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Evaluation of commercial multi-drug oral fluid devices to identify 39 new amphetamine-designer drugs
Legal Medicine 16 (2014) 106–109
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
Evaluation of commercial multi-drug oral fluid devices to identify 39 new amphetamine-designer drugs Maria Nieddu a, Lucia Burrai a, Claudia Trignano b, Gianpiero Boatto a,⇑ a b
Dipartimento di Chimica e Farmacia, Università di Sassari, Via Muroni 23/a, 07100 Sassari, Italy Dipartimento di Scienze Biomediche, Università di Sassari, Via Matteotti 16, 07100 Sassari, Italy
a r t i c l e
i n f o
Article history: Received 19 July 2013 Received in revised form 4 November 2013 Accepted 3 December 2013 Available online 12 December 2013 Keywords: Oral fluid devices (OFDs) Legal highs Amphetamine Designer drugs Immunoassay screening
a b s t r a c t Recently, the diffusion on the black market of new psychoactive substances not controlled and often sold as ‘legal highs’, is exponentially increasing in Europe. Generally, the first analysis for these drugs involves an immunoassay screening in urine or plasma. Actually, there is growing interest in the use of oral fluid (OF) as alternative specimen over conventional biological fluids for drug testing, because of the significant advantages, as a non-invasive collection under direct observation without undue embarrassment or invasion of privacy, and a good correlation with plasma analytical data. Few assays have been developed for detection of new psychoactive compounds in biological samples, so it is important to investigate how they may or may not react in pre-existing commercial immunoassays. In this paper, two different multi-drugs oral fluid screen devices (OFDs) (ScreenÒ Multi-Drug OFD and GIMA One Step Multi-Line Screen Test OFD) were evaluated to determine the cross-reactivity of thirty-nine new amphetamine designer drugs, including twelve substances officially recognized as illicit by italian legislation. Crossreactivity towards most drugs analyzed was <1 in assays targeting amphetamine (AMP) or methamphetamine (MET). Only two (p-methoxyamphetamine and p-methoxymethamphetamine) of all tested amphetamines gave a positive result. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Recently, the diffusion on the drug market of new psychoactive substances not controlled and often sold as ‘legal highs’ is exponentially increasing in Europe. ‘Legal highs’ are substances of synthetic or natural origin having psychotropic properties. More than 280 legal highs and other new psychoactive synthetic drugs are actually monitored by EU drug agency [1]. Generally, the first analysis for these drugs involves an immunoassay screening in urine or plasma. If the screening test is positive, additional tests by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) must be performed. Actually, there is growing interest in the use of oral fluid (OF) as alternative specimen over conventional biological fluids (urine, blood) for drug testing. The great advantages of these matrix are a non-invasive collection under direct observation without undue embarrassment or invasion of privacy, and a good correlation with plasma analytical data [2–4]. Drug concentrations in OF reflect the free, unbound parent drug and lipophilic metabolites circulating in the blood. Since these are the forms of the drug which cross the blood–brain barrier and effect performance and behavior, OF is a ⇑ Corresponding author. Tel.: +39 079228725; fax: +39 079228727. E-mail addresses: [email protected] (M. Nieddu), [email protected] (L. Burrai), [email protected] (C. Trignano), [email protected] (G. Boatto). 1344-6223/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2013.12.001
good specimen for detecting drug involvement in driving behavior or impairment of performance. Drug and lipophilic metabolite concentrations in OF are a function of the drug’s pKa, plasma, and OF pH and the fraction of drug bound to OF and plasma protein [2]. Italian legislation on traffic safety has recently accepted oral fluid as a possible alternative biological sample for toxicological analysis [5]. The national guidelines establishing how to perform the analysis and the practical issues regarding which device and cut-off values should be used are however still undetermined. There have been numerous studies regarding the performance characteristics of commonly used OFDs for known abuse drugs [6–9]. Most of these studies reported good agreement between screening and confirmatory results. Differently, few assays have been developed for detection of new psychoactive compounds; it is important to investigate how new ‘legal highs’ may or may not react in pre-existing and broad diffused commercial immunoassay kits. The main objective of this study is to evaluate whether two commercial OFDs (ScreenÒ Multi-Drug OFD and GIMA One Step Multi-Line Screen Test OFD) can be an effective alternative for screening thirty-nine new amphetamine designer drugs. The ability and degree of reliability of these devices in spiked samples are examined. Between the thirty-nine amphetamine compounds analyzed, twelve (PMA, PMMA, 2,5-DMA, DOB, DOM, DOET, 2C-B, 2C-I, 2C-T-2, 2C-T-7, TMA, TMA-2) are already classified as
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psychotropic substances by Italian legislation [10,11], so their consume and diffusion are punishable by law. A low cross-reactivity towards these compounds may result in these abused substances not being detected in forensic case samples.
