function one only (parent compound, low energy), the five urine samples were then screened for possible metabolites of AKB-48, including 11 major metabolites suggested by Gandhi et al.²⁸ (Table 1). Only one metabolite was detected in all five urine samples, 1-adamantylamine, which we named adamantyl-type NOID marker.

Table 1.

Summary of possible AKB-48 metabolites used for screening five test patient urine samples by UPLC-Tof using NOID specific method.

Metabolite ID/description	Elemental composition	Precursor ion (m/z + 1H)
Monohydroxylation (M17)²⁸	C₂₃H₃₁N₃O₂	382.2487
Dihydroxylation (M5)²⁸	C₂₃H₃₁N₃O₃	398.2441
Dihydroxylation (M7)²⁸	C₂₃H₃₁N₃O₃	398.2437
Dihydroxylation (M10)²⁸	C₂₃H₃₁N₃O₃	398.2440
Dihydroxylation (M15)²⁸	C₂₃H₃₁N₃O₃	398.2443
Trihydroxylation (M1)²⁸	C₂₃H₃₁N₃O₄	414.2391
Trihydroxylation (M4)²⁸	C₂₃H₃₁N₃O₄	414.2386
Trihydroxylation (M9)²⁸	C₂₃H₃₁N₃O₄	414.2387
Dihydroxylation and glucuronide conjugation (M13)²⁸	C₂₉H₃₉N₃O₉	558.2799
Monohydroxylation and glucuronide conjugation (M6)²⁸	C₂₉H₂₉N₃O₈	574.2753
Dihydroxylation + ketone formation (M3)²⁸	C₂₃H₂₉N₃O₈	412.2232
1-Adamantylamine	C₁₀H₁₇N	152.1439
Adamantane	C₁₀H₁₆	137.1330
1-Adamantanol	C₁₀H₁₆O	153.1279
Adamantinecarboxylic acid	C₁₁H₁₆O₂	181.1229
Adamantenediol	C₁₀H₁₆O₂	169.1229
3-Amino-1-adamantinol	C₁₀H₁₇NO	168.1388
2-Methyl-2-adamantinol	C₁₁H₁₈O	167.1436

Validation UPLC-Tof method for adamantyl-type NOID marker

For the UPLC-Tof general drug screening method, the limit of detection of the adamantyl-type NOID marker determined using drug-spiked urines collected from 10 different volunteers was <2 μg/L for detection of the parent compound only, and 5 μg/L for parent compound and qualifier ion.

Validation of LC-MS/MS method for adamantyl-type NOID marker

The limit of quantitation (LOQ) defined as the lowest drug-spiked urine calibrator concentration with a signal to noise >10, minimum of 10 data points across each peak, drug concentration ± 20% of nominal value (%accuracy) and %CV < 20% (n = 5) for our adamantyl-type NOID marker was <2 μg/L. To enable UPLC-Tof confirmation the positive cut-off threshold for the routine LC-MS/MS screening method for adamantyl-type NOID marker was set above the LOQ at 5 μg/L. The coefficient of determination was R²> 0.99 (0–100 μg/L). Carryover at 10,000 μg/L was <1 μg/L and below the LOQ. At 10,000 μg/L no cross-talk was observed between quantifier and qualifier MRM for the adamantyl-type NOID marker and the IS rimantadine. Inter-assay precision at ±25% of the 5 μg/L positive cut-off threshold (n = 10 analysed over 2 weeks) was 9.9% and 11.1%, respectively. Matrix effects (ME) were assessed according to the method of Gandhi et al.²⁸ Absolute matrix effects (AME), true recovery and process efficiency were all within our acceptable target for a screening method of 100 ± 30%.

