Journal of Chromatography A, 1372 (2014) 145–156
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Screening for new psychoactive substances in hair by ultrahigh performance liquid chromatography–electrospray ionization tandem mass spectrometry夽 Sabina Strano-Rossi a,∗ , Sara Odoardi a , Marco Fisichella a , Luca Anzillotti a , Rossella Gottardo b , Franco Tagliaro b a b
Institute of Public Health, Section of Legal Medicine, Catholic University of Sacred Heart, Rome, Italy Department of Public Health and Community Medicine, Section of Legal Medicine, University of Verona, Verona, Italy
a r t i c l e
i n f o
Article history: Received 2 September 2014 Received in revised form 28 October 2014 Accepted 31 October 2014 Available online 6 November 2014 Keywords: Hair analysis NPS Synthetic cannabinoids Amphetamine-type substances UHPLC–MS/MS Forensic toxicology
a b s t r a c t In the latest years, many new psychoactive substances (NPS) from several drug classes have appeared in the illicit drug market. Their rapid, sensitive and specific identification in biological fluids is hence of great concern for clinical and forensic toxicologists. Here is described a multi-analyte method for the determination of NPS, pertaining to different chemical classes (synthetic cannabinoids, synthetic cathinones, ketamine, piperazines and amphetamine-type substances—ATS) in human hair using ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) in electrospray ionization mode. We focused on a sample preparation able to extract the different classes of NPS. About 30 mg of hair was decontaminated and incubated overnight under sonication in different conditions depending on the type of analytes to be extracted: (a) with 300 L of HCOOH 0.1% for cathinones, piperazines and ATS; (b) with 300 L of MeOH for synthetic cannabinoids. Ten microliter of the extracts were then injected in UHPLC–ESI–MS/MS in MRM mode. The LODs varied from 2 pg/mg to 20 pg/mg. The method was linear in the range from the LOQ to 500 pg/mg and showed acceptable precision (%RSD < 15) and accuracy (%E < 15) for all the analytes. The method was finally applied on 50 samples from real forensic cases (driving license re-granting, postmortem toxicological analyses, workplace drug testing). In three samples we detected synthetic cannabinoids, in four samples cathinones or ephedrines, in two samples ketamine. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In the last decade many NPS with different chemical structures have appeared in the illicit drug market. These substances belong to different drug classes, including synthetic cannabinoids, synthetic cathinones, ketamines, phenethylamines, piperazines, substances not pertaining to any of these groups and plant-based materials. The easy distribution of NPS through the e-commerce and in the smart shops favored their rapid spreading worldwide. According to the 2013 World Drug Report, the number of NPS reported by member states to the United Nations Office on Drugs and Crime (UNODC) rose from 166 at the end of 2009 to 252 by mid-2012 [1],
夽 “Presented at 38th International Symposium on Capillary Chromatography and 11th GCxGC Symposium, 18 - 23 May 2014, Riva del Garda, Italy.” Item Group Code IG002313. ∗ Corresponding author. Tel.: +39 0630156098; fax: +39 063051168. E-mail addresses: [email protected], [email protected] (S. Strano-Rossi). http://dx.doi.org/10.1016/j.chroma.2014.10.106 0021-9673/© 2014 Elsevier B.V. All rights reserved.
an increase of more than 50 per cent. For the first time, the number of NPS exceeded the total number of substances under international control. Their identification in biological fluids/tissues is hence of great concern for forensic and clinical toxicologists, in order to evaluate the spread of NPS among population, and to diagnose intoxications and impairment due to the use of these substances. Analytical methods were developed for the identification of NPS in biological fluids, such as oral fluid [2–4], blood, plasma or serum [5–9], urine [10–12]. Head or body hair is a useful alternative biological matrix, allowing the determination of drugs that accumulate in keratinized tissues. Moreover hair samples permit a retrospective evaluation of the drug use history corresponding to several months before the actual sampling moment, depending essentially on hair length; this makes hair analysis a valuable tool to evaluate the spread of chronic use of drugs in a specific population. Other advantages linked to hair analysis are the easy, not invasive sample collection and the difficult sample adulteration. Furthermore, in hair (and in keratinized matrices in general) parent, un-metabolized drugs accumulate prevalently, in comparison with the corresponding
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metabolites [13]. Conversely, studies performed both in vivo and in vitro on some of the synthetic cannabinoids showed that they are extensively metabolized, and in many cases no parent compound is detectable in urine [14–18]. Thus, whereas the determination of synthetic cannabinoids in urine and blood will therefore be faced with the issue of identifying several metabolites often not well known, hair analysis can be focussed directly on the parent drug. Nevertheless, to date, only few studies deal with the determination of NPS in hair [19–24]. Two studies reported in the recent literature for the determination of synthetic cannabinoids used extraction methods based on incubation in concentrated sodium hydroxide solutions, providing the dissolution of the keratin matrix [19,20]. However, these procedures require a further extraction of the analytes from the aqueous solution, which increases the complexity of sample handling. Rust et al. described a two-step extraction of hair, the first one with absolute ethanol and the second with acidified ethanol for the screening of cathinones and piperazines [22]; the extracts were then evaporated to dryness and re-dissolved in mobile phase. They reported that the double extraction was necessary in order to extract all the compounds with adequate recovery. In another study, the analysis of cahinone, cathine and norephedrine was performed by incubation in an acidic aqueous solution and subsequent solid-phase extraction of the incubation mixture and derivatization prior to GC/MS analysis [25]. The present work was aimed at the development of a simple, high-throughput UHPLC–MS/MS screening method in MRM mode for the determination of NPS of different classes in hair matrix. The described method can be useful not only in the forensic investigation of NPS-related addiction histories, but also in epidemiological studies on the spread of NPS among specific safety-sensitive social groups, such as drivers and workers.
2. Materials and methods 2.1. Chemicals and reagents 1-[(5-Fluoropentyl)-1H-indol-3-yl]-(naphthalen-1-yl)methanone (AM22011), (2-Iodophenyl)(1-{[(2S)-1-methyl-2-piperidinyl]methyl}-1H-indol-3-yl)methanone (AM2233), [1-(5-fluoropentyl)-1H-indol-3-yl](2-iodophenyl)-methanone (AM694), 1– naphthalenyl[4-(pentyloxy)-1-naphthalenyl]-methanone (CB13), (2-methyl-1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-007), (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH-015), (1-butyl-2-methyl-1H-indol-3-yl)(1naphthyl)methanone (JWH-016), (1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-018), (1-hexyl-1H-indol-3-yl)-1naphthalenyl-methanone (JWH-019), 1-naphthalenyl(1-pentyl1H-pyrrol-3-yl) methanone (JWH-030), (1-butyl-1H-indol-3yl)-1-naphthalenyl-methanone (JWH-073), (4-methoxy-1-naphthalenyl)(1-pentyl-1H-indol-3-yl)methanone (JWH-081), (4-methoxy-1-naphthalenyl)(2-methyl-1-pentyl-1H-indol-3-yl)methanone (JWH-098), (4-methyl-1-naphthalenyl)(1-pentyl-1H-indol3-yl)methanone (JWH-122), (1-hexyl-5-phenyl-1H-pyrrol-3-yl) (1-naphthalenyl)methanone (JWH-147), {1-[2-(4-morpholinyl) ethyl]-1H-indol-3-yl}(1-naphthalenyl)methanone (JWH-200), 2(4-methoxyphenyl)-1-(1-phentyl-1H-indol-3-yl)-ethanone (JWH201), 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (JWH-250), 2-(2-methylphenyl)-1-(1-pentyl-1H-indol-3yl)-ethanone (JWH-251), 2-(3-methoxyphenyl)-1-(1pentyl-1H-indol-3yl)-ethanone (JWH-302), (5-(2-fluorophenyl)-1-pentylpyrrol-3yl)-naphthalen-1-yl-methanone (JWH-307), (4-chloronaphthalen1yl)(1-pentyl-1H-indole-3-yl)-methanone (JWH-398), (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)-methanone (RCS4), 1-(1-(2cyclohexylethyl)-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (RCS8), (4-methoxyphenyl)[(2-methyl)-1-[2-(4-morpholinyl)
ethyl]-1H-indol-3-yl]-methanone (WIN48,098), 1-(3-chlorophenyl)piperazine (1mCPP), 1-(3,4-dimethylphenyl)-2-(methylamino)propan-1-one (3,4-dimethylmethcathinone or 3,4-DMMC), 1(4-fluorophenyl)propan-2-amine (4-fluoroamphetamine or 4FA), 1-(4-methylphenyl)propan-2-amine (4-methyl amphetamine or 4MA), 2-(ethylamino)-1-(4-methylphenyl)propan-1-one (4methylethcathinone or 4MEC), benzylpiperazine (BZP), 2-(methylamino)-1-phenylbutan-1-one (buphedrone), 1-(1,3-benzodioxol5-yl)-2-(methylamino)butan-1-one (butylone), (1S,2S)-2-amino1-phenyl-propan-1-ol (cathine), (2S)-2-amino-1-phenyl-1-propanone (cathinone), (1R,2S)-2-(methylamino)-1-phenylpropan1-ol (ephedrine), 2-(ethylamino)-1-phenylpropan-1-one (ethylcathinone), 1-(1,3-benzodioxol-5-yl)-2-(ethylamino)propan-1one (ethylone), 1-(4-fluorophenyl)-2-(methylamino)propan-1one (flephedrone), 2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one (ketamine), 1-(1,3-benzodioxol-5-yl)-N-methyl-2-butanamine (methylbenzodioxolylbutanamine or MBDB), 6,7dihydro-5H-cyclopenta[f][1,3]benzodioxol-6-amine (5,6-methylenedioxy-2-aminoindane or MDAI), 1-(1,3-benzodioxol-5-yl)2-pyrrolidin-1-yl-pentan-1-one (methylenedioxypyrovalerone or MDPV), (RS)-1-(4-methylphenyl)-2-methylaminopropan-1-one (mephedrone), 1-(4-methoxyphenyl)-2-(methylamino)propan-1one (methedrone), 1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one (methylone), 1-[4-(methylthio)phenyl]propan-2amine (4-methylthioamphetamine or MTA), 1-naphthalen-2-yl2-pyrrolidin-1-yl-pentan-1-one (naphyrone), 2-(methylamino)-1phenylpentan-1-one (pentedrone), 1-(1,3-benzodioxol-5-yl)-2(methylamino)pentan-1-one (pentylone), (1S,2S)-2-(methylamino)-1-phenylpropan-1-ol (pseudoephedrine), 1-phenyl-2propanamine D5 (amphetamine D5), ketamine D4, methylone D4, tetrahydrocannabinol-D3 (THC D3), JWH 210-D9 were supplied from LGC standards (Milan, Italy). Water, acetonitrile, formic acid, acetone and methanol were purchased from 3V-Chemicals (Rome, Italy); ammonium formate was from Agilent (Agilent Technologies, Santa Clara, CA, USA). Tween 80, sodium hydroxide, hexane and ethyl acetate were from Sigma (Milan, Italy). All reagents and solvents were of LC/MS grade. Standard compounds were stored according to supplier recommendations until their use. 2.2. Sample preparation Two aliquots of 30 mg of hair were washed with 3 mL × 3 of a solution of TWEEN 80 × 0.1% for 3 min each, rinsed three times with 5 mL of distilled water and finally twice with 1 mL of acetone. After drying, each sample was cut with scissors into small pieces of 1 mm. Two different extractions of analytes from keratin matrix were tested: incubation under sonication overnight at 45 ◦ C with (A) 300 L of methanol; (B) 300 L of HCOOH 0.1%. The optimized procedure was as follows. For the extraction of synthetic cannabinoids, one aliquot of hair samples was added with 10 L of internal standard JWH 210-D9 (1 g/mL), 300 L of methanol and incubated under sonication overnight at 45 ◦ C. For the extraction of cathinones, ketamine, piperazines, stimulants and ATS, 30 mg of hair samples were added with 10 L of a mixture of internal standards amphetamine D5, ketamine D4 and methylone D4 (1 g/mL), 300 L HCOOH 0.1% and incubated under sonication overnight at 45 ◦ C. 2.3. Preparation of calibration curves Individual methanolic stock solutions containing 1 mg/mL of each of the listed standards were used to prepare two working mixtures of standards at 1 g/mL:
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MIX-CANNAB, containing AM22011, AM2233, AM694, CB13, JWH-007, JWH-019, JWH-015, JWH-016, JWH-018, JWH-030, JWH081, JWH-098, JWH-122, JWH-147, JWH-201, JWH-251, JWH-302, JWH-307, JWH-398, JWH-073, JWH-200, JWH-250, RCS4, RCS8, WIN48,098. MIX-STIMULANTS, containing mCPP, DMMC, 4FA, 4MA, 4MEC, BZP, buphedrone, butylone, cathinone, cathine, ephedrine, ethylcathinone, ethylone, flephedrone, ketamine, MBDB, MDAI, MDPV, mephedrone, metamfepramone, methedrone, methylone, MTA, naphyrone, pentedrone, pentylone, pseudoephedrine. A further dilution of each of the two mixtures at a concentration of 0.1 g/mL was prepared in methanol for the set-up of the lower points of calibration curves (5, 10 and 20 ng/pg). Stock and working solutions were stored at −20 ◦ C until use. Three drug-free hair samples were obtained from laboratory staff and used for the preparation of calibration curves and for matrix effect studies. Hair samples were previously tested with the described methods. No interferences were found with the investigated analytes. Calibration curves were prepared at the concentrations of 5, 10, 20, 50, 100 and 500 pg/mg as follows: for each of the calibration points 1.5–3–6–1.5–3–15 L of mixture of standards 0.1 g/mL (for 5, 10 and 20 ng/pg) or 1 g/mL (for 50, 100 and 500 pg/mg) were spiked with 10 L of mixture of internal standards and diluted in 100 L of methanol, then added to 30 mg of finely cut hair, vortexed and let dry before the extraction procedure. 2.4. UHPLC–MS/MS equipment and method The UHPLC instrument was an Agilent 1290 Infinity system: binary pump with integrated vacuum degasser, high performance well-plate autosampler and thermostatted column compartment modules. The detection system was an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a Jet-Stream electrospray ionization source. The column was a superficially porous Kinetex C18 column (2.6 m, 100 × 2.1 mm from Phenomenex, Bologna, Italy). The column temperature was set at 40 ◦ C and injection volume was 10 L. The mobile phases used were: (A) 5 mM ammonium formate containing 0.1% formic acid and (B) methanol/acetonitrile 1:1 with 0.1% of formic acid. Because of too different chemical structures, two different analytical methods were developed for the identification of synthetic cannabinoids (method cannabinoids) and for cathinones and other stimulants (method stimulants). The mobile phase gradient was from 45% to 100% B within 12 min, plus 3 min of equilibration, for cannabinoids analysis, and from 0% B to 90% B within 11 min, plus 3 min of equilibration for stimulants. For both methods the flow rate was set to 350 L/min and the eluate was introduced into the mass spectrometer by means of electrospray ionization (ESI) Jet-Stream in the positive mode. The optimized MS parameters were as follows: capillary voltage was set to 4000 V, the ion source was heated up to 350 ◦ C and nitrogen was used as nebulizing and collision gas at 12 L/min and 40 psi, respectively; EM voltage was set to +1000 V and nozzle voltage at 2000 V. The detector operated in Multiple Reaction Monitoring (MRM) mode. Specific MRM transitions and collision energies were determined through a series of experiments: determination of the optimal MRM transitions and respective collision energies for all compounds was carried out by consecutive injections analyses of the individual standards at a concentration of 1 g/mL, by using a specific Agilent optimizer software (Mass Hunter Optimizer). The optimal transitions, respective collision energies and retention times (RT) of all compounds are summarized in Table 1a (cannabinoids) and 1b (stimulants).
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2.5. Method validation The method was validated according to FDA guidelines [26]. The parameters studied are listed below. 2.5.1. Specificity The specificity was determined by analyzing 10 negative hair samples from laboratory personnel and verifying the absence of peaks that could interfere with the substances of interest. Satisfactory specificity was established if the signals relative to endogenous compounds did not interfere with those of any analyte of interest in terms of characteristic transitions and RT. Furthermore, specificity of the method was evaluated by analysing samples spiked with 100 pg/mg of compounds with analogue structures and with common illicit and therapeutic drugs (such as amphetamine and methamphetamine, MDMA, MDA, cocaine and metabolites, opiates, benzodiazepines, phosphodiesterase 5 inhibitors and antipsychotics). 2.5.2. Limit of detection LOD and limit of quantitation LOQ The LOD was figured out at a concentration value giving a S/N > 3 for at least three transitions for each substance. The LOQ was considered the concentration value giving a S/N > 10 for three transitions and acceptable accuracy and precision (%CV and %E 50%, ±25% for ions with relative
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Table 1 (a) Synthetic cannabinoids: MRM transitions, respective collision energies and fragmentor voltages applied, and retention times (Rt ). Analyte
MRM transitions
Collision energy
Fragmentor (V)
Rt (min)
AM2201 AM2233 AM694 CB13 JWH-007 JWH-015 JWH-016 JWH-018 JWH-019 JWH-030 JWH-073 JWH-081 JWH-098 JWH-122 JWH-147 JWH-200 JWH-201 JWH-250/JWH-302 JWH-251 JWH-307 JWH-398 RCS4 RCS8 WIN48098
360 → 155, 144, 232 459 → 112, 362, 98 436 → 231, 292, 203 369 → 155, 241, 299 356 → 155, 127, 228 328 → 200, 155, 127 342 → 155, 127,214 342 → 155, 127, 214 356 → 155, 127, 228 292 → 127, 155, 94 328 → 200, 155, 127 372 → 185, 268, 214 386 → 228, 185, 157 35677.0 → 169, 214, 141 382 → 127, 155 385 → 155, 127, 114 336 → 121, 149, 135 336 → 144, 130, 200 320 → 214, 143, 105 386 → 258, 155, 127 376 → 189,214,161 322 → 135,107,92 376 → 91, 144, 121 379 → 77, 107, 114
10 10 30, 40 (203) 10 20, 50 (127) 30 10 10 20, 50 (127) 30 50 20 20, 50 (157) 18 50 20 20 30 20 10, 50 (127) 20 50 50 50
135 150 135 100 100 100 135 135 100 150 45 100 100 20 100 40 100 45 100 135, 100 (127) 100 100 100 80
4.0 1.1 3.6 7.1 5.1 4.3 4.7 4.9 5.4 4.1 4.6 5.2 5.3 5.4 6.2 1.6 4.3 4.4 4.9 5.9 5.8 4.4 5.5 1.2
(b) Cathinones and other stimulants: MRM transitions, respective collision energies and fragmentor voltages applied, and retention times (Rt ) Analyte
MRM transitions
Collision energy
Fragmentor (V)
Rt (min)
1mCPP 3,4 DMMC 4-FA 4-MA 4-MEC Benzylpiperazine Buphedrone Butylone Cathine/norephedrine Cathinone Ephedrine Ethcathinone Ethylone Flephedrone Ketamine MBDB MDAI MDPV Mephedrone Methedrone Methylone MTA Naphyrone Pentedrone Pentylone Pseudoephedrine
197 → 154, 140, 119 192 → 174, 159, 115 154 → 109, 137, 121 150 → 105, 133, 120 192 → 144, 130, 119 177 → 91, 85, 65 178 → 160, 132, 91 222 → 174, 204, 161 152 → 134,117, 115 150 → 105, 117, 90 166 → 148, 117, 115 178 → 160, 133, 105 222 → 174, 204, 72 182 → 164,149, 123 238 → 220, 179, 124 208 → 135, 177, 147 178 → 161, 131, 103 276 → 126, 175, 135 178 → 160, 145, 91 194 → 176, 161, 145 208 → 160, 190, 132 182 → 165, 137, 117 282 → 211, 141, 126 192 → 174, 132, 91 236 → 188, 218, 175 166 → 148, 117, 115
20 10, 50 (115) 10, 14 (109) 20, 10 (133) 30 20 10 10 5, 17, 25 30 9, 17, 25 10 10 10 10, 14, 26 10, 14 (135) 10, 18, 30 26, 18 (175) 8, 20, 36 10 10 10, 18, 14 10 10 10 9, 17, 25
135 50 40 100,150 (120) 100 80, 120 (91) 20 100 66 100 81 50 100 20 45 45 45 45 10 25 135 45 50 25 100 81
7.8 7.1 5.6 7.2 6.9 3.4 6.4 6.4 3.9/3.6 4.5 4.5 5.7 5.7 5.4 7.5 7.4 5.2 7.8 7.2 6.8 6.1 7.7 8.1 7.3 7.6 4.8
intensities between 10 and 50% and ±50% for ions with relative intensities 0.99 for all the analytes, and showing acceptable precision (%RSD < 15) and accuracy (%E < 15%) for all analytes. The results of the studies on the matrix effect are shown in Table 2, in which a value of 100% means no matrix effect, whereas 100% signal enhancement. Matrix effect was moderate for cathinones and stimulants, ranging from a 39% of ion suppression at low concentrations to 11% of ion enhancement. For synthetic cannabinoids, ion suppression was higher, but still acceptable, ranging from 80% to 20%. No significant interferences with the peaks from endogenous components of the hair matrix were found. Structural analogues and common medicaments did not interfere with the analytes of interest. No memory effect was observed, after the injection of 500 and 1000 pg/mg spiked sample. Figs. 1 and 2 show the extracted ionic chromatograms of blank samples spiked with synthetic cannabinoids and cathinones and other stimulants, respectively, at 100 pg/mg. Consecutive extractions performed in duplicate on authentic samples performed to evaluate the extraction efficiency demonstrated, in case of synthetic cannabinoids, that aqueous formic acid solution did not extract detectable amount of these analytes from an authentic sample positive for JWH-018 and JWH-073. On the contrary the hair extraction using methanol gave a mean yield of 80% for the cannabinoids in the first extraction, 19% in the second, 1% in the third and traces below LOQ in the fourth. The analysis performed after dissolving the keratin by an alkaline digestion showed that no residual synthetic cannabinoids were present in hair matrix after the methanolic extractions. In the case of two samples containing pseudoephedrine and cathine, the extraction in acidic aqueous solution gave a mean recovery of analytes of
Fig. 1. Extracted ionic chromatogram of a blank hair sample spiked with synthetic cannabinoids at 100 pg/mg.