2. Materials and methods 2.1. Chemicals and reagents Hydrochloride salt forms of p-methoxyamphetamine (PMA), p-methoxymethamphetamine (PMMA), 2,5-dimethoxy-amphetamine (2,5-DMA), 2,5-dimethoxy-4-bromoamphetamine (DOB), 2, 5-dimethoxy-4-chloroamphetamine (DOC), 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-iodoamphetamine (DOI), 2,5-dimethoxy-4-methylamphetamine (DOM), 2,5-dimethoxy-4nitroamphetamine (DON), 2,5-dimethoxy-4-propylamphetamine (DOPR), 2,5-dimethoxy-4-bromophenethylamine (2C-B), 2,5-dimethoxy-4-iodophenethylamine (2C-I), 2,5-dimethoxy-4nitrophenethylamine (2C-N), 2,5-dimethoxy-4-methylphenethylamine (2C-M), 2,5-dimethoxy-4-methylthiophenethylamine (2CT), 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), 2,5-dimethoxy-4cyclo hexylthiophenethylamine (2C-T-5), 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), 2,5-dimethoxy-4-(2-methoxyethyl) thiophenethylamine (2C-T-13), 2,5-dimethoxy-4-isobutylthiophenethylamine (2C-T-17), 2,5-dimethoxy-4-methylthioamphetamine (ALEPH), 2,5-dimethoxy-4-ethylthioamphetamine (ALEPH-2),
2,5-dimethoxy-4-cyclohexylthioamphetamine (ALEPH-5), 2,5-dimethoxy-4-propylthioamphetamine (ALEPH-7), 2,5-dimethoxy-4-cyclopropylmethylthioamphetamine (ALEPH-8), 2,5-dime thoxy-4-(2-methoxyethyl) thioamphetamine (ALEPH-13), 2,5dimethoxy-4-isobutylthioamphetamine (ALEPH-17), 3,4,5-trimeth oxyamphetamine (TMA), 2,4,5-trimethoxyamphetamine (TMA-2), 2,3,4-trimethoxyamphetamine (TMA-3), 2,4,6-trimethoxyamphet amine (TMA-6), 3,4-methylenedioxy-N-isopropylamphetamine (MDIP), 3,4-methylenedioxy-N-benzylamphetamine(MDBZ), 3,4-methylenedioxy-N-cyclopropylmethylamphetamine (MDCPM), 2-(3, 4-methylenedioxyphenyl)-2-methoxyethylamine (BOH), 4methyl-2, 5-dimethoxy-b-hydroxyphenethylamine (BOHD), 2-(4bromo-2, 5-dimethylenedioxyphenyl)-2-methoxyethylamine (BOB), 2-(2,5-dimethoxy-4methylphenyl)-2-methoxyethylamine (BOD) were synthesized in our laboratory according to the methods of Shulgin and Shulgin [12]. Aqueous stock solutions of all the mentioned amphetamines were prepared at 10 lg/ml, and they were diluted to appropriate concentrations (10; 20; 50; 100; 200; 1000; 2000; 5000 ng/ml) with a drug-free pooled oral fluid sample collected by 10 volunteers. 2.2. Immunoassay OF tests Oral fluid screening tests were performed using ScreenÒ MultiDrug OFD (SCREEN ITALIA Srl, Perugia, Italy), and GIMA One Step Multi-Line Screen Test OFD (GIMA Spa, Gessate, Milan, Italy), according to the manufacturer’s instructions [13,14]. These multi-drug tests are made to detect amphetamine (AMP), cocaine
Table 1 Cross-reactivities of thirty-nine amphetamine designer drugs on ScreenÒ test. Analyte
p-Methoxyamphetamine (PMA) p-Methoxymethamphetamine (PMMA) 2,5-Dimethoxyamphetamine (2,5-DMA) 2,5-Dimethoxy-4-bromoamphetamine (DOB) 2,5-Dimethoxy-4-chloroamphetamine (DOC) 2,5-Dimethoxy-4-ethylamphetamine (DOET) 2,5-Dimethoxy-4-iodoamphetamine (DOI) 2,5-Dimethoxy-4-methylamphetamine (DOM) 2,5-Dimethoxy-4-nitroamphetamine (DON) 2,5-Dimethoxy-4-propylamphetamine (DOPR) 2,5-Dimethoxy-4-bromophenethylamine (2C-B) 2,5-Dimethoxy-4-iodophenethylamine (2C-I) 2,5-Dimethoxy-4-nitrophenethylamine (2C-N) 2,5-Dimethoxy-4-methylphenethylamine (2C-M) 2,5-Dimethoxy-4-methylthiophenethylamine (2C-T) 2,5-Dimethoxy-4-ethylthiophenethylamine (2C-T-2) 2,5-Dimethoxy-4-isopropylthiophenethylamine (2C-T-4) 2,5-Dimethoxy-4-cyclohexylthiophenethylamine (2C-T-5) 2,5-Dimethoxy-4-n-propylthiophenethylamine (2C-T-7) 2,5-Dimethoxy-4-(2-methoxyethyl)thiophenethylamine (2C-T-13) 2,5-Dimethoxy-4-i-butylthiophenethylamine (2C-T-17) 2,5-Dimethoxy-4-methylthioamphetamine (ALEPH) 2,5-Dimethoxy-4-ethylthioamphetamine (ALEPH-2) 2,5-Dimethoxy-4-cyclohexylthioamphetamine (ALEPH-5) 2,5-Dimethoxy-4-n-propylthioamphetamine (ALEPH-7) 2,5-Dimethoxy-4-cyclopropylmethylthioamphetamine (ALEPH-8) 2,5-Dimethoxy-4-(2-methoxyethyl) thioamphetamine (ALEPH-13) 2,5-Dimethoxy-4-isobutylthioamphetamine (ALEPH-17) 3,4,5-Trimethoxyamphetamine (TMA) 2,4,5-Trimethoxyamphetamine (TMA-2) 2,3,4-Trimethoxyamphetamine (TMA-3) 2,4,6-Trimethoxyamphetamine (TMA-6) 3,4-Methylenedioxy-N-isopropylamphetamine (MDIP) 3,4-Methylenedioxy-N-benzylamphetamine(MDBZ) 3,4-Methylenedioxy-N-cyclopropylmethylamphetamine (MDCPM) 2-(3,4-Methylenedioxyphenyl)-2-methoxyethylamine (BOH) 2-(4-Bromo-2,5-dimethylenedioxyphenyl)-2-methoxyethylamine (BOB) 2-(2,5-Dimethoxy-4methylphenyl)-2-methoxyethylamine (BOD) 2-(2,5-Dimethoxy-4methylphenyl)-2-idroxyethylamine (BOHD)
% Cross-reactivity AMP
MET
25 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
<1 250 <5 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
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Table 2 Cross-reactivities of thirty-nine amphetamine designer drugs on GIMA test. Analyte
% Cross-reactivity