Urine screening results

From July 2014 to February 2015 we received 14,465 urine samples from 7232 individuals for urine drugs of abuse screening by LC-MS/MS. Of these samples 29 (0.2%) were rejected after the integrity check (creatinine concentration <1.0 mmol/L) indicated adulteration. The majority of our workload (86%) are clinical requests with 6% originating from our own Accident & Emergency (A&E) and inpatients, 16.5% from other hospitals, 63% from mental health trusts and 0.7% other. The remaining 13.8% of requests were for private testing for medico-legal purposes such as workplace testing. More samples were received from males 10,711 (74%) compared to females 3601 (24.9%) with 153 (1.1%) sex not stated. The mean age of patients tested was 34.6 years.

Of the 14,436 urine samples screened 296 (2.05% total workload) tested positive for the adamantyl-type NOID marker and these results were also confirmed by UPLC-Tof analysis. Overall detection rates for the adamantyl-type NOID marker from the different requesting sources were 2.5% from our own Trust, 1.4% other hospitals, 2.4% mental health and 0.9% private testing. The total monthly positive detection rates for the adamantyl-type NOID marker from July 2014 to February 2015 range from 1.1% to 2.9% and are summarized in Table 2.

Table 2.

Summary of LC-MS/MS urine screen %positive monthly detection rates for adamantyl-type NOID marker.

Month	%positive detection rate for adamantyl-type NOID marker	Total samples tested
July 2014	1.1	1602
August	2.4	1719
September	1.5	1904
October	1.6	2057
November	1.8	1898
December	2.9	1776
January 2015	2.7	1909
February	2.4	1571
Total	2.0	14,436

Overall, 180 individuals tested positive for the adamantyl-type NOID marker which is 2.5% of the total 7232 individuals tested over the eight months. The majority of these 180 individuals (86.5%) were male. The mean age was 37 years (range 3 days to 56 years). The youngest patient to test positive was a three-day-old male infant with this request querying intrauterine drug exposure which we confirmed. Repeat testing was responsible for the remaining 116 positive adamantyl-type NOID marker results we reported. One 53-year-old male tested positive for the adamantyl-type NOID marker on 19 different occasions. It was confirmed by the case worker that this individual is a regular user of ‘Black Mamba’ a common generic street name used for any herb type legal high which is typically added to smoking products.

Discussion

In January 2014, we received patient samples as well as suspect materials for screening for NPS from a secure mental health facility, where there was a high suspicion that patients were abusing a synthetic cannabinoid (NOID) type legal high. By testing the suspected legal high product (Herbal Haze II), and contents of associated hand-rolled cigarettes, we were able to confirm the suspicion of NOID involvement with the detection of third generation NOIDS, including the adamantyl-type NOIDS AKB-48, 5F-AKB-48 and STS-135. None of these adamantyl-type NOIDS were detected in the patient urine samples. This is not unexpected as NOIDS are known to undergo extensive metabolism.^27,28 We then used UPLC-MS/Tof to also screen for 18 potential adamantyl-type NOID metabolites, seven of which were proposed by us, and 11 by Gandhi et al.²⁸ The goal of this study was to identify any adamantyl-type NOID metabolite(s) with potential as a screening marker. Only one metabolite, 1-adamantylamine (C₁₀H₁₇N), was detected in all five urine samples from the test case. Using our method we failed to detect any of the 11 major metabolites proposed by Gandhi et al.²⁸ Likewise Gandhi et al.²⁸ failed to identify our proposed metabolite 1-adamantylamine.

There are several major differences between our metabolite study and that of Gandhi et al.²⁸ In addition to the extraction method, Gandhi et al.²⁸ used human hepatocytes incubated in a certified Cerilliant standard of 10 µmol/L AKB-48 at 37℃ for up to 3 h. For our metabolite study we tested random urine from actual patients smoking a legal high herbal blend of different adamantyl-type NOIDS (AKB-48, 5F-AKB-48 and STS-135), any of which could be responsible for yielding the 1-adamantylamine we detected, either through metabolism or as a breakdown of the legal high product during smoking. The 1-adamantylamine may also be present as an artefact of manufacturing, and indeed was detected in Herbal Haze II when re-analysed at high concentration (1 mg/mL). This highlights the advantage of using real patient samples when investigating potential markers for screening in biological samples.