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Fig. 2. Extracted ionic chromatogram of a blank hair sample spiked with cathinones and other stimulants at 100 pg/mg.
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Fig. 2. (Continued ).
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Table 2 (a) Limits of detection, quantification, matrix effect and linearity (curve equation and correlation coefficient). Analyte
LOD (pg/mg)
LOQ (pg/mg)
AM 2201 AM 2233 AM 694 CB 13 JWH 007 JWH 015 JWH 016 JWH 018 JWH 019 JWH 030 JWH 073 JWH 081 JWH 098 JWH 122 JWH 147 JWH 200 JWH 201 JWH 250 JWH 251 JWH 307 JWH 398 RCS 4 RCS 8 WIN 48098
10 10 10 10 5 2 5 5 5 10 10 5 5 10 10 5 2 10 10 10 5 10 5 5
10 20 20 20 10 10 10 10 20 10 20 20 20 20 20 10 10 20 20 20 10 20 10 20
Matrix effect (%) 20 pg/mg
100 pg/mg
500 pg/mg
54 55 43 19 42 40 22 79 20 53 35 29 21 20 35 85 31 33 24 56 21 36 21 54
18 63 19 25 32 21 34 40 21 59 21 35 22 17 20 31 22 11 24 40 26 19 21 31
74 78 80 36 40 59 34 65 41 83 49 40 26 24 38 84 52 46 28 68 36 33 29 64
Curve equation
R2
Y = 5.3838x − 0.0944 Y = 15.4985x + 0.0198 Y = 66.621x + 0.7893 Y = 6.9128x − 0.0394 Y = 24.7994x − 0.1830 Y = 65.1670x − 0.0726 Y = 15.7673x + 0.2960 Y = 15.4202x + 1.7730 Y = 23.4544x − 0.7867 Y = 28.8562x + 0.1030 Y = 55.0970x + 2.0676 Y = 101.222x + 1.0237 Y = 21.5370x − 0.0761 Y = 8.7546x + 0.5079 Y = 24.4850x + 0.1507 Y = 137.32x + 1.8611 Y = 118.77x + 0.7966 Y = 57.3728x + 0.6330 Y = 11.1112x + 0.3522 Y = 6.6492x − 0.0061 Y = 16.9521x + 0.5360 Y = 7.2949x + 0.1339 Y = 68.2567x − 0.8070 Y = 58.6963x + 0.9665
0.9937 0.9971 0.9940 0.9965 0.9999 0.9989 0.9994 0.9998 0.9989 0.9964 0.9914 0.9999 0.9989 0.9967 0.9989 0.9960 0.9996 0.9996 0.9977 0.9979 0.9999 0.9980 0.9972 0.9991
(b) Limits of detection, quantification, matrix effect and linearity (curve equation and correlation coefficient) Analyte
LOD (pg/mg)
LOQ (pg/mg)
Matrix effect (%) 20 pg/mg
1mcPP 3,4 DMMC 4-FA 4-MA 4-MEC Benzylpiperazine Buphedrone Butylone Cathinone Cathine Ephedrine Ethcathinone Ethylone Flephedrone Ketamine MBDB MDAI MDPV Mephedrone Methedrone Methylone MTA Naphyrone Pentedrone Pentylone Pseudoephedrine
5 5 2 5 5 5 20 5 10 2 2 20 2 5 2 2 2 2 2 2 2 2 10 2 2 2
20 20 5 10 20 20 20 20 20 10 5 20 5 10 10 5 5 5 20 20 20 20 20 20 20 5
59 81 73 94 84 71 81 71 93 59 89 84 88 92 90 79 66 66 81 81 78 59 52 76 75 100
88% in the first extraction, 10% in the second extraction and less than 2% in the third and fourth extractions. In one sample positive for ketamine, the recovery was about 90% in the first extraction using formic acid, less than 10% in the second and traces below LOQ in the third extraction. No residual analytes were detectable after dissolution of the keratin. The developed method was hence applied on 50 authentic samples from forensic cases, in order to evaluate its suitability for the screening of NPSs in hair samples. Three samples resulted positive for more than one synthetic cannabinoids: in one case JWH-073 (below LOQ), JWH-018 (20 pg/mg), JWH-081 (470 pg/mg) and AM694 (30 pg/mg) were determined. In another case, JWH-073
100 pg/mg
500 pg/mg
81 95 91 90 103 92 102 92 111 93 100 100 101 101 98 96 90 93 103 102 94 95 91 96 86 97
94 99 98 100 104 98 105 93 99 95 104 105 104 104 98 99 97 97 108 106 105 98 90 90 86 104
Curve equation
R2
Y = 0.4080x − 0.0005 Y = 2.3287x − 0.0094 Y = 1.6977x − 0.0287 Y = 0.2463x + 0.0033 Y = 0.7932x − 0.0152 Y = 1.6601x − 0.0536 Y = 0.8909x − 0.0210 Y = 3.2404x − 0.0327 Y = 1.4547x − 0.093 Y = 0.0665x + 0.0009 Y = 3.5259x − 0.0147 Y = 1.1433x − 0.0231 Y = 0.0178x − 0.0005 Y = 0.5410x − 0.0120 Y = 3.7588x − 0.0686 Y = 7.6680x − 0.0017 Y = 0.9695x − 0.0076 Y = 1.2270x − 0.0213 Y = 0.9659x − 0.0150 Y = 1.2653x − 0.0217 Y = 0.2950x − 0.0075 Y = 0.4780x − 0.0024 Y = 0.1196x − 0.0043 Y = 0.6603x − 0.0106 Y = 1.9607x − 0.0265 Y = 7.6167x + 0.0965
0.9992 0.9999 0.9997 0.9999 0.9999 0.9994 0.9999 0.9996 0.9963 0.9985 0.9999 0.9989 0.9999 0.9981 0.9977 0.9985 0.9992 0.9990 0.9987 0.9986 0.9976 0.9982 0.9938 0.9978 0.9972 0.9942
was detected at 2.1 ng/mg and JWH-018 at 90 pg/mg. The third case showed the presence of JWH-073, JWH-018 and JWH-081 in traces below LOQ. Three samples were positive for cathinones: MDPV at a concentration of 50 pg/mg in one sample, 4-MEC in two samples, at a concentration below LOQ in one case and at 26 pg/mg in another case. In two samples was detected ketamine at 390 and 200 pg/mg, respectively. In two samples were detected pseudoephedrine and cathine at 1.1 ng/mg and 1.5 ng/mg and 120 and 100 pg/mg, respectively. For analytes at concentrations above the calibration curve, analyses were repeated using a lesser amount of hair samples (10 mg). The sample containing ketamine showed also the presence of
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Table 3 (a) Intraday and interday reproducibility (expressed as relative standard deviation) and accuracy at low, medium and high concentrations. Analyte
AM 2233 AM 694 CB 13 JWH-007 JWH-015 JWH-016 JWH-018 JWH-019 JWH-030 JWH-073 JWH-081 JWH-098 JWH-122 JWH-147 JWH-200 JWH-201 JWH-250 JWH-251 JWH-302 JWH-307 JWH-398 RCS 4 RCS 8 WIN 48098
20 pg/mg
100 pg/mg
%RSD intraday
%RSD interday
3 3 5 4 3 9 8 10 4 4 7 11 9 2 4 4 8 10 3 9 5 10 7 5
8 10 12 8 2 7 10 7 10 11 1 9 7 13 13 10 12 7 5 9 9 12 15 5
E% −1 7 11 2 4 1 4 −3 7 −2 0.4 −3 −2 14 17 −7 6 2 12 9 −1 0.3 6 10
500 pg/mg
%RSD intraday
%RSD interday
2 3 3 6 1 2 5 2 2 2 2 3 3 1 7 2 4 3 2 2 3 1 2 5
2 11 8 6 9 8 6 7 6 4 10 11 11 4 10 11 0.4 12 4 7 10 9 7 6
E% 9 −3 −6 3 −2 1 0.1 6 −7 8 2 9 9 −4 −12 11 7 3 −6 3 13 7 −4 −1
%RSD intraday 2 0.3 1 1 2 1 2 0.3 1 1 1 1 2 1 1 1 1 3 1 1 1 1 2 1
%RSD interday
E% −1 1 1 −0.2 1 −0.1 0.2 −1 1 −1 −0.2 −1 −1 1 2 −2 −0.4 −0.2 1 0.3 −2 −1 1 1
3 2 6 1 1 1 2 3 1 3 1 2 2 1 6 2 −2 2 1 1 1 2 2 2
(b) Intraday and interday reproducibility (expressed as relative standard deviation) and accuracy at low, medium and high concentrations Analyte
1mcPP 3,4 DMMC 4-FA 4-MA 4-MEC Benzylpiperazine Buphedrone Butylone Cathinone Cathine Ephedrine Ethcathinone Ethylone Flephedrone Ketamine MBDB MDAI MDPV Mephedrone Methedrone Methylone MTA Naphyrone Pentedrone Pentylone Pseudoephedrine
20 pg/mg
100 pg/mg
%RSD intraday
%RSD interday
E%
12 10 5 10 4 11 7 12 14 15 14 12 14 12 14 1 6 13 6 13 12 11 17 16 16 3
3 15 15 6 13 11 15 12 6 13 7 2 14 5 13 13 8 14 9 12 6 12 14 1 9 9
14 12 7 −3 11 0.3 −10 −5 −2 −13 −7 −11 −3 −7 11 10 −8 8 −5 −7 5 8 9 −3 7 9
500 pg/mg
%RSD intraday 0.2 12 15 4 1 0.4 3 8 13 15 13 2 11 2 7 1 3 7 1 1 9 8 8 10 7 10
ephedrine, pseudoephedrine and norephedrine. Fig. 3 shows the extracted ionic chromatogram of a hair where JWH-081, JWH-018, JWH-073 and AM694 were detected. Fig. 4 shows the extracted ionic chromatogram of a hair sample from a forensic case showing the presence of ketamine and ephedrines (ephedrine, pseudoephedrine and norephedrine). 4. Discussion One of the challenges of identification of NSP in biological samples is represented by the need of recognizing, detecting and discriminating the largest number of substances, in some cases structurally correlated to each other, which can potentially be taken
%RSD interday 12 13 6 11 3 7 8 7 10 9 11 5 9 6 6 4 8 1 4 3 1 5 4 15 10 3
E% −4 −9 −11 −1 0.3 −5 2 −12 −5 −5 −7 1 −8 −3 6 4 −2 5 0.2 −1 −9 4 −3 −8 −3 3
%RSD intraday 3 3 3 2 1 7 1 2 6 3 5 1 4 3 3 2 0.3 4 1 1 2 4 2 3 2 2
%RSD interday 3 8 1 1 3 3 1 2 2 2 1 1 1 0.2 4 1 1 1 1 1 1 1 7 8 3 6
E% 2 1 1 −0.1 −0.4 5 −1 2 0.5 −1 −0.5 −1 1 −0.3 2 −1 0.01 −0.3 −0.3 −0.4 1 −1 6 1 2 1
by an individual. Hence, the approach of choice is the determination of a large number of analytes using a single instrumental method and a minimum of sample pretreatment. At present, although a few immunochemical assays can detect some NPS, chromatographymass spectrometry is the gold standard for the detection of a sufficiently vast panel of drugs employing the minimum number of analytical runs. Taking into consideration these issues, in the present work an UHPLC–MS/MS screening approach for the determination of NPS of different chemical classes in hair matrix has been developed and validated. The original goal of the present study was the development of a single and rapid analytical method for the screening of NPS belonging to different chemical classes (synthetic
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Fig. 3. Extracted ionic chromatogram of an authentic hair sample positive for JWH-081, JWH-018, JWH-073 and AM694.