p-Methoxyamphetamine (PMA) p-Methoxymethamphetamine (PMMA) 2,5-Dimethoxyamphetamine (2,5-DMA) 2,5-Dimethoxy-4-bromoamphetamine (DOB) 2,5-Dimethoxy-4-chloroamphetamine (DOC) 2,5-Dimethoxy-4-ethylamphetamine (DOET) 2,5-Dimethoxy-4-iodoamphetamine (DOI) 2,5-Dimethoxy-4-methylamphetamine (DOM) 2,5-Dimethoxy-4-nitroamphetamine (DON) 2,5-Dimethoxy-4-propylamphetamine (DOPR) 2,5-Dimethoxy-4-bromophenethylamine (2C-B) 2,5-Dimethoxy-4-iodophenethylamine (2C-I) 2,5-Dimethoxy-4-nitrophenethylamine (2C-N) 2,5-Dimethoxy-4-methylphenethylamine (2C-M) 2,5-Dimethoxy-4-methylthiophenethylamine (2C-T) 2,5-Dimethoxy-4-ethylthiophenethylamine (2C-T-2) 2,5-Dimethoxy-4-isopropylthiophenethylamine (2C-T-4) 2,5-Dimethoxy-4-cyclohexylthiophenethylamine (2C-T-5) 2,5-Dimethoxy-4-n-propylthiophenethylamine (2C-T-7) 2,5-Dimethoxy-4-(2-methoxyethyl)thiophenethylamine (2C-T-13) 2,5-Dimethoxy-4-i-butylthiophenethylamine (2C-T-17) 2,5-Dimethoxy-4-methylthioamphetamine (ALEPH) 2,5-Dimethoxy-4-ethylthioamphetamine (ALEPH-2) 2,5-Dimethoxy-4-cyclohexylthioamphetamine (ALEPH-5) 2,5-Dimethoxy-4-n-propylthioamphetamine (ALEPH-7) 2,5-Dimethoxy-4-cyclopropylmethylthioamphetamine (ALEPH-8) 2,5-Dimethoxy-4-(2-methoxyethyl) thioamphetamine (ALEPH-13) 2,5-Dimethoxy-4-isobutylthioamphetamine (ALEPH-17) 3,4,5-Trimethoxyamphetamine (TMA) 2,4,5-Trimethoxyamphetamine (TMA-2) 2,3,4-Trimethoxyamphetamine (TMA-3) 2,4,6-Trimethoxyamphetamine (TMA-6) 3,4-Methylenedioxy-N-isopropylamphetamine (MDIP) 3,4-Methylenedioxy-N-benzylamphetamine(MDBZ) 3,4-Methylenedioxy-N-cyclopropylmethylamphetamine (MDCPM) 2-(3,4-Methylenedioxyphenyl)-2-methoxyethylamine (BOH) 2-(4-Bromo-2,5-dimethylenedioxyphenyl)-2-methoxyethylamine (BOB) 2-(2,5-Dimethoxy-4methylphenyl)-2-methoxyethylamine (BOD) 2-(2,5-Dimethoxy-4methylphenyl)-2-idroxyethylamine (BOHD)
(COC), methamphetamine (MET), tetrahydrocannabinol (THC), phencyclidine (PCP), and opiates (OPI), and are based on the principle of competitive binding: drugs which may be present in the oral fluid specimen compete against their respective drug conjugate for binding sites on their specific antibody. A drug, if present in the oral fluid specimen below its cutoff concentration, is not able to saturate the binding site of its specific antibody. Measurements were performed without the need of instruments. Oral fluid samples have been collected in a clean and dry container and analyzed at room temperature (25 °C) within 1 h from the specimen collection. The sponge end of the collector has been vertically immersed into the oral fluid sample until the sponge is completely saturated. Results were read at 10 min. The absence or the presence of a colored line, even a faint line, in the test line region, indicates a positive or a negative result, respectively; the presence of a colored line in the control line region is required to validate both negative and positive results. Each measurement was performed in triplicate (n = 3). The cross-reactivity was calculated using Eq. (1), where C50 was the concentration of the drug necessary to produce a positive response, equivalent to 50 ng/ml of target drug. For this purpose, the concentration of 50 ng/ml was chosen as the manufacturer’s recommended cutoff:
% Cross reactivity ¼
50 100 C 50
ð1Þ
AMP
MET
50 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
<1 25 <5 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
3. Results and discussion The cross-reactivity profiles of the thirty-nine amphetamines studied on two commercial OFDs are shown in Tables 1 and 2. All tests gave overlapped results with minimal variations regarding PMA and PMMA, as we expected considering PMA and PMMA are p-substituted analogous of amphetamine and methamphetamine, and cross-reacted to AMP and MET assay, respectively. Regarding ScreenÒ Multi-Drug device, PMA showed cross-reactivity four times lower than amphetamine (% cross-reactivity = 25) whereas PMMA had a cross-reactivity two-half times greater than methamphetamine (% cross-reactivity = 250). However, tests conducted by GIMA device showed slightly different % cross-reactivity, 50 for PMA and 25 for PMMA, corresponding to a concentration of 100 and 200 ng/ml, respectively. Concentrations of 5000 ng/ml were not sufficient to produce a positive response for all other amphetamines analyzed. This clearly demonstrates that although these kits are extremely effective for the target drugs for which they were intended (amphetamine and methamphetamine), they cannot be used to reliably identify all designers drugs studied, even at concentrations that would greatly exceed those expected in case samples [15]. These results demonstrate that some substances officially considered illicit by Italian law (2,5-DMA, DOET, DOM, 2C-B, 2C-I, 2C-T-2, 2C-T-7, TMA and TMA-2) [10,11] and recently appeared in clandestine market, can give negative results with screening
M. Nieddu et al. / Legal Medicine 16 (2014) 106–109