NOIDS are far more potent than cannabis.⁴ As demonstrated in Figure 1 in the test case, only about 5% of the cigarette contents are legal high material. Legal high products are sold as not for human consumption and as such do not include instructions for use and this presents a particular risk to new users unfamiliar with the potency of NOIDS, as they are more likely to unintentionally overdose. Often the only information available to clinicians on what a patient may have taken is the packaging, which can be misleading. This uncertainty in identifying what an individual has taken further highlights the need for relevant NPS screening, such as our admanatyl-type NOID marker, to support both patient diagnosis and management.

Our admanatyl-type NOID marker has now been in routine use for over eight months. Overall the positive detection rate was 2.0%. To put this into the context over the same period the positive detection rate for amphetamine, metamphetamine plus ecstasy (MDMA and MDA) was 1.7%. According to the most recent FEWS report the most popular NOIDs currently on sale in UK in 2014 were the adamantyl-type NOIDS.²³

Our adamantyl-type marker can easily be incorporated into a routine urine LC-MS/MS screen with minimal financial implications. This enables us to report results in a clinically relevant time frame of hours rather than days. Whilst our marker is well suited for clinical use the inability to identify the actual parent adamantyl-NOID may make it unsuitable for forensic use.

In developing our adamantyl-type NOID marker we have taken a pragmatic approach and we do not have any data on the positive or false negative rate for example. Clinical details from the patient group under study are notoriously unreliable and herbal legal high products contain a blend of NOIDS. An important part of ongoing validation of all our methods includes feedback from users. For example there is a possibility that patients maybe on legitimate medication that contains an adamanatyl group, though this has not been observed so far. What is significant from our routine screening of 14,436 urine samples is we have yet to receive a single challenge to any of the positive adamantyl-type NOID marker positive results we have reported. Many questions still remain unanswered about our admanatyl-type NOID marker including the detection window in urine, which adamantyl-type NOIDS our marker is specific for, and if it is a metabolite or artefact of manufacturing, and we are continuing to work with our clinicians to help determine these.

In conclusion we have developed and demonstrated the use of a marker for the most common third generation NOIDS currently in use in the UK and used it to look for NOID use in over 13,000 patient urines. This marker can easily be incorporated into routine LC-MS/MS drug screening panel alongside classic drugs of abuse making it ideal for clinical use where accurate and rapid results are needed.

Footnotes

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Both authors run a clinical service in the United Kingdom for Toxicology.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Sandwell & West Birmingham Hospital NHS Trust, Department of Clinical Biochemistry.

Ethical approval

Not applicable.

Guarantor

LF and JB.

Contributorship

LF undertook the research and wrote the first draft. JB edited the manuscript.

References

Fantegrossi

Moran

Radominska-Pandya

. Distinct pharmacology and metabolism of K2 synthetic cannabinoids compared to Δ9-THC: mechanism underlying greater toxicity? Life Sci 2014; 97: 45–54.

Thakur

Nikas

Mariyannis

. CB1 cannabinoid receptor ligands. Mini Rev Med Chem 2005; 5: 631–640.

Aston

Wright

McPartland

. Cannabinoid CB1 and CB2 receptor ligand specificity and the development of CB-2 selective agonists. Curr Med Chem 2008; 15: 1428–1443.

Atwood

Huffman

Straiker

. JWH018, a common constituent of spice herbal blend is a potent and efficacious cannabinoid receptor agonist. Br J Pharmacol 2010; 160: 585–593.

LaPoint

James

Moran

. Severe toxicity following synthetic cannabinoid ingestion. Clin Toxicol 2011; 45: 1186–1194.

Simmons

Cookman

Kang

. Three cases of “spice” exposure. Clin Toxicol 2011; 49: 431–433.

Cohen

Morrison

Grrenberg

. Clinical presentation of intoxication due to synthetic cannabinoids. Paediatrics 2012; 129: e1064–e1067.