cannabinoids, cathinones, ketamine, amphetamine-type stimulants ATS). Unfortunately, the preliminary results obtained during the development of the UHPLC–MS/MS method and the hair sample pretreatment showed that two different procedures were needed to obtain optimal recoveries and sensitivities for the different classes of compounds. Synthetic cannabinoids could be better separated and detected using a gradient of mobile phases starting with a high percent of organic component (phase B); furthermore, their
extraction from keratins had to be performed using an organic solvent, as they are practically insoluble in water. Extraction tests performed on authentic samples using the acidified water showed almost no recovery. On the contrary, the separation of cathinones and ATS were enhanced starting the chromatographic run with 100% phase A. Also, an extraction from the hair matrix with acidic aqueous solution produced a very clean extract with a negligible matrix effect. Consequently two separate extractions and analytical
Fig. 4. Extracted ionic chromatogram of an authentic hair sample showing the presence of ketamine, ephedrine, norephedrine and pseudoephedrine.
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methods for the determination of cannabinoids and cathinones and other stimulants were used. The proposed analytical approach hence includes two different extractions and chromatographic procedures in UHPLC–MS/MS in order to obtain the best separations and sensitivities for the different classes of analytes. In our hands, the main advantage of the splitted procedures is the simplicity of sample preparation and the rapidity of the chromatographic runs. In fact, although a double analysis is needed for cannabinoids and stimulants, the sample extraction can be performed in parallel, after which the extraction medium can be directly injected into the UHPLC–MS/MS system, with analytical runs lasting less than 15 min for both chromatographic separations. LODs and LOQs obtained using this approach were similar to those reported in the recent literature for the determination of synthetic cannabinoids, where it was performed an incubation in concentrated sodium hydroxide solution, that provides a dissolution of the keratin matrix, with a higher complexity of sample handling [19,20]. In those case LODs obtained were comparable, being around 10 pg/mg, and LOQs ranged from 10 to 20 pg/mg [19] or slightly better, probably due to the different analytical instrumentation used [20]. Similarly, the LODs obtained by Rust et al. performing a double ethanolic extraction of hair, are similar to those obtained in our study, ranging from 10 to 50 pg/mg [22]. Using the extraction of cathinones and stimulants in acidic water, better sensitivities for cathinones than those reported by Rust et al. was found, probably caused by a lower ion suppression. Moreover, this sample preparation was simpler. In fact, the aqueous extract from hair incubation was very “clean”, providing a negligible matrix effect. On the contrary, synthetic cannabinoids, required a solvent extraction with, in some cases, a more pronounced ion suppression. However, the sensitivities obtained were adequate for the detection of these analyte at low pg/mg levels. One of the main issues in hair analysis is the difficulty in evaluating the performance of the extraction procedure. This can be evaluated only on authentic positive samples. Lacking any certified reference hair with known drug content, consecutive extractions were performed (in duplicate) on authentic samples which demonstrated, in case of synthetic cannabinoids, that aqueous formic acid solution does not extract detectable amounts of these analytes from an authentic sample positive for JWH-018 and JWH-073. On the contrary, methanol extraction gave satisfactory extractions yields (mean 80%) for the cannabinoids already in the first extraction. On the other hand, acidic aqueous extraction of cathinones and other stimulants gave a mean recovery of about 90% from the first extraction. The studies performed on authentic samples to evaluate the practical application of the method in the “real world”, showed the suitability of the proposed analytical method for the identification of NPS in hair. Interestingly, the samples positive for synthetic cannabinoids resulted negative for natural cannabinoids; in the same way, both the samples positive for synthetic cathinones were negative for other drugs of abuse. This phenomenon is in agreement with the theory that in many cases consumers shift from traditional drugs to different NPS, in order to escape the toxicological screenings performed on the drivers and in the workplace.
5. Conclusions Hair analysis is a valuable tool for the identification of substance abuse among the populations, showing a particularly high potential for the identification of NPS users. Hair analysis for NPS,
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however, is still in an early stage of development, particularly on the toxicological screening side. The proposed method allows for the identification of synthetic cannabinoids, cathinones and other stimulants in hair samples with a simple sample pre-treatment, thanks to the use of UHPLC–MS/MS. Notwithstanding a rough preanalytical phase, this high-throughput chromatography enables the identification of a large panel of NPS belonging to different classes with satisfactory sensitivity, as it was demonstrated also on authentic samples. On the grounds of the presented data, hair analysis with UHPLC–MS/MS can become be ideal tool for the surveillance of the spreading of the NPS in the population. Acknowledgments This study was performed under the framework of the project “Nepra”, funded by the Italian Antidrug Policies Department, Rome. References [1] UNODC, World Drug Report 2013 (United Nations publications, Sale No. E.13.XI.6, UNODC, 2013, http://www.unodc.org/unodc/secured/ wdr/wdr2013/World Drug Report 2013.pdf (last accessed July 7th, 2014). [2] C. Coulter, M. Garnier, C. Moore, Synthetic cannabinoids in oral fluid, J. Anal. Toxicol. 35 (2011) 424–430. [3] S. Strano-Rossi, L. Anzillotti, E. Castrignano, F.S. Romolo, M. Chiarotti, Ultra high performance liquid chromatography–electrospray ionization-tandem mass spectrometry screening method for direct analysis of designer drugs, spice and stimulants in oral fluid, J. Chromatogr. A 1258 (2012) 37–42. [4] S. Kneisel, V. Auwarter, J. Kempf, Analysis of 30 synthetic cannabinoids in oral fluid using liquid chromatography–electrospray ionization tandem mass spectrometry, Drug Test Anal. 5 (2013) 657–669. [5] M.J. Swortwood, D.M. Boland, A.P. DeCaprio, Determination of 32 cathinone derivatives and other designer drugs in serum by comprehensive LC–QQQ–MS/MS analysis, Anal. Bioanal. Chem. 405 (2013) 1383–1397. [6] A. Wohlfarth, W. Weinmann, S. Dresen, LC–MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum, Anal. Bioanal. Chem. 396 (2010) 2403–2414. [7] S. Dresen, S. Kneisel, W. Weinmann, R. Zimmermann, V. Auwarter, Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples, J. Mass Spectrom. 46 (2011) 163–171. [8] D. Ammann, J.M. McLaren, D. Gerostamoulos, J. Beyer, Detection and quantification of new designer drugs in human blood: Part 2—Designer cathinones, J. Anal. Toxicol. 36 (2012) 381–389. [9] J. Ammann, J.M. McLaren, D. Gerostamoulos, J. Beyer, Detection and quantification of new designer drugs in human blood: Part 1—Synthetic cannabinoids, J. Anal. Toxicol. 36 (2012) 372–380. [10] M. Sundstrom, A. Pelander, V. Angerer, M. Hutter, S. Kneisel, I. Ojanpera, A highsensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC–HR–TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine, Anal. Bioanal. Chem. 405 (2013) 8463–8474. [11] M. Concheiro, S. Anizan, K. Ellefsen, M.A. Huestis, Simultaneous quantification of 28 synthetic cathinones and metabolites in urine by liquid chromatography–high resolution mass spectrometry, Anal. Bioanal. Chem. 405 (2013) 9437–9448. [12] C. Bell, C. George, A.T. Kicman, A. Traynor, Development of a rapid LC–MS/MS method for direct urinalysis of designer drugs, Drug Test Anal. 3 (2011) 496–504. [13] A.C. Moffat, D. Osselton, B. Widdop, E.G.C. Clarke, Methodology and analytical techniques. Hair analysis, Clarke’s analysis of drugs and poisons, in: Pharmaceuticals Body Fluids and Postmortem Material, Pharmaceutical Press, London, 2004. [14] T. Sobolevsky, I. Prasolov, G. Rodchenkov, Detection of JWH-018 metabolites in smoking mixture post-administration urine, Forensic Sci. Int. 200 (2010) 141–147. [15] A. Grigoryev, A. Melnik, S. Savchuk, A. Simonov, V. Rozhanets, Gas and liquid chromatography–mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking mixtures, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 879 (2011) 2519–2526. [16] P. Adamowicz, D. Zuba, K. Sekula, Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine, Forensic Sci. Int. 233 (2013) 320–327. [17] P. Kavanagh, A. Grigoryev, A. Melnik, S. Savchuk, A. Simonov, V. Rozhanets, Detection and tentative identification of urinary phase I metabolites of phenylacetylindole cannabimimetics JWH-203 and JWH-251, by GC–MS and LC–MS/MS, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 934 (2013) 102–108.
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[18] A. Grigoryev, P. Kavanagh, A. Melnik, The detection of the urinary metabolites of 1-[(5-fluoropentyl)-1H-indol-3-yl]-(2-iodophenyl)methanone (AM-694), a high affinity cannabimimetic, by gas chromatography–mass spectrometry, Drug Test Anal. 5 (2013) 110–115. [19] R. Gottardo, D. Sorio, G. Musile, E. Trapani, C. Seri, G. Serpelloni, F. Tagliaro, Screening for synthetic cannabinoids in hair by using LC–QTOF MS: a new and powerful approach to study the penetration of these new psychoactive substances in the population, Med. Sci. Law 54 (2014) 22–27. [20] A. Salomone, C. Luciano, D. Di Corcia, E. Gerace, M. Vincenti, Hair analysis as a tool to evaluate the prevalence of synthetic cannabinoids in different populations of drug consumers, Drug Test Anal. 6 (2014) 126–134. [21] M. Hutter, S. Kneisel, V. Auwarter, M.A. Neukamm, Determination of 22 synthetic cannabinoids in human hair by liquid chromatography–tandem mass spectrometry, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 903 (2012) 95–101. [22] K.Y. Rust, M.R. Baumgartner, A.M. Dally, T. Kraemer, Prevalence of new psychoactive substances: a retrospective study in hair, Drug Test Anal. 4 (2012) 402–408. [23] M. Martin, J.F. Muller, K. Turner, M. Duez, V. Cirimele, Evidence of mephedrone chronic abuse through hair analysis using GC/MS, Forensic Sci. Int. 218 (2012) 44–48.