tests commonly used. These data are significantly important from forensic and toxicological point of view because a negative result is not examined with additional test of confirm. For this reason, when circumstances surrounding the case suggest the usage of drugs, also a negative result must be investigated by instrumental techniques based on mass spectrometric detection. 4. Conclusions This study verifies the cross-reactivities of thirty-nine new amphetamine-designer drugs on two commercial OFDs (ScreenÒ Multi-Drug OFD and GIMA One Step Multi-Line Screen Test OFD). The present data can offer useful information when it is needed to explain the screening results in drug testing laboratories: results showed cross-reactivity <1 towards most drugs analyzed in assays targeting amphetamine (AMP) or methamphetamine (MET). Only two (p-methoxyamphetamine and p-methoxymethamphetamine) of the thirty-nine amphetamines tested gave a positive result. For identifying these drugs, the screening tests commercially available are often not sufficient. This evidences the need to develop new kits that enable to control the misuse of new amphetamine-like drugs. Acknowledgement The authors are grateful to English Centre of Sassari University for the English editing of the manuscript. References [1] European Drug Report. Trends and developments. Lisbon: EMCDDA; 2013. [2] Spiehler V, Baldwin D, Hand C. Cut-off concentrations for drugs of abuse in saliva for DUI, DWI or other driving-related crimes. Probl Forensic Sci 2000;XLII:160–8. [3] Kintz P, Samyn N. Use of alternative specimens: drugs of abuse in saliva and doping agents in hair. Ther Drug Monit 2002;24:239–46.
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[4] Gjerde H, Verstraete AG. Estimating equivalent cutoff thresholds for drugs in blood and oral fluid using prevalence regression: a study of tetrahydrocannabinol and amphetamine. Forensic Sci Int 2011;212:26–30. [5] Italian Decree ‘‘Legge 29 luglio 2010, n. 120. Disposizioni in materia di sicurezza stradale. Art. 33: modifiche agli articoli 186 e 187 e introduzione dell’articolo186-bis del decreto legislativo n. 285 del 1992’’ [Italian Official Gazette No. 175/ 2010]. [6] Strano-Rossi S, Castrignanò E, Anzillotti L, Serpelloni G, Mollica R, Tagliaro F, et al. Evaluation of four oral fluid devices (dds1, drugtest 50001, drugwipe 5+1 and rapidstat1) for on-site monitoring drugged driving in comparison with UHPLC–MS/MS analysis. Forensic Sci Int 2012;221:70–6. [7] Souza DZ, Boehl PO, Comiran E, Prusch DS, Zancanaro I, Fuentefria AM, et al. Which amphetamine-type stimulants can be detected by oral fluid immunoassays? Ther Drug Monit 2012;34:98–109. [8] Pehrsson A, Blencowe T, Vimpari K, Impinen A, Gunnar T, Lillsunde P. Performance evaluation of the DrugWipeÒ 5/5+ on-site oral fluid screening device. Int J Legal Med 2011;125:675–83. [9] Wille SM, Samyn N, Ramírez-Fernández Mdel M, De Boeck G. Evaluation of onsite oral fluid screening using Drugwipe-5 (+), RapidSTAT and Drug Test 5000 for the detection of drugs of abuse in drivers. Forensic Sci Int 2010;198: 2–6. [10] Decree of the Italian Republic President, no. 309/1990. ‘‘Testo unico delle leggi in materia di disciplina degli stupefacenti e sostanze psicotrope, prevenzione, cura e riabilitazione dei relativi stati di tossicodipendenza’’ – ‘‘Law for the discipline of abuse drugs and psychotropic substances, prevention, treatment and rehabilitation of drug addiction – Italian’’ [Italian Official Gazette No. 25/ 1990, Suppl. Ord. No. 67]. [11] Italian Decree ‘‘Provvedimento no. 99/cu del 30/10/2007, Intesa, ai sensi dell’articolo 8, comma 6, della legge 5 giugno 2003, n. 131, in materia di accertamento di assenza di tossicodipendenza – Italian’’. ‘‘Measure no. 99/cu del 30/10/2007, Agreement for the assessment of a drug addiction status, according to the article 8, comma 6, of the law n. 131 of05/06/2003’’ [Italian Official Gazette No. 266/ 2007]. [12] Shulgin A, Shulgin A. Pihkal, a chemical love story. Berkeley (CA): Transform Press; 1991. [13] Package insert. ScreenÒ Multi-drug OFD Oral Fluid Drug Screen Device for AMP/COC/THC/MET/OPI/PCP. SCREEN ITALIA Srl, Perugia, Italy. [14] Package insert. GIMA One Step Multi-Line Screen Test OFD for AMP/COC/THC/ MET/OPI/PCP. GIMA Spa, Gessate, Milan, Italy. [15] Nakanishi K, Miki A, Zaitsu K, Kamata H, Shima N, Kamata T, et al. Crossreactivities of various phenethylamine-type designer drugs to immunoassays for amphetamines, with special attention to the evaluation of the one-step urine drug test Instant-View™, and the EmitÒ assays for use in drug enforcement. Forensic Sci Int 2012;217:174–81.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
Evaluation of commercial multi-drug oral fluid devices to identify 39 new amphetamine-designer drugs Maria Nieddu a, Lucia Burrai a, Claudia Trignano b, Gianpiero Boatto a,⇑ a b
Dipartimento di Chimica e Farmacia, Università di Sassari, Via Muroni 23/a, 07100 Sassari, Italy Dipartimento di Scienze Biomediche, Università di Sassari, Via Matteotti 16, 07100 Sassari, Italy
a r t i c l e
i n f o