Harris

Brown

. synthetic cannabinoid intoxication: a case series and review. J Emerg Med 2013; 44: 360–366.

Hermanns-Clausen

Kneisel

Szabo

. Acute toxicity due to the confirmed consumption of synthetic cannabinoids: clinical and laboratory findings. Addiction 2013; 108: 534–544.

10.

Papanti

Schifano

Botteon

. “Spiceophrenia”: a systematic overview of “spice”-related psychopathological issues and a case report. Hum Psychopharmacol 2013; 28: 379–389.

11.

Rodgman

Kinzie

Leimbach

. Bad Mojo: use of the new marijuana leads to more and more ED visits for acute psychosis. Am J Emerg Med 2011; 29: 232–232.

12.

Benford

Caplan

. Psychiatric sequelae of Spice, K2, and synthetic cannabinoid receptor agonists. Psychosomatics 2011; 52: 295–295.

13.

Trecki

Gerona

Schwartz

. Synthetic cannabinoid-related illnesses and deaths. N Engl J Med 2015; 373: 103–107.

14.

Law

Schier

Martin

. Notes from the field: increase in reported adverse health effects related to synthetic cannabinoid use – United States, January–May 2015. MMWR 2015; 64: 618–619.

15.

Advisory Council on the Misuse of Drugs (ACMD), Consideration of the Major Cannabinoid Agonists. Home Office, London, 2009. https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/119149/acmd-report-agonists.pdf (accessed January 2015).

16.

Howlett

. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacol Rev 2002; 54: 161–202.

17.

Dargan

Husdson

Ramsey

. The impact of changes in UK classification of the synthetic cannabinoid receptor agonists in ‘Spice’. Int J Drug Policy 2011; 22: 274–277.

18.

Change To The Misuse Of Drugs Act 1971, 2013. https://www.gov.uk/government/publications/change-to-the-misuse-of-drugs-act-1971 (accessed January 2015).

19.

Uchiyama

Kawamura

Kikura-Hanajiri

. Identification of two new-type synthetic cannabinoids, N-(1-adamantyl)-1-pentyl-1H-indole-3-carboxamide (APICA) and N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (APINACA), and detection of five synthetic cannabinoids, AM-1220, AM-2233, AM-1241, CB-13 (CRA-13), and AM-1248, as designer drugs in illegal products. Forensic Toxicol 2012; 30: 114–125.

20.

Advisory council misuse of drugs Third Generation Synthetic cannabinoids. Home Office, London, 2014. https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/380161/CannabinoidsReport.pdf (accessed January 2015).

21.

Hayden

Spyker

AMDA

Hoffman

. Comparative single-dose pharmacokinetics of amantadine hydrochloride and rimantadine hydrochloride in young and elderly adults. Antimicrob Agents Chemother 1985; 28(2): 216–221.

22.

Liu

Obando

Liao

. The many faces of the adamantyl group in drug design. Eur J Med Chem 2011; 46: 1949–1963.

23.

Annual Report on the Home Office Forensic Early Warning System (FEWS) A system to identify New Psychoactive Substances in the UK. ISBN: 978-1-78246-462-4 Published by Home Office©. Crown Copyright 2014.

24.

Lindigkeit

Boeheme

Eiserloh

. Spice: a never ending story? Forensic Sci Int 2009; 191: 58–63.

25.

Grabenauer

Krol

Wiley

. Analysis of synthetic cannabinoids using high-resolution mass spectrometry and mass defect filtering: implication for non-targeted screening of designer drugs. Anal Chem 2013; 84: 5574–5581.

26.

Shanks

Behonik

Dahn

. Identification of novel third generation synthetic cannabinoids in products by ultra-performance liquid chromatography and time of flight mass spectrometry. J Anal Toxicol 2013; 37: 517–525.

27.

Kneisel

Westphal

Bisel

. Identification and structural characterisation of the synthetic cannabinoid 3-(1-adamantonyl)-1-pentylindole as ana additive in ‘herbal incense’. J Mass Spectrom 2012; 47: 195–200.