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Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Screening for new psychoactive substances in hair by ultrahigh performance liquid chromatography–electrospray ionization tandem mass spectrometry夽 Sabina Strano-Rossi a,∗ , Sara Odoardi a , Marco Fisichella a , Luca Anzillotti a , Rossella Gottardo b , Franco Tagliaro b a b
Institute of Public Health, Section of Legal Medicine, Catholic University of Sacred Heart, Rome, Italy Department of Public Health and Community Medicine, Section of Legal Medicine, University of Verona, Verona, Italy
a r t i c l e
i n f o
Article history: Received 2 September 2014 Received in revised form 28 October 2014 Accepted 31 October 2014 Available online 6 November 2014 Keywords: Hair analysis NPS Synthetic cannabinoids Amphetamine-type substances UHPLC–MS/MS Forensic toxicology
a b s t r a c t In the latest years, many new psychoactive substances (NPS) from several drug classes have appeared in the illicit drug market. Their rapid, sensitive and specific identification in biological fluids is hence of great concern for clinical and forensic toxicologists. Here is described a multi-analyte method for the determination of NPS, pertaining to different chemical classes (synthetic cannabinoids, synthetic cathinones, ketamine, piperazines and amphetamine-type substances—ATS) in human hair using ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) in electrospray ionization mode. We focused on a sample preparation able to extract the different classes of NPS. About 30 mg of hair was decontaminated and incubated overnight under sonication in different conditions depending on the type of analytes to be extracted: (a) with 300 L of HCOOH 0.1% for cathinones, piperazines and ATS; (b) with 300 L of MeOH for synthetic cannabinoids. Ten microliter of the extracts were then injected in UHPLC–ESI–MS/MS in MRM mode. The LODs varied from 2 pg/mg to 20 pg/mg. The method was linear in the range from the LOQ to 500 pg/mg and showed acceptable precision (%RSD < 15) and accuracy (%E < 15) for all the analytes. The method was finally applied on 50 samples from real forensic cases (driving license re-granting, postmortem toxicological analyses, workplace drug testing). In three samples we detected synthetic cannabinoids, in four samples cathinones or ephedrines, in two samples ketamine. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In the last decade many NPS with different chemical structures have appeared in the illicit drug market. These substances belong to different drug classes, including synthetic cannabinoids, synthetic cathinones, ketamines, phenethylamines, piperazines, substances not pertaining to any of these groups and plant-based materials. The easy distribution of NPS through the e-commerce and in the smart shops favored their rapid spreading worldwide. According to the 2013 World Drug Report, the number of NPS reported by member states to the United Nations Office on Drugs and Crime (UNODC) rose from 166 at the end of 2009 to 252 by mid-2012 [1],
夽 “Presented at 38th International Symposium on Capillary Chromatography and 11th GCxGC Symposium, 18 - 23 May 2014, Riva del Garda, Italy.” Item Group Code IG002313. ∗ Corresponding author. Tel.: +39 0630156098; fax: +39 063051168. E-mail addresses: [email protected], [email protected] (S. Strano-Rossi). http://dx.doi.org/10.1016/j.chroma.2014.10.106 0021-9673/© 2014 Elsevier B.V. All rights reserved.
an increase of more than 50 per cent. For the first time, the number of NPS exceeded the total number of substances under international control. Their identification in biological fluids/tissues is hence of great concern for forensic and clinical toxicologists, in order to evaluate the spread of NPS among population, and to diagnose intoxications and impairment due to the use of these substances. Analytical methods were developed for the identification of NPS in biological fluids, such as oral fluid [2–4], blood, plasma or serum [5–9], urine [10–12]. Head or body hair is a useful alternative biological matrix, allowing the determination of drugs that accumulate in keratinized tissues. Moreover hair samples permit a retrospective evaluation of the drug use history corresponding to several months before the actual sampling moment, depending essentially on hair length; this makes hair analysis a valuable tool to evaluate the spread of chronic use of drugs in a specific population. Other advantages linked to hair analysis are the easy, not invasive sample collection and the difficult sample adulteration. Furthermore, in hair (and in keratinized matrices in general) parent, un-metabolized drugs accumulate prevalently, in comparison with the corresponding
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metabolites [13]. Conversely, studies performed both in vivo and in vitro on some of the synthetic cannabinoids showed that they are extensively metabolized, and in many cases no parent compound is detectable in urine [14–18]. Thus, whereas the determination of synthetic cannabinoids in urine and blood will therefore be faced with the issue of identifying several metabolites often not well known, hair analysis can be focussed directly on the parent drug. Nevertheless, to date, only few studies deal with the determination of NPS in hair [19–24]. Two studies reported in the recent literature for the determination of synthetic cannabinoids used extraction methods based on incubation in concentrated sodium hydroxide solutions, providing the dissolution of the keratin matrix [19,20]. However, these procedures require a further extraction of the analytes from the aqueous solution, which increases the complexity of sample handling. Rust et al. described a two-step extraction of hair, the first one with absolute ethanol and the second with acidified ethanol for the screening of cathinones and piperazines [22]; the extracts were then evaporated to dryness and re-dissolved in mobile phase. They reported that the double extraction was necessary in order to extract all the compounds with adequate recovery. In another study, the analysis of cahinone, cathine and norephedrine was performed by incubation in an acidic aqueous solution and subsequent solid-phase extraction of the incubation mixture and derivatization prior to GC/MS analysis [25]. The present work was aimed at the development of a simple, high-throughput UHPLC–MS/MS screening method in MRM mode for the determination of NPS of different classes in hair matrix. The described method can be useful not only in the forensic investigation of NPS-related addiction histories, but also in epidemiological studies on the spread of NPS among specific safety-sensitive social groups, such as drivers and workers.
2. Materials and methods 2.1. Chemicals and reagents 1-[(5-Fluoropentyl)-1H-indol-3-yl]-(naphthalen-1-yl)methanone (AM22011), (2-Iodophenyl)(1-{[(2S)-1-methyl-2-piperidinyl]methyl}-1H-indol-3-yl)methanone (AM2233), [1-(5-fluoropentyl)-1H-indol-3-yl](2-iodophenyl)-methanone (AM694), 1– naphthalenyl[4-(pentyloxy)-1-naphthalenyl]-methanone (CB13), (2-methyl-1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-007), (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH-015), (1-butyl-2-methyl-1H-indol-3-yl)(1naphthyl)methanone (JWH-016), (1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-018), (1-hexyl-1H-indol-3-yl)-1naphthalenyl-methanone (JWH-019), 1-naphthalenyl(1-pentyl1H-pyrrol-3-yl) methanone (JWH-030), (1-butyl-1H-indol-3yl)-1-naphthalenyl-methanone (JWH-073), (4-methoxy-1-naphthalenyl)(1-pentyl-1H-indol-3-yl)methanone (JWH-081), (4-methoxy-1-naphthalenyl)(2-methyl-1-pentyl-1H-indol-3-yl)methanone (JWH-098), (4-methyl-1-naphthalenyl)(1-pentyl-1H-indol3-yl)methanone (JWH-122), (1-hexyl-5-phenyl-1H-pyrrol-3-yl) (1-naphthalenyl)methanone (JWH-147), {1-[2-(4-morpholinyl) ethyl]-1H-indol-3-yl}(1-naphthalenyl)methanone (JWH-200), 2(4-methoxyphenyl)-1-(1-phentyl-1H-indol-3-yl)-ethanone (JWH201), 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (JWH-250), 2-(2-methylphenyl)-1-(1-pentyl-1H-indol-3yl)-ethanone (JWH-251), 2-(3-methoxyphenyl)-1-(1pentyl-1H-indol-3yl)-ethanone (JWH-302), (5-(2-fluorophenyl)-1-pentylpyrrol-3yl)-naphthalen-1-yl-methanone (JWH-307), (4-chloronaphthalen1yl)(1-pentyl-1H-indole-3-yl)-methanone (JWH-398), (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)-methanone (RCS4), 1-(1-(2cyclohexylethyl)-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (RCS8), (4-methoxyphenyl)[(2-methyl)-1-[2-(4-morpholinyl)
ethyl]-1H-indol-3-yl]-methanone (WIN48,098), 1-(3-chlorophenyl)piperazine (1mCPP), 1-(3,4-dimethylphenyl)-2-(methylamino)propan-1-one (3,4-dimethylmethcathinone or 3,4-DMMC), 1(4-fluorophenyl)propan-2-amine (4-fluoroamphetamine or 4FA), 1-(4-methylphenyl)propan-2-amine (4-methyl amphetamine or 4MA), 2-(ethylamino)-1-(4-methylphenyl)propan-1-one (4methylethcathinone or 4MEC), benzylpiperazine (BZP), 2-(methylamino)-1-phenylbutan-1-one (buphedrone), 1-(1,3-benzodioxol5-yl)-2-(methylamino)butan-1-one (butylone), (1S,2S)-2-amino1-phenyl-propan-1-ol (cathine), (2S)-2-amino-1-phenyl-1-propanone (cathinone), (1R,2S)-2-(methylamino)-1-phenylpropan1-ol (ephedrine), 2-(ethylamino)-1-phenylpropan-1-one (ethylcathinone), 1-(1,3-benzodioxol-5-yl)-2-(ethylamino)propan-1one (ethylone), 1-(4-fluorophenyl)-2-(methylamino)propan-1one (flephedrone), 2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one (ketamine), 1-(1,3-benzodioxol-5-yl)-N-methyl-2-butanamine (methylbenzodioxolylbutanamine or MBDB), 6,7dihydro-5H-cyclopenta[f][1,3]benzodioxol-6-amine (5,6-methylenedioxy-2-aminoindane or MDAI), 1-(1,3-benzodioxol-5-yl)2-pyrrolidin-1-yl-pentan-1-one (methylenedioxypyrovalerone or MDPV), (RS)-1-(4-methylphenyl)-2-methylaminopropan-1-one (mephedrone), 1-(4-methoxyphenyl)-2-(methylamino)propan-1one (methedrone), 1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one (methylone), 1-[4-(methylthio)phenyl]propan-2amine (4-methylthioamphetamine or MTA), 1-naphthalen-2-yl2-pyrrolidin-1-yl-pentan-1-one (naphyrone), 2-(methylamino)-1phenylpentan-1-one (pentedrone), 1-(1,3-benzodioxol-5-yl)-2(methylamino)pentan-1-one (pentylone), (1S,2S)-2-(methylamino)-1-phenylpropan-1-ol (pseudoephedrine), 1-phenyl-2propanamine D5 (amphetamine D5), ketamine D4, methylone D4, tetrahydrocannabinol-D3 (THC D3), JWH 210-D9 were supplied from LGC standards (Milan, Italy). Water, acetonitrile, formic acid, acetone and methanol were purchased from 3V-Chemicals (Rome, Italy); ammonium formate was from Agilent (Agilent Technologies, Santa Clara, CA, USA). Tween 80, sodium hydroxide, hexane and ethyl acetate were from Sigma (Milan, Italy). All reagents and solvents were of LC/MS grade. Standard compounds were stored according to supplier recommendations until their use. 2.2. Sample preparation Two aliquots of 30 mg of hair were washed with 3 mL × 3 of a solution of TWEEN 80 × 0.1% for 3 min each, rinsed three times with 5 mL of distilled water and finally twice with 1 mL of acetone. After drying, each sample was cut with scissors into small pieces of 1 mm. Two different extractions of analytes from keratin matrix were tested: incubation under sonication overnight at 45 ◦ C with (A) 300 L of methanol; (B) 300 L of HCOOH 0.1%. The optimized procedure was as follows. For the extraction of synthetic cannabinoids, one aliquot of hair samples was added with 10 L of internal standard JWH 210-D9 (1 g/mL), 300 L of methanol and incubated under sonication overnight at 45 ◦ C. For the extraction of cathinones, ketamine, piperazines, stimulants and ATS, 30 mg of hair samples were added with 10 L of a mixture of internal standards amphetamine D5, ketamine D4 and methylone D4 (1 g/mL), 300 L HCOOH 0.1% and incubated under sonication overnight at 45 ◦ C. 2.3. Preparation of calibration curves Individual methanolic stock solutions containing 1 mg/mL of each of the listed standards were used to prepare two working mixtures of standards at 1 g/mL:
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MIX-CANNAB, containing AM22011, AM2233, AM694, CB13, JWH-007, JWH-019, JWH-015, JWH-016, JWH-018, JWH-030, JWH081, JWH-098, JWH-122, JWH-147, JWH-201, JWH-251, JWH-302, JWH-307, JWH-398, JWH-073, JWH-200, JWH-250, RCS4, RCS8, WIN48,098. MIX-STIMULANTS, containing mCPP, DMMC, 4FA, 4MA, 4MEC, BZP, buphedrone, butylone, cathinone, cathine, ephedrine, ethylcathinone, ethylone, flephedrone, ketamine, MBDB, MDAI, MDPV, mephedrone, metamfepramone, methedrone, methylone, MTA, naphyrone, pentedrone, pentylone, pseudoephedrine. A further dilution of each of the two mixtures at a concentration of 0.1 g/mL was prepared in methanol for the set-up of the lower points of calibration curves (5, 10 and 20 ng/pg). Stock and working solutions were stored at −20 ◦ C until use. Three drug-free hair samples were obtained from laboratory staff and used for the preparation of calibration curves and for matrix effect studies. Hair samples were previously tested with the described methods. No interferences were found with the investigated analytes. Calibration curves were prepared at the concentrations of 5, 10, 20, 50, 100 and 500 pg/mg as follows: for each of the calibration points 1.5–3–6–1.5–3–15 L of mixture of standards 0.1 g/mL (for 5, 10 and 20 ng/pg) or 1 g/mL (for 50, 100 and 500 pg/mg) were spiked with 10 L of mixture of internal standards and diluted in 100 L of methanol, then added to 30 mg of finely cut hair, vortexed and let dry before the extraction procedure. 2.4. UHPLC–MS/MS equipment and method The UHPLC instrument was an Agilent 1290 Infinity system: binary pump with integrated vacuum degasser, high performance well-plate autosampler and thermostatted column compartment modules. The detection system was an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a Jet-Stream electrospray ionization source. The column was a superficially porous Kinetex C18 column (2.6 m, 100 × 2.1 mm from Phenomenex, Bologna, Italy). The column temperature was set at 40 ◦ C and injection volume was 10 L. The mobile phases used were: (A) 5 mM ammonium formate containing 0.1% formic acid and (B) methanol/acetonitrile 1:1 with 0.1% of formic acid. Because of too different chemical structures, two different analytical methods were developed for the identification of synthetic cannabinoids (method cannabinoids) and for cathinones and other stimulants (method stimulants). The mobile phase gradient was from 45% to 100% B within 12 min, plus 3 min of equilibration, for cannabinoids analysis, and from 0% B to 90% B within 11 min, plus 3 min of equilibration for stimulants. For both methods the flow rate was set to 350 L/min and the eluate was introduced into the mass spectrometer by means of electrospray ionization (ESI) Jet-Stream in the positive mode. The optimized MS parameters were as follows: capillary voltage was set to 4000 V, the ion source was heated up to 350 ◦ C and nitrogen was used as nebulizing and collision gas at 12 L/min and 40 psi, respectively; EM voltage was set to +1000 V and nozzle voltage at 2000 V. The detector operated in Multiple Reaction Monitoring (MRM) mode. Specific MRM transitions and collision energies were determined through a series of experiments: determination of the optimal MRM transitions and respective collision energies for all compounds was carried out by consecutive injections analyses of the individual standards at a concentration of 1 g/mL, by using a specific Agilent optimizer software (Mass Hunter Optimizer). The optimal transitions, respective collision energies and retention times (RT) of all compounds are summarized in Table 1a (cannabinoids) and 1b (stimulants).
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2.5. Method validation The method was validated according to FDA guidelines [26]. The parameters studied are listed below. 2.5.1. Specificity The specificity was determined by analyzing 10 negative hair samples from laboratory personnel and verifying the absence of peaks that could interfere with the substances of interest. Satisfactory specificity was established if the signals relative to endogenous compounds did not interfere with those of any analyte of interest in terms of characteristic transitions and RT. Furthermore, specificity of the method was evaluated by analysing samples spiked with 100 pg/mg of compounds with analogue structures and with common illicit and therapeutic drugs (such as amphetamine and methamphetamine, MDMA, MDA, cocaine and metabolites, opiates, benzodiazepines, phosphodiesterase 5 inhibitors and antipsychotics). 2.5.2. Limit of detection LOD and limit of quantitation LOQ The LOD was figured out at a concentration value giving a S/N > 3 for at least three transitions for each substance. The LOQ was considered the concentration value giving a S/N > 10 for three transitions and acceptable accuracy and precision (%CV and %E 50%, ±25% for ions with relative
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Table 1 (a) Synthetic cannabinoids: MRM transitions, respective collision energies and fragmentor voltages applied, and retention times (Rt ). Analyte
MRM transitions
Collision energy
Fragmentor (V)
Rt (min)
AM2201 AM2233 AM694 CB13 JWH-007 JWH-015 JWH-016 JWH-018 JWH-019 JWH-030 JWH-073 JWH-081 JWH-098 JWH-122 JWH-147 JWH-200 JWH-201 JWH-250/JWH-302 JWH-251 JWH-307 JWH-398 RCS4 RCS8 WIN48098
360 → 155, 144, 232 459 → 112, 362, 98 436 → 231, 292, 203 369 → 155, 241, 299 356 → 155, 127, 228 328 → 200, 155, 127 342 → 155, 127,214 342 → 155, 127, 214 356 → 155, 127, 228 292 → 127, 155, 94 328 → 200, 155, 127 372 → 185, 268, 214 386 → 228, 185, 157 35677.0 → 169, 214, 141 382 → 127, 155 385 → 155, 127, 114 336 → 121, 149, 135 336 → 144, 130, 200 320 → 214, 143, 105 386 → 258, 155, 127 376 → 189,214,161 322 → 135,107,92 376 → 91, 144, 121 379 → 77, 107, 114
10 10 30, 40 (203) 10 20, 50 (127) 30 10 10 20, 50 (127) 30 50 20 20, 50 (157) 18 50 20 20 30 20 10, 50 (127) 20 50 50 50
135 150 135 100 100 100 135 135 100 150 45 100 100 20 100 40 100 45 100 135, 100 (127) 100 100 100 80
4.0 1.1 3.6 7.1 5.1 4.3 4.7 4.9 5.4 4.1 4.6 5.2 5.3 5.4 6.2 1.6 4.3 4.4 4.9 5.9 5.8 4.4 5.5 1.2
(b) Cathinones and other stimulants: MRM transitions, respective collision energies and fragmentor voltages applied, and retention times (Rt ) Analyte
MRM transitions
Collision energy
Fragmentor (V)
Rt (min)
1mCPP 3,4 DMMC 4-FA 4-MA 4-MEC Benzylpiperazine Buphedrone Butylone Cathine/norephedrine Cathinone Ephedrine Ethcathinone Ethylone Flephedrone Ketamine MBDB MDAI MDPV Mephedrone Methedrone Methylone MTA Naphyrone Pentedrone Pentylone Pseudoephedrine
197 → 154, 140, 119 192 → 174, 159, 115 154 → 109, 137, 121 150 → 105, 133, 120 192 → 144, 130, 119 177 → 91, 85, 65 178 → 160, 132, 91 222 → 174, 204, 161 152 → 134,117, 115 150 → 105, 117, 90 166 → 148, 117, 115 178 → 160, 133, 105 222 → 174, 204, 72 182 → 164,149, 123 238 → 220, 179, 124 208 → 135, 177, 147 178 → 161, 131, 103 276 → 126, 175, 135 178 → 160, 145, 91 194 → 176, 161, 145 208 → 160, 190, 132 182 → 165, 137, 117 282 → 211, 141, 126 192 → 174, 132, 91 236 → 188, 218, 175 166 → 148, 117, 115
20 10, 50 (115) 10, 14 (109) 20, 10 (133) 30 20 10 10 5, 17, 25 30 9, 17, 25 10 10 10 10, 14, 26 10, 14 (135) 10, 18, 30 26, 18 (175) 8, 20, 36 10 10 10, 18, 14 10 10 10 9, 17, 25
135 50 40 100,150 (120) 100 80, 120 (91) 20 100 66 100 81 50 100 20 45 45 45 45 10 25 135 45 50 25 100 81
7.8 7.1 5.6 7.2 6.9 3.4 6.4 6.4 3.9/3.6 4.5 4.5 5.7 5.7 5.4 7.5 7.4 5.2 7.8 7.2 6.8 6.1 7.7 8.1 7.3 7.6 4.8
intensities between 10 and 50% and ±50% for ions with relative intensities 0.99 for all the analytes, and showing acceptable precision (%RSD < 15) and accuracy (%E < 15%) for all analytes. The results of the studies on the matrix effect are shown in Table 2, in which a value of 100% means no matrix effect, whereas 100% signal enhancement. Matrix effect was moderate for cathinones and stimulants, ranging from a 39% of ion suppression at low concentrations to 11% of ion enhancement. For synthetic cannabinoids, ion suppression was higher, but still acceptable, ranging from 80% to 20%. No significant interferences with the peaks from endogenous components of the hair matrix were found. Structural analogues and common medicaments did not interfere with the analytes of interest. No memory effect was observed, after the injection of 500 and 1000 pg/mg spiked sample. Figs. 1 and 2 show the extracted ionic chromatograms of blank samples spiked with synthetic cannabinoids and cathinones and other stimulants, respectively, at 100 pg/mg. Consecutive extractions performed in duplicate on authentic samples performed to evaluate the extraction efficiency demonstrated, in case of synthetic cannabinoids, that aqueous formic acid solution did not extract detectable amount of these analytes from an authentic sample positive for JWH-018 and JWH-073. On the contrary the hair extraction using methanol gave a mean yield of 80% for the cannabinoids in the first extraction, 19% in the second, 1% in the third and traces below LOQ in the fourth. The analysis performed after dissolving the keratin by an alkaline digestion showed that no residual synthetic cannabinoids were present in hair matrix after the methanolic extractions. In the case of two samples containing pseudoephedrine and cathine, the extraction in acidic aqueous solution gave a mean recovery of analytes of
Fig. 1. Extracted ionic chromatogram of a blank hair sample spiked with synthetic cannabinoids at 100 pg/mg.