Article history: Received 19 July 2013 Received in revised form 4 November 2013 Accepted 3 December 2013 Available online 12 December 2013 Keywords: Oral fluid devices (OFDs) Legal highs Amphetamine Designer drugs Immunoassay screening
a b s t r a c t Recently, the diffusion on the black market of new psychoactive substances not controlled and often sold as ‘legal highs’, is exponentially increasing in Europe. Generally, the first analysis for these drugs involves an immunoassay screening in urine or plasma. Actually, there is growing interest in the use of oral fluid (OF) as alternative specimen over conventional biological fluids for drug testing, because of the significant advantages, as a non-invasive collection under direct observation without undue embarrassment or invasion of privacy, and a good correlation with plasma analytical data. Few assays have been developed for detection of new psychoactive compounds in biological samples, so it is important to investigate how they may or may not react in pre-existing commercial immunoassays. In this paper, two different multi-drugs oral fluid screen devices (OFDs) (ScreenÒ Multi-Drug OFD and GIMA One Step Multi-Line Screen Test OFD) were evaluated to determine the cross-reactivity of thirty-nine new amphetamine designer drugs, including twelve substances officially recognized as illicit by italian legislation. Crossreactivity towards most drugs analyzed was <1 in assays targeting amphetamine (AMP) or methamphetamine (MET). Only two (p-methoxyamphetamine and p-methoxymethamphetamine) of all tested amphetamines gave a positive result. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Recently, the diffusion on the drug market of new psychoactive substances not controlled and often sold as ‘legal highs’ is exponentially increasing in Europe. ‘Legal highs’ are substances of synthetic or natural origin having psychotropic properties. More than 280 legal highs and other new psychoactive synthetic drugs are actually monitored by EU drug agency [1]. Generally, the first analysis for these drugs involves an immunoassay screening in urine or plasma. If the screening test is positive, additional tests by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) must be performed. Actually, there is growing interest in the use of oral fluid (OF) as alternative specimen over conventional biological fluids (urine, blood) for drug testing. The great advantages of these matrix are a non-invasive collection under direct observation without undue embarrassment or invasion of privacy, and a good correlation with plasma analytical data [2–4]. Drug concentrations in OF reflect the free, unbound parent drug and lipophilic metabolites circulating in the blood. Since these are the forms of the drug which cross the blood–brain barrier and effect performance and behavior, OF is a ⇑ Corresponding author. Tel.: +39 079228725; fax: +39 079228727. E-mail addresses: [email protected] (M. Nieddu), [email protected] (L. Burrai), [email protected] (C. Trignano), [email protected] (G. Boatto). 1344-6223/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.legalmed.2013.12.001
good specimen for detecting drug involvement in driving behavior or impairment of performance. Drug and lipophilic metabolite concentrations in OF are a function of the drug’s pKa, plasma, and OF pH and the fraction of drug bound to OF and plasma protein [2]. Italian legislation on traffic safety has recently accepted oral fluid as a possible alternative biological sample for toxicological analysis [5]. The national guidelines establishing how to perform the analysis and the practical issues regarding which device and cut-off values should be used are however still undetermined. There have been numerous studies regarding the performance characteristics of commonly used OFDs for known abuse drugs [6–9]. Most of these studies reported good agreement between screening and confirmatory results. Differently, few assays have been developed for detection of new psychoactive compounds; it is important to investigate how new ‘legal highs’ may or may not react in pre-existing and broad diffused commercial immunoassay kits. The main objective of this study is to evaluate whether two commercial OFDs (ScreenÒ Multi-Drug OFD and GIMA One Step Multi-Line Screen Test OFD) can be an effective alternative for screening thirty-nine new amphetamine designer drugs. The ability and degree of reliability of these devices in spiked samples are examined. Between the thirty-nine amphetamine compounds analyzed, twelve (PMA, PMMA, 2,5-DMA, DOB, DOM, DOET, 2C-B, 2C-I, 2C-T-2, 2C-T-7, TMA, TMA-2) are already classified as
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psychotropic substances by Italian legislation [10,11], so their consume and diffusion are punishable by law. A low cross-reactivity towards these compounds may result in these abused substances not being detected in forensic case samples.