28.

Gandhi

Zhu

Pang

. First characterization of AKB-48 metabolism, a novel synthetic cannabinoid, using human hepatocytes and high-resolution mass spectrometry. AAPS J 2013; 15: 1091–1098.

29.

Arnston

Ofsa

Lancaster

. Validation of a novel immunoassay for the detection of synthetic cannabinoids and metabolites in urine specimens. J Anal Toxicol 2013; 37: 284–290.

30.

Kronstrand

Brinkhagen

Birath-Karlsson

. LC-QTof_MS as a superior strategy to immunoassay for the comprehensive analysis of synthetic cannabinoids in urine. Anal Bioanal Chem 2014; 406: 3599–3609.

31.

Sobolevsky

Prasolov

Rodchenkov

. Detection of JWH-018 metabolites in smoking mixture post-administration urine. Forensic Sci Int 2010; 200: 141–147.

32.

Hudson

Ramsey

. The emergence and analysis of synthetic cannabinoids. Drug Test Anal 2011; 3: 466–478.

33.

Yanes

Lovett

. High-throughput bioanalytical method for analysis of synthetic cannabinoid metabolites in urine using salting-out sample preparation and LC-MS/MS. J Chromatogr B 2012; 909: 42–50.

34.

De Jager

Warner

Henman

. Lc-MS/MS method for quantitation of metabolites of eight commonly used synthetic cannabinoids in human urine – an Australian perspective. J Chromatogr B 2012; 897: 22–31.

35.

Kröner

Specncer

. A pragmatic approach to detect SPICE-metabolites in urine with HPLC-MS/MS. Toxichem Kimtech 2013; 80: 375–380.

36.

Rosano

Wood

Ihenetu

. Drug screening in medical casework by high resolution mass spectrometry (UPLC-MS^E-Tof). J Anal Toxicol 2013; 37: 580–593.

37.

European Workplace Drug Testing Society. European guidelines for legally defensible workplace drug testing (urine drug testing), v1.0 2002. http//www.ewdts.org/data/uploads/documents/wedtsguidelines.pdf (accessed January 2015).

38.

Guidance for Industry Bioanalytical method validation. US department of Health and human services, substance Abuse and mental health services administration, Rockville, MD, 2001. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf (accessed January 2015).

39.

European union decision 2002/657/EC 17.08.2002: commission decision laying down performance criteria for the analytical methods to be used for certain substances and residues thereof in live animals and animal products. Off J Eur Comm 2002; 221: 8–32.

40.

Peters

Drummer

Musshoff

. Validation of new methods. For Sci Int 2007; 165: 216–224.

41.

Whitmire

Ammermann

Lisio

. LC-MS/MS bioanalysis method development, validation, and sample analysis: points to consider when conducting nonclinical and clinical studies in accordance with current regulatory guidelines. J Anal Bioanal Tech 2011, pp. S4–001.

42.

Scientific working group for forensic toxicology (SWGTOX) standard practices for method validation in forensic toxicology. SWGTOX Doc 003, Revision 1, 2013. http://www.nyc.gov/html/ocme/downloads/pdf (accessed January 2015).

1-Adamantylamine a simple urine marker for screening for third generation adamantyl-type synthetic cannabinoids by ultra-performance liquid chromatography tandem mass spectrometry

Abstract

Background

Methods

Results

Conclusion

Keywords

Introduction

Experimental

Chemicals and solutions

Validation samples

Routine urine samples for drugs of abuse screening

Preparation of non-biological samples

Preparation of urine samples

UPLC-Tof analysis

UPLC-MS/MS urine drug screen

Results

Validation samples

Validation UPLC-Tof method for adamantyl-type NOID marker

Validation of LC-MS/MS method for adamantyl-type NOID marker

Urine screening results

Discussion

Footnotes

Declaration of conflicting interests

Funding

Ethical approval

Guarantor

Contributorship

References