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Fig. 2. Extracted ionic chromatogram of a blank hair sample spiked with cathinones and other stimulants at 100 pg/mg.
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Fig. 2. (Continued ).
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Table 2 (a) Limits of detection, quantification, matrix effect and linearity (curve equation and correlation coefficient). Analyte
LOD (pg/mg)
LOQ (pg/mg)
AM 2201 AM 2233 AM 694 CB 13 JWH 007 JWH 015 JWH 016 JWH 018 JWH 019 JWH 030 JWH 073 JWH 081 JWH 098 JWH 122 JWH 147 JWH 200 JWH 201 JWH 250 JWH 251 JWH 307 JWH 398 RCS 4 RCS 8 WIN 48098
10 10 10 10 5 2 5 5 5 10 10 5 5 10 10 5 2 10 10 10 5 10 5 5
10 20 20 20 10 10 10 10 20 10 20 20 20 20 20 10 10 20 20 20 10 20 10 20
Matrix effect (%) 20 pg/mg
100 pg/mg
500 pg/mg
54 55 43 19 42 40 22 79 20 53 35 29 21 20 35 85 31 33 24 56 21 36 21 54
18 63 19 25 32 21 34 40 21 59 21 35 22 17 20 31 22 11 24 40 26 19 21 31
74 78 80 36 40 59 34 65 41 83 49 40 26 24 38 84 52 46 28 68 36 33 29 64
Curve equation
R2
Y = 5.3838x − 0.0944 Y = 15.4985x + 0.0198 Y = 66.621x + 0.7893 Y = 6.9128x − 0.0394 Y = 24.7994x − 0.1830 Y = 65.1670x − 0.0726 Y = 15.7673x + 0.2960 Y = 15.4202x + 1.7730 Y = 23.4544x − 0.7867 Y = 28.8562x + 0.1030 Y = 55.0970x + 2.0676 Y = 101.222x + 1.0237 Y = 21.5370x − 0.0761 Y = 8.7546x + 0.5079 Y = 24.4850x + 0.1507 Y = 137.32x + 1.8611 Y = 118.77x + 0.7966 Y = 57.3728x + 0.6330 Y = 11.1112x + 0.3522 Y = 6.6492x − 0.0061 Y = 16.9521x + 0.5360 Y = 7.2949x + 0.1339 Y = 68.2567x − 0.8070 Y = 58.6963x + 0.9665
0.9937 0.9971 0.9940 0.9965 0.9999 0.9989 0.9994 0.9998 0.9989 0.9964 0.9914 0.9999 0.9989 0.9967 0.9989 0.9960 0.9996 0.9996 0.9977 0.9979 0.9999 0.9980 0.9972 0.9991
(b) Limits of detection, quantification, matrix effect and linearity (curve equation and correlation coefficient) Analyte
LOD (pg/mg)
LOQ (pg/mg)
Matrix effect (%) 20 pg/mg
1mcPP 3,4 DMMC 4-FA 4-MA 4-MEC Benzylpiperazine Buphedrone Butylone Cathinone Cathine Ephedrine Ethcathinone Ethylone Flephedrone Ketamine MBDB MDAI MDPV Mephedrone Methedrone Methylone MTA Naphyrone Pentedrone Pentylone Pseudoephedrine
5 5 2 5 5 5 20 5 10 2 2 20 2 5 2 2 2 2 2 2 2 2 10 2 2 2
20 20 5 10 20 20 20 20 20 10 5 20 5 10 10 5 5 5 20 20 20 20 20 20 20 5
59 81 73 94 84 71 81 71 93 59 89 84 88 92 90 79 66 66 81 81 78 59 52 76 75 100
88% in the first extraction, 10% in the second extraction and less than 2% in the third and fourth extractions. In one sample positive for ketamine, the recovery was about 90% in the first extraction using formic acid, less than 10% in the second and traces below LOQ in the third extraction. No residual analytes were detectable after dissolution of the keratin. The developed method was hence applied on 50 authentic samples from forensic cases, in order to evaluate its suitability for the screening of NPSs in hair samples. Three samples resulted positive for more than one synthetic cannabinoids: in one case JWH-073 (below LOQ), JWH-018 (20 pg/mg), JWH-081 (470 pg/mg) and AM694 (30 pg/mg) were determined. In another case, JWH-073
100 pg/mg
500 pg/mg
81 95 91 90 103 92 102 92 111 93 100 100 101 101 98 96 90 93 103 102 94 95 91 96 86 97
94 99 98 100 104 98 105 93 99 95 104 105 104 104 98 99 97 97 108 106 105 98 90 90 86 104
Curve equation
R2
Y = 0.4080x − 0.0005 Y = 2.3287x − 0.0094 Y = 1.6977x − 0.0287 Y = 0.2463x + 0.0033 Y = 0.7932x − 0.0152 Y = 1.6601x − 0.0536 Y = 0.8909x − 0.0210 Y = 3.2404x − 0.0327 Y = 1.4547x − 0.093 Y = 0.0665x + 0.0009 Y = 3.5259x − 0.0147 Y = 1.1433x − 0.0231 Y = 0.0178x − 0.0005 Y = 0.5410x − 0.0120 Y = 3.7588x − 0.0686 Y = 7.6680x − 0.0017 Y = 0.9695x − 0.0076 Y = 1.2270x − 0.0213 Y = 0.9659x − 0.0150 Y = 1.2653x − 0.0217 Y = 0.2950x − 0.0075 Y = 0.4780x − 0.0024 Y = 0.1196x − 0.0043 Y = 0.6603x − 0.0106 Y = 1.9607x − 0.0265 Y = 7.6167x + 0.0965
0.9992 0.9999 0.9997 0.9999 0.9999 0.9994 0.9999 0.9996 0.9963 0.9985 0.9999 0.9989 0.9999 0.9981 0.9977 0.9985 0.9992 0.9990 0.9987 0.9986 0.9976 0.9982 0.9938 0.9978 0.9972 0.9942
was detected at 2.1 ng/mg and JWH-018 at 90 pg/mg. The third case showed the presence of JWH-073, JWH-018 and JWH-081 in traces below LOQ. Three samples were positive for cathinones: MDPV at a concentration of 50 pg/mg in one sample, 4-MEC in two samples, at a concentration below LOQ in one case and at 26 pg/mg in another case. In two samples was detected ketamine at 390 and 200 pg/mg, respectively. In two samples were detected pseudoephedrine and cathine at 1.1 ng/mg and 1.5 ng/mg and 120 and 100 pg/mg, respectively. For analytes at concentrations above the calibration curve, analyses were repeated using a lesser amount of hair samples (10 mg). The sample containing ketamine showed also the presence of
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Table 3 (a) Intraday and interday reproducibility (expressed as relative standard deviation) and accuracy at low, medium and high concentrations. Analyte
AM 2233 AM 694 CB 13 JWH-007 JWH-015 JWH-016 JWH-018 JWH-019 JWH-030 JWH-073 JWH-081 JWH-098 JWH-122 JWH-147 JWH-200 JWH-201 JWH-250 JWH-251 JWH-302 JWH-307 JWH-398 RCS 4 RCS 8 WIN 48098
20 pg/mg
100 pg/mg
%RSD intraday
%RSD interday
3 3 5 4 3 9 8 10 4 4 7 11 9 2 4 4 8 10 3 9 5 10 7 5
8 10 12 8 2 7 10 7 10 11 1 9 7 13 13 10 12 7 5 9 9 12 15 5
E% −1 7 11 2 4 1 4 −3 7 −2 0.4 −3 −2 14 17 −7 6 2 12 9 −1 0.3 6 10
500 pg/mg
%RSD intraday
%RSD interday
2 3 3 6 1 2 5 2 2 2 2 3 3 1 7 2 4 3 2 2 3 1 2 5
2 11 8 6 9 8 6 7 6 4 10 11 11 4 10 11 0.4 12 4 7 10 9 7 6
E% 9 −3 −6 3 −2 1 0.1 6 −7 8 2 9 9 −4 −12 11 7 3 −6 3 13 7 −4 −1
%RSD intraday 2 0.3 1 1 2 1 2 0.3 1 1 1 1 2 1 1 1 1 3 1 1 1 1 2 1
%RSD interday
E% −1 1 1 −0.2 1 −0.1 0.2 −1 1 −1 −0.2 −1 −1 1 2 −2 −0.4 −0.2 1 0.3 −2 −1 1 1
3 2 6 1 1 1 2 3 1 3 1 2 2 1 6 2 −2 2 1 1 1 2 2 2
(b) Intraday and interday reproducibility (expressed as relative standard deviation) and accuracy at low, medium and high concentrations Analyte
1mcPP 3,4 DMMC 4-FA 4-MA 4-MEC Benzylpiperazine Buphedrone Butylone Cathinone Cathine Ephedrine Ethcathinone Ethylone Flephedrone Ketamine MBDB MDAI MDPV Mephedrone Methedrone Methylone MTA Naphyrone Pentedrone Pentylone Pseudoephedrine
20 pg/mg
100 pg/mg
%RSD intraday
%RSD interday
E%
12 10 5 10 4 11 7 12 14 15 14 12 14 12 14 1 6 13 6 13 12 11 17 16 16 3
3 15 15 6 13 11 15 12 6 13 7 2 14 5 13 13 8 14 9 12 6 12 14 1 9 9
14 12 7 −3 11 0.3 −10 −5 −2 −13 −7 −11 −3 −7 11 10 −8 8 −5 −7 5 8 9 −3 7 9
500 pg/mg
%RSD intraday 0.2 12 15 4 1 0.4 3 8 13 15 13 2 11 2 7 1 3 7 1 1 9 8 8 10 7 10
ephedrine, pseudoephedrine and norephedrine. Fig. 3 shows the extracted ionic chromatogram of a hair where JWH-081, JWH-018, JWH-073 and AM694 were detected. Fig. 4 shows the extracted ionic chromatogram of a hair sample from a forensic case showing the presence of ketamine and ephedrines (ephedrine, pseudoephedrine and norephedrine). 4. Discussion One of the challenges of identification of NSP in biological samples is represented by the need of recognizing, detecting and discriminating the largest number of substances, in some cases structurally correlated to each other, which can potentially be taken
%RSD interday 12 13 6 11 3 7 8 7 10 9 11 5 9 6 6 4 8 1 4 3 1 5 4 15 10 3
E% −4 −9 −11 −1 0.3 −5 2 −12 −5 −5 −7 1 −8 −3 6 4 −2 5 0.2 −1 −9 4 −3 −8 −3 3
%RSD intraday 3 3 3 2 1 7 1 2 6 3 5 1 4 3 3 2 0.3 4 1 1 2 4 2 3 2 2
%RSD interday 3 8 1 1 3 3 1 2 2 2 1 1 1 0.2 4 1 1 1 1 1 1 1 7 8 3 6
E% 2 1 1 −0.1 −0.4 5 −1 2 0.5 −1 −0.5 −1 1 −0.3 2 −1 0.01 −0.3 −0.3 −0.4 1 −1 6 1 2 1
by an individual. Hence, the approach of choice is the determination of a large number of analytes using a single instrumental method and a minimum of sample pretreatment. At present, although a few immunochemical assays can detect some NPS, chromatographymass spectrometry is the gold standard for the detection of a sufficiently vast panel of drugs employing the minimum number of analytical runs. Taking into consideration these issues, in the present work an UHPLC–MS/MS screening approach for the determination of NPS of different chemical classes in hair matrix has been developed and validated. The original goal of the present study was the development of a single and rapid analytical method for the screening of NPS belonging to different chemical classes (synthetic
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Fig. 3. Extracted ionic chromatogram of an authentic hair sample positive for JWH-081, JWH-018, JWH-073 and AM694.