2. Materials and methods 2.1. Chemicals and reagents Hydrochloride salt forms of p-methoxyamphetamine (PMA), p-methoxymethamphetamine (PMMA), 2,5-dimethoxy-amphetamine (2,5-DMA), 2,5-dimethoxy-4-bromoamphetamine (DOB), 2, 5-dimethoxy-4-chloroamphetamine (DOC), 2,5-dimethoxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-iodoamphetamine (DOI), 2,5-dimethoxy-4-methylamphetamine (DOM), 2,5-dimethoxy-4nitroamphetamine (DON), 2,5-dimethoxy-4-propylamphetamine (DOPR), 2,5-dimethoxy-4-bromophenethylamine (2C-B), 2,5-dimethoxy-4-iodophenethylamine (2C-I), 2,5-dimethoxy-4nitrophenethylamine (2C-N), 2,5-dimethoxy-4-methylphenethylamine (2C-M), 2,5-dimethoxy-4-methylthiophenethylamine (2CT), 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), 2,5-dimethoxy-4cyclo hexylthiophenethylamine (2C-T-5), 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), 2,5-dimethoxy-4-(2-methoxyethyl) thiophenethylamine (2C-T-13), 2,5-dimethoxy-4-isobutylthiophenethylamine (2C-T-17), 2,5-dimethoxy-4-methylthioamphetamine (ALEPH), 2,5-dimethoxy-4-ethylthioamphetamine (ALEPH-2),
2,5-dimethoxy-4-cyclohexylthioamphetamine (ALEPH-5), 2,5-dimethoxy-4-propylthioamphetamine (ALEPH-7), 2,5-dimethoxy-4-cyclopropylmethylthioamphetamine (ALEPH-8), 2,5-dime thoxy-4-(2-methoxyethyl) thioamphetamine (ALEPH-13), 2,5dimethoxy-4-isobutylthioamphetamine (ALEPH-17), 3,4,5-trimeth oxyamphetamine (TMA), 2,4,5-trimethoxyamphetamine (TMA-2), 2,3,4-trimethoxyamphetamine (TMA-3), 2,4,6-trimethoxyamphet amine (TMA-6), 3,4-methylenedioxy-N-isopropylamphetamine (MDIP), 3,4-methylenedioxy-N-benzylamphetamine(MDBZ), 3,4-methylenedioxy-N-cyclopropylmethylamphetamine (MDCPM), 2-(3, 4-methylenedioxyphenyl)-2-methoxyethylamine (BOH), 4methyl-2, 5-dimethoxy-b-hydroxyphenethylamine (BOHD), 2-(4bromo-2, 5-dimethylenedioxyphenyl)-2-methoxyethylamine (BOB), 2-(2,5-dimethoxy-4methylphenyl)-2-methoxyethylamine (BOD) were synthesized in our laboratory according to the methods of Shulgin and Shulgin [12]. Aqueous stock solutions of all the mentioned amphetamines were prepared at 10 lg/ml, and they were diluted to appropriate concentrations (10; 20; 50; 100; 200; 1000; 2000; 5000 ng/ml) with a drug-free pooled oral fluid sample collected by 10 volunteers. 2.2. Immunoassay OF tests Oral fluid screening tests were performed using ScreenÒ MultiDrug OFD (SCREEN ITALIA Srl, Perugia, Italy), and GIMA One Step Multi-Line Screen Test OFD (GIMA Spa, Gessate, Milan, Italy), according to the manufacturer’s instructions [13,14]. These multi-drug tests are made to detect amphetamine (AMP), cocaine
Table 1 Cross-reactivities of thirty-nine amphetamine designer drugs on ScreenÒ test. Analyte
p-Methoxyamphetamine (PMA) p-Methoxymethamphetamine (PMMA) 2,5-Dimethoxyamphetamine (2,5-DMA) 2,5-Dimethoxy-4-bromoamphetamine (DOB) 2,5-Dimethoxy-4-chloroamphetamine (DOC) 2,5-Dimethoxy-4-ethylamphetamine (DOET) 2,5-Dimethoxy-4-iodoamphetamine (DOI) 2,5-Dimethoxy-4-methylamphetamine (DOM) 2,5-Dimethoxy-4-nitroamphetamine (DON) 2,5-Dimethoxy-4-propylamphetamine (DOPR) 2,5-Dimethoxy-4-bromophenethylamine (2C-B) 2,5-Dimethoxy-4-iodophenethylamine (2C-I) 2,5-Dimethoxy-4-nitrophenethylamine (2C-N) 2,5-Dimethoxy-4-methylphenethylamine (2C-M) 2,5-Dimethoxy-4-methylthiophenethylamine (2C-T) 2,5-Dimethoxy-4-ethylthiophenethylamine (2C-T-2) 2,5-Dimethoxy-4-isopropylthiophenethylamine (2C-T-4) 2,5-Dimethoxy-4-cyclohexylthiophenethylamine (2C-T-5) 2,5-Dimethoxy-4-n-propylthiophenethylamine (2C-T-7) 2,5-Dimethoxy-4-(2-methoxyethyl)thiophenethylamine (2C-T-13) 2,5-Dimethoxy-4-i-butylthiophenethylamine (2C-T-17) 2,5-Dimethoxy-4-methylthioamphetamine (ALEPH) 2,5-Dimethoxy-4-ethylthioamphetamine (ALEPH-2) 2,5-Dimethoxy-4-cyclohexylthioamphetamine (ALEPH-5) 2,5-Dimethoxy-4-n-propylthioamphetamine (ALEPH-7) 2,5-Dimethoxy-4-cyclopropylmethylthioamphetamine (ALEPH-8) 2,5-Dimethoxy-4-(2-methoxyethyl) thioamphetamine (ALEPH-13) 2,5-Dimethoxy-4-isobutylthioamphetamine (ALEPH-17) 3,4,5-Trimethoxyamphetamine (TMA) 2,4,5-Trimethoxyamphetamine (TMA-2) 2,3,4-Trimethoxyamphetamine (TMA-3) 2,4,6-Trimethoxyamphetamine (TMA-6) 3,4-Methylenedioxy-N-isopropylamphetamine (MDIP) 3,4-Methylenedioxy-N-benzylamphetamine(MDBZ) 3,4-Methylenedioxy-N-cyclopropylmethylamphetamine (MDCPM) 2-(3,4-Methylenedioxyphenyl)-2-methoxyethylamine (BOH) 2-(4-Bromo-2,5-dimethylenedioxyphenyl)-2-methoxyethylamine (BOB) 2-(2,5-Dimethoxy-4methylphenyl)-2-methoxyethylamine (BOD) 2-(2,5-Dimethoxy-4methylphenyl)-2-idroxyethylamine (BOHD)
% Cross-reactivity AMP
MET
25 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
<1 250 <5 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
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Table 2 Cross-reactivities of thirty-nine amphetamine designer drugs on GIMA test. Analyte
% Cross-reactivity
p-Methoxyamphetamine (PMA) p-Methoxymethamphetamine (PMMA) 2,5-Dimethoxyamphetamine (2,5-DMA) 2,5-Dimethoxy-4-bromoamphetamine (DOB) 2,5-Dimethoxy-4-chloroamphetamine (DOC) 2,5-Dimethoxy-4-ethylamphetamine (DOET) 2,5-Dimethoxy-4-iodoamphetamine (DOI) 2,5-Dimethoxy-4-methylamphetamine (DOM) 2,5-Dimethoxy-4-nitroamphetamine (DON) 2,5-Dimethoxy-4-propylamphetamine (DOPR) 2,5-Dimethoxy-4-bromophenethylamine (2C-B) 2,5-Dimethoxy-4-iodophenethylamine (2C-I) 2,5-Dimethoxy-4-nitrophenethylamine (2C-N) 2,5-Dimethoxy-4-methylphenethylamine (2C-M) 2,5-Dimethoxy-4-methylthiophenethylamine (2C-T) 2,5-Dimethoxy-4-ethylthiophenethylamine (2C-T-2) 2,5-Dimethoxy-4-isopropylthiophenethylamine (2C-T-4) 2,5-Dimethoxy-4-cyclohexylthiophenethylamine (2C-T-5) 2,5-Dimethoxy-4-n-propylthiophenethylamine (2C-T-7) 2,5-Dimethoxy-4-(2-methoxyethyl)thiophenethylamine (2C-T-13) 2,5-Dimethoxy-4-i-butylthiophenethylamine (2C-T-17) 2,5-Dimethoxy-4-methylthioamphetamine (ALEPH) 2,5-Dimethoxy-4-ethylthioamphetamine (ALEPH-2) 2,5-Dimethoxy-4-cyclohexylthioamphetamine (ALEPH-5) 2,5-Dimethoxy-4-n-propylthioamphetamine (ALEPH-7) 2,5-Dimethoxy-4-cyclopropylmethylthioamphetamine (ALEPH-8) 2,5-Dimethoxy-4-(2-methoxyethyl) thioamphetamine (ALEPH-13) 2,5-Dimethoxy-4-isobutylthioamphetamine (ALEPH-17) 3,4,5-Trimethoxyamphetamine (TMA) 2,4,5-Trimethoxyamphetamine (TMA-2) 2,3,4-Trimethoxyamphetamine (TMA-3) 2,4,6-Trimethoxyamphetamine (TMA-6) 3,4-Methylenedioxy-N-isopropylamphetamine (MDIP) 3,4-Methylenedioxy-N-benzylamphetamine(MDBZ) 3,4-Methylenedioxy-N-cyclopropylmethylamphetamine (MDCPM) 2-(3,4-Methylenedioxyphenyl)-2-methoxyethylamine (BOH) 2-(4-Bromo-2,5-dimethylenedioxyphenyl)-2-methoxyethylamine (BOB) 2-(2,5-Dimethoxy-4methylphenyl)-2-methoxyethylamine (BOD) 2-(2,5-Dimethoxy-4methylphenyl)-2-idroxyethylamine (BOHD)
(COC), methamphetamine (MET), tetrahydrocannabinol (THC), phencyclidine (PCP), and opiates (OPI), and are based on the principle of competitive binding: drugs which may be present in the oral fluid specimen compete against their respective drug conjugate for binding sites on their specific antibody. A drug, if present in the oral fluid specimen below its cutoff concentration, is not able to saturate the binding site of its specific antibody. Measurements were performed without the need of instruments. Oral fluid samples have been collected in a clean and dry container and analyzed at room temperature (25 °C) within 1 h from the specimen collection. The sponge end of the collector has been vertically immersed into the oral fluid sample until the sponge is completely saturated. Results were read at 10 min. The absence or the presence of a colored line, even a faint line, in the test line region, indicates a positive or a negative result, respectively; the presence of a colored line in the control line region is required to validate both negative and positive results. Each measurement was performed in triplicate (n = 3). The cross-reactivity was calculated using Eq. (1), where C50 was the concentration of the drug necessary to produce a positive response, equivalent to 50 ng/ml of target drug. For this purpose, the concentration of 50 ng/ml was chosen as the manufacturer’s recommended cutoff:
% Cross reactivity ¼
50 100 C 50
ð1Þ
AMP
MET
50 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
<1 25 <5 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1
3. Results and discussion The cross-reactivity profiles of the thirty-nine amphetamines studied on two commercial OFDs are shown in Tables 1 and 2. All tests gave overlapped results with minimal variations regarding PMA and PMMA, as we expected considering PMA and PMMA are p-substituted analogous of amphetamine and methamphetamine, and cross-reacted to AMP and MET assay, respectively. Regarding ScreenÒ Multi-Drug device, PMA showed cross-reactivity four times lower than amphetamine (% cross-reactivity = 25) whereas PMMA had a cross-reactivity two-half times greater than methamphetamine (% cross-reactivity = 250). However, tests conducted by GIMA device showed slightly different % cross-reactivity, 50 for PMA and 25 for PMMA, corresponding to a concentration of 100 and 200 ng/ml, respectively. Concentrations of 5000 ng/ml were not sufficient to produce a positive response for all other amphetamines analyzed. This clearly demonstrates that although these kits are extremely effective for the target drugs for which they were intended (amphetamine and methamphetamine), they cannot be used to reliably identify all designers drugs studied, even at concentrations that would greatly exceed those expected in case samples [15]. These results demonstrate that some substances officially considered illicit by Italian law (2,5-DMA, DOET, DOM, 2C-B, 2C-I, 2C-T-2, 2C-T-7, TMA and TMA-2) [10,11] and recently appeared in clandestine market, can give negative results with screening
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tests commonly used. These data are significantly important from forensic and toxicological point of view because a negative result is not examined with additional test of confirm. For this reason, when circumstances surrounding the case suggest the usage of drugs, also a negative result must be investigated by instrumental techniques based on mass spectrometric detection. 4. Conclusions This study verifies the cross-reactivities of thirty-nine new amphetamine-designer drugs on two commercial OFDs (ScreenÒ Multi-Drug OFD and GIMA One Step Multi-Line Screen Test OFD). The present data can offer useful information when it is needed to explain the screening results in drug testing laboratories: results showed cross-reactivity <1 towards most drugs analyzed in assays targeting amphetamine (AMP) or methamphetamine (MET). Only two (p-methoxyamphetamine and p-methoxymethamphetamine) of the thirty-nine amphetamines tested gave a positive result. For identifying these drugs, the screening tests commercially available are often not sufficient. This evidences the need to develop new kits that enable to control the misuse of new amphetamine-like drugs. Acknowledgement The authors are grateful to English Centre of Sassari University for the English editing of the manuscript. References [1] European Drug Report. Trends and developments. Lisbon: EMCDDA; 2013. [2] Spiehler V, Baldwin D, Hand C. Cut-off concentrations for drugs of abuse in saliva for DUI, DWI or other driving-related crimes. Probl Forensic Sci 2000;XLII:160–8. [3] Kintz P, Samyn N. Use of alternative specimens: drugs of abuse in saliva and doping agents in hair. Ther Drug Monit 2002;24:239–46.
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