cannabinoids, cathinones, ketamine, amphetamine-type stimulants ATS). Unfortunately, the preliminary results obtained during the development of the UHPLC–MS/MS method and the hair sample pretreatment showed that two different procedures were needed to obtain optimal recoveries and sensitivities for the different classes of compounds. Synthetic cannabinoids could be better separated and detected using a gradient of mobile phases starting with a high percent of organic component (phase B); furthermore, their
extraction from keratins had to be performed using an organic solvent, as they are practically insoluble in water. Extraction tests performed on authentic samples using the acidified water showed almost no recovery. On the contrary, the separation of cathinones and ATS were enhanced starting the chromatographic run with 100% phase A. Also, an extraction from the hair matrix with acidic aqueous solution produced a very clean extract with a negligible matrix effect. Consequently two separate extractions and analytical
Fig. 4. Extracted ionic chromatogram of an authentic hair sample showing the presence of ketamine, ephedrine, norephedrine and pseudoephedrine.
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methods for the determination of cannabinoids and cathinones and other stimulants were used. The proposed analytical approach hence includes two different extractions and chromatographic procedures in UHPLC–MS/MS in order to obtain the best separations and sensitivities for the different classes of analytes. In our hands, the main advantage of the splitted procedures is the simplicity of sample preparation and the rapidity of the chromatographic runs. In fact, although a double analysis is needed for cannabinoids and stimulants, the sample extraction can be performed in parallel, after which the extraction medium can be directly injected into the UHPLC–MS/MS system, with analytical runs lasting less than 15 min for both chromatographic separations. LODs and LOQs obtained using this approach were similar to those reported in the recent literature for the determination of synthetic cannabinoids, where it was performed an incubation in concentrated sodium hydroxide solution, that provides a dissolution of the keratin matrix, with a higher complexity of sample handling [19,20]. In those case LODs obtained were comparable, being around 10 pg/mg, and LOQs ranged from 10 to 20 pg/mg [19] or slightly better, probably due to the different analytical instrumentation used [20]. Similarly, the LODs obtained by Rust et al. performing a double ethanolic extraction of hair, are similar to those obtained in our study, ranging from 10 to 50 pg/mg [22]. Using the extraction of cathinones and stimulants in acidic water, better sensitivities for cathinones than those reported by Rust et al. was found, probably caused by a lower ion suppression. Moreover, this sample preparation was simpler. In fact, the aqueous extract from hair incubation was very “clean”, providing a negligible matrix effect. On the contrary, synthetic cannabinoids, required a solvent extraction with, in some cases, a more pronounced ion suppression. However, the sensitivities obtained were adequate for the detection of these analyte at low pg/mg levels. One of the main issues in hair analysis is the difficulty in evaluating the performance of the extraction procedure. This can be evaluated only on authentic positive samples. Lacking any certified reference hair with known drug content, consecutive extractions were performed (in duplicate) on authentic samples which demonstrated, in case of synthetic cannabinoids, that aqueous formic acid solution does not extract detectable amounts of these analytes from an authentic sample positive for JWH-018 and JWH-073. On the contrary, methanol extraction gave satisfactory extractions yields (mean 80%) for the cannabinoids already in the first extraction. On the other hand, acidic aqueous extraction of cathinones and other stimulants gave a mean recovery of about 90% from the first extraction. The studies performed on authentic samples to evaluate the practical application of the method in the “real world”, showed the suitability of the proposed analytical method for the identification of NPS in hair. Interestingly, the samples positive for synthetic cannabinoids resulted negative for natural cannabinoids; in the same way, both the samples positive for synthetic cathinones were negative for other drugs of abuse. This phenomenon is in agreement with the theory that in many cases consumers shift from traditional drugs to different NPS, in order to escape the toxicological screenings performed on the drivers and in the workplace.
5. Conclusions Hair analysis is a valuable tool for the identification of substance abuse among the populations, showing a particularly high potential for the identification of NPS users. Hair analysis for NPS,
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however, is still in an early stage of development, particularly on the toxicological screening side. The proposed method allows for the identification of synthetic cannabinoids, cathinones and other stimulants in hair samples with a simple sample pre-treatment, thanks to the use of UHPLC–MS/MS. Notwithstanding a rough preanalytical phase, this high-throughput chromatography enables the identification of a large panel of NPS belonging to different classes with satisfactory sensitivity, as it was demonstrated also on authentic samples. On the grounds of the presented data, hair analysis with UHPLC–MS/MS can become be ideal tool for the surveillance of the spreading of the NPS in the population. Acknowledgments This study was performed under the framework of the project “Nepra”, funded by the Italian Antidrug Policies Department, Rome. References [1] UNODC, World Drug Report 2013 (United Nations publications, Sale No. E.13.XI.6, UNODC, 2013, http://www.unodc.org/unodc/secured/ wdr/wdr2013/World Drug Report 2013.pdf (last accessed July 7th, 2014). [2] C. Coulter, M. Garnier, C. Moore, Synthetic cannabinoids in oral fluid, J. Anal. Toxicol. 35 (2011) 424–430. [3] S. Strano-Rossi, L. Anzillotti, E. Castrignano, F.S. Romolo, M. Chiarotti, Ultra high performance liquid chromatography–electrospray ionization-tandem mass spectrometry screening method for direct analysis of designer drugs, spice and stimulants in oral fluid, J. Chromatogr. A 1258 (2012) 37–42. [4] S. Kneisel, V. Auwarter, J. Kempf, Analysis of 30 synthetic cannabinoids in oral fluid using liquid chromatography–electrospray ionization tandem mass spectrometry, Drug Test Anal. 5 (2013) 657–669. [5] M.J. Swortwood, D.M. Boland, A.P. DeCaprio, Determination of 32 cathinone derivatives and other designer drugs in serum by comprehensive LC–QQQ–MS/MS analysis, Anal. Bioanal. Chem. 405 (2013) 1383–1397. [6] A. Wohlfarth, W. Weinmann, S. Dresen, LC–MS/MS screening method for designer amphetamines, tryptamines, and piperazines in serum, Anal. Bioanal. Chem. 396 (2010) 2403–2414. [7] S. Dresen, S. Kneisel, W. Weinmann, R. Zimmermann, V. Auwarter, Development and validation of a liquid chromatography–tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples, J. Mass Spectrom. 46 (2011) 163–171. [8] D. Ammann, J.M. McLaren, D. Gerostamoulos, J. Beyer, Detection and quantification of new designer drugs in human blood: Part 2—Designer cathinones, J. Anal. Toxicol. 36 (2012) 381–389. [9] J. Ammann, J.M. McLaren, D. Gerostamoulos, J. Beyer, Detection and quantification of new designer drugs in human blood: Part 1—Synthetic cannabinoids, J. Anal. Toxicol. 36 (2012) 372–380. [10] M. Sundstrom, A. Pelander, V. Angerer, M. Hutter, S. Kneisel, I. Ojanpera, A highsensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC–HR–TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine, Anal. Bioanal. Chem. 405 (2013) 8463–8474. [11] M. Concheiro, S. Anizan, K. Ellefsen, M.A. Huestis, Simultaneous quantification of 28 synthetic cathinones and metabolites in urine by liquid chromatography–high resolution mass spectrometry, Anal. Bioanal. Chem. 405 (2013) 9437–9448. [12] C. Bell, C. George, A.T. Kicman, A. Traynor, Development of a rapid LC–MS/MS method for direct urinalysis of designer drugs, Drug Test Anal. 3 (2011) 496–504. [13] A.C. Moffat, D. Osselton, B. Widdop, E.G.C. Clarke, Methodology and analytical techniques. Hair analysis, Clarke’s analysis of drugs and poisons, in: Pharmaceuticals Body Fluids and Postmortem Material, Pharmaceutical Press, London, 2004. [14] T. Sobolevsky, I. Prasolov, G. Rodchenkov, Detection of JWH-018 metabolites in smoking mixture post-administration urine, Forensic Sci. Int. 200 (2010) 141–147. [15] A. Grigoryev, A. Melnik, S. Savchuk, A. Simonov, V. Rozhanets, Gas and liquid chromatography–mass spectrometry studies on the metabolism of the synthetic phenylacetylindole cannabimimetic JWH-250, the psychoactive component of smoking mixtures, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 879 (2011) 2519–2526. [16] P. Adamowicz, D. Zuba, K. Sekula, Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine, Forensic Sci. Int. 233 (2013) 320–327. [17] P. Kavanagh, A. Grigoryev, A. Melnik, S. Savchuk, A. Simonov, V. Rozhanets, Detection and tentative identification of urinary phase I metabolites of phenylacetylindole cannabimimetics JWH-203 and JWH-251, by GC–MS and LC–MS/MS, J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 934 (2013) 102–108.